Je Remercie

Fernanda De Carvalho-Niebel

Valérie Hocher

Gabrielle Tichtinsky

Kevin Magne

Jean-Malo Couzigou

Katharina Schiessl

Jeoffrey George

Lucien Sahl

Frederic Boyer

Anelia Iantcheva

Kirankumar S Mysore

Jiangqi Wen

Vladimir Zhukov

Giles E D Oldroyd

Pascal Ratet

Systemic Acquired Resistance SEM Scanning Electron Microscopy SIFT Sorting Intolerant From Tolerant SM Spikelet Meristems SNP Single Nucleotide Polymorphism TALE THREE AMINO-ACID LOOP EXTENTION TGA

Keywords: Root nodule symbiosis, indeterminate nodule, meristematic domain, nodule identity, fix minus, evolution, development, NBCL gene, class II KNOX TF

INTRODUCTION

p. 1. Legume crops in sustainable agriculture p. 2. Generalities on plant / nitrogen-fixing bacteria symbiosis p. 3. The different nodule structures and their evolution p. 4. Recognition between legume and rhizobia p. 5. The Nod factor-dependent nodulation signaling pathway p. 6. The bacterial infection p. 7. Building an indeterminate nodule with a persistent meristem in Medicago p. 8. The NOOT-BOP-COCH-LIKE genes are key regulators of the symbiotic organ identity p. 9. The role of the NBCL genes in plant development p. 9.1. The shoot apical meristem regulation p. 9.2. The meristem to organ boundary regulation p. 9.3. The role of BOP in lateral organ initiation and patterning p. 9.4. The role of BOP in flower initiation and patterning p. 9.5. Differentiation of the MTOB into abscission zone and functioning p. 9.6. Other roles of BOPs unrelated to boundaries p. 10. Objectives of the PhD project p. 11. Manuscript structure and content p. CHAPTER I. p. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity CHAPTER II. p. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development CHAPTER III. p. The key regulators of the symbiotic identity MtNODULE-ROOT1 and MtNODULE-ROOT2 interact with the TGA bZIP transcriptional factor MtPERIANTHIA-LIKE CHAPTER IV. p. NOOT-BOP-COCH-LIKE1 is essential for multiple aspects of Lotus japonicus development CHAPTER V. p. The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops CHAPTER VI. p. Role of the Brachypodium distachyon NBCL genes BdNPR5-LIKE and BdNPR6-LIKE in development DISCUSSION p. REFERENCES p. Francisco Madueno Ana Berbel-tornero pour m'avoir accueilli dans leur laboratoire et formé à la microscopie électronique afin d'étudier le développement des primordia floraux des mutants de lotier et Cristina Ferrandiz pour son aide dans l'interprétation des résultats. Je souhaite remercier aussi Katharina Schiessl pour toutes ses contributions au projet NOOT. Je remercie Jean-Malo Couzigou pour nos collaborations, nos discussions scientifiques et le partage de connaissances concernant les mutants nbcl chez les légumineuses. Je remercie aussi Frédérique Guinel pour ses conseils et interprétations concernant l'anatomie des faisceaux vasculaires des nodules. Je souhaite remercier les membres de mon comité de thèse, Fabienne Cartieaux, Frédérique Débellé, Véronique Pautot et Marianne Delarue pour leur disponibilité et tous les conseils qu'ils m'ont prodigués pour mener à bien mes

Pascal m'a donné l'opportunité d'intégrer son équipe de recherche « Contrôle génétique de la symbiose » au 1 avril 2014 en tant qu'ingénieur d'étude. Pascal, m'a donné ma chance et m'a permis de mettre un premier pied dans le milieu de la recherche et pour cela je ne le remercierai jamais assez. Je le remercie également de m'avoir attribué 45 mois de CDD, qui m'ont permis d'entreprendre avec son approbation, la réalisation de mon doctorat suite aux conseils de Benjamin Gourion que je remercie aussi grandement. Je tiens également à remercier chaleureusement Mme Jacqui Shykoff qui a accepté que j'intègre son école doctorale afin que je réalise cette thèse.

Je remercie l'Agence National de la Recherche pour le financement de l'ensemble des travaux de ma thèse.

Je tiens à remercier tous les membres de l'équipe « Contrôle génétique de la symbiose ». Je remercie Marie Garmier pour sa bonne humeur quotidienne, sa gentillesse ainsi que pour les discussions scientifiques que nous avons eues régulièrement à propos des NPR. Je tiens à remercier Mlle Sophie Massot, pour son humour, sa bonne humeur, sa zénitude, et son dévouement au laboratoire et plus généralement à l'Institut. Sophie a joué un grand rôle pour le laboratoire et dans le déroulement de ma thèse. Non seulement elle a assuré l'approvisionnement de tout ce dont le laboratoire avait besoin mais elle a également assuré un énorme soutien moral auprès de nombreux doctorants y compris moi. Je remercie Nathalie Rezé pour sa gentillesse ainsi que pour le solide soutien technique qu'elle apporte quotidiennement dans le laboratoire. Je remercie tous les doctorants qui ont réalisé et qui réalisent leur thèse dans le laboratoire de Pascal, Elhosseyn Ait salem, Shengbin Liu ainsi qu'Andressa Reis sans oublier Fathi Berrabah qui m'a guidé lorsque je faisais mes premiers pas hésitants au laboratoire. Je remercie sincèrement Fathi pour toutes nos discussions philosophiques, pour tous les moments de distractions que nous avons eus au laboratoire et bien sûr pour nos échanges scientifiques. Je tiens à remercier Jeoffrey George, Blandine Broquet et Juliette Laude dont j'ai encadré les stages et qui m'ont apporté une aide non négligeable durant ma thèse.

Je souhaite remercier les différents collaborateurs qui ont participé à ces travaux. Je remercie Marion Dalmais et Abdelhafid Bendahmane pour m'avoir accueilli dans leur laboratoire afin que puisse rechercher les mutants TILLING de pois et de Brachypodium qui ont servi dans cette étude. Je remercie également Rafael Costa, Christine Saffray, Christelle Troadec et Fabien Marcel qui m'ont formé au TILLING-NGS. Je remercie Guillaume Beaumont et Brahim Mania les bioinformatiens qui ont développé le pipeline bioinformatique pour le traitement des données NGS. Je remercie Richard Sibout et Sébastien Antelme pour nous avoir fourni les graines des mutants de Brachypodium ainsi que Christine Le Signor et Richard Thompson pour nous avoir fourni les graines des mutants de pois. Je remercie Stig Uggerhøj Andersen pour nous avoir fourni les graines de mutant de lotier. Je tiens à remercier Crescent. Beans of legume provide mainly carbohydrates, proteins but also are source of lipids, fibers, mineral elements and vitamins. For efficient growth, plants require energy provided by the photosynthesis, water, and mineral macro and micro-elements. Among the macro-elements, nitrogen is often limiting for plant growth. The earth atmosphere is mainly composed by nitrogen and dioxygen (N 2 , 79 %; 0 2 , 21 %) but atmospheric nitrogen is highly stable and not directly usable by plants. On earth, active nitrogen sources have four origins, lightings of thunderstorms, combustion process, industrial processes and the biological symbiotic fixation.

List of abbreviations

In 1909, Fritz Haber and Carl Bosh invented the industrial process allowing ammonia production from N 2 . As in lightings of thunderstorms, this process consists in breaking the triple bond between the two nitrogen atoms to allow them to interact with hydrogen and to produce NH 3 . The industrial production of ammonia is an energivorous process still used nowadays to produce nitrogen fertilizers for agriculture.

Legume crops represent an alternative to the use of nitrogen-containing fertilizers because of their capacity to enter in symbiosis with nitrogen fixing rhizobia. Legume plants can be used in rotation with other crops of interest as intermediary cultures. These intermediary cultures decrease the pathogens pressure and are generally destroyed and buried at the end of the culture cycle to provide organic material and nitrogen (green fertilizers) for the subsequent crops. This rotation can increase yield with less industrial fertilizers.

Furthermore, aboveground organs of intermediary crops can be harvested to serve as forage for livestock feeding [START_REF] Schneider | Les légumineuses pour des systèmes agricoles et alimentaires durables[END_REF].

Generalities on plant / nitrogen-fixing bacteria symbiosis

Plant from bryophytes (mosses, hornworts and liverworts), pteridophytes (the water fern Azolla), gymnosperms (cycads) and angiosperms (Gunnera) are able to establish a nitrogen fixing symbiosis with cyanobacteria also called Nostoc [START_REF] Rai | Cyanobacteria in Symbiosis[END_REF][START_REF] Adams | Symbiotic interactions[END_REF]. These interactions are ancestral and by contrast to the nitrogen-fixing root nodule symbioses, plant-cyanobacteria interactions do not establish specialized symbiotic organs INTRODUCTION 6 such as nodules. In plant-cyanobacteria interactions, mature filaments of cyanobacteria differentiate in heterocysts that are specialized cells in which nitrogen fixation occurs [START_REF] Costa | Sequence based data supports a single Nostoc strain in individual coralloid roots of cycads[END_REF]. As an example, Cycas form highly specialized types of lateral roots that are called coralloid roots to host symbiotic cyanobacteria [START_REF] Adams | Symbiotic interactions[END_REF][START_REF] Brenner | Cycads: evolutionary innovations and the role of plant-derived neurotoxins[END_REF].

The plant host roots are infected intracellularly and the cyanobacteria are found in the cyanobacterial zones which are specific cortical cell layers inside the coralloid roots [START_REF] Costa | Sequence based data supports a single Nostoc strain in individual coralloid roots of cycads[END_REF]).

In angiosperms, some species belonging to the Rosids I clade [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF] are also able to use the atmospheric nitrogen by entering in symbiosis with soil nitrogen-fixing bacteria such as gram -rhizobia and gram + actinobacteria. The particularity of this clade is the ability of the plants to host their symbionts into a specialized nodule organ developing from the plant roots. In nodules, these bacteria are able to convert, via the nitrogenase enzyme, the atmospheric nitrogen (N 2 ) into ammonia (NH 4 + ), a nitrogen form directly usable by the plant. The plant will use this ammonia to synthesize the molecules required for its metabolism (nucleic acids, amino-acids and proteins).

The different nodule structures and their evolution

The nodulation ability is confined in the Rosid I clade [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms[END_REF]. The current way of thinking is that the root nodule predisposition acquisition has evolved once, between 70 and 100 million years ago (MYA; [START_REF] Doyle | Phylogenetic perspectives on the origins of nodulation[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF], from the ancestral and widespread arbuscular mycorrhizas symbiosis which itself appeared 400 MYA. Among the Rosids I clade, plants can form dedicated organs called nodules in order to host their symbionts [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF]. Among this clade, many different types of nodule structures have been reported [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Sprent | Legume Evolution: Where Do Nodules and Mycorrhizas Fit In[END_REF][START_REF] Sprent | Evolution and diversity of legume symbiosis[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF][START_REF] Sprent | Biogeography of nodulated legumes and their nitrogen-fixing symbionts[END_REF]. Basically, the morphology of a nodule depends on the nature of the meristem (persistent/non-persistent), on the mode of infection (crack entry/infection threads), on the type of bacteroid compartimentation (fixation thread/symbiosome), on the position of the vascularization (peripheral/central) and on the presence or not of interstitial cells in the infected tissues. The genetic bases underlying the nodule morphology diversity is currently poorly understood.

In the Leguminosae family the majority of the species are able to form nodules. The Legume Phylogeny Working Group (LPWG) recently proposed an updated Legumes classification based on chloroplast and nuclear markers [START_REF] Lpwg | Legume phylogeny and classification in the 21st century: progress, prospects and lessons[END_REF][START_REF] Lpwg | A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny[END_REF]Sprent et al., INTRODUCTION 7 2017). Until now, the Leguminosae family was subdivided in 3 subfamilies: Caesalpinioideae, Mimosoideae and Papilionoideae. These subfamilies classification has been revised and from now Leguminosae consists in six subfamilies: The Duparquetioideae, the Cercidoideae, the Detarioideae, the Dialioideae, the Caesalpinioideae that includes now the mimosoid clade and the Papilionoideae (Fig. 1a). By contrast to the Papilionoideae and Caesalpinioideae subfamilies, the relationships between the other legume subfamilies are unresolved and form for instance a polytomy (node with more than 2 branches). Despite this recent progress in legume classification, the legume systematic is not achieved and still in progress. Among the Leguminosae subfamilies, only the Caesalpinioideae also called the Mimosoideae-Caesalpinieae-Cassieae (MCC) and the Papilionoideae contain legume species forming nodules (Fig. 1).

In the Caesalpinioideae sub-family, there are mainly trees, shrubs and few herbaceous species that are adapted to tropical regions. In these woody species, ceasalpinioid nodules (formerly astragaloid) are the predominant nodule form. Ceasalpinioid nodules are simple or branched and present indeterminate NM (Fig. 2a). The caesalipinoid nodules show both infected and uninfected cells and bacteria are with some exception retained in modified infection threads (ITs) called fixation threads (Sprent, 2006;Sprent et James, 2007). This type of nodule has the characteristic to be the largest type of nodule and to be lignified in the outer layers [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF]Sprent et James, 2007).

Species belonging to the Mimosoid clade (formerly the Mimosoideae sub-family) are also mainly woody. In this sub-family, nodules are all indeterminate with varying degrees of branching (Fig. 2b). In the Mimosoid clade, infections mainly occur via root hairs, involved ITs and bacteroids are released into symbiosomes [START_REF] Sprent | Evolving ideas of legume evolution and diversity: a taxonomic perspective on the occurrence of nodulation[END_REF]. In this clade fixation threads are absent and the central infected tissues display interstitial cells.

The Papilionoideae is the most diversified subfamily in terms of nodule morphology.

In this sub-family (Fig. 1b), the species belonging to the Astragaleae/Hedysareae, Cicereae, Trifolieae, Fabeae and Medicago clades belong to the monophyletic Inverted Repeat Lacking Clade (IRLC). This clade is characterized by the loss of one of the two 25-kb inverted repeats in the chloroplast genome. Legume belonging to the IRLC forms indeterminate nodules with a persistent nodule meristem (NM) generated from the root inner cortex. Indeterminate nodules have an elongated shape and are able to branch resulting in more or less complex fanlike nodules (Fig. 2c). These nodules are initiated after root-hair infection, involved ITs, bacteroids are released in symbiosomes and undergo terminal differentiation. The nitrogenfixing tissues of these nodules contain a mixture of infected and uninfected cells [START_REF] Sprent | Legume Evolution: Where Do Nodules and Mycorrhizas Fit In[END_REF] [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF]. e, Aeschynomenoid or Dalbergioid nodules typical of the Papilionoideae showing small and spherical nodules always associated with lateral roots (black arrows) (Aeschynomene americana, [START_REF] Sprent | Biogeography of nodulated legumes and their nitrogen-fixing symbionts[END_REF]. f, Lupinoid or genistoid nodule typical of the Papilionoideae showing the girdling of the root (Lupinus pilosus, [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF]. g, Crotalarioid nodule showing fan-like multilobed indeterminate structure (Crotalaria monteiroi, [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF]. h, Parasponia nodule with a central vasculature and peripheral infected cells (Parasponia andersonii, [START_REF] Behm | Parasponia : A novel system for studying mutualism stability[END_REF]. i, Actinorhizal nodules with central vasculature and peripheral infected cortical tissue (Casuarina sp.). m, indicate the presence of a persistent apical nodule meristem. NPAAA, non-protein amino acid-accumulating clade; IRLC, Inverted Repeat Lacking Clade.

Outside of the IRLC but still in the monophyletic non-protein amino acidaccumulating (NPAAA) clade in which plant are characterized by the accumulation of canavanine in the seeds, the species belonging to the Phaseoleae, Psoraleeae and Loteae tribes form determinate nodules (desmodioid nodules) without a persistent NM (Fig. 1b). In these groups the nodule primordium and the NM originate from the outer root cortical cells [START_REF] Hirsch | Developmental biology of legume nodulation[END_REF]. Due to the arrest of the NM mitotic activity, determinate nodules have thus determinate growth [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Sprent | Evolution and diversity of legume symbiosis[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. Usually, determinate nodules are more or less spherical, in contrast to the indeterminate nodules that have elongated shape (Fig. 2d). In this type of nodule, infection is made via root hairs and infected tissues contain interstitial cells [START_REF] Sprent | Legume Evolution: Where Do Nodules and Mycorrhizas Fit In[END_REF]. In addition, by contrast to indeterminate nodule, determinate nodules do not branch and often display lenticels. The lenticels are external porous tissues favoring gas exchanges. In the Phaseoleae tribe, species such as Glycin max Still in the Papilionoideae sub-family, outside of the NPAAA clade, other types of nodules exist. The aeschynomenoid or dalbergioid nodule type is found in species of the Dalbergieae tribe such as in Arachis, Aeschynomene, Stylosanthes and Adesmia (Fig. 1b). In aeschynomenoid nodules, the NM is determinate resulting in small and oblate nodules that develop at the axil of a lateral root [START_REF] Dart | Infection and development of leguminous nodules[END_REF][START_REF] Sprent | Nodulation as a taxonomic tool[END_REF][START_REF] Tajima | Developmental changes in peanut root structure during root growth and root-structure modification by nodulation[END_REF].

Aeschynomenoid nodules are often numerous and in terms of shape, they look-like determinate nodules but are generally smaller and have often no lenticels (Fig. 2e).

Aeschynomenoid nodules infection occurs by crack entry, does not present ITs and the infected tissues do not present interstitial cells [START_REF] Sprent | Legume Evolution: Where Do Nodules and Mycorrhizas Fit In[END_REF].

The lupinoid or genistoid nodules type is found for example in Lupinus sp. L. (Fig. 1b). Lupinoid nodules surround the host plant root and display many lateral meristems of indeterminate growth. These nodules are roughly spherical in shape, quite large and collarshaped [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Sprent | Evolution and diversity of legume symbiosis[END_REF]. Lupinoid nodules are unusual and the girdling of the root is characteristic (Fig. 2f). As for the aeschynomedoid nodules, lupinoid nodules are initiated by a crack entry infection mechanism and do not form ITs but the central nodule tissue is entirely filled of infected cells.

The crotalarioid group is phylogenetically close to the genistoid and can be found in both herbaceous and woody species of the Papilionoideae family (Fig. 1b; [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF].

These nodules are often multilobed, each lobe has indeterminate meristems and they form fan-like nodules without root hair infection or ITs [START_REF] Renier | Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features[END_REF]. Despite the presence of indeterminate meristems, [START_REF] Renier | Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features[END_REF] described that crotalarioid nodules of Crotalaria podocarpa have a limited growth, relayed by lateral branching and emergence of new lobes (Fig. 2g). Among the Crotalariae tribe, some species form efficient symbiotic nodules with the Methylobacterium genus [START_REF] Sy | Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes[END_REF][START_REF] Renier | Nodulation of Crotalaria podocarpa DC. by Methylobacterium nodulans displays very unusual features[END_REF].

Still in the Rosid I clade, but outside of the Leguminosae family, the nitrogen-fixing rhizobium symbiosis ability has also been acquired in the Parasponia genus that belongs to the Cannabaceae family [START_REF] Trinick | Symbiosis between Rhizobiumand the non-legume, Trema aspera[END_REF]Op den Camp et al., 2011). Parasponia appeared to be a relatively young host for rhizobia since the genus Trema, phylogenetically closely linked to Paraponia, is non-symbiotic and also because endosymbionts enter in plant via crack entry which is considered as a less-sophisticated infection mode [START_REF] Behm | Parasponia : A novel system for studying mutualism stability[END_REF]. Furthermore, rhizobia are hosted in thread-like structures called fixation threads that branch in the host cells. Interestingly, the nodules of Parasponia consist in a central vascular bundle and peripheral infected cell zones. These nodules strongly look-like to actinorhizal nodules in terms of organization (See below). Anatomically, the Parasponia nodules are related to lateral roots [START_REF] Behm | Parasponia : A novel system for studying mutualism stability[END_REF] and it seems that these nodules harbors different apical meristems likely indeterminate (Fig. 2h).

Actinorhizal plants are able to form nodules infected by filamentous Gram + actinobacteria called Frankia. Actinorhizal plants comprise approximately 260 species INTRODUCTION 13 heterodimers but also the formation of homo and heterodimers between LysM and Leucine Rich Repeat (LRR) Receptor-Like Kinase (RLK). Nod factors perception by the plant will trigger a complex signaling pathway inducing simultaneously two main processes required for nodulation: The plant root hair infection by the rhizobia that allow a subsequent root cortex infection and the dedifferentiation/proliferation of the root cortical cells below the point of infection. In addition, the perception of specific bacterial exopolysaccharides by other LysM receptor kinase appears also important for plant-bacteria recognition [START_REF] Kelly | Conditional requirement for exopolysaccharide in the Mesorhizobium-Lotus symbiosis[END_REF]Kawaharada et al., 2017a).

The Nod factor-dependent nodulation signaling pathway

NF perception by the NF receptor/NF perceptor complex, in association with the LRR-RLK MtDOES NOT MAKE INFECTIONS 2 (MtDMI2, LjSYMRK in L. japonicus) leads to the activation of a signaling cascade in the epidermal cells. The 3-hydroxy-3methylglutaryl-CoA reductase (HMGR) able to associates with MtDMI2/LjSYMRK, is involved in mevalonate production and may functions as secondary messenger to transduce the signal to nucleus (Fig. 3; Ziepfel and [START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. The root nodule signaling pathway first involves a series of nuclear channels: the K + permeable channel MtDOES NOT MAKE INFECTIONS1 (MtDMI1, LjPOLLUX in Lotus) required for K + transport from nucleus to nuclear envelop [START_REF] Venkateshwaran | The recent evolution of a symbiotic ion channel in the legume family altered ion conductance and improved functionality in calcium signaling[END_REF]; the Ca 2+ permeable channel MtCYCLIC NUCLEOTIDE-GATED CHANNEL15 (MtCNGC15) required for the release of Ca 2+ from nuclear envelope/ER stores toward the nucleoplasm [START_REF] Charpentier | Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations[END_REF], the sarco/endoplasmic reticulum calcium-dependent ATPase (SERCA) required for the Ca 2+ pump back from nucleus toward the nuclear envelope (MtMCA8, [START_REF] Capoen | Nuclear membranes control symbiotic calcium signaling of legumes[END_REF], as well as other nucleoporins (Fig. 3). The regulation of these nuclear channels allows the release of Ca 2+ into the nucleus and the coordination of calcium oscillations (Fig. 3; [START_REF] Ehrhardt | Calcium spiking in plant root hairs responding to Rhizobium nodulation signals[END_REF].

The calcium oscillations are decoded by MtDOES NOT MAKE INFECTIONS3 (MtDMI3) a CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE (CCaMK, [START_REF] Levy | A putative Ca2+ and calmodulin-dependent protein kinase required for bacterial and fungal symbioses[END_REF][START_REF] Mitra | A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning[END_REF] which is able to phosphorylate MtINTERACTING PROTEIN OF DMI3 (MtIPD3, LjCYCLOPS in Lotus) a nuclear coiled-coil transcriptional activator protein [START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF]Horvath et al., 2011). MtIPD3/LjCYCLOPS is a central regulator of the symbiotic signaling and directly binds the promoter of the RWP-RK-type transcription factor NODULE INCEPTION (NIN, [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[END_REF][START_REF] Kalo | Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators[END_REF][START_REF] Murakami | Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation[END_REF][START_REF] Smit | NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription[END_REF][START_REF] Marsh | Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase[END_REF][START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF] and the promoter of the APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription-factor ERF REQUIRED FOR NODULATION 1 (ERN1; [START_REF] Andriankaja | AP2-ERF transcription factors mediate nod factor-dependent MtENOD11 activation in root hairs via a novel cis-regulatory motif[END_REF][START_REF] Middleton | An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction[END_REF][START_REF] Cerri | The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus[END_REF], to activate their expressions. and NODULATION SIGNALING PATHWAY 2 (NSP1 and NSP2; [START_REF] Kalo | Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators[END_REF][START_REF] Smit | NSP1 of the GRAS protein family is essential for rhizobial Nod factor-induced transcription[END_REF][START_REF] Murakami | Positional cloning identifies Lotus japonicus NSP2, a putative transcription factor of the GRAS family, required for NIN and ENOD40 gene expression in nodule initiation[END_REF][START_REF] Heckmann | Lotus japonicus nodulation requires two GRAS-domain regulators, one of which is functionally conserved in a non-legume[END_REF][START_REF] Hirsch | GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula[END_REF] and DELLAs [START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF]Fonouni-Farde et al., 2016a, 2017), also act downstream the calcium spiking signalization. NSP1 and NSP2 form a complex which via NSP1 DNA-binding activity binds the promoter of the NF inducible genes NIN, ERN1 and ENOD11 [START_REF] Hirsch | GRAS proteins form a DNA binding complex to induce gene expression during nodulation signaling in Medicago truncatula[END_REF], and NSP2 can interact with DELLA which can also binds ERN1 promoter (Fonouni-Farde et al., 2016a). NSP1, NSP2 and DELLA potentially associate with MtIPD3/LjCYCLOPS in a bigger regulatory complex to regulate the root nodule symbiosis [START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF]. In addition, the DELLA1-mediated gibberellin signaling regulates the cytokinin response which promotes the early nodulation genes NSP2 and ERN1 [START_REF] Fonouni-Farde | DELLA1-mediated gibberellin signaling regulates cytokinin-dependent symbiotic nodulation[END_REF].

NIN is able to directly activate the MtNUCLEAR FACTOR-YA1 subunit gene (MtNF-YA1, formerly called MtHAEM ADHESION PROTEIN2-1, MtHAP2-1 in Medicago) that encodes a CCAAT box-binding transcription factor. Both NIN and NF-Y play roles in the process of reactivation of the cortical cell divisions and in addition MtNF-YA1 is involved in ITs progression [START_REF] Soyano | NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF][START_REF] Combier | MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula[END_REF][START_REF] Laporte | The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection[END_REF]. NIN also activates the expression of MtCYTOKININ RESPONSE 1 (MtCRE1, LjLHK1 in Lotus).

MtCRE1 perceives cytokinins, activates the cytokinin pathway in the root cortex and promotes nodule organogenesis [START_REF] Vernié | The NIN transcription factor coordinates diverse nodulation programs in different tissues of the Medicago truncatula root[END_REF]. MtLEAFY-PETIOLE (MtLEP) is an ERF transcription factor homologous to MtPUCHI1 and MtPUCHI2, induced during early step of symbiosis. In Mtnf-ya1 mutant, MtLEP expression is reduced and in wild-type this gene is expressed in the dividing cells of pericycle, endodermis and inner cortex of the nodule primordia. In mature nodule, MtLEP expression pattern partially overlaps with the expression of MtNF-YA1 in nodule meristem. MtLEP seems required for nodule initiation and meristem formation, and might by dependent of the MtNF-YA1 regulation (Ripodas et al., unpublished data).

The AP2-ERF transcription factor, ERN1 is required for ITs formation and nodule primordia development [START_REF] Andriankaja | AP2-ERF transcription factors mediate nod factor-dependent MtENOD11 activation in root hairs via a novel cis-regulatory motif[END_REF][START_REF] Middleton | An ERF transcription factor in Medicago truncatula that is essential for Nod factor signal transduction[END_REF][START_REF] Kawaharada | The Ethylene Responsive Factor Required for Nodulation 1 (ERN1) Transcription factor is required for infection-thread formation in Lotus japonicus[END_REF]. By contrast to Lotus that has only one ERN1 copy [START_REF] Cerri | The ERN1 transcription factor gene is a target of the CCaMK/CYCLOPS complex and controls rhizobial infection in Lotus japonicus[END_REF], in M.

truncatula ERN1 has been duplicated from ERN2. ERN1 and ERN2 are partially redundant and are required to coordinate the rhizobial infection and the nodule organogenesis since primordia are no longer formed in the Mtern1ern2 double mutant (Cerri et al., 2012;[START_REF] Cerri | The symbiosis-related ERN transcription factors act in concert to coordinate rhizobial host root infection[END_REF].

The bacterial infection

In root hair cells bacterial infection, the rhizobia first attach to the plant root hair.

Plant lectins, which are particularly abundant at the root hair tip, enhance the rhizobia attachment through specific binding of bacterial polysaccharides [START_REF] Dazzo | Specific phases of root hair attachment in the Rhizobium trifolii clover symbiosis[END_REF][START_REF] Smit | Molecular mechanisms of attachment of Rhizobium bacteria to plant roots[END_REF][START_REF] Diaz | Genomic requirements of Rhizobium for nodulation of white clover hairy roots transformed with the pea lectin gene[END_REF]. Once the NFs produced by the rhizobia are perceived by the host plant, the cytoplasmically dense root hair tip starts to swell, a calcium gradient is established and calcium influx are induced leading to the root hair deformation (Fig. 4.1; [START_REF] Miwa | Analysis of nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus[END_REF]. NFs induce the root hair curling around the attached bacteria and form a special structure called a shepherd's crook. Bacteria entrapped in the curled root hair proliferate and form an infection foci resulting in an accumulation of NFs [START_REF] Goormachtig | Rhizobium infection: lessons from the versatile nodulation behaviour of water-tolerant legumes[END_REF][START_REF] Miwa | Analysis of nod-factor-induced calcium signaling in root hairs of symbiotically defective mutants of Lotus japonicus[END_REF]. At the infection foci, rhizobial entry is enabled by the root hair cell wall degradation leading to the invagination of bacteria inside a plant-derived intracellular tunnel growing inside the root hair cell: the infection thread (IT; Fig. 4.2).

The IT lumen is similar to intercellular space [START_REF] Brewin | Plant cell wall remodelling in the rhizobium-legume symbiosis[END_REF] and at its growing tips, the rhizobia continue to divide and form a column of bacteria. The IT progression is accompanied by cytoplasmic streaming, reorientation of the endoplasmic reticulum and movement of the nucleus and vacuole. The IT progression allows the rhizobia to reach the root cortical cells for a subsequent invasion (Fig. 4.3;[START_REF] Gage | Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes[END_REF][START_REF] Miller | Lipochito-oligosaccharide nodulation factors stimulate cytoplasmic polarity with longitudinal endoplasmic reticulum and vesicles at the tip in vetch root hairs[END_REF][START_REF] Sieberer | Cytoarchitecture and pattern of cytoplasmic streaming in root hairs of Medicago truncatula during development and deformation by nodulation factors[END_REF].

Concomitantly to the epidermal events leading to the rhizobial infection, cortical cells are activated and prepare to rhizobial invasion. In the cortex, nuclei and cytoplasm delocalize to the center of the cells and align to guide the progression of ITs. This cortex cells rearrangement is called the cytoplasmic bridge or pre-infection thread (PIT; Fig. 4.4;[START_REF] Niwa | Responses of a model legume Lotus japonicus to lipochitin oligosaccharide nodulation factors purified from Mesorhizobium loti JRL501[END_REF][START_REF] Timmers | Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements[END_REF][START_REF] Van Brussel | Induction of pre-infection thread structures in the leguminous host plant by mitogenic lipooligosaccharides of Rhizobium[END_REF].

Root cortical cells finally undergo dedifferentiation, division and start to form the nodule primordium. Once the ITs reach these newly divided cortical cells, endocytocis occur at the tip of the ITs. Rhizobia are then released into a plant derived plasma-membrane and become an intracellular organelle-like structure called symbiosome. In the symbiosome, the bacteria differentiate into bacteroids and reduce atmospheric nitrogen (Fig. 4.5; [START_REF] Roth | Bacterium release into host cells of nitrogenfixing soybean nodules the symbiosome membrane comes from three sources[END_REF][START_REF] Brewin | Plant cell wall remodelling in the rhizobium-legume symbiosis[END_REF].

The epidermal cells infection program and the cortical cells organogenetic program must be tightly coordinated to allow the inception of nodules correctly invaded by bacteria.

These two processes involve a common set of molecular actors but they can be partially dissociated. Indeed, overexpressions or constitutive activations of most of the molecular actors that act in the cortical processes are sufficient to induce the formation of nodule primordia even in absence of rhizobia, NF or NF receptors. In addition, in many legumerhizobia symbiosis, the bacteria are able to overcome the root epidermal cell barrier by entering the root through collapsed zones, generally at the base of the emerging lateral roots.

This non-root hair infection process, in which NFs production is not a prerequisite, is called the crack entry. As an example, some Bradirhizobium species that do not produce NF use crack entry to infect their hosts Aeschynomene sp. [START_REF] Giraud | Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia[END_REF]. Once rhizobia are below the root epidermis, in contact with cortical cells, ITs can form and bacteria can be released in the host cells. Note that the ITs formation is also not absolutely required for INTRODUCTION 18

Building an indeterminate nodule with a persistent meristem in Medicago

By contrast to a lateral root formation, in which cell mitotic activities are initiated in the root pericycle cells (Herbach et al., 2014), nodule mitotic cell cycle reactivation starts in the root cortical and pericycle cells [START_REF] Timmers | Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements[END_REF][START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF]. Below the infection site, the root pericycle, endodermis and cortical cell layers will together contribute to the formation of the nodule primordium and the cluster of dividing cortical cells will serve for bacteria accommodation. [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF] define that, once the meristem at the tip of the nodule primordium starts to function, the nodule primordium becomes a young nodule. [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF], also produced the Medicago truncatula indeterminate nodule fate map that precisely describes the origin of the different nodule tissues. They showed that cells layers at the base of the nodules derive from the inner cortical layers, root endodermis and pericycle.

Endodermis and pericycle cells will differentiate into uninfected tissues at the base of the nodule, the inner cortical cells will differentiate into 8 infected cell layers and the third cortical cell layer will give rise to the NM (Fig. 5). This apical meristem, once active, adds cells to the different nodule tissues throughout the nodule lifespan [START_REF] Hadri | Diversity of root nodulation and rhizobial infection processes[END_REF]Lotocka et al., 2012). There are mainly two types of cells derived from the NM: bacteria infected and uninfected cells. The nodule central zone derived from the NM activity presents a gradient of age from young (close to the NM) to old toward the root from which originates the nodule. Usually, the infection initiates front of a xylem pole (most frequent events) but infections can also initiate facing to a phloem pole (occasionally), when vascular traces initiate from two different xylem poles (Fig. 6;[START_REF] Hirsch | Developmental biology of legume nodulation[END_REF][START_REF] Heidstra | Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF].

Figure 6 | Indeterminate nodule organization

General anatomical features of the indeterminate root nodules. Infection events occur generally in front of a xylem pole but sometimes the infection occurs facing a phloem pole [START_REF] Hirsch | Developmental biology of legume nodulation[END_REF][START_REF] Heidstra | Ethylene provides positional information on cortical cell division but is not involved in Nod factor-induced root hair tip growth in Rhizobium-legume interaction[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. Here the nodule developed from an infection opposite a phloem pole. The indeterminate nodule show 6 different zones from young to old (right to left). The zone I consists in an uninfected nodule meristem. Discrete cell domains add cells for growth of various nodule tissues. The zone II corresponds to the infection thread penetration zone. The inter-zone II/III is enriched in large amyloplasts. The zone III corresponds to the nitrogen fixation zone. The zone IV corresponds to the senescence zone and the last zone V corresponds to a saprophytic zone. Zones II-V represent stages of differentiation of the bacteroid-containing tissue. This tissue is surrounded with cortical layers (from outer to inner layer): parenchymatous outer cortex (OC) of non-specialized cells, cortical endodermis (CE), which is a monolayer of lignified cells that together with specialized cells located within the inner cortex (IC) contribute to the maintenance of micro-oxic conditions necessary for nitrogenase activity. The CE is continuous with the root endodermis. Vascular system of the nodule consists of inner cortex-INTRODUCTION 20 cortex, the nodule cortical endodermis and finally the nodule parenchymatous outer cortex (Fig. 6;[START_REF] Bond | Origin and developmental morphology of root nodules of Pisum Sativum[END_REF][START_REF] Van De Wiel | Nodulin gene-expression and Enod2 localization in effective, nitrogenfixing and ineffective, bacteria-free nodules of alfalfa[END_REF][START_REF] Brewin | Development of the legume root nodule[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]Lotocka et al., 2012). The uninfected inner cortex tissue contains the nodule vascular bundles (NVB) that are ontologically related to root [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] and develop through the activity of independent nodule vascular meristems (NVM, Fig. 6; [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF].

The auxin reporter DR5:GUS construct is expressed in the NM and orthologs of Arabidopsis root regulators such as MtWUSCHEL-RELATED-HOMEOBOX5 (MtWOX5) and

MtPLETHORA1-4 (MtPLT1-4) genes were also found to be expressed in the NM suggesting that a root-like derived program is active in the NM [START_REF] Osipova | Features of the expression of a meristem-specific WOX5 gene during nodule organogenesis in legumes[END_REF][START_REF] Osipova | WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. MtPLT1-4 genes have been shown to be functionally redundant and required for primordia formation, nodule development and NM formation and maintenance. Using the root meristem markers DR5, MtWOX5, MtPLT1, MtPLT2, MtPLT3, MtPLT4 and the cytokinins reporter gene construct TCS, distinct gene expression signatures were observed in the NM and revealed its composite nature. The NM consists in fact in a distinct nodule central meristem (NCM) surrounded by multiple NVM domains [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. MtPLT1 and MtPLT2 are detected in NVM but not in NCM while MtPLT3 and MtPLT4 expression levels were comparable in both NCM and NVM. These authors highlighted that the NCM is characterized by a high cytokinin content, a lower auxin content and by the expression of MtPLT3 and MtPLT4. In NVM, they report a high auxin content and the expression of MtPLT1 and MtPLT2. They suggest that an auxin/PLT-directed root-like developmental program has been recruited to control each NVM. In mature indeterminate nodules, MtWOX5 is expressed in NCM and extends to the NVM forming a star-like pattern and DR5:GUS constructs were expressed predominantly in NVM inside the NM (Fig. 7; [START_REF] Osipova | Features of the expression of a meristem-specific WOX5 gene during nodule organogenesis in legumes[END_REF][START_REF] Osipova | WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. The auxin/cytokinin phytohormone balance plays a central role in the organogenesis in plant. In nodule, cytokinins play a major role since mutant in the cytokinins receptor MtCYTOKININ RESPONSE 1 (MtCRE1) lose the ability to produce nodule primordia [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF]. Furthermore, the exogenous application of cytokinins on root induces the expression of nodule primordia associated genes such as ENOD40 [START_REF] Fang | Studying early nodulin gene ENOD40 expression and induction by nodulation factor and cytokinin in transgenic alfalfa[END_REF][START_REF] Mathesius | Temporal and spatial order of events during the induction of cortical cell divisions in white clover by Rhizobium leguminosarum bv. trifolii inoculation or localized cytokinin addition[END_REF]. Other phytohormones such as brassinosteroids (BRs) and gibberellins have been described to positively regulate the nodulation [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF].

Other genes are involved in the nodule primordia development and potentially play a role in the NM establishment. For example, the deregulation of the MtDMI2 activity leads to hyper-activation of the nodule organogenesis program (Saha et al., 2014). In absence of rhizobia, the gain-of function mutation of the CCaMK (MtDMI3), of the phosphorylated LjCYCLOPS (MtIPD3) and of the DELLA1 induce spontaneous nodule formation, morphologically similar to rhizobia-colonized nodules (Gleason et al., 2006;Tirichine et al., 2006;[START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF][START_REF] Fonouni-Farde | DELLA-mediated gibberellin signalling regulates Nod factor signalling and rhizobial infection[END_REF]. The overexpression of NIN induces spontaneous nodule-like structures with central vasculature [START_REF] Soyano | NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF]. The NUCLEAR FACTOR-YA1 (NF-YA1) regulated by the microRNA169, is required for NM establishment, functioning and persistence [START_REF] Combier | MtHAP2-1 is a key transcriptional regulator of symbiotic nodule development regulated by microRNA169 in Medicago truncatula[END_REF][START_REF] Laporte | The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection[END_REF] and its overexpression also induces spontaneous nodule-like structures with central vasculature [START_REF] Soyano | NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF]. The RESPONSE REGULATOR 9 (RR9) that acts downstream in the cytokinin signaling pathway was reported as involved in the control of the nodule primordium development (Op den Camp et al., 2011). et al., 2011;[START_REF] Osipova | WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] Also, members of the class II MtKNOX genes family are also involved in the nodule organogenesis. Three class II KNOX transcription factors, MtKNOX3, MtKNOX5 and MtKNOX9 are induced during nodulation. [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF] first showed that as for NIN and NF-Y [START_REF] Soyano | NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF], MtKNOX3 over-expression leads to spontaneous nodulelike structure with central vasculature. MtKNOX3 is required for the nodule organogenesis since it induces the expression of MtADENYLATE ISOPENTHENYL TRANSFERASE 3 (MtIPT3) and MtLONELY GUY 2 (MtLOG2) that are genes involved in cytokinin signaling [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF]. Later, it was also shown using RNAi approaches that MtKNOX3, 5

and 9 are redundant and play roles in determining the nodules size, its boundaries and shapes [START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. Thus, these genes belong to the gene network involved in the symbiotic nodule development.

Despite the existence of many candidate genes, the molecular mechanism underlying the creation of the nodule primordia, the formation, the development and the maintenance of the NM and its sub-domains (NVM/NCM), and how root developmental program have been recruited to build the nodule remains poorly understood. PsCOCHLEATA (PsCOCH) genes [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Genes belonging to the NBCL clade encode transcriptional co-factors containing Bric-a-Brac Tramtrack and Broad complex/POx virus and Zinc finger (BTB/POZ) and ankyrin repeat domains.

The NOOT-BOP-COCH-LIKE

In addition to the roles of NBCL genes in the development of the whole plant organs (see next sections), in legume forming indeterminate nodules, this clade of genes was recruited for nodule formation and appears particularly important to establish properlyorganized indeterminate nodules. Indeed, in M. truncatula and P. sativum nbcl mutants, the nodule infection process is not altered but the nodule development is drastically impacted [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. To be more precise, nodules of nbcl mutant plants are characterized by the growth of ectopic roots arising from the NVM, suggesting that the control of the root identity in the NVM is impaired [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF]. This nodule to root conversion is asynchronous and can occur early during nodule formation or later when nodules are mature [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

This nodule to root conversion phenotype is, to our knowledge, only associated to plant carrying mutation in NBCL genes making these genes key regulators of the symbiotic organ development and identity.

Interestingly, some studies have reported that increasing the temperature can triggered similar nodule to root conversions or calli structures in Medicago sativa and various Trifolium sp. [START_REF] Dart | Infection and development of leguminous nodules[END_REF][START_REF] Day | Root formation by legume nodules[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF]. There is no evidence explaining these phenotypes but [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF] suggested that NBCL protein activity could be thermosensitive. Also Glycine max determinate nodules inoculated with Bradyrhizobium japonicum mutant strains (ΔphyR, ΔecfG) also exhibited nodule to root conversion [START_REF] Gourion | The PhyR-sigma (EcfG) signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum[END_REF]. PhyR and ecfG are bacterial response regulators involved in resistance to various stresses. Similarly, in Phaseolus vulgaris, inoculation with Rhizobium elti mutant strain presenting an auxotrophy for lysin (lysA) also resulted in nodule forming ectopic roots [START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF]. These mutant bacteria were thus classed in Root INDucer (RIND) bacterial mutants by [START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF]. Both authors emitted the hypothesis that these RIND bacterial mutants may alter phytohormone levels in nodules and eventually disturbed the proliferation of the different nodule tissues leading to an asynchronous development of NCM and NVM [START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF][START_REF] Gourion | The PhyR-sigma (EcfG) signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum[END_REF].

The role of the NBCL genes in plant development

The studies on the roles of NBCL genes begun with the discovery of the Pea Pscochleata (Pscoch) mutant affected in stipule formation [START_REF] Wellensiek | Neutronic mutations in peas[END_REF][START_REF] Blixt | Linkage studies in Pisum VII. The manifestation of the genes cri, and coch, and the doublerecessive in Pisum[END_REF].

The NBCL nature of the associated gene remained however unknown for a long time. Later PsCOCH1 was shown as required for the P. sativum inflorescence development and flower organ identity acquisition [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] and only recently its role on nodule development and identity maintenance was described [START_REF] Voroshilova | A unique pea (Pisum sativum L.) mutant impaired in nodule, leaf and flower development[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Zhukov | Gene COCHLEATA in Pisum sativum L. and its ortholog NOOT in Medicago truncatula Gaertn. control meristem fate of nitrogen fixing nodules. Biologiyanauka XXI veka (Biology-the Science of 21[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. The molecular characterization of the PsCOCH1 gene lately revealed that PsCOCH1 was orthologous to the AtBOP genes of A. thaliana [START_REF] Zhukov | Cloning of Symbiotic Pea (Pisum sativum L.) Genes using legume genome synteny[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

The Arabidopsis AtBOP1/2 genes are so far, the best characterized NBCL genes in the literature. The corresponding mutants have intensively been studied since the beginning of the XXI century and the roles of the AtBOP1/2 genes have been reviewed by [START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF]Hepworth andPautot, 2015 and[START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF].

The AtBOPs are essential genes involved in the establishment and the functioning of crucial frontiers between meristematic domains and lateral organs called meristem to organ boundaries (MTOB, reviewed in [START_REF] Zadnikova | How boundaries control plant development[END_REF]Simon, 2014, Hepworth andPautot, 2015;[START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF]. These MTOB are specific zones possessing their own genetic programs which basically consist in the repression the cell proliferation and in enhancing the differentiation of the adjacent lateral organs. One major characteristic of MTOB establishment is the inatypical phytohormones contents. In MTOB, the redirection of the auxin efflux carrier proteins PINFORMED1 (PIN1) leads to a low auxin content and in addition MTOB present a low level of BRs. The main roles of AtBOP1/2 genes in Arabidopsis development and meristem functioning are described in the next 6 paragraphs.

The shoot apical meristem regulation

In Arabidopsis, the shoot apical meristem (SAM) consists in a cluster of few hundred family that promote boundary formation and suppress organ initiation [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Takeda | CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells[END_REF]. The tri-meric complex containing, the LATERAL ORGAN BOUNDARY DOMAIN (LBD)-TF JAGGED LATERAL ORGANS (JLO), the myeloblastosis oncoprotein (MYB)-domain TF ASYMMETRIC LEAVES1 (AS1) and the LBD-TF ASYMMETRIC LEAVES2 (AS2) abolish the expression of class I KNOX genes via an epigenetic regulatory mechanism. The KNOX genes repression seems to involve the recruitment of the chromatin-remodeling protein HIstone Regulator A (HIRA; [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hamant | Plant development: a TALE story[END_REF][START_REF] Li | The putative RNA-Dependent RNA Polymerase RDR6 acts synergistically with ASYMMETRIC LEAVES1 and 2 to repress BREVIPEDICELLUS and microRNA165/166 in Arabidopsis leaf development[END_REF][START_REF] Guo | Direct repression of KNOX loci by the ASYMMETRIC LEAVES1 complex of Arabidopsis[END_REF][START_REF] Phelps-Durr | Maize ROUGH SHEATH2 and its Arabidopsis orthologue ASYMMETRIC LEAVES1 interact with HIRA, a predicted histone chaperone, to maintain knox gene silencing and determinacy during organogenesis[END_REF] and of the POLYCOMB REPRESSIVE COMPLEX2 (PRC2) through the JLO, AS1 and AS2 complex [START_REF] Lodha | The ASYMMETRIC LEAVES complex maintains repression of KNOX homeobox genes via direct recruitment of polycomb-repressive complex2[END_REF]. The chromatin compaction inhibits KNOX genes transcription and allows the meristematic cells to turn-off their stem cell state [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Rast | Arabidopsis JAGGED LATERAL ORGANS acts with ASYMMETRIC LEAVES2 to coordinate KNOX and PIN expression in shoot and root meristems[END_REF]. The (MYB)-domain TF LATERAL ORGAN FUSION1 (LOF1)

undifferentiated
and LATERAL ORGAN FUSION2 (LOF2) appears also involved in the restriction of cell division and in organ separation. Since MTOB are present at the base of all plant organs, they play a crucial role for subsequent lateral organs differentiation and development. Thus, BOP1/2 which are MTOB key regulators, directly influence the developmental process of many organs. In Arabidopsis, AtBOP1/2 genes are functionally redundant. The characterization of AtBOP single and double mutants showed that these genes are involved in stipule formation, proximal/distal leaf patterning, leaf growth determinacy, internode elongation, floral meristem identity acquisition, inflorescence initiation, flower patterning, floral abscission and recently they were shown to be involved in photo/thermo-morphogenesis [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Jun | BLADE-ON-PETIOLE1 Coordinates Organ Determinacy and Axial Polarity in Arabidopsis by Directly Activating ASYMMETRIC LEAVES2[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]Khan et al., 2012;[START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF][START_REF] Zhang | BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance[END_REF]. The roles of NBCL genes and the developmental processes involving them have been reviewed in [START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF].

The role of BOP in lateral organ initiation and patterning

The establishment of the MTOB is a first developmental step allowing the subsequent elaboration of the lateral organs. Auxin plays a central regulatory role for organ patterning following directed transport by PIN efflux proteins. In the leaf founder cells, an auxindependent signaling downregulates the meristem activity of class I KNOX STM and BP/KNAT1 genes in order to promote leaf determinacy. Reactivation of class I KNOX genes can occurs to enhance the leaf complexity, in compound leaves for example.

At the base of leaves and stipules, NBCL genes participate to the time-dependent inhibition of the blastozone activity, a marginal (by opposition to apical SAM) meristematic region at the base of organs. NBCLs exclude blade-promoting transcription factors expression and enhance determinacy [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF].

In Arabidopsis, BOP1/2 restrict class I KNOX expression to pattern the leaf. In the leaf petiole, likely via TGACG-type basic leucine zipper transcription factors (TGA bZIP TFs), BOP1 activates AS2 that forms a complex with AS1 and JLO to directly repress class I KNOX [START_REF] Jun | BLADE-ON-PETIOLE1 Coordinates Organ Determinacy and Axial Polarity in Arabidopsis by Directly Activating ASYMMETRIC LEAVES2[END_REF]. In addition BOPs inhibit blade outgrowth by repressing JAGGED (JAG).

The BOPs also induce genes involved in the adaxial fate and polarity of the leaf [START_REF] Ha | BLADE-ON-PETIOLE1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes[END_REF] via for example the repression of the abaxial fate determinant YABBY (YAB) (reviewed in [START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF].

Other factors, such as CINCINNATA (CIN)-like TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) or CIN-TCPs promote leaf growth and regulate leaf shape complexity by contributing to the negative regulation of CUC and KNOX activity [START_REF] Koyama | TCP transcription factors control the morphology of shoot lateral organs via negative regulation of the expression of boundary-specific genes in Arabidopsis[END_REF][START_REF] Koyama | TCP transcription factors regulate the activities of ASYMMETRIC LEAVES1 and miR164, as well as the auxin response, during differentiation of leaves in Arabidopsis[END_REF].

The role of BOP in flower initiation and patterning

After SAM to inflorescence meristem (IM) transition, members of the BEL1-LIKE (BELL) HOMEODOMAIN transcription factors such as PENNYWISE (PNY) and POUNDFOOLISH (PNF) are required for IM maintenance and flower initiation. For flowering, PNY, PNF and STM restrict the BOP1/2, ATH1 and KNAT6 regulatory module, by direct repression of BOP1/2, in MTOB domain flanking the IM to maintain the meristem integrity and stem cell maintenance [START_REF] Ragni | Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis inflorescences[END_REF]Khan et al., 2012a,b;[START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF]. Preventing the expression of BOP1/2-KNAT6-ATH1 allows the accumulation of FLOWERING LOCUST (FT) and bZIP transcription factor FD, the formation of the complex FT-FD, the expression of floral meristem identity genes such as LEAFY (LFY), CAULIFLOWER (CAL) and APETALA1 (AP1) and thus flowering (Fig. 9a; [START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF]. In the Atpnypnf mutant, the mis-repression of BOPs during flowering represses the meristem activity, induces the expression of jasmonic acid biosynthesis genes, resulting in the accumulation of JA. This antagonizes the gibberellin pathway and blocks the accumulation of FT and FD that are required for floral meristem gene induction and flowering (Fig. 9a; [START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF]. Other regulator such as UNUSUAL FLORAL ORGANS (UFO) which is a co-activator of LFY is involved in the flower boundaries establishment (Fig. 9b; [START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF].

BOP1 and BOP2 are also involved in the floral meristem identity patterning and as LFY and AP1, BOPs are considered as class A homeotic genes which antagonize class C in outer whorls [START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF].

Differentiation of the MTOB into abscission zone and functioning

Aerial organs shedding is an important genetically-controlled process that leads to the separation of damaged, senescent or mature above-ground organs from the plant body. Under proper developmental, hormonal and environmental stimuli, MTOB will specialize into abscission zones (AZs) dedicated to the detachment of lateral organs. AZs consist in small cytoplamiscally dense cell layers that, in response to abscission signals, undergo cell wall dissolution, via cell-wall modifying and hydrolyzing enzymes (Reviewed in [START_REF] Roberts | Abscission, dehiscence, and other cell separation processes[END_REF][START_REF] Estornell | Elucidating mechanisms underlying organ abscission[END_REF][START_REF] Yoon | The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscission-zone development and inhibiting lignin biosynthesis[END_REF].

The abscission regulation of the Arabidosis floral organs is well described. Before the abscission process occurs, boundary genes, especially BOPs genes are essential to promote the differentiation and formation of AZs. This process is potentially mediated through the activation of the ARABIDOPSIS THALIANA HOMEOBOX GENE1 (ATH1), KNAT2 and KNAT6 (Fig. 10; [START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF]. The formation of AZs is a prerequisite for the effectiveness of the downstream process leading to abscission. Indeed, in many dicots including legume plants, it has been shown that many mutants lacking NBCL functions do not form AZs and do not lose aerial organs [START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF][START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. Once differentiated the AZ cells are able to respond to abscission-related hormone signals such as ethylene, jasmonic acid and abscisic acid which execute a positive regulation on the abscission process in contrast to auxin, BRs and gibberellin which are negative regulators of this process (Fig. 10).

Figure 10 | Current model of the four steps abscission process in floral AZs

The abscission process is defined by four key stages: I-AZ cells differentiation (pink). II-AZ cells acquire competence to respond to hormone inductive signals (red). III-activation of abscission and detachment (red).

IV-differentiation of a protective layer over the scar (blue). Red lettering, SAM-leaf boundary genes. Question marks, hypothetical pathway. IAA, auxin; BR, brassinosteroid; GA, gibberellin; ET, ethylene; JA, jasmonic acid; ABA, abscissic acid. Adapted from [START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Leibfried | WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators[END_REF][START_REF] Niederhuth | Letting go is never easy: abscission and receptor-like protein kinases[END_REF].

Later, perception of the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)

peptides by the receptor-like kinase HAESA and HAESA-LIKE2 (HAE-HSL2; [START_REF] Cho | Regulation of floral organ abscission in Arabidopsis thaliana[END_REF]Stenvik et al., 2008a, Stenvik et al., 2008b;[START_REF] Aalen | IDA: a peptide ligand regulating cell separation processes in Arabidopsis[END_REF] triggers a signaling pathway involving the downregulation of BP/KNAT1 and the upregulation of KNAT2 and KNAT6, leading to the accumulation of hydrolytic enzymes secretion and cell separation (Fig. 10). After separation, the cells exposed to the external environment will be protected by PATHOGENESIS-RELATED genes induction and by the apposition of impermeable compounds such as waxes, lignin, and suberin (Fig. 10).

Other roles of BOPs unrelated to boundaries

BOPs are involved in the trade-off between development and defense. Indeed, while

AtBOP genes are overall associated to plant developmental processes, they were also shown to be involved in the regulation of plant defenses, especially in the methyl jasmonate induced resistance [START_REF] Canet | The BLADE-ON-PETIOLE genes of Arabidopsis are essential for resistance induced by methyl jasmonate[END_REF]. In planta, BOPs and NPR1 interact with TGA bZIP TFs which function in redundancy. The BOPs and NPR1 are in competition for these TGA bZIP TFs which are required for JA induced resistance [START_REF] Canet | The BLADE-ON-PETIOLE genes of Arabidopsis are essential for resistance induced by methyl jasmonate[END_REF].

Also, a role for AtBOPs genes was found in root organ development in which BP/STM promote xylem differentiation through the repression of BOP1 and BOP2 [START_REF] Liebsch | Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl[END_REF][START_REF] Woerlen | Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDICELLUS is essential for differentiation of secondary xylem in Arabidopsis root[END_REF].

Objectives of the PhD project.

Previously in the laboratory, two legume NBCL genes have been characterized. These two genes, MtNOOT1 in Medicago and PsCOCH1 in pea, are the closest orthologs of the AtBOPs genes. In legumes, the functions of MtNOOT1 and PsCOCH1 are conserved, and in addition to the function described in A. thaliana, which is not able to establish root nodule symbiosis, these NBCL genes have been recruited to participate to the indeterminate nodule development and identity [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. The laboratory has also shown that NBCL functions are conserved for the abscission ability in legumes. Lastly, the team has identified a second NBCL copy in legumes. These Medicago and pea NBCL paralogs were called MtNOOT2 and PsCOCH2, respectively.

During my PhD work, I aimed to explore in more detailed the role of NBCL1 genes in nodule identity using two indeterminate nodule forming species which are Medicago and pea.

The study of NBCL1 genes using two different legume species could allow us to make robust interpretations on the roles of these genes in the context of legume-rhizobia symbiosis.

Furthermore, the role of NBCL1 genes has never been studied in a context of determinate To better understand the role and the functioning of the legume NBCL genes in nodule development and identity, in development and in abscission, the laboratory was funded by the ANR grant NOOT (Projet ANR-14-CE19-0003-01).

Manuscript structure and content

The present PhD work has mainly followed the objectives of the ANR project and this manuscript will consist in six chapters written as scientific papers. Each chapter of this thesis will contain its own introduction, results, conclusion and material and methods sections. Brachypodium distachyon. This grass species is a close relative of wheat and barley but has not been selected for the non-abscission character or for particular inflorescence architectures that restrict seed shedding. This article has been submitted for publication in Plant Physiology journal but rejected in its present form.

Finally, this manuscript will end with a more general discussion summarizing the main points of the different studies and trying to highlight some generalities about the NBCL genes. In this general discussion, I will also discuss the conservation of NBCL genes during plant evolution and their recurrent recruitment for the establishment and specialization of new organs, here for example, the root nodule. The discussion will also highlight that NBCL were not recruited alone but with their original interacting partners and finally form a conserved regulatory module. In the discussion, I will also propose new perspectives to continue this PhD work and to improve our understanding of the NBCL roles and functioning in plant development.

Introduction

Establishment of the symbiotic interaction between legume plants and Rhizobia occurs when the soil nitrogen source is limiting and results in the formation of the root nodule where rhizobia fix nitrogen [START_REF] Ferguson | Molecular Analysis of Legume Nodule Development and Autoregulation[END_REF][START_REF] Oldroyd | Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants[END_REF]. Bacteria invade their host through structures called infection threads (IT). Once the IT has reached the newly dividing nodule cortex cells, bacteria tightly enclosed by a plasmalemma-derived membrane are released into the plant cells, differentiate in a N 2 fixing form call bacteroids, and reside in the symbiotic cell as an organelle-like structure called the symbiosome [START_REF] Jones | How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model[END_REF][START_REF] Oldroyd | Coordinating Nodule Morphogenesis with Rhizobial Infection in Legumes[END_REF][START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF].

Genetic studies that led to the discovery of the plant genes necessary for early steps of the plant-rhizobium interaction (recognition, infection and organogenesis) have shown that some of these genes are necessary for both the root nodule symbiosis (RNS) and the arbuscular mycorrhizal (AM) symbiosis (common symbiosis signaling pathway). The AM symbiosis is established in the vast majorityof vascular land plant species indicating an early origin in plant evolution, whereas nodulating plants are restricted to a subclade of Eurosid I including the Fabaceae family [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF]. It is thus generally accepted that the early signaling pathway necessary for RNS has been acquired from AM symbiosis [START_REF] Smith | Mycorrhizal Symbiosis[END_REF][START_REF] Bonfante | Mechanisms underlying beneficial plantfungus interactions in mycorrhizal symbiosis[END_REF][START_REF] Humphreys | Mutualistic mycorrhiza-like symbiosis in the most ancient group of land plants[END_REF]. The genes necessary for these early steps of the interaction belong to the Nod factor (NodF) signaling pathway [START_REF] Kouchi | How Many Peas in a Pod? Legume Genes Responsible for Mutualistic Symbioses Underground[END_REF]Horvath et al., 2011;[START_REF] Ovchinnikova | IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp[END_REF][START_REF] Oldroyd | Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants[END_REF][START_REF] Udvardi | Transport and Metabolism in Legume-Rhizobia Symbioses[END_REF].

This NodF signal is a modified lipo-chitooligosacharide produced by the rhizobia and specifically recognized by the host plant to initiate RNS. The identification of the lipo-chitooligosacharide Myc factors [START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF] structurally similar to the rhizobial NodF, is in agreement with a common origin of the two symbiotic signaling pathways. In addition to their role in triggering symbiotic interactions, the Myc and Nod factors can stimulate lateral root branching [START_REF] Lerouge | Symbiotic hostspecificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal[END_REF][START_REF] Olah | Nod factors and a diffusible factor from arbuscular mycorrhizal fungi stimulate lateral root formation in Medicago truncatula via the DMI1/DMI2 signalling pathway[END_REF][START_REF] Maillet | Fungal lipochitooligosaccharide symbiotic signals in arbuscular mycorrhiza[END_REF][START_REF] Herrbach | Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula[END_REF] suggesting that the original function of these molecules might have been related to modification of root morphogenesis in order to facilitate symbiotic interactions.

Medicago truncatula nodules belong to the indeterminate nodule form, which consist of a meristematic zone at the nodule apex, (Zone I), an infection zone (Zone II), a nitrogen fixing zone (Zone III) and a senescent zone in older nodules (Zone IV) (Vasse et al., 1990). In the nitrogen fixing zone, each symbiotic cell can host a spectacular density of nitrogen fixing bacteria. [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF] have produced a nodule fate map that precisely describes the origin of the different nodule tissues and thus the origin of the cells that compose the different nodule zones including the peripheral tissues. This study allowed a more precise analysis of mutant phenotypes and facilitates our understanding of the origin of the nodule organ. [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF] and [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] further described this nodule organization and the genes expressed in the nodule apical (meristematic) region. The apical region in indeterminate nodules couples meristematic areas of different origin, the nodule central meristem (NCM) and the nodule vasculature meristems (NVM).Several root associated genes are differentially expressed between these two meristematic domains. For example, an auxin maximum response, monitored using the DR5 reporter, is associated with early lateral root and nodule organogenesis [START_REF] Suzaki | Positive and negative regulation of cortical cell division during root nodule development in Lotus japonicus is accompanied by auxin response[END_REF][START_REF] Herrbach | Lateral root formation and patterning in Medicago truncatula[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. However, symbiotic nodule organ and lateral root formation have different requirements for cytokinins. Indeed, activation of the Lotus japonicus cytokinin response receptor LjLOTUS HISTIDINE KINASE 1 (LjLHK1) is sufficient to induce "spontaneous nodule" formation (in the absence of Rhizobia) and the corresponding M. truncatula CYTOKININ RESPONSE RECEPTOR 1, (MtCRE1)

antagonistically controls nodule and lateral root formation [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Murray | A Cytokinin Perception Mutant Colonized by Rhizobium in the Absence of Nodule Organogenesis[END_REF][START_REF] Tirichine | A Gain-of-Function Mutation in a Cytokinin Receptor Triggers Spontaneous Root Nodule Organogenesis[END_REF].

The genetic approaches used to understand plant/bacteria recognition and nodule organogenesis have generated mainly nodule-deficient (nod -) plants or plants developing nonfunctional (fix -) nodules. Nod -mutants have allowed for the identification of genes necessary for the first steps of the interaction (plant-bacteria recognition and plant infection) and nodule organogenesis. In addition, plant or bacterial mutants can result in an altered nodule development. For example Mtlumpy infection (Mtlin1), Mtvapyrin 2 (Mtvpy2) and nodules developed by Sinorhizobium meliloti exopolysaccharides Y (SmexoY) mutant [START_REF] Kuppusamy | LIN, a Medicago truncatula gene required for nodule differentiation and persistence of rhizobial infections[END_REF][START_REF] Guan | Rhizobial Infection Is Associated with the Development of Peripheral Vasculature in Nodules of Medicago truncatula[END_REF] have a central vasculature instead of peripheral vascular tissue.

Similarly, the over-expression of the MtKNOTTED1-LIKE HOMEOBOX 3 (MtKNOX3) gene in M. truncatula [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF], LjNODULE INCEPTION (LjNIN) gene in L. japonicus

and LjNUCLEAR FACTOR-Y SUBUNIT (LjNF-Y) gene in L. japonicus, without any bacterial infection [START_REF] Soyano | NODULE INCEPTION directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF], also results in the formation of nodule-like structures with central vasculature. In these mutants a defect in infection and/or NCM functioning results in a missynchronization between the NVM and the NCM development [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. This suggests that NVM and NCM development must be tightly coordinated to enable the establishment of a wild-type nodule.

We previously identified a M. truncatula mutant named Mtnodule root (Mtnoot) with a phenotype similar to the Pisum sativum cochleata (Pscoch) mutant, that develops roots from the apical position of the nodule [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF]. These organ identity shifts are asynchronous and can occur early during nodule formation [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

They correspond to a homeosis and represent an interesting genetic model to understand the identity and the origin of the symbiotic organ [START_REF] Hofer | Developmental specialisations in the legume family[END_REF]. The detailed characterization of the Mtnoot1 and Pscoch1 mutants has unraveled that the ectopic roots do not derive from the NCM but originate from the NVM initials. This suggests that the nodule vascular strands are ontologically related to roots, which is in agreement with studies of Osipova et al.

(2012), [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF] and [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. The MtNOOT1 and PsCOCH1 genes belong to the NBCL gene clade [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] and are orthologs to the Arabidopsis thaliana BLADE-ON-PETIOLE1/2 (AtBOP1/2) genes that participate in the gene regulatory network required for the boundary patterning within and between organs throughout the plant lifespan [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF].

Accordingly, MtNOOT1 and MtCOCH1 may coordinate the meristematic activities in the nodule apical region probably by delimiting the cellular domains of the nodule central meristem and the nodule vascular meristems to ensure a harmonious nodule development.

Here we report the identification and the characterization of the MtNOOT1 paralogous gene MtNODULE ROOT2 (MtNOOT2) in M. truncatula. MtNOOT2 belongs to a new NBCL subclade which is distinct from the one of MtNOOT1. We provide evidences that the MtNOOT2 gene plays a key role in the establishment and maintenance of the nodule identity. The Mtnoot1noot2 double mutant exhibits a complete nodule to root reversion, is unable to develop a nodule with the indeterminate nodule organization or to host its symbiotic partner and thereby to carry-out a successful N 2 fixing process. In addition, the double Mtnoot1noot2 forms root-like structures from aborted primordia when inoculated with with infection -rhizobia (exo -kps -mutant, [START_REF] Putnoky | Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction[END_REF]. In this study, we show that nodule identity reversion is also accompanied by drastic gene expression reprogramming, especially for gene involved in symbiosis, in defense reaction and in root development. Furthermore, the expression of members of the class II MtKNOTTED1-LIKE HOMEOBOX genes described as regulators of the symbiotic nodule development [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF][START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF], was also modified in the Mtnoot mutant contexts.

Results

The 1).

The phylogenetic analysis revealed that the Legume NBCL proteins form a specific clade distinctly divided in two sub-clades containing the NBCL1 and the NBCL2 Legume proteins

Neighbor-Joining tree (Fig. 1). This dual NBCL clades model is also supported by a Maximum-Likelihood phylogenetic tree analysis. Furthermore using the available transcriptomic data released from the oneKP sequencing project (https://db.cngb.org/blast4onekp/), only

Codariocalyx motorius, a nodulating Fabaceae close to Glycine max, was found to possess two 2). This suggests that during evolution, legumes have retained two distinct NBCL sub-clades by contrast to the closely related non-symbiotic species, highlighting differences in the NBCL sub-clades evolution in the different plant families. Pooideae species were aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The phylogenetic tree was built using the neighbor-joining method with the MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The tree was drawn according to the evolutionary distances, based on the proportion of amino-acid differences between sequences and was established using pairwise distance analysis on MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. Bootstrap method was used and set at 10,000 replicates for the phylogenetic analysis. The tree was rooted using the more distant

MtNOOT2 is early induced during the symbiotic process

In order to better understand the role of the MtNOOT genes, their expression level in M.

truncatula R-108 primary roots, root apical meristem (RAM) and nodules was determined by real-time quantitative RT-PCR (qRT-PCR). According to previous studies, MtNOOT1 transcripts accumulated in primary roots and nodules by contrast to RAM where transcripts accumulate less [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]; Fig. 2 A). In the primary root, once inoculated with Sinorhizobium medicae WSM419, MtNOOT1 gene expression decreased slightly during the early stages of the symbiotic interaction at 2 and 5 days post inoculation (dpi) in comparison to uninoculated primary roots. Such decrease of the MtNOOT1 gene expression was also observed by [START_REF] Herrbach | Nod factors potentiate auxin signaling for transcriptional regulation and lateral root formation in Medicago truncatula[END_REF] on roots 10h after Nod factors treatment. Later, when nodules are visible at 8, 12, 16 and 21 dpi, MtNOOT1 expression increased to the level detected in the uninoculated primary roots (Fig. 2 A). In contrast, MtNOOT2 expression remains undetectable in uninoculated primary roots, in RAM and during the early steps of the symbiotic interaction at 2 and 5 dpi. Once nodule primordia begin to be visible, at 8 dpi, MtNOOT2 transcripts begin to accumulate. After this stage, MtNOOT2 transcripts continued to accumulate through nodule development from 8 to 21 dpi (Fig. 2 B). MtNOOT1 and MtNOOT2 expression are thus up-regulated from the early nodule primordium stage at 8 dpi and their expressions increased during the nodule development at 12, 16 and 21 dpi (Fig. 2 A and B). We also notice that in nodule, MtNOOT1 transcripts accumulate higher compared to MtNOOT2 (Supplemental Fig. 2). is expressed in roots but repressed during nodule development (Fig. 3). The MtPR10 gene was induced early after the primary root inoculation (2 dpi), decreased quickly at 5 dpi and became undetectable from 8 dpi onwards which is in agreement with previously published expression data (Bourcy et al., 2013b). The early symbiotic MtNIN gene is expressed in nodule primordia (8 dpi) and strongly induced at 12 and 15 dpi. MtLEGH1 and the late induced MtNCR001 are detected first at 12 dpi and their expression increased until 21 dpi (Fig. 3). Together, these experiments showed that MtNOOT2 is expressed like a symbiotic gene and that its expression pattern is most similar to the expression pattern of the early nodulation gene MtNIN.

Supplemental

These results indicate that MtNOOT1 and MtNOOT2 gene expression behaviors are clearly different meaning that they may play different roles during nodule organogenesis.

MtNOOT1 is expressed in nodule vascular bundles

To precisely define the spatial and temporal expression pattern of the MtNOOT1 gene during nodule development, promoterMtNOOT1:GUS (ProNOOT1:GUS) expressing transgenic plants were created [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. In these transgenic plants, the ProNOOT1:GUS is expressed constitutively in root stele [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. The nodule primordia developmental stage III-IV is characterized by anticlinal divisions in C3 and endodermis and periclininal division in endodermis and pericycle (Fig. 4 A; [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF]. The stage V is recognizable by the presence of anticlinal divisions in C2 and the presence of a multiple cell layers derived from the C3 (Fig. 4 B; [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF]. During nodule primordia developmental stages III-IV and V described above, the ProNOOT1:GUS fusion is repressed and remains undetectable in root stele where the future nodule will take place probably to allow the new nodule vascular bundles outgrowth (Fig. 4 A-B). At stage VI, when approximatively six cell layers derived from the endodermis and pericycle cells are formed at the basal part of the nodule primordia (Fig. 4 C; [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF], the ProNOOT1:GUS expression starts to be visible in endodermis and pericycle derived cell layers that will later give rise to the nodule vascular bundle, pericycle, and endodermis tissues. Nodule primordium to functional nodule transition occurs once the nodule meristem starts to function (Fig. 4 D;[START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF], from this transition point, a strong expression of ProNOOT1:GUS fusion is detected in the vascular bundles that form at the nodule periphery. Then, during nodule development, the ProNOOT1:GUS expression become highly dynamic (Supplemental Fig. 3 Detailed observations at the NVM/NCM boundary region show that ProNOOT1:GUS fusion associate mostly to the differentiated nodule vascular pericycle and endodermis cells, as previously described in transverse sectioned nodule vasculature (Fig. 4 G-H; [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Note that the ProNOOT1:GUS expression is never observed in NCM, nodule infection zone, nodule fixation zone, nodule parenchyma, endodermis and cortex tissues (Fig. 4 C-H).

Thus, based on previous studies describing the anatomy of indeterminate nodule vascular bundles [START_REF] Pate | Ultrastructure and functioning of the transport system of the leguminous root nodule[END_REF][START_REF] Pate | Transport in Plants II: Tissues and Organs. Chapter 6. Transport in symbiotic systems fixing nitrogen[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF] the expression of the ProNOOT1:GUS expression is restricted to the nodule vascular bundles, to the nodule vascular pericycle and to the nodule vascular endodermis cells (Fig. 4 G and H). The MtNOOT1 gene expression pattern precisely defines new territories derived from the root pericycle and endodermis cells inside the symbiotic organ.

MtNOOT2 is expressed in the nodule central meristem

In order to study the spatial and temporal expression pattern of MtNOOT2 in mature nodules a promoterMtNOOT2:GUS fusion (ProNOOT2:GUS) was transformed into M.

truncatula Jemalong J5 by hairy roots transformation and into R-108 by stable transformation. In both ecotypes similar expression patterns were observed for ProNOOT2:GUS. During nodule primordia developmental stage V, expression of ProNOOT2:GUS was associated with C3 and C4 derived cell layers (Fig. 4 I; [START_REF] Xiao | Fate map of Medicago truncatula root nodules Fate map of Medicago truncatula root nodules[END_REF]. It is interesting to note that the expression of ProNOOT2:GUS is detected in C3 derived cell layers that will later become the NCM.

Once the nodule organization is established, the ProNOOT2:GUS staining is detected from the NCM to the nodule vascular bundle apex and results in a star-like pattern (Fig. 4 J-L; Supplemental Fig. 3 panel B I-VI). Thin longitudinal sections at the NVM/NCM boundary region show that ProNOOT2:GUS fusion associate to the NCM, to the apical nodule vascular pericycle cells, to the apical nodule vascular endodermis cells and to the cells around the nodule vascular bundle apex (Fig. 4 L). Thus, the ProNOOT2:GUS expression pattern suggests that the MtNOOT2 gene is predominantly expressed in the NCM and extends its expression domain towards NVM. The ProNOOT2:GUS expression pattern which is mostly associated to the NCM is therefore distinct from the ProNOOT1:GUS expression pattern which is restricted to the nodule vascular bundles (Fig. 4; Supplemental Fig. 3). Taken together, these results indicate also that the ProNOOT1:GUS and ProNOOT2:GUS patterns are complementary and connected at the NVM/ NCM junction (Fig. 4 H and L).

The MtNOOT2 gene expression pattern is dependent on the nodule identity

Using transgenic plants expressing the ProNOOT2:GUS fusion crossed with Mtnoot1 mutants, we determined the ProNOOT2:GUS expression pattern in Mtnoot1 mutant nodules context. In Mtnoot1 young and no yet converted nodules, the ProNOOT2:GUS expression pattern associate to the NCM (Fig. 5 B) as in wild-type R-108 background (Fig. 5 A), but in addition the ProNOOT2:GUS fusion is expressed at the base of the nodule where MtNOOT1 is normally expressed (Fig. 5 B). During Mtnoot1 nodule to root identity shift, the ProNOOT2:GUS expression is conserved in NCM but is never observed in the emerging ectopic roots (Fig. 5 C).

In older Mtnoot1 nodules, unconverted nodule lobes continue to express the ProNOOT2:GUS fusion in the NCM, at the base of some nodules and furthermore an extended ProNOOT2:GUS CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 50 expression can be observed in nodule vasculatures (Fig. 5 D). By contrast, in fully converted nodules, the apical and basal expression of ProNOOT2:GUS disappears (Fig. 5 D,E). However, nodule primordia formed on ectopic roots that emerge from these converted nodules, correctly express the ProNOOT2:GUS reporter fusion (Fig. 5 E).

The study of the ProNOOT2:GUS expression pattern in the Mtnoot1 context indicates that the MtNOOT2 gene expression pattern depends of the nodule identity integrity. We thus constructed the Mtnoot1noot2 double mutant line.

Indeterminate nodules resulting from the interaction between wild-type M. truncatula and S. medicae can be grouped in two nodule types represented by the uni-lobed (55%) and multilobed (45%) nodule (Fig. 6 A, B, G). In contrast, in the Mtnoot1 mutant we found nodules that can be grouped into four nodule types. The uni-lobed and multi-lobed nodule types represent only 14% and 35% of the nodules formed, respectively. The third nodule type consists in nodule to root conversion [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] and represents 44% of the nodules. Note that these nodule to root conversion events occur asynchronously (Fig. 6 G). The fourth nodule type (7% of the nodules) consists in successive nodule to root conversions coupled with rhizobia reinfection events generating a complex and disorganized nodule and root organ aggregate that we call "nodule-like structure" (Fig. 6 G). Nodules to root conversion events or nodule-like structures were not observed in Mtnoot2 single mutants that exhibit only uni-lobed (52%) and multi-lobed (48%) wild-type nodule forms (Fig. 6 G). By contrast, the double mutant Mtnoot1noot2 displays few uni-lobed (1%) and multi-lobed (4%) nodule forms (Fig. 6 G). In Mtnoot1noot2, the majority (95%) of nodules developing on root system consist in complex and disorganized nodule-like structures (Fig. 6 C,D,E,G). This severe and complete reversion of nodule to root phenotype was found in three independent Mtnoot1noot2 double mutant lines. In these three Mtnoot1noot2 double mutant lines, aerial developmental modifications were observed but only those related to the symbiosis are described in this study. Mtnoot1noot2 mutant symbiotic structures often exhibit a fasciation of the nodule vascular bundle apex resulting in aberrant and unstable plurivascularized roots that emerge from the apical part of the nodule and which later dichotomize (Fig. 6 C and D). As already observed for the Mtnoot1 nodule ectopic roots [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF], the Mtnoot1noot2 ectopic roots appear to be functional since bacterial infection events can induce new nodule primordia formation (Fig. 6 D) and give rise to the development of new nodule like-structure (Fig. 6 E). Thus the redundant action of the two MtNOOT genes is not 52 required for successful infection but required for proper nodule development and robust nodule identity maintenance through root identity repression. The N 2 fixation capacity of the whole nodule populations of wild-type, single and double mutant plants were assessed by Acetylene Reduction Assay (ARA; [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. The nitrogenase activities of the single mutants, Mtnoot1 (15 nmol C 2 H 4 .h -1 . plant -1 ) and Mtnoot2 (11 nmol C 2 H 4 .h -1 . plant -1 ) are not significantly different from the wild-type plant nitrogenase activity (17 nmol C 2 H 4 .h -1 . plant -1 ). On the other hand, the nitrogenase activity of the double mutant

Mtnoot1noot2 (0,3 nmol C 2 H 4 .h -1 . plant -1
) is completely abolished compared to the wild-type plant (Fig. 6 H). This fix -phenotype was identical in the three independent Mtnoot1noot2 double mutant lines. In these mutant nodules, the bacterial infection level is reduced, phenolics characteristic of defense reactions accumulate and figures of degradation are present (Supplemental Fig. 4, Berrabah et al., 2015). This indicates that the complete loss of nodule identity in the Mtnoot1noot2 double mutant does not allow the establishment of a functional symbiosis. In contrast in the Mtnoot1 mutant, the wild-type level of N 2 fixation activity can be explained by the presence of completely functional uni-lobed (14%) and multi-lobed (35%) nodules and by nodules converted lately during nodule development and that are colonized by rhizobia and fix+. These can compensate the partial nodule loss of identity of the early converted nodule forms (Fig. 6 G and H, Supplemental Fig. 4). Taken together, these results indicate that the loss of MtNOOT1 and MtNOOT2 functions leads to the complete loss of nodule identity and results in the absence of N 2 symbiotic fixation.

The robust nodule identity MtNOOT-dependent maintenance is thus required to guarantee an efficient and sustainable symbiotic process.

CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity

55

Inoculation with infection -rhizobial strain triggers root-like structures from aborted nodule primordia inMtnoot1noot2.

Surface polysaccharide mutants are often deficient in infection and induce fix -bump-like structures blocked at the primordial stage. In some experiments they have been shown to be able to induce nodule like primordia structures with central vasculature [START_REF] Guan | Rhizobial Infection Is Associated with the Development of Peripheral Vasculature in Nodules of Medicago truncatula[END_REF] that did not not develop to mature nodules or roots. Wild-type R-108 plants inoculated with wild-type the rhizobia S. meliloti strain Rm41, Rm41 kps -or Rm41 exo -single mutant form normal nodules (Fig. 7 A, B, C, E, F and G). By contrast, when wild-type R-108 plants were inoculated with the Rm41exo -kps -double mutant, aborted nodule primordia blocked early in the symbiotic process were observed as previously described in the literature (Fig. 7 D and H; [START_REF] Putnoky | Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction[END_REF].

Mtnoot1noot2 double mutant infected with wild-type Rm41, Rm41 kps -or Rm41 exo -single mutant form nodules that all converted into roots (Fig. 7 I, J, K, M, N and O). When

Mtnoot1noot2 plants were inoculated with Rm41exo -kps -, root-like structures developed from most of the aborted nodule primordia (Fig. 7 L and P). In addition, these root-like structures showed an enlarged base and multiple central vasculatures (stained with methylene blue)

suggesting that they originated from nodules. This suggests that MtNOOT genes are necessary to delimit and control the growth of root vasculature derived tissues and that in the noot mutants ectopic roots can emerge from aborted nodule primordia. The expression of the Histidine-Kinase MtCYTOKININ RESPONSE1 (MtCRE1) gene encoding a cytokinin receptor essential for the symbiotic interaction [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF][START_REF] Plet | MtCRE1-dependent cytokinin signaling integrates bacterial and plant cues to coordinate symbiotic nodule organogenesis in Medicago truncatula[END_REF] is not significantly affected in the single mutants even if its expression is reduced in the double mutant nodules (Fig. 8 C). The expression of the early nodulin MtNIN gene coding for a master symbiotic regulator is not affected in single mutants but significantly reduced in the Mtnoot1noot2 background (Fig. 8 C).

The (Bourcy et al., 2013a;Bourcy et al., 2013b;Berrabah et al., 2014a, Berrabah et al., 2014b, Berrabah et al., 2015).

In et al., 2013b;[START_REF] Berrabah | Multiple steps control immunity during the intracellular accommodation of rhizobia[END_REF]. These defense-related gene up-regulations are in agreement with both the defense-related phenolic compounds accumulation and the figure of degradation observed in Mtnoot1 and Mtnoot1noot2 mutant nodules (Supplemental Fig. 4).

These results demonstrate that the partial loss of symbiotic identity in Mtnoot1 mutants is linked both to symbiotic gene expression down-regulation and to defense-related genes expression up-regulation and that this phenomenon is enhanced in Mtnoot1noot2. Mtnoot1 and Mtnoot1noot2 mutant nodules clearly show a shift in their immune status potentially linked to the fix -phenotype of Mtnoot1noot2.

The organ identity switch correlates with a change in the expression of RAM genes

The nodule apical region is a complex meristematic zone [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] with a NCM surrounded by several NVM ontologically related to RAM [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF]. It was previously described that ectopic roots emerging from Mtnoot1/Pscoch1 mutant nodules originate from the nodule vasculature initials [START_REF] Vandenbosch | Nodule Initiation Elicited by Noninfective Mutants of Rhizobium phaseoli[END_REF][START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

In order to investigate how these different meristematic regions are affected during the development of the mutant organs, we performed qRT-PCR on the Medicago homologs of three key regulators previously shown to be involved in the establishment and maintenance of the RAM in A. thaliana. These key regulators are MtWUSHEL-LIKE HOMEOBOX5 (MtWOX5) a homeodomain transcription factor expressed in the root quiescent center [START_REF] Osipova | WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation[END_REF], MtCRINKLY4 (MtACR4) a receptor-kinase putative ortholog to AtCRINKLY4, known to control AtWOX5 activity through the perception of MtCLAVATA3/EMBRYO-SURROUNDING REGION (CLE) peptides in Arabidopsis thaliana [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF] and MtSHORT ROOT (MtSHR) a GRAS family transcription factor putative ortholog to AtSHORT ROOT described as required for stem cell maintenance in A. thaliana [START_REF] Levesque | Whole-genome analysis of the SHORT-ROOT developmental pathway in Arabidopsis[END_REF][START_REF] Cui | An evolutionarily conserved mechanism delimiting SHR movement defines a single layer of endodermis in plants[END_REF]. The expression of all three RAM marker genes are induced in the Mtnoot1 and/or Mtnoot1noot2 nodules in comparison to wild-type nodules, while in Mtnoot2 the expression remains unchanged relative to wild-type (Fig. 9 A). Thus, in agreement with a nodule to root identity shift the expression of these root identity genes is induced in Mtnoot1 and Mtnoot1noot2 nodules. In A. thaliana, AtPLETHORA AP2-like ethylene-responsive transcription factors are involved in auxin-mediated positive regulation of cell division and proliferation during root development [START_REF] Aida | The PLETHORA Genes Mediate Patterning of the Arabidopsis Root Stem Cell Niche[END_REF]Mähönen et al., 2014). In M. truncatula, four MtPLETHORA genes have been found to be expressed in nodules. MtPLT1 and MtPLT2 are predominantly expressed in the NVM and MtPLT3 and MtPLT4 are predominantly expressed in the NCM [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. Using qRT-PCR, MtPLETHORA gene expression kinetics were carried-out during R-108/WSM419 symbiosis. In mature R-108 nodules, MtPLT3/MtPLT4

were significantly up-regulated relative to uninoculated RAM while MtPLT1/MtPLT2 showed low expression levels relative to uninoculated RAM (Supplemental Fig. 5). It is noteworthy, that

MtPLT2 expression was up-regulated in the nodule primordia at 8 dpi compared to uninoculated primary root (Supplemental Fig. 5), suggesting that MtPLT2 expression may be required during organogenesis, but has to be strictly controlled in mature wild-type nodules, where MtPLT2 are required for the initiation and the patterning of organs and for the meristem establishment and maintenance [START_REF] Hake | The role of KNOX genes in plant development[END_REF][START_REF] Hay | KNOX genes: versatile regulators of plant development and diversity[END_REF]. The roles of class II KNOX TFs are less understood but they have been suggested to function antagonistically to the class I KNOX TFs in A. thaliana [START_REF] Furumizu | Antagonistic Roles for KNOX1 and KNOX2 Genes in Patterning the Land Plant Body Plan Following an Ancient Gene Duplication[END_REF]. In M. truncatula, three members of the class II MtKNOX genes, MtKNOX3, MtKNOX5 and MtKNOX9 belonging to the MtKNAT3/4/5-like subclass genes are constitutively expressed in the root stele including pericycle and endodermis (Di Giacomo et al., 2016 Supplemental Fig. 8) and up-regulated in nodules [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF].

Because of their potential importance during nodule meristem establishment and maintenance, class II MtKNOX gene expressions were investigated in Mtnoot mutant nodules using qRT-PCR. To summarize, once nodule identity is lost, the ProKNOX9:GUS fusion is de novo expressed in nodule vascular bundles as in a root organ [START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF] S3). These results

suggest that the MtNOOT1 gene needs to be repressed at the beginning of the nodule establishment in order to allow the formation of new vascular domains derived from the preexisting root vasculature.

From the late primordia developmental stages, MtNOOT1 gene expression is induced and the ProNOOT1:GUS expression associates to the nodule vascular tissues and more precisely to the differentiating tissues derived from the root pericycle/endodermis cell layers. Our data therefore suggest that once nodule vascular bundles are created MtNOOT1 is reactivated to control the growth and define the boundaries of the new vascular tissues inside the symbiotic nodule. The ProNOOT1:GUS expression was also associated to the vascular tissue differentiation as the strongest expression is detected below the NVM and decreased in fully differentiated tissues. This suggests a role for this gene in the differentiation of the symbiotic vascular tissues, which is in agreement with a role for AtBOP1/2 in promoting cell differentiation in the leaf base CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 67 [START_REF] Jun | BLADE-ON-PETIOLE1 Coordinates Organ Determinacy and Axial Polarity in Arabidopsis by Directly Activating ASYMMETRIC LEAVES2[END_REF] and in the floral shoot [START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF] in A. thaliana. Note here that this fusion represents a good tool as nodule vascular tissue marker.

On the other hand, the ProNOOT2:GUS fusion is expressed early in the pre-NCM in nodule primordia and once nodule develop, the ProNOOT2:GUS fusion associates mostly to the NCM and surrounding tissue.

The expression pattern of the two genes reveals their distinct but complementary roles.

The expression of the two fusions connects at the NVM/NCM boundary regions that correspond to the region where the ectopic roots emerge in Mtnoot1 and Mtnoot1noot2. Based on the common localization observed for the ProNOOT1:GUS and the ProNOOT2:GUS fusions at the NVM/NCM junction and on the fact that proteins containing BTB/POZ and ANKYRIN motifs are able to form homodimers and heterodimers [START_REF] Mou | Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF] These results suggest that a precise spatial pattern of expression of these two genes is necessary to define the symbiotic identity of the different territories of this symbiotic organ.

The identity shift of the symbiotic organ

The characteristic of the Mtnoot1 and Mtnoot1noot2 mutant is a phenotypic and molecular change in the identity of the symbiotic organ. The phenotype of the double Mtnoot1noot2 mutant is increased compared to Mtnoot1 and results in an early and complete reversion of the nodule identity into a root identity. This highlights the important complementary and redundant functions of these two genes during nodule development.

Inoculation of M. truncatula with rhizobia mutated in surface polysaccharide production results in the failure of the rhizobial infection process and leads in the Mtnoot1noot2 mutant to the formation of root-like structures with central vasculature. This result supports well our hypothesis that MtNOOT genes repress the root developmental program. Indeed, in this double mutant, the vasculatures established in the very early stages of nodule development will give rise to the central vasculature of a root-like structure in absence of the sustained rhizobial infection.

This process represents a homeosis characteristic of the homeotic mutants. Homeotic mutants often represent a step back in evolution and highlight the evolutionary pathway giving rise to a new organ. The nodule to root reversion phenotype thus suggests that nodule vasculature is ontologically related to roots. One characteristic of some of the mutant ectopic roots is their multiple (at least double) vasculatures, resulting in a fasciation of the nodule vascular bundles. In the double mutants some root structures can contain up to six vascular bundles inside one root.

We can hypothesise that these root-like structures could represent intermediate structures between a wild-type root and a nodule. If this has been the case during evolution, one crucial step in the acquisition of a symbiotic nodule organ was to acquire the capacity to form multiple vascular bundle initials. This might still persists at the early stages of mutant nodule development and allow for the formation of root-like structures with multiple vasculatures. In this model NOOT(s) might define precisely the territories that will be at the origin at the vascular tissue. It can be hypothesised that NOOT1 may restrict the vasculature domain in the root to allow the formation of one vascular bundle and extend this domain in the case of the nodule to allow at least two contiguous vascular traces.

The shift in organ identity observed at the phenotypic level is correlated with a decrease in symbiotic gene expression. The expression of the early symbiotic gene MtNIN is less affected than the expression of late symbiotic genes in the Mtnoot1 and Mtnoot1noot2 mutants suggesting a reduced impact of the mutation at the primordium stage when the nodule organization in different zones has not yet taken place. Considering the role of the NBCL genes in boundary formation (i.e. cell territories formation) we can speculate that the MtNOOT genes play a more important role when the differentiation of the different nodule zones takes place. This suggests that at the primordia stage the complete symbiotic identity may have not yet taken place and can still be reversible to root identity. Our results also suggest that the rhizobia infection possibly through Nod factor production, is necessary to trigger and/or maintain the symbiotic identity during the early stages of organogenesis.

In parallel to the down-regulation of symbiotic gene expression, the expression of defense-related marker genes was up-regulated and the infected cells containing bacteroids undergo degeneration and a senescence-like process suggesting an increased immune status in these mutant organs. The fix -phenotype, together with both the increased expression of defenserelated genes and the decreased expression of symbiotic marker genes, shows that the symbiotic identity acquisition during nodule development is probably a key point for hosting the bacterial symbiont [START_REF] Gourion | Rhizobiumlegume symbioses: the crucial role of plant immunity[END_REF]. This suggests that the symbiotic organ identity allows a change in immunity that permits the chronic high level of bacterial infections necessary for the efficient nitrogen fixation.

The expression analysis of the root meristematic markers MtWOX5, MtACR4, MtSHR and MtPLT2 suggests that the auxin-related root-derived developmental program recruited for the functioning of the nodule vascular bundle initials is up-regulated in Mtnoot1 and Mtnoot1noot2

and results in a nodule to root organ identity switch (homeosis). Investigation of the MtWOX5,

MtACR4, MtSHR and MtPLT2 gene expression patterns via GUS fusion in R-108 compared to

Mtnoot backgrounds will allow a better understanding of the nodule to root homeosis. In wildtype nodule, the expression of MtPLT2 is mostly associated to the NVM [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] and appears strictly controlled from nodule primordia establishment to the subsequent stages of nodule lifespan. In Mtnoot1 and Mtnoot1noot2, MtPLT2 expression is strongly up-regulated, in agreement with the hypothesis that the auxin-related root-derived developmental program recruited to initiate the nodule vascular bundle formation is re-activated in the mutants. In A. thaliana, the AtBOP genes participate to the definition of domain within and between organs to ensure their correct developments [START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF]. In M.

truncatula indeterminate nodule, at the NVM/NCM boundaries, MtNOOT genes may indirectly control the expression domain of RAM-related genes and coordinate their complex expression patterns [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] to allow an harmonious development of the nodule in order to acquire a robust symbiotic organ identity.

This double Mtnoot1noot2 mutant represents an interesting genetic tool for future studies designed to understand nodule origin, organogenesis, development and identity.

The role of the class II MtKNOX genes in nodule identity

In A. thaliana, one of the roles of AtBOP genes in meristem-to-organ-boundary and at the base of lateral organ is to repress cell proliferation through the repression of class I AtKNOTTED1-LIKE HOMEOBOX1/2/6 genes (AtKNAT1, AtKNAT2 and AtKNAT6; [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana[END_REF][START_REF] Zadnikova | How boundaries control plant development[END_REF]). An antagonistic role during development between the class I and class II KNOX identity genes has been proposed in A. thaliana [START_REF] Furumizu | Antagonistic Roles for KNOX1 and KNOX2 Genes in Patterning the Land Plant Body Plan Following an Ancient Gene Duplication[END_REF]. This antagonism may act via the regulation of phytohormones metabolism.

Recently in M. truncatula, class II KNOX TFs were shown to be important for nodule development [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF][START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. The class II MtKNOX3, MtKNOX5 and MtKNOX9 genes are induced during symbiosis, appear to be functionally redundant and contribute to the regulation of proper nodule size, boundary and shape (Di [START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. The MtKNOX3 gene appears essential for the expression of the MtADENYLATE ISOPENTHENYL TRANSFERASE 3 (MtIPT3) and MtLONELY GUY 2 (MtLOG2), two key genes encoding enzymes involved in the cytokinin signaling during nodule development [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF].

As observed for the expression of the RAM genes, the expression of the class II MtKNOX

Material and methods

Bacterial strains and growth conditions. Sinorhizobium medicae WSM419 strain [START_REF] Howieson | Acid tolerance in the Rhizobium meliloti-Medicago symbiosis[END_REF], Sinorhizobium meliloti Rm41 [START_REF] Kondorosi | Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: Identification of nodulation genes[END_REF], S. meliloti Rm41 kps - (PP711, Becquart-de [START_REF] Becquart-De Kozak | Role of the K-antigen subgroup of capsular polysaccharides in the early recognition process between Rhizobium meliloti and alfalfa leaves[END_REF], S. meliloti Rm41 exo -(AK631, [START_REF] Putnoky | Rhizobium fix genes mediate at least two communication steps in symbiotic nodule development[END_REF] and S. meliloti Rm41 exo -kps -(PP666, [START_REF] Putnoky | Rhizobium meliloti lipopolysaccharide and exopolysaccharide can have the same function in the plant-bacterium interaction[END_REF] were cultivated in YEB medium [START_REF] Krall | Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens[END_REF] for 24 h at 30 °C before inoculation. The S. medicae WSM419 chloramphenicol and tetracycline resistant strains holding eGFP constructions provided by Dr.

Gabriella Endre (Biological Research Centre, Szeged, Hungary) were used to inoculate M.

truncatula plants to localize rhizobia inside nodule. Agrobacterium rhizogenese A4TC24 was used for hairy-root production. The strain was grown on YEB medium [START_REF] Krall | Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens[END_REF] with appropriated antibiotics.

Plant material and growth conditions. Medicago truncatula Gaertn ecotypes R108 [START_REF] Hoffmann | A New Medicago truncatula Line with Superior in Vitro Regeneration, Transformation, and Symbiotic Properties Isolated Through Cell Culture Selection[END_REF] and its corresponding mutants Mtnoot1 (Tnk507, [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] Tnk507, this study) were cultivated on a mixture of perlite and sand (1/2, v/v) or on buffered nodulation medium (BNM) in vitro plate [START_REF] Ehrhardt | Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors[END_REF] solidified with Kalys agar 7 g.L -1 and supplemented with 0,5 µM of 2-aminoethoxyvinyl glycine (AVG). Classically, for M. truncatula nodulation, S. medicae WSM419 or S. meliloti Rm41 were used for inoculation as described in [START_REF] Berrabah | Multiple steps control immunity during the intracellular accommodation of rhizobia[END_REF]. Plants were grown in a controlled environmental chamber with 16/8 h light-dark cycle, 24/24 °C day-night temperature, relative humidity 60 % and 200 µE light intensity. Plants cultivated in sand perlite mixture were watered with 1 g.L -1 N-free nutritive solution (NPK 0-15-40; PLANT PROD).

Acetylene reduction assay. Acetylene reduction assays (ARA) were performed on individual plants inoculated with S. medicae WSM 419 (35dpi) with a protocol modified from [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. Basically, the nodulated root system of one plant was placed in 21 mL glass vial sealed with rubber septa in presence of 200 µL of water. Acetylene gas (500 µL) was injected CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 73 into each vial and 2 h incubation was performed. For each sample, 200 µL of gas were injected.

Ethylene production was measured by gas chromatography, using a 7820A Gas Chromatograph from Agilent Technologies (Massy, France) equipped a GS-Alumina column (50 m x 0,53 mm) with hydrogen as carrier gas. Column temperature and gas flow were adjusted at 120 °C and at 7,5 mL.min -1 , respectively. The experiment represents three independent biological replicates of nine plants for each Mtnoot mutant and R-108 plant. sequences and origin of oligonucleotides used for qRT-PCR are described in Supplemental Table 3. Oligonucleotide sequences and origin of oligonucleotides used for genotyping are given in Supplemental Table 3.

Gene Expression

Mtnoot

Construction of the promoter:GUS reporter fusions. For the promoter MtNOOT2:GUS reporter, the 2534 bp of the MtNOOT2 promoter region including the 5' untranslated region plus the 15 first amino acids of the MtNOOT2 protein was amplified with the with Phusion polymerase (Thermo Fisher Scientific) using the reverse primers

(5'GGATCCAGGTAGTCAAGGGAGAGAGATCTT3') and the forward primer

(5'CGCCCCATTGCCATGCCTATATATC3'). The amplified fragment was cloned into pGEM-T Easy vector (Promega) and sequenced from each extremities. The fragment corresponding to this promoter region was cloned in the pPR97 vector [START_REF] Szabados | New plant promoter and enhancer testing vectors[END_REF] as a BamH1-EcoR1 fragment and resulted in a translational fusion to the GUS gene. The resulting promoter-GUS region was sequenced for confirmation. For the promoter MtKNOX9:GUS-terminator MtKNOX9 reporter, sequence information for the promoter and terminator region of Medtr4g116545 was obtained from Medicago truncatula Mt4.0v1 genome via https://phytozome.jgi.doe.gov [START_REF] Goodstein | Phytozome: a comparative platform for green plant genomics[END_REF]. Sequences were synthesized by GeneArt (Life Technology) as level 0 modules for Golden Gate cloning [START_REF] Weber | A modular cloning system for standardized assembly of multigene constructs[END_REF] (2001). Thereafter, the transgenic roots were transferred on BNM plate supplemented with 0,5 µM of AVG during three days before inoculation with S. medicae WSM419 (15 mL per plate).

Nodules were collected every two days from 4 dpi to 15 dpi for subsequent histochemical GUS Phylogenetic Analysis. Full-length NBCL protein sequences of different Fabaceae, Brassicaceae, Salicaceae, Solanaceae, Cucurbitaceae and Pooideae species were aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] and the phylogenetic tree was built using the neighbor-joining method with the MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The tree was drawn according to the evolutionary distances, based on the proportion of amino-acid differences between sequences and was established using pairwise distance analysis on MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF].

Bootstrap method was used and set at 10.000 replicates for the phylogenetic analysis.

Light Microscopy and Sample Preparation. Histochemical staining for GUS activity was performed as previously described [START_REF] Pichon | Rhizobium meliloti Elicits Transient Expression of the Early Nodulin Gene ENOD12 in the Differentiating Root Epidermis of Transgenic Alfalfa[END_REF]. Samples were pre-fixed in cold acetone 90 % during 1 h at -20 °C. Samples were infiltrated 30 min under vacuum (≈500 mm Hg) in Xgluc staining buffer (phosphate buffer pH: 7,2 (50 mM), potassium ferri-cyanide (1 mM), potassium ferro-cyanide (1 mM), sodium dodecyl sulfate 0,1 %, EDTA (1 mM), 5-bromo-4chloro-3-indolyl-beta-D-glucuronic acid (X-gluc) containing cyclohexylammonium (CHA) salt

(1,25 mM) and incubate at 37 °C, overnight, under darkness. Samples were fixed in phosphate buffer pH: 7,2 (100 mM), glutaraldehyde 1 %, formaldehyde 4 % during 2 h under vacuum (≈ 500 mm Hg).

For vascular bundle methylene blue staining [START_REF] Truchet | The Rhizobium-legumes symbiosis Two methods to discriminate between nodules and other root-derived structures[END_REF], whole root systems were infiltrated 15 min under vacuum (≈ 500 mm Hg) in sodium hypochlorite (6 %) and left for 15 min under atmospheric pressure. Three rinsing were performed with sterile water, root system were incubated in potassium permanganate (0,04 %) for 1 h and wash with sterile water. Tissues were stained with methylene blue (0,01 %) for 20 min, cleared with sodium hypochlorite (6 %)

for approximately 2 min and finally washed with sterile water.

For semi thin sections (60 µm thickness), samples were embedded in agarose (6 %) and sliced with a vibratome (VT1200S, LEICA) in Tris-HCl pH: 7,5 (50 mM). Observations were done using an AZ100 Macroscope (NIKON) and pictures were acquired with the NIS element software (NIKON) and treated with ImageJ software or observed and captured using a microscope BX53

(OLYMPUS) and the cellSens Standard software (OLYMPUS).

Sample sections embedded in Technovit resine were treated essentially as described in Van de [START_REF] Van De Velde | Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula[END_REF]. In brief, fixed samples were infiltrated 15 min under vacuum (≈500mm Hg) in sodium cacodylate buffer pH: 7 (0,05 M), glutaraldehyde 1 % and formaldehyde 4% and (TC-65, LEICA). 7 µm and 5 µm thickness sections were respectively made for GUS staining counter-stained with ruthenium red (0,05 %) samples and toluidine blue (0,02 %) stained samples. Section were stained 10 min by immersion in ruthenium red (0,05 %) or toluidine blue (0,02 %) and observed with a microscope BX53 (OLYMPUS) and acquired using cellSens Standard software.

Accession numbers

Accession numbers for all the genes and proteins described in this work are listed in Supplemental Table S1.

Supplemental table S2. Legume and non-legume NBCL protein identity/similarity matrix Similarity/identity matrix of the 32 full-length NBCL protein sequences used to draw the neighbor-joining phylogenetic tree shown Fig. 1 was carried-out using MATrix Global Alignment Tool software version 2.01 [START_REF] Campanella | MatGAT: An application that generates similarity / identity matrices using protein or DNA sequences[END_REF].

Abstract

Legume plants have the unique ability to enter in symbiosis with free-living bacteria called rhizobia. The legume-rhizobia symbiosis is characterized by the formation of a symbiotic organ on roots allowing the rhizobia accommodation and the fixation of the atmospheric nitrogen.

The indeterminate nodule is a de-novo generated, highly structured organ that grows continuously, and this growth is governs by a set of apical meristems that are ontologically not related. The main central meristem is generated from the root cortex and is surrounded by multiple peripheral meristems originating from root pericycle and endodermis. The correct synchronization and coordination of these meristems and the maintenance of their identities ensure the proper development of the nodule and the success of the symbiosis.

Few legume mutants impaired in the nodule identity maintenance have been reported until now but the NOOT-BOP-COCH-LIKE1 (NBCL1) genes are known as key regulators of the symbiotic nodule identity. In M. truncatula and Pisum sativum, the MtNODULE ROOT1 and the PsCOCHLEATA1 genes, respectively, are strictly required to maintain the identity of the nodule and to avoid ectopic root growth from the nodule peripheral meristems. Outside these NBCL1 genes, the molecular actors involved in the nodule identity maintenance and the underlying molecular mechanisms governing this identity remains poorly investigated.

Here we report the identification and the characterization of a new member of the legumespecific NBCL2 clade, the second NBCL gene of pea PsCOCHLEATA2 as involved in nodule development and identity. Previously, in Medicago, the Mtnoot2 mutant was shown to have no symbiotic phenotype however the Mtnoot1noot2 double mutant presents exacerbated phenotypes leading to a fix minus phenotype. Here, in pea, we showed that the Pscoch2 single mutant presents also no symbiotic phenotype however the Pscoch1coch2 double mutant has supernumerary and poorly developed nodules converted to roots with reduced nitrogen fixation performances. Also, we found that PsCOCH2 gene is involved in the patterning of aerial organs such as stipules, leaves and flowers.

In this study, we uncover a new molecular actor involved in the still poorly understood regulation of the nodule identity and our findings support the fact that legume specific NBCL2

genes play important roles in the indeterminate nodule development, identity establishment and maintenance.

Introduction

Pisum sativum (P. sativum) is a legume species characterized by a unique developmental plasticity that has been intensively used to understand for example the genetic network underlying the regulation of compound leaves development. P. sativum nodes, as most of dicots, present a trilacunar nodal anatomy [START_REF] Sinnott | Investigations on the Phylogeny of the Angiosperms. 3. Nodal Anatomy and the Morphology of Stipules[END_REF]Kumar et al., 2009a). One compound leaf and two foliaceous stipules initiate independently from the main vascular trace and from the lateral vascular traces, respectively, at each nodes. The leaf of P. sativum is imparipinnately compound and constituted by a petiole, up to three pairs of proximal pinnae leaflets, up to four pairs of distal pinnae tendrils and a terminal tendril [START_REF] Hofer | The genetic control of patterning in pea leaves[END_REF][START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF]Kumar et al., 2009a). The basal pair of foliaceous stipules arising from node at the base of the leaf are leaflet-like organs developmentally autonomous regarding to the leaf.

All these organs are initiated from lateral organogenesis-competent zone termed marginal blastozone by opposition to the apical blastozone [START_REF] Hagemann | Organogenetic capacity of leaves: The significance of marginal blastozones in angiosperms[END_REF].

PsUNIFOLIATA (PsUNI) gene is orthologous to the AtLEAFY (AtLFY) gene of Arabidopsis thaliana (A. thaliana). PsUNI is a master regulator in the gene network underlying lateral organ development that act through the promotion of the marginal blastozone activity and regulate the indeterminacy status of the leaf primordia during its development to pattern the compound leaf architecture [START_REF] Eriksson | Erbkomplexe des Rotklees und der Erbsen[END_REF][START_REF] Marx | Tendrilled acacia (tac): an allele at the uni locus[END_REF][START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF][START_REF] Weigel | LEAFY controls floral meristem identity in Arabidopsis[END_REF][START_REF] Hofer | The effect of uni on leaf shape[END_REF][START_REF] Hofer | UNIFOLIATA regulates leaf and flower morphogenesis in pea[END_REF][START_REF] Hofer | The genetic control of patterning in pea leaves[END_REF][START_REF] Moyroud | LEAFY blossoms[END_REF]. Thus Psuni mutant results in pleiotropic phenotypes in leaf and flower patterning and presents a reduced leaf complexity and a proliferating sepalloid and carpelloid structures [START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF][START_REF] Hofer | UNIFOLIATA regulates leaf and flower morphogenesis in pea[END_REF]. Other genes are involved in the leaf primordium organogenesis and in the determination of the compound leaf architecture. Depending on the leaf portions, the negative regulation of PsUNI expression by PsTENDRIL-LESS (PsTL) promotes the distal and terminal tendrils identity [START_REF] De Vilmorin | A case of gametic coupling in Pisum[END_REF], PsUNI repression by PsAFILA (PsAF) gene promotes the proximal leaflet identity [START_REF] Kujala | Felderbse beiwelcher die ganze Blattspreite in Ranken umgewandelt ist[END_REF][START_REF] Goldenberg | afila, a new mutation in pea (Pisum sativum L.)[END_REF] and PsUNI repression by PsCOCHLEATA1 (PsCOCH1) gene confers the stipule identity [START_REF] Wellensiek | Neutronic mutations in peas[END_REF][START_REF] Blixt | Linkage studies in Pisum VII. The manifestation of the genes cri, and coch, and the doublerecessive in Pisum[END_REF][START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF][START_REF] Kumar | Regulation of stipule development by COCHLEATA and STIPULE-REDUCED genes in Pisum sativum[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. The Pscoch1 mutant displays a wide range of stipule phenotypes that can be explain by different reasons: i) The different genetic backgrounds studied influence considerably other factors involved in the meristematic cell strength of a given organ primordia that will drive the formation and the size of lateral organ [START_REF] Kumar | Regulation of stipule development by COCHLEATA and STIPULE-REDUCED genes in Pisum sativum[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. ii) In a single given genotype, it exists a phenomenon called hyper-heterostipulation characterized by both the asymmetry of a pair of stipules are mostly absent, in nodes 6 to 7 stipules are often strap-like, elliptical or spoon-like, from node 8 to 11 stipules can be compounds behaving as leaf-blade but lacking stipules themself, above the 12 th node the complexity was reduced and stipule were more spoon-like or occasionally thread-like. In addition along the stem 25 % of the stipule pair showed asymmetry [START_REF] Blixt | Linkage studies in Pisum VII. The manifestation of the genes cri, and coch, and the doublerecessive in Pisum[END_REF]Gourley, 1999;[START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Similarly to leaf, in stipules PsCOCH1 inhibits PsUNI to prevent blastozone fate in stipule primordia and generate simple foliaceous stipules [START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF]. In stipule primordia, PsCOCH1 functions as a repressor of the leaf-blade program through the repression of PsUNI to avoid the formation of a more or less compound leaf-like structure (Kumar et al., 2009a). The PsCOCH1 gene is thus a master regulator of the stipule initiation, development and determinacy [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF]. In agreement with this role, in situ hybridization experiment showed that PsCOCH1 transcripts accumulated at the base of the developing leaf were stipules are formed [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

In the Psstipule reduced (Psst) mutant, stipules do not expand and presents small non-peltate stipules. It has been suggested that PsST is required for the maintenance and/or the cell proliferation of meristematic cell in the stipule primordium (Pellew and Swerdrup, 1923;[START_REF] Kumar | Regulation of stipule development by COCHLEATA and STIPULE-REDUCED genes in Pisum sativum[END_REF]. In addition, the double mutant Pscoch1st does not form stipule laminae suggesting that PsST gene acts redundantly with PsCOCH1 for the initiation of stipule primordia and the development of normal peltate stipules [START_REF] Blixt | Linkage studies in Pisum VII. The manifestation of the genes cri, and coch, and the doublerecessive in Pisum[END_REF][START_REF] Marx | A suit of mutants that modify pattern formation in pea leaves[END_REF][START_REF] Gourlay | An Analysis of Genes Involved in Pea Compound Leaf Development[END_REF][START_REF] Kumar | Regulation of stipule development by COCHLEATA and STIPULE-REDUCED genes in Pisum sativum[END_REF].

CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development

85

PsCOCH1 is also required for the correct P. sativum inflorescence development and for flower organ identity acquisition [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Wild-type P.

sativum produces generally two typical legume type flowers by panicle. These flowers are organized in four whorls showing five sepals, five petals, ten stamens and one carpel. The five sepals are fused at their bases, the five petals consist in one adaxial large standard, two lateral wings and two short abaxial petals fused on their adjacent edges forming the keel. Nine of the anthers are fused and formed a staminal tube enclosing the central carpel, and the tenth adaxial anther is unfused. The carpel and the anthers are themselves enclosed in the keel [START_REF] Hole | Development and control of the number of flowers per node in Pisum sativum[END_REF][START_REF] Tucker | Overlapping organ initiation and common primordial in flowers of Pisum sativum (Leguminosae: Papilionoideae)[END_REF][START_REF] Ferrandiz | Flower Development in Pisum sativum: From the War of the Whorls to the Battle of the Common Primordia[END_REF].

As for stipules, the Pscoch1 mutant displays a range of phenotypes from normal flowers to open ones showing supernumerary organs at each whorl with abnormal organ fusion and aberrant organ mosaic [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Pscoch1 flowers show an increased number of sepals, sepal/petal mosaic in sepal whorl, an increased number of wings and keels, wings/keel mosaic in petal whorl and narrower petal bases. The aberrant petal organization and identity defects lead to open flower structures in which stigma and anthers are not correctly enclosed in keel. This open flower phenotype leads to the quick drying of the pollen that largely reduced self-fertility. The number and the development of stamens are also affected and more or less ten abnormally fused anthers are present. In addition, up to 4 occasionally fused carpels maybe present in Pscoch1 mutant [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF]. Due to this partial sterility, the Pscoch1 mutant tends to produce more lateral branches and to develop larger and thicker leaves in smaller number compared to wild-type P. sativum due to an increased cell number per organ leading to a general Pscoch1 enhanced growth, size and biomass [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Sharma | COCHLEATA controls leaf size and secondary inflorescence architecture via negative regulation of UNIFOLIATA (LEAFY ortholog) gene in garden pea Pisum sativum[END_REF]. In the Pscoch1 mutant, the floral bracts are also to be modified and the phenotypes appear again genotype-dependent. [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF] described a collar leafy bract tissues with crenulate edge surrounding the pedicel base (10 %) while [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] described bracts as more complex in terms of length.

Pscoch1 has pleiotropic effects on stipules, leaves and flowers development but abnormalities were also described in nodules [START_REF] Voroshilova | A unique pea (Pisum sativum L.) mutant impaired in nodule, leaf and flower development[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Zhukov | Gene COCHLEATA in Pisum sativum L. and its ortholog NOOT in Medicago truncatula Gaertn. control meristem fate of nitrogen fixing nodules. Biologiyanauka XXI veka (Biology-the Science of 21[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Nodule are nitrogen-fixing organs initiated from the root cortical cells in response to telluric rhizobia signals called nod factors [START_REF] Kouchi | How Many Peas in a Pod? Legume Genes Responsible for Mutualistic Symbioses Underground[END_REF]Horvath et al., 2011;[START_REF] Ovchinnikova | IPD3 Controls the Formation of Nitrogen-Fixing Symbiosomes in Pea and Medicago Spp[END_REF][START_REF] Oldroyd | Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants[END_REF][START_REF] Udvardi | Transport and Metabolism in Legume-Rhizobia Symbioses[END_REF]. P.

sativum forms indeterminate nodules with an apical persistent meristem. The Pscoch1 homeotic mutant presents a nodule identity perturbation characterized by the presence of abnormal nodule vascular bundle-derived roots arising from the apical part of the nodule. Again, Pscoch1 shows a range of nodule phenotypes from wild-type nodules to nodule to root converted nodule to complex nodule/root hybrid structures [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Thus, in addition to its role in stipule, leaf and flower determinacy, PsCOCH1 function has been recruited during evolution to coordinate the maintenance of the nodule identity and its developmental program, to define the domains in nodules and to repress the root identity in nodule vascular bundle initials [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Once cloned [START_REF] Zhukov | Cloning of Symbiotic Pea (Pisum sativum L.) Genes using legume genome synteny[END_REF] [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

NBCL and NPR1-LIKE genes encode transcriptional co-factors known to interact preferentially with TGACG sequence-specific binding BASIC LEUCINE ZIPPER transcription factors (TGA bZIP TFs; [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF][START_REF] Després | The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors[END_REF][START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF][START_REF] Després | The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Boyle | The BTB/POZ Domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function[END_REF][START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF][START_REF] Canet | The BLADE-ON-PETIOLE genes of Arabidopsis are essential for resistance induced by methyl jasmonate[END_REF], with growth regulator Arabidospsis LSH1 and Oryza G1 proteins (ALOG) containing DNA-binding domain [START_REF] Xu | Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators[END_REF] and may also play a role in the regulation/degradation of their interacting partners through the BTB/POZ domain as cullin-based E3 ligase adaptator for ubiquitin-dependent degradation by the 26S proteasome [START_REF] Pintard | Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family[END_REF][START_REF] Gingerich | Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/ broad complex ubiquitin-ligase gene family in rice[END_REF][START_REF] Zhang | BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance[END_REF]. NBCL genes are above all known to be involved in the establishment and the regulation of the plant boundaries, that are zones dedicated to the separation between meristems and lateral organs through the repression of cell proliferation and the promotion of the adjacent lateral organs differentiation (Aida andTasaka, 2006a, 2006b;[START_REF] Barton | Twenty years on: The inner workings of the shoot apical meristem, a developmental dynamo[END_REF][START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF].

In M. truncatula, we previously described that both MtNOOT1 and its paralog MtNOOT2 Transcript accumulation differences between young and more developed organs such as leaves and flowers suggests a preponderant role for these NBCL genes during early organ development.

are
Similarly in nodes, in leaves and in flowers organs, PsCOCH1 transcripts were always more abundant relative to PsCOCH2 transcripts in accordance with the PsGEA data (Fig. 1b; Fig. 2).

PsCOCH1 and PsCOCH2 transcripts were detected in young seeds and their abundances increased during seed development. Together RNAseq data and qRT-PCR gene expression analysis show coherent results and reveal that PsCOCH1 and PsCOCH2 are often co-expressed in the different organs analyzed with PsCOCH1 expression often higher relative to PsCOCH2.

93

Following PsCOCH1 gene mutations genotyping, homozygous mutants were obtained for families Ps3092, Ps2702 and Ps2058. The mutations in lines Ps2240 and Ps547 were not found (Table 1). For the PsCOCH2 gene, homozygous mutant were obtained for the family Ps1178.

Plants from line Ps3340 carried the mutation but were lost because of the deleterious impact of other mutations on fertility (Table 1). S4), we were able to explain the lack of phenotype for Ps2702 and Ps2058. In Ps2702, the D248N point mutation modified the amino acid property from a negatively charged to a polar uncharged side chain but the mutation was on a low conserved amino acid position (Supplementary figure S4). In Ps2058, the T413I point mutation modified also the amino acid property from a polar uncharged to hydrophobic side chain but the mutation was this time on an unconserved amino acid position. In Ps3092, presenting a C114Y mutation, the thiol to aromatic hydrophobic side chain modified the amino acid property at a highly conserved amino acid position (Supplementary figure S4) but this mutation does not impact the protein functions since no coch1 expected phenotypes were observed in the homozygous mutant. Ps1178 KO mutant line was finally obtained at homozygous state. This Pscoch2 allele was backcrossed 3 times as single mutant and the plants form backcross 2 were used to construct the double mutant Pscoch1coch2 (Table 1).

Pscoch2 single mutant has no symbiotic phenotype

In M. truncatula, the Mtnoot2 single mutant does not present any symbiotic phenotype (Magne et al., 2017a, unpublished). To discover the phenotype associated to Pscoch2 mutation and to confirm or refute the constant absence of phenotype associated to the nbcl2 mutations in indeterminate nodules and aerial organs, the mutant line Pscoch2 Ps1178 was used. Selffertilized progeny of two heterozygous lines for the PsCOCH2 locus revealed that PsCOCH2 segregated following mendelian law (Table 2). Pscoch2 Ps1178 homozygous mutants were nodulated using R. leguminosarum P221 and nodule phenotypes were compared to PsCaméor.

Twenty-seven days after inoculation with rhizobia, the Pscoch2 Ps1178 mutant nodules presented a typical wild-type indeterminate nodule shape and were not different from wild-type

Pscaméor. Moreover, the nodule to root conversion phenotype typical of nbcl1 mutants was not observed in Pscoch2 mutant nodules (Fig. 4). The symbiotic process and performances are altered in Pscoch1coch2 double mutant

In M. truncatula, the Mtnoot2 mutation has no detectable effect on the symbiotic process.

By contrast, the double mutant Mtnoot1noot2 has an exacerbated Mtnoot1 nodule to root phenotype compared to Mtnoot1 single mutant leading to a complete loss of nodule identity and the loss of the nitrogen fixation ability [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]Magne et al., 2017a, unpublished).

Pscoch1 single mutant to already presents a very strong nodule to root phenotype (≈90 % of nodule conversion; [START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF] which does not impaired the symbiotic performance of pea (Fergusson and Reid, 2005). In order to know if the Pscoch2 mutation increases the Pscoch1 nodule to root phenotype and if it causes deleterious effects on the nodulation process, we thus generated the double mutant Pscoch1coch2 by cross fertilization using the mutant KO alleles Pscoch1 FN3185/1325 backcrossed one time in caméor background and Pscoch2 Ps1178 backcrossed 2 times in caméor background. The self-fertilized progenies of seven independent F1 heterozygous lines for both the PsCOCH1 and the PsCOCH2 locus revealed that PsCOCH1 and PsCOCH2 locus segregated as two independent monogenic recessive loci and followed a mendelian dihybridism distribution 9:3:3:1 in the F2 generation (Table 3). Pscaméor, Pscoch1 and Pscoch2 formed in average one hundred nodules that are mostly converted in Pscoch1 (Fig. 5f). Surprisingly, while Pscaméor and Pscoch2 produced in average forty nodule primordia per plant, very few nodule primordia were observed in Pscoch1 (Fig. 5f).

Pscoch1coch2 double mutants present supernumerary nodules and nodule primordia, whith in average two hundred nodules that is twice as much as Pscaméor, Pscoch1 and Pscoch2 and a hundred of nodule primordia that is also twice as much as Pscaméor and Pscoch2 (Fig. 5f).

Symbiotic performance were assessed in Pscoch mutants and normalized by plant. The ARA revealed that all the mutants were fix+, however once normalized per nodule, the experiment showed that the Pscoch1coch2 nitrogen fixation is significantly reduced relative to wild-type (Fig. 5g,h). Leaves from first nodes of Pscoch mutants were compared to Pscaméor. The large majority of these leaves were looking like the wild-type with two proximal leaflets and distal tendrils (Fig. 6a). In Pscoch1, we found few leaves that showed small defects in leaf patterning, often limited to an additional proximal leaflet (Fig. 6b). Pscoch2, as in previous observations, presented no particular phenotypes (Fig. 6c). In Pscoch1coch2 most of the leaves did not present patterning defects, however some leaves presented an increased leaf complexity. In these cases, Pscoch1coch2 leaves presented more than two proximal leaflets and in addition the distal part of the leaf was often reiterating compound leaves or leaflets (Fig. 6d).

On later nodes, once leaves become compound, all the leaves in the different Pscoch mutants showed wild-type phenotype with at least two pairs of proximal leaflets, a pair of distal tendrils and a terminal tendril (Fig. 6e,f,g). Occasionally, the Pscoch1coch2 mutant showed leaf patterning defects such as dichotomy in leaves along the proximal distal axis (Fig. 6h), dichotomy along stem were also observed (data not shown). Analysis of stipule morphology revealed that Pscaméor and Pscoch2 have wild-type peltate stipules (Fig. 6i,k).

Pscoch1 stipules morphology are drastically affected as previously described in the literature and show a range of stipule phenotypes, such as the absence of stipules, thread-like stipules, leaflet-like stipules or compound leaf-like stipules (Fig. 6j). The same kind of modifications were observed in the Pscoch1coch2 double mutant (Fig. 6i). The stipule modifications and the penetrance of this phenotype are already particularly strong in Pscoch1 and it is difficult to assess if this phenotype is increased in the Pscoch1coch2 double mutant. To investigate if Pscoch2 increased the stipule phenotype due to Pscoch1, the stipules morphology was checked node by node form 1 to 13 in Pscaméor, Pscoch1, Pscoch2 and Pscoch1coch2. The analysis revealed that Pscaméor and Pscoch2 present only wild-type peltate stipule (Fig. 6i,k;

Table 4). Pscoch1 presented mainly an absence of stipule from nodes 1 to 5, thread-like stipules from nodes 6 to 7 and leaflet-like stipules from nodes 8 to 13 (Fig. 6j; Table 4). Pscoch1coch2

presented mainly an absence of stipule from nodes 1 to 9, thread-like stipules at nodes 10 and 100 leaflet-like stipules from nodes 11 to 13 (Fig. 6l; Table 4). These results show that the absence of stipules is prolonged along the nodes of the double mutant and thus suggests that the stipules alteration is increased in Pscoch1coch2 double mutant compared to Pscoch1 (Table 4). supernumerary sepals (Fig. 7h). In the third whorl, petals of the two flowers co-exist resulting in a complex corolla showing supernumerary petals. Roughly at the center of these kinds of flowers, two petals that look-like to wings were fused along their adaxial side indicating the site of fusion between the two flower corollas (Fig. 7h). Medicago. In the Mtnoot1noot2 double mutant, stipule morphology alterations are enhanced and characterized by stipule to compound-leaf conversion, highly reduced stipules or stipule absence (Couzigou et al., unpublished) while in Mtnoot1 single mutant, stipules display reduced digitation number and reduced size [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Further analysis will allow to describe more precisely all these modifications.

CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development

106

PsCOCH2 may participate to the regulation of the floral patterning in pea

Similarly to the strong penetrance of the Pscoch1 mutation in nodules and stipules development and identity, Pscoch1 flowers are highly altered in their organization and identity [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. As previously mentioned for other characters, the Pscoch2 single mutant does not present any particular floral anomaly. Thus, to determine if

PsCOCH2 is also involved in the floral patterning and identity acquisition in pea, we have To find most of true mutants with high confidence, the binomial law was used to statistically determine the smallest variant significant frequency in a TILLING NGS population and was set at 0,7%. Bioinformatic analysis. Variants validation was carried-out using a two dimensional pooling strategy (X-Y). The two dimensional coordinates of a given mutation allow to recover the coordinate of the mutated family on individual plate. The bioinformatic analysis was performed using SENTINEL software [START_REF] Bendahmane | SENTINEL, SOFTWARE dedicated to TILLING by NGS Analysis[END_REF]. Mutation characterization.

Mutations were characterized by the reference nucleotide, the variant nucleotide, the position of the mutation on the amplicon, on the gDNA, on the coding sequence, by the corresponding amino Mismatch detection by ENDO1. To confirm uncertain mutations obtained by TILLING-NGS, single nucleotide polymorphism (SNP) detection assays were performed using the mismatch specific endonuclease ENDO1 [START_REF] Triques | Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea[END_REF]. Briefly targeted amplicons were first amplified with specific primers tailed by illumina adaptators and reamplified using both NGS-M13 oligonucleotide adaptators and universal M13 fluorescent oligonucleotides M13F700 and M13R800 labeled at the 5'end with infra-red dye IRD700 and IRD800 respectively (LI-COR®, Lincoln, NE, USA). Equimolar quantities of PCR products from mutant and wild-type were mixed, heated and cooled for denaturation and hybridization respectively, to generate DNA heteroduplex. DNA heteroduplex were digested by ENDO1 enzymes [START_REF] Triques | Characterization of Arabidopsis thaliana mismatch specific endonucleases: application to mutation discovery by TILLING in pea[END_REF]

P. sativum DNA extraction.

Plant genomic DNA was extracted from leaves as described in [START_REF] Dellaporta | A plant DNA minipreparation: Version II[END_REF] with some modifications. Young leaves were frozen in liquid nitrogen and grinded with a ball grinder (Retsch, MM400) two times 30 seconds with a frequency of 25.s-1. 600µL of fresh DNA extraction buffer (NaCl (350mM), Tris-HCl pH: 7,6 (10mM), Urea (7M), N-Lauroyl sarcosine (2%), EDTA (50mM), aqueous phenol pH: 7,9 (6,5%)) were added to the powder. The suspension was vortexed, incubated 5 min at 50°C and shaken 10 min at room temperature. 700 µL of aqueous phenol pH: 7,9; chloroform; iso-amylalcohol (25:24:1) were added. Suspension was vortexed, centrifugated 10min at 14000rpm and aqueous phase was collected. 700 µL of chloroform; iso-amylalcohol (24:1) were add on aqueous phase. Suspension was vortexed, centrifuged 5min at 14000rpm, aqueous phase was collected and DNA precipitation was carried-out adding cold sodium acetate 3 M; isopropanol (0,1:1). After a 10min centrifugation at 14000 rpm the DNA pellet was washed with 500 µL of ethanol 70 %. After a 5 min centrifugation at 14000 rpm the DNA pellet was dried, resuspended in 100 µL of sterile water and heated 5 min at 65°C. RNase treatment was finally performed with 5 µL of 1mg.mL -1

RNase (Roche). S6.

respectively.

In M. truncatula and P. Sativum indeterminate nodules, these pairs of NBCL genes are important for the nodule identity and the correct establishment of the symbiotic process, however the gene network controlling the nodule identity is completely unknown and the interacting partner of NBLC proteins have not been identify.

Here we report the identification and the characterization of the MtPERIANTHIA-LIKE gene, the closest ortholog of the Arabidopsis AtPERIANTHIA in Medicago, as a first interacting partner of the NBCL proteins in a context of root nodule symbiosis.

We show that MtPERIANTHIA-LIKE is a nodule-specific gene early induced during nodulation. It encodes a TGA basic leucine zipper transcription factor (TGA bZIP TF) that directly interacts with both MtNOOT1 and MtNOOT2 in vivo. However, Mtpan-like single mutant showed no symbiotic defects and the Mtnootpan-like double mutants did not present exacerbate Mtnoot mutants phenotypes. This suggested that MtPAN-LIKE is not strictly essential for the nodule identity establishment and maintenance or that its function could be ensured by other MtPAN-LIKE paralogs.

Nethertheless, our results revealed that a pre-existing mechanism involving the NBCL/TGAbZIP TF regulatory module has possibly been recruited for the regulation of the root nodule symbiosis. We have thus unrevealed a part of a probably complex regulatory mechanism underlying the regulation of the nodule identity.

Based on the existence of a such NBCL/TGA bZIP regulatory module in nodule, it will henceforth be possible to investigate the targets of MtPAN-LIKE to improve our comprehension of the nodule identity MtNOOT-dependent pathway. [START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF]. In plants and animals, the BTB/POZ domains serve as dimerization interface and are also Cullin-3 adaptors that provide substrate specificity for Cullin-3 based E3 ubiquitin ligases for subsequent ubiquitin-dependent degradation by the 26S proteasome [START_REF] Pintard | Cullin-based ubiquitin ligases: Cul3-BTB complexes join the family[END_REF][START_REF] Van Den Heuvel | Protein degradation: CUL-3 and BTB-partners in proteolysis[END_REF][START_REF] Gingerich | Large-scale, lineage-specific expansion of a bric-a-brac/tramtrack/ broad complex ubiquitin-ligase gene family in rice[END_REF][START_REF] Zhang | BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance[END_REF]. On the other hand, the four conserved ankyrin repeats are known to mediate interactions with TGACG sequence specific binding (Shindler et al., 1992;[START_REF] Foster | Plant bZIP proteins gather at ACGT elements[END_REF] basic leucin zipper transcription factors (TGA bZIP TFs; [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF]Deprès et al., 2000;[START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF].

Introduction

In A. thaliana, the members of the TGA bZIP TF family belong to a distinct subclade in the bZIP super family and participate in many development and stress response processes [START_REF] Jakoby | bZIP transcription factors in Arabidopsis[END_REF]. In A. thaliana, there are ten TGA bZIP TFs which are divided into five clades according to their sequences and functions [START_REF] Gatz | From pioneers to team players: TGA transcription factors provide a molecular link between different stress pathways[END_REF][START_REF] Gutsche | The N-terminus of the floral Arabidopsis TGA transcription factor PERIANTHIA mediates redox-sensitive DNA binding[END_REF].

TGA1 and TGA4 group in class I, TGA2, TGA5 and TGA6 group in class II, TGA3 and TGA7 group in class III, TGA9 and TGA10 group in class IV and TGA8/PERIANTHIA groups in class V. The TGA bZIP TFs are known to perform their activities by binding cisregulatory DNA elements that comprise at least TGACG core recognition sequence.

The A. thaliana AtNPR1 is a well characterized regulator of the plant immunity involved in localized (LAR) and systemic (SAR) acquired resistance [START_REF] Cao | Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance[END_REF]Delany et al., 1995;[START_REF] Wang | A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants[END_REF][START_REF] Ding | Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana[END_REF]. In the AtNPR1 signaling model, the accumulation of salicylic acid (SA) phytohormone induces the expression of AtNPR1 [START_REF] Ryals | The Arabidopsis NIM1 protein shows homology to the mammalian transcription factor inhibitor IB[END_REF][START_REF] Yu | Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression[END_REF]. In the cytosol AtNPR1 forms oligomers and the SA modify the intracellular redox status that results in the conversion of AtNPR1 into active monomers [START_REF] Mou | Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes[END_REF] able to penetrate in the nucleus. Once in the nucleus, AtNPR1

interacts with a panel of TGA bZIP TFs such as TGA2, TGA5, TGA6, TGA3 and TGA7 [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF]Deprès et al., 2000;[START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF][START_REF] Boyle | The BTB/POZ Domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function[END_REF]. AtNPR1/TGA bZIP TF complexes are able to bind SA-responsive elements and to promote the expression of defense-related genes [START_REF] Eckardt | A new twist on systemic acquired resistance: redox control of the NPR1-TGA1 interaction by salicylic acid[END_REF][START_REF] Dong | NPR1, all things considered[END_REF]. The turnover of AtNPR1 proteins is regulated via AtNPR3 and AtNPR4 Cullin-3 adaptor proteins for degradation via proteasome (Petroski and Deshaies, 2005;[START_REF] Spoel | Proteasome-mediated turnover of the transcription coactivator NPR1 plays dual roles in regulating plant immunity[END_REF][START_REF] Fu | NPR3 and NPR4 are receptors for the immune signal salicylic acid in plants[END_REF][START_REF] Ding | Abscisic acid promotes proteasome-mediated degradation of the transcription coactivator NPR1 in Arabidopsis thaliana[END_REF].

The A. thaliana AtBLADE-ON-PETIOLE1 and AtBLADE-ON-PETIOLE2 (AtBOP1/2) represent the best characterized NBCL genes. These two genes are functionally redundant and are essential regulators in the gene network allowing the complex regulation of plant boundaries (Aida andTasaka, 2006a, 2006b;[START_REF] Barton | Twenty years on: The inner workings of the shoot apical meristem, a developmental dynamo[END_REF][START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF]. AtBOP1/2 genes are involved in stipule formation, proximal/distal leaf patterning, leaf growth determinacy, internode elongation, floral meristem identity acquisition, inflorescence initiation, flower patterning, floral abscission and were recently shown to be involved in photo/thermo-morphogenesis [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]Khan et al., 2012;[START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF][START_REF] Zhang | BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance[END_REF]. AtBOP1/2 genes were originally described for the leafy petiole phenotype observed in Atbop1 and Atbop1bop2 mutants [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF]2004;[START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF] and for the abnormal long leaves and inflorescence defects of Atbop1bop2, resulting in serrated cauline leaves, loss of floral organ abscission and floral organization defects [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF]. Wild-type A. thaliana flowers present a high degree of radial symmetry in which four types of organ initiated from floral meristem are arranged in four distinct concentric whorls. The wild-type A. thaliana tetramerous flower is composed by four sepals, four petals, six stamens and two carpels (from outer to inner whorls). In Atbop1bop2, flowers show supplementary floral organs leading to asymmetric flowers. Flowers that are often subtended by a small floral bract contain usually five sepals, four petals, six or seven stamens and two carpels [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF].

Similarly, AtTGA8/PERIANTHIA (AtPAN) gene encoding a TGA bZIP TF is also required for the correct patterning of A. thaliana flower and regulates the initiation of floral organ primordia. Atpan mutant phenotypes mostly impact the flower and affect specifically the floral organs number leading to the formation of pentamerous (five sepals, five petals and five stamens) flowers instead of tetramerous one [START_REF] Running | Mutations in the PERIANTHIA gene of Arabidopsis specifically alter floral organ number and initiation pattern[END_REF][START_REF] Meyerowitz | Genetic control of cell division patterns in developing plants[END_REF][START_REF] Chuang | The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Gutsche | The N-terminus of the floral Arabidopsis TGA transcription factor PERIANTHIA mediates redox-sensitive DNA binding[END_REF].

The Atpan mutant displays also elongated petioles, curled leaves and twisted rosettes when exposed to long day conditions [START_REF] Maier | The bZIP transcription factor PERIANTHIA: a multifunctional hub for meristem control[END_REF].

AtBOP1/2 proteins were shown to directly interact with the AtPAN, with the TGA bZIP TFs interacting with AtNPR1 and moreover with TGA1 and TGA4 that are not AtNPR1 partners [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF]. AtBOP1-AtBOP2-AtPAN were later described as forming a nuclear complex controlling the sepal number explaining the overlapping perianth patterning defects observed in Atbop1bop2 double mutant and Atpan [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]. Such NBCL/TGA bZIP TF interactions were also described in the Solanaceae Nicotiana tabacum. In this plant, the corolla abscission zone establishment requires the function of NtBOP2 which is also able to interact with TGA bZIP TFs supporting that such NBCL/TGA bZIP TF interactions are biologically relevant [START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF]. Also, it indicates that this NBCL/TGA bZIP TF module has been recruited several times during plant evolution.

In legume species forming nitrogen fixing nodules, a pair of NBCL genes is generally expressed in nodules. In Medicago truncatula and Pisum sativum, MtNOOT1/2 and PsCOCH1/2, respectively are orthologous to the AtBOP1/2 genes [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]Magne et al., 2017a, unpublished). Indeterminate nodules of M. truncatula and P. sativum are characterized by the presence of an apical meristem which is surrounded by several peripheral vascular meristematic zones derived from the root. In Mnoot1 and Pscoch1 single mutants, nodules display a partial loss of identity characterized by the outgrowth of ectopic roots arising from nodule vascular bundle apex [START_REF] Voroshilova | A unique pea (Pisum sativum L.) mutant impaired in nodule, leaf and flower development[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. In this particular context of symbiotic organs, NBCL genes appear to have been recruited to maintain the nodule identity through the repression of the peripheral vascular bundle root identity [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] themselves and with each other. We constructed the Mtnoot1pan-like and the Mtnoot2panlike double mutants and initiated their phenotypic and molecular characterization. In agreement with the transcriptomic data of Roux et al., 2014 (data not shown), qRT-PCR gene expression analysis of the 13 M. truncatula TGA bZIP TFs showed that they were all expressed in M. truncatula R-108 mature nodules excepted the second ortholog of AtPAN, MtTGA8-LIKE2, which was not enough expressed to be clearly detected in the nodule (Fig. 1b). This TGA bZIP TF transcriptional analysis revealed that our candidate gene MtPAN-LIKE is well expressed in nodule relative to the other MtTGA bZIP TFs and is the fourth and MtNOOT2 (Fig. 3). MtNOOT1, MtNOOT2 and MtPAN-LIKE seems to be expressed at the same times (Fig. 3) and in the same tissues during nodulation (Fig. 4), supporting well our hypothesis in which MtPAN-LIKE could represent a potential interacting partner of MtNOOT1 and/or MtNOOT2.

Results

Identitfication of the M. truncatula nodule specific gene

Isolation and characterization of M. truncatula Mtperianthia-like Tnt1 insertional mutant.

In the present study, to discover if Mtpan-like mutant exhibits nodule to root phenotypes, different M. truncatula Tnt1 insertional mutant lines for MtPAN-LIKE gene were used and compared to the Mtnoot Tnt1 mutants.

MtNOOT1 and MtNOOT2 encode BTB/POZ and four ankyrin repeats domains which are protein/protein interaction domains. The insertion lines Tnk507 [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]Magne et al., 2017a, unpublished) and NF5464 (Magne et al., 2017a, unpublished) are located in the BTB/POZ domain of MtNOOT1 and MtNOOT2 respectively (Fig. 5a,b).

MtPAN-LIKE gene displays eleven exons and ten introns and encodes a type D TGA bZIP TF (Fig. 5c; [START_REF] Jakoby | bZIP transcription factors in Arabidopsis[END_REF][START_REF] Bentsink | Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis[END_REF][START_REF] Wang | Genome-wide analysis of the basic leucine zipper (bZIP) transcription factor gene family in six legume genomes[END_REF]. described as affecting seed dormancy [START_REF] Bentsink | Cloning of DOG1, a quantitative trait locus controlling seed dormancy in Arabidopsis[END_REF]. In its C-terminus, MtPAN-LIKE encodes also two glutamine-rich regions (22%, exon 10 and 11) with 6/29 glutamine residues at 346-374 and 5/21 glutamine residues at 417-437 accordinding to [START_REF] Chuang | The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[END_REF] These glutamine residues may serves as transcriptional activation domains [START_REF] Chuang | The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[END_REF][START_REF] Courey | Analysis of Sp1 in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif[END_REF]. Two insertion lines were isolated for the gene MtPAN-LIKE, NF12413 and NF10706 located in the third exon and in the third intron, respectively (Fig. 5c).

In M. truncatula cv. Jemalong A17, expressed sequence tag (EST) revealed the presence of four MtPAN-LIKE alternative transcripts (Medtr5G054900.1, Medtr5G054900.2, Medtr5G054900.3 and Medtr5G054900.4) due to the presence of tree alternative ATG start codon and an alternative splicing site of 12 bp located between exon 7 and 8 (Fig. 6a). We checked the existence of these alternative transcripts in M. truncatula R-108 by qRT-PCR and failed to demonstrate their existence (Fig. 6b). qRT-PCR transcript analysis, MtPAN-LIKE cDNA cloning and sequencing showed that M. truncatula R-108 expresses only one MtPAN-LIKE transcript which is the equivalent of the M. truncatula cv. Jemalong A17 Medtr5G054900.2 without the 12 bp deletion (Fig. 6c). qRT-PCR gene expression analysis on the Mtpan-like NF12413 mutant line showed that the Tnt1 insertion impacts the MtPAN-LIKE transcription relative to R-108 in nodule and indicates that this line represents a true KO mutant (Fig. 6d). Characterization of both Mtpan-like NF10706 (intronic insertion) and NF12413

(exonic insertion) did not reveal any particular aerial phenotype in agreement with the absence of MtPAN-LIKE expression in aerial organs (Fig. 2). Global observations of nodules at 35 dpi did not reveal any mutant phenotype nor nodule organization defect relative to wildtype R-108 nodules. The nitrogen fixation efficiencies of Mtpan-like mutants were assessed by acetylene reduction assay (ARA; [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF] and did not reveal significant differences relative to R-108 nodules (Fig. 7). This result indicates that MtPAN-LIKE gene alone is not essential for the nodule formation nor for nodule identity maintenance. The subsequent experiments will be focused on line Mtpan-like NF12413 because of the exonic insertion of the Tnt1 retro-element. with the ability of NBCL and NPR1-LIKE proteins to dimerize and to interact with TGA bZIP TFs described in the literature. In A. thaliana, AtBOP1 and AtBOP2 homo and heterodimerize [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Canet | The BLADE-ON-PETIOLE genes of Arabidopsis are essential for resistance induced by methyl jasmonate[END_REF][START_REF] Jun | BLADE-ON-PETIOLE1 Coordinates Organ Determinacy and Axial Polarity in Arabidopsis by Directly Activating ASYMMETRIC LEAVES2[END_REF], AtNPR1 forms homodimers [START_REF] Mou | Inducers of plant systemic acquired resistance regulate NPR1 function through redox changes[END_REF], in N. tabacum, NtBOP2 is able to form homodimer (Wu et al., CHAPTER III. The key regulators of the symbiotic identity MtNODULEROOT1 and MtNODULEROOT2 interact with the TGA bZIP transcriptional factor MtPERIANTHIA-LIKE 129 2012) and in S. lycopersicum, SlBOP1, SlBOP2 and SlBOP3 homo and hetero-dimerize [START_REF] Xu | Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators[END_REF]. Moreover, AtBOP1/AtBOP2 interact with AtPAN [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF]Xu et al., 2010), NtBOP2 interact with a set of TGA bZIP TF such TGA1a, TGA2.2, TGA2.1, PG13 and RSG [START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF] and AtNPR1 interacts with TGA1, TGA2, TGA3, TGA5, TGA6, and TGA7 [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF][START_REF] Després | The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors[END_REF][START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF][START_REF] Després | The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1[END_REF][START_REF] Boyle | The BTB/POZ Domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function[END_REF]. To confirm these interactions in planta, we undertook an original strategy based on the transient expression of a protein fused to the green fluorescent protein (GFP) together with another protein without GFP. This approach can work if the tested proteins have distinct subcellular localizations. In this case, the interaction between the two proteins will result in a delocalization of the GFP signal. We constructed p35S:NOOT1, p35S:NOOT2, p35S:PAN-LIKE fused or not to GFP. The p35S:GFP construct served as positive control to assess the experiment efficiency and the viral protein helper component-proteinase (HC-Pro) was used as RNA silencing inhibitor to improve the transient expression [START_REF] Mallory | A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco[END_REF].

Single constructions were first tested alone in tobacco leaf epidermal cells, p35S:GFP showed a nucleo/cytoplasmic GFP signal (Fig. 10a), p35S:NOOT1, p35S:NOOT2 and p35S:PAN-LIKE showed no GFP signal (Fig. 10b,c,d), p35S:NOOT1:GFP and p35S:NOOT2:GFP showed a nucleo/cytoplasmic GFP signal (Fig. 10e,f) and p35S:PAN-LIKE:GFP showed a GFP signal restricted to the nucleus (Fig. 10g). MtPAN-LIKE is involve in the nodule identity MtNOOT1/2-dependent pathway [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]Magne et al., 2017a, unpublished) we initiated genetic approaches to uncover the relationship existing between MtNOOT1, MtNOOT2 and MtPAN-LIKE.

We constructed Mtnoot1pan-like and Mtnoot2pan-like double mutants. F2 progenies from four F1 Mtnoot1pan-like double heterozygous mutants and from three F1 Mtnoot2panlike double heterozygous mutants segregated following dihybridism inheritance ratio and showed the MtNOOT1, MtNOOT2 and MtPAN-LIKE loci reciprocal independence (9:3:3:1; X²: 5.717 and X²: 6.007, respectively; Tables 1 and2). according Magne et al., 2017a, unpublished) and the remaining nodules were uni-or multilobed (≈50 %-50 %). Mtnoot1noot2 showed almost only nodules converted into roots (Fig. 12a) as previously described (Magne et al., 2017a, unpublished). The proportion of nodule types observed in Mtnoot1pan-like and Mtnoot2pan-like double mutants was not different from those of their corresponding Mtnoot single mutants suggesting that there is no additional phenotype due to the Mtpan-like mutation in association with the single Mtnoot mutations (Fig. 12a). In addition ARA showed that all mutant lines were fix + except Mtnoot1noot2 double mutant which was completely fix -(Fig. 12b), as previously described (Magne et al., 2017a, unpublished). MtPAN-LIKE gene appears facultative in the nodule identity MtNOOTdependent pathway and more generally for nitrogen fixing symbiosis since the Mtpan-like mutation alone and in association with Mtnoot1 or Mtnoot2 does not impact the symbiotic performance by contrast to the drastic symbiotic performance defect observed in Mtnoot1noot2 (Fig. 12a,b). The loss of nodule identity is characterized by a nodule to root homeosis and by drastic molecular shifts such as symbiotic marker gene expression reduction, and both defense and root identity marker genes expression induction (Magne et al., 2017a, unpublished). To better characterized Mtnootpan-like mutants, qRT-PCR expression analysis were performed in mutant nodules using symbiotic, defense and root identity marker genes (Fig. 13a,b,c). MtNIN, MtNCR001 and MtLEGH1 symbiotic genes expression analysis showed that all the mutants correctly expressed symbiotic genes except Mtnoot1noot2

showing a low level expression (Fig. 13a). We noted that in Mtnoot1 and Mtnoot1pan-like Then we investigated if other TGA bZIP TF genes are over-expressed in the Mtpanlike mutant background to compensate the loss of expression of the MtPAN-LIKE gene. For this, the expression of the 13 TGA bZIP TF genes was analyzed in the nodules of Mtnoot1, Mtnoot2 and Mtpan-like single mutants and compared to wild-type R-108 nodules (Fig. 14).

mutants
In Mtpan-like background, the Mtpan-like gene expression is significantly reduced but no other TGA bZIP TF gene are over-expressed. However the expression of MtTGA2/5/6-LIKE 1 is slightly reduced and the expression of MtTGA1/4-LIKE3 gene is two times less important than in the wild-type nodules (Fig. 14). Intriguingly, when we look at the TGA bZIP TF gene expressions in Mtnoot1 mutant in which the MtPAN-LIKE gene expression is reduced, we also found a similar drastic reduction of the MtTGA1/4-LIKE 3 expression.

In Mtnoot1, MtTGA2/5/6-LIKE 1 and MtTGA9-LIKE 1 are slightly up-regulated and MtTGA3/7-LIKE 2 gene expression is reduced (Fig. 14). In the Mtnoot2 mutant background, the MtPAN-LIKE gene expression is not affected and surprisingly the MtTGA1/4-LIKE3 gene expression, that was reduced in Mtnoot1 and in Mtpan-like, remained similar to the level of expression in R-108 nodules. Mtnoot2 mutation affects the expression of MtTGA3/7-LIKE2

and MtTGA9-LIKE1 (Fig. 14). These experiments also show that MtTGA8-LIKE 2 gene expression is always below the detection level and that MtTGA2/5/6-LIKE2, MtTGA3 MtTGA 2/5/6-LIKE2. These class II TGA bZIP TFs are known to be important for the SAdependent NPR1 pathway immunity response in A. thaliana [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF]Deprès et al., 2000;[START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF]. It is known that part of the immune status in nodule is repressed via the action of MtDNF2 and SYMCRK in order to accommodate the high number of rhizobia needed to establish an efficient nitrogen fixing symbiosis (Bourcy et al., 2013a;Bourcy et al., 2013b;Berrabah et al., 2014a, Berrabah et al., 2014b, Berrabah et al., 2015).

The closest ortholog of AtPAN in M. truncatula corresponds to the nodule specific

MtPAN-LIKE gene

Here, we overall identified the closest ortholog of AtPAN in M. truncatula and named it MtPAN-LIKE gene. In Arabidopsis, AtPAN is known to interact with AtBOP1 and AtBOP2

to regulate the perianth patterning [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]. The phenotypic analysis of the Mtpan-like mutant did not reveal any aerial phenotype in agreement with the absence of expression of this gene in the vegetative and reproductive part of the plant. Surprisingly, despite the nodule-specific expression pattern of this gene, phenotypic analysis of the Mtpan-like nodules did not revealed particular mutant phenotypes or nitrogen fixation defect. Thus we conclude that MtPAN-LIKE gene alone was not essential for the nodule formation neither for the nodule identity maintenance.

MtNOOT1, MtNOOT2 are able to interact with MtPAN-LIKE in vivo

In addition to the common spatial and temporal gene expression pattern of MtNOOT1, MtNOOT2 and MtPAN-LIKE genes, using transient expression of protein-GFP fusion in tobacco leaf epidermal cells, we demonstrated that MtNOOT1:GFP and MtNOOT2:GFP are both localized in the nucleus and the cytoplasm and that the MtPAN-LIKE:GFP localization is restricted to the nucleus. These localizations are in agreement with the localization described in the literature for BTB/POZ and ankyrin repeat co-transcriptional factor and TGA bZIP TF. Indeed, AtNPR1-GFP [START_REF] Kinkema | Nuclear localization of NPR1 is required for activation of PR gene expression[END_REF], AtBOP1-GFP [START_REF] Jun | BLADE-ON-PETIOLE1 Coordinates Organ Determinacy and Axial Polarity in Arabidopsis by Directly Activating ASYMMETRIC LEAVES2[END_REF], AtBOP2-GFP [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF], PpBOP1-GFP [START_REF] Saleh | MicroRNA534a control of BLADE-ON-PETIOLE 1 and 2 mediates juvenile-to-adult gametophyte transition in Physcomitrella patens[END_REF], NtBOP2-GFP [START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF], and SlBOP1-GFP, SlBOP2-GFP, SlBOP3-GFP [START_REF] Xu | Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators[END_REF] were all found in both cytosol and nucleus. In addition AtPAN was described as localized in the nucleus [START_REF] Chuang | The PERIANTHIA gene encodes a bZIP protein involved in the determination of floral organ number in Arabidopsis thaliana[END_REF]. Given the spatial localization of these proteins at a subcellular level, these results indicate that interaction between nucleo-cytoplasmic MtNOOT proteins and nuclear TGA bZIP TF are possible. Interestingly, in their works, [START_REF] Maier | The bZIP transcription factor PERIANTHIA: a multifunctional hub for meristem control[END_REF] showed that despite that the Atperianthia mutant does not display drastic developmental defect, AtPAN is intimately connected to the shoot apical meristem regulatory network. The authors described that AtPAN functions as an important interface (multifunctional hub) in plant stem-cell control by genetically interacting between the components of both the cytokinin and the auxin pathways.

It is highly interesting that AtPAN can play such a role in the shoot apical meristem regulation and we can thus hypothesize that MtPAN-LIKE plays a similar role in the nodule by ensuring the regulation of the nodule central meristem and/or the nodule peripheral meristem.

The proNOOT1:GUS and the proNOOT2:GUS expression patterns have been described as complementary. The proNOOT1:GUS expression pattern associate mainly to the nodule vascular bundle tips and the proNOOT2:GUS expression pattern associate to the nodule central meristem. The proNOOT1:GUS and the proNOOT2:GUS expression patterns overlap at the junctions between NVM and NCM (Fig. 15; Magne et al., 2017a, unpublished). Data from [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF] indicate that MtPAN-LIKE is mostly expressed in the apical part of the nodule, similarly to the MtNOOT genes (Fig. 4). Despite the incertitude concerning the precise MtPAN-LIKE gene expression pattern in the nodule, the protein interactions we described between MtNOOT1, MtNOOT2 and MtPAN-LIKE suggest that MtPAN-LIKE gene should at least be expressed in nodule vascular bundles, in the nodule central meristem and at the NVM / NCM boundary region to interact with both of MtNOOT proteins (Fig. 15). On the other hand, the presence of MtPAN-LIKE transcripts outside of these tissues cannot be excluded. We can also imagine that an eventual MtNOOT1/MtNOOT2/MtPAN-LIKE complex can be formed at the NVM / NCM boundary region where both MtNOOT proteins should be present (Fig. 15) as described for the perianth patterning regulation involving BOP1/BOP2/PAN in Arabidopsis [START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF].

According to the derepression of MtPLETHORA2-LIKE (auxin pathway) and the downregulation of MtKNOX3 (cytokinin pathway) previously observed in the Mtnoot1 and the Mtnoot1noot2 double mutant (Magne et al., 2017a, unpublished), on the findings of the present study and on the works of [START_REF] Maier | The bZIP transcription factor PERIANTHIA: a multifunctional hub for meristem control[END_REF], it is tempting to speculate that MtPAN-LIKE can participate to the regulation network that ensure the co-existence and the maintenance of the different meristems present in the nodule by modulating components of the cytokinin and the auxin phytohormone pathways. 2008; GenBank: BAG50070.1). The bZIP-M43 gene is described as lately induced during nodulation [START_REF] Asamizu | A positive regulatory role for LjERF1 in the nodulation process is revealed by systematic analysis of nodule-associated transcription factors of Lotus japonicus[END_REF] and is also found as strongly expressed in the determinate nodule in the Lotus japonicus Gene Expression Atlas (LjGEA;Hogslund et al., 2009;chr5.CM0909.32_at). Also in P. sativum, PsCam014568 is found as strongly induced in

Material and methods

Plant material and growth conditions. Medicago truncatula Gaertn ecotypes R108 [START_REF] Hoffmann | A New Medicago truncatula Line with Superior in Vitro Regeneration, Transformation, and Symbiotic Properties Isolated Through Cell Culture Selection[END_REF] and its corresponding mutants Mtnoot1 (Tnk507, [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF], Mtnoot2 (NF5464, this study), Mtpan-like (NF12413, this study), Mtnoot1pan-like (Tnk507 crossed with NF12413), Mtnoot2pan-like (NF5464 crossed with NF12413) and Mtnoot1noot2 (NF2717 crossed with NF5464, this study) were grown on a mixture of perlite and sand (2/1, v/v) or on buffered nodulation medium (BNM) in vitro plate [START_REF] Ehrhardt | Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors[END_REF] solidified with Kalys agar 7 g.L -1 and supplemented with 0,5 µM of 2aminoethoxyvinyl glycine (Sigma-aldrich). Classically, for M. truncatula nodulation, S. medicae WSM419 were used for inoculation as described in [START_REF] Berrabah | Multiple steps control immunity during the intracellular accommodation of rhizobia[END_REF]. Plants were grown in a controlled environmental chamber with 16/8 h light-dark cycle, 24/24 °C day-night temperature, relative humidity 60 % and 200 µE light intensity. Plants were watered three times a week (2 times with water / 1 times with nutritive solution in alternance)

with N-free nutritive solution (Plant prod, NPK 0-15-40) for nodulation.

Rhizobial strain, growth conditions and inoculation. Sinorhizobium medicae WSM419 strain chloramphenicol resistant [START_REF] Howieson | Acid tolerance in the Rhizobium meliloti-Medicago symbiosis[END_REF], were grown on YEB medium [START_REF] Krall | Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens[END_REF] for 24 h at 30 °C before inoculation. For in vitro and sand/perlite nodulation, M. truncatula seedlings were inoculated with 1 mL and 25 mL of S. medicae suspension at OD: 0,1, respectively.

Yeast strain and growth conditions. AH109 yeast strain [START_REF] James | Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast[END_REF]Holtz, unpublished) was used for Y2H approaches. AH109 were grown on solid YPD agar (Y1500, Sigma-aldrich) for 2 day at 30 °C or in liquid YPD Broth (Y1375, Sigma-aldrich) at 30 °C, under 180 rpm agitation. Co-transformed AH109 yeast strains were grown for 2 day at 30 °C on minimal SD -L -W agar (Clontech, 630412, 630417) or for 16-18 h at 30 °C in 5 mL of minimal SD -L -W Broth (Clontech, ST0047) under 230-270 rpm agitation until OD 600nm >1,5.

Agrobacterium tumefaciens strain and growth conditions. Agrobacterium tumefaciens strain EHA105 and GV3.101 rifampycin resistant were used for protein transient expression assays in tobacco leaf epidermic cells. EHA105 were grown overnight at 28 °C under 200 rpm agitation in 5 mL of YEB [START_REF] Krall | Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens[END_REF] supplemented with kanamycin 50 µg/mL when transformed with pCAMBIA13102 .

Phylogenetic tree construction. Full-length M. truncatula TGACG type bZIP transcription factor protein sequences were obtained by BLAST search on NCBI (https://blast.ncbi.nlm.nih.gov). TGACG type bZIP transcription factor protein sequences were aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] and the phylogenetic tree was built using maximum likelihood-based method using the Jones-Taylor-Thornton (JTT) substitution model with the MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF].

M. truncatula DNA extraction.

Young leaves were frozen in liquid nitrogen and grinded with a ball grinder (Retsch, MM400) two times 30 seconds with a frequency of 25.s -1 . 600 µL of fresh DNA extraction buffer (NaCl (350 mM), Tris-HCl pH: 7,6 (10 mM), Urea (7 M), N-Lauroyl sarcosine (2 %), EDTA (50 mM), aqueous phenol pH: 7,9 (6,5 %)) were added to the powder. The suspension was vortexed, incubated 5 min at 50 °C and shaken 10 min at room temperature. 700 µL of aqueous phenol pH: 7,9; chloroform; iso-amylalcohol (25:24:1) were added. Suspension was vortexed, centrifugated 10 min at 14000 rpm and aqueous phase was collected. 700 µL of chloroform; iso-amylalcohol (24:1) were add on aqueous phase.

Suspension was vortexed, centrifuged 5 min at 14000 rpm, aqueous phase was collected and DNA precipitation was carried-out adding cold sodium acetate 3 M; isopropanol (0,1:1).

After a 10min centrifugation at 14000 rpm the DNA pellet was washed with 500 µL of ethanol 70 %. After a 5 min centrifugation at 14000 rpm the DNA pellet was dried, resuspended in 100 µL of sterile water and heated 5 min at 65 °C. RNase treatment was finally performed with 5 µL of 1 mg.mL -1 RNase (Roche).

M. truncatula mutants genotyping. M. truncatula noot1 (TNK507), Mtnoot2 (NF5464),

Mtpan-like (NF12413) and Mtpan-like (NF10706) mutant plants homozygous for a Tnt1 retroelement insertion were genotyped by semi-quantitative PCR. Detailed information concerning oligonucleotides used for genotyping are provides supplemental table 1.

M. truncatula cross-fertilization. The M. truncatula Mtnoot1 (Tnk507) and M. truncatula Mtnoot2 (NF5464) mutant plants homozygous for a Tnt1 retroelement insertion were crossed independently with M. truncatula Mtpan-like (NF12413) mutant plants homozygous for a Tnt1 retroelement insertion. For each crossing Mtnoot1 or Mtnoot2 plant served as pollen receiver female plant and Mtpan-like served as pollen donor male plant. Briefly, stamens holding fresh pollen were first collected from male donor plant at an advanced flower development stage before flower aperture. Under magnifying glass, receiver plant flowers were selected when sepals represent two-thirds of the whole flower length, opened from the standard petal and the anthers were removed. Fresh sticky pollen from male donor plant was dropped on the female plant receiver stigma and the flower was delicately reclosed.

Following cross-fertilization, female receiver flowers were protected from desiccation using a 50 mL Falcon tube and removed two days after pollination.

Acetylene reduction assay

Acetylene reduction assays (ARA) were performed on individual plants inoculated with S. medicae WSM419 (35 dpi) with a protocol modified from [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. Basically, the nodulated root system of one plant was placed in 21 mL glass vial sealed with rubber septa in presence of 200 µL of water to avoid dessication. Acetylene gas (500 µL) was injected into each vial and 2 h incubation was performed. For each sample, 200 µL of gas were injected. Ethylene production was measured by gas chromatography, using a 7820A

Gas Chromatograph (Agilent Technologies) equipped with a GS-Alumina column (50 m x 0,53 mm) with hydrogen as carrier gas. Column temperature and gas flow were adjusted at 120 °C and at 7,5 mL.min -1 , respectively. The experiment represents three independent biological replicates of nine plants for each Mtnoot mutant and R-108 plant.

Gene Expression Analyses

Total RNA extractions were performed from frozen tissues with TRIzol® reagent (Ambion).

RNA samples were treated with the TURBO DNA-free™ Kit (Ambion) according to the manufacturer's recommendations. Full-length cDNA were synthesized using SuperScript™ sequences and origin of oligonucleotides used for qRT-PCR are described in supplemental table 2.

Vector construction for Yeast-two-hybrid assay. cDNA amplification. MtNOOT1 (1452 bp), MtNOOT2 (1479 bp) and MtPAN-LIKE (1410 bp) sequences were amplified from cDNA of R-108 nodule (51 dpi) by PCR using High Fidelity Taq DNA polymerase (EUROBIO GAETHF004D). Information concerning oligonucleotides used for cDNA amplification are given in supplemental table 3. PCR products were sequenced by Sanger (www.eurofinsgenomics.eu). Cloning. 3' adenine were added using Dream Taq DNA polymerase (Thermo Fisher Scientific Inc.), PCR products were purified with GeneJET PCR Purification Kit (Thermo Fisher Scientific Inc.), cloned in pGEM-T Easy vector using pGEM-T Easy Vector System I (Promega) and introduced in One Shot® TOP10

Chemically Competent E. coli (Thermo Fisher Scientific Inc.). pGEM-T Easy vectors containing construction were sequenced by Sanger using T7 and SP6 universal oligonucleotides (Supplemental Table 3). Sub-cloning in pGADT7 AD and pGBKT7 vectors. MtNOOT1, MtNOOT2 and MtPAN-LIKE full length cDNA were respectively excised by digestion from pGEM-T easy vector using the following restriction enzyme combinations NdeI/BamHI, NdeI/XmaI and SfiI/BamHI (New England Biolabs). Inserts from pGEM-T easy vectors and linearized pGADT7 AD and pGBKT7 destination vectors (Clontech Laboratories, Inc) were purified with NucleoSpin ® Gel and PCR Clean-up (Macherey nagel). Each construction was sub-cloned using T4 DNA ligase (Thermo Fisher Scientific Inc.) in both pGADT7 AD and pGBKT7 destination vectors and introduced in One Shot® TOP10. pGADT7 AD:MtNOOT1, pGADT7 AD:MtNOOT2, pGADT7 AD:MtPAN-LIKE, pGBKT7: MtNOOT1, pGBKT7: MtNOOT2 and pGBKT7:MtPAN-LIKE construction were sequenced by Sanger. Sequences obtained were aligned on their respective reference sequences using ClustalW online alignment software (www.genome.jp/tools/clustalw/), cDNA sequences and orientations in their respective vectors were checked. Information concerning oligonucleotides used for sequencing in pGADT7 AD and pGBKT7 are provided in supplemental table 3 .

AH109 Yeast co-transformation. Yeast transformations were performed using the LiAC method [START_REF] Gietz | Transforming yeast with DNA[END_REF] with some modifications. Fresh AH109 liquid culture is carried-out during 24 h until DO 600nm reach 2,6. The culture is diluted in fresh pre-warmed liquid YPD at OD 600nm = 0,2-0,25 and grown for 3 to 6 h to obtain 0,4 < OD 600nm < 0,6. 50 mL of the culture was centrifuged 5 min at 3500 rpm, resuspended in 25 mL of sterile water and centrifuged 5 min at 3500 rpm, resuspended in 1 mL of freshly-prepared lithium acetate (LiAc) 0,1 M, pH: 7,5 (L-4158, sigma-aldrich), transferred in a 2 mL tube and full speed centrifuged for 15 sec and finally resuspended in 400 µL of LiAc 0,1 M, pH: 7,5. Cotransformations were carried-out in 2 mL tube, in presence of 250 ng of each vector, 50 µL of yeast cell suspension, 240 µL of sterile PEG 50 % (p-3640, Sigma-aldrich), 36 µL of sterile LiAc 1 M, pH: 7,5, 64 µL of sterile water and 10 µL of 10 mg/mL denaturated single stranded deoxyribonucleic acid from salmon testes (D7656, Sigma-aldrich). Mixtures were vortexed for 30 sec, incubated for 30 min at 30 °C under 200 rpm agitation and the tubes were inverted every 10 min. Yeast cells were incubated at 42 °C during 30 min, centrifuged at 7000 rpm for 15 sec, resuspended in 200 µL of sterile water and grown for 4-7 days on minimal SD -L -W agar to select co-transformed yeast colonies.

Y2H growth assay. Protein/protein interactions was carried-out by assessing the growth fitness of co-transformed AH109. Co-transformed AH109 were grown in 1 mL of minimal SD -L -W Broth in a 2 mL 96 well plate (greiner bio-one, Masterblock®, 780271) sealed with adhesive gas permeable seals (THERMO-SCIENTIFIC, AB-0718) overnight and under agitation. The overnight cultures were diluted 20 and 100 times in sterile water, 5 µL of each dilution were dropped on both minimal SD -L -W -H ( 630412, 630419, CLONTECH) and minimal SD -L -W -H -A ( 630412, 630428, CLONTECH) media supplemented with 0, 1, 2,5, 5, 10, 25 mM of 3-amino-1,2,4-triazole (3-AT, Sigma-aldrich, A8056).

Y2H β-galactosidase assay. The β-galactosidase assay using ONPG as substrate was carriedout following manufacturer's recommendation (Protocol PT3024-1, Clontech Laboratories, Inc). Briefly, from 2 mL of overnight co-transformed AH109, a suspension is carried-out in YPD broth to OD 600nm = 0,3. The suspension was grown for 3-6 h under shaking until mid-log phase between 0,5 < OD600nm < 0,8 and the exact OD 600 was recorded. Three 1,5 mL tubes were filled 1,5 mL of culture, centrifuged at 14000 rpm for 30 sec wash with 1,5 mL of Z buffer [Na 2 HPO 4 .7H 2 0 (16,1 g/L); NaH 2 PO 4 .H 2 O (5,5 g/L); KCl (0,75 g/L); MgSO 4 .7H 2 0 (0,246 g/L); pH: 7], centrifuged at 14000 rpm for 30 sec and resuspended in 375 µL of Z buffer, the concentration factor was 4-fold. 100 µL of cell suspension are broken open by 3 successive freeze/thaw cycles and 700 µL of Z buffer/β-mercaptoethanol (0,27 %) were added, a blank was set up with 100 µL of Z buffer. 160 µL of freshly prepared o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma Aldrich, N-1127) dissolved in sterile Z buffer (4 mg/mL) were immediately added to the reactions. Reactions were incubated at 30°C during 1 h until the yellow coloration develops and stopped by adding 400 µL of Na 2 CO 3 1 M.

Reactions were centrifuged for 10 min at 14000 rpm and supernatants absorbance were measured at OD 420nm relative to the blank using a spectrophotometer. The linear range of the assay is comprised between 0,02 < OD 420nm < 1,0. β-galactosidase activity is expressed in βgalactosidase units or Miller units. 1 β-galactosidase unit corresponds to the amount which hydrolyzes 1 µmol of ONPG in o-nitrophenol and D-galactose per min and per cell (Miller, 1972;Miller, 1992). β-galactosidase units= (1000 x OD 420nm )/ [time of incubation in min x (0,1 mL x concentration factor) x OD 600nm of 1 mL of culture].

Vector construction for protein/GFP transient expression assays. cDNA amplification.

MtNOOT1 (1452 bp), MtNOOT2 (1479 bp) and MtPAN-LIKE (1410 bp) sequences were amplified using Q5® High-Fidelity DNA Polymerase (New England Biolabs, M0491S) from pGEM-T easy using different pairs of oligonucleotides allowing to clone cDNA sequences fused or instead of the mGFP5 sequence. Information concerning oligonucleotides used for cDNA amplification are given in supplemental table 4. Cloning. PCR products were purified using GeneJET PCR Purification Kit (Thermo Fisher Scientific Inc.). PCR products and pCAMBIA1302 binary vector (www.cambia.org/daisy/cambia/585.html; GenBank:

AF234298.1) were digested using appropriate digestion enzymes (NcoI Fast Digest, Thermo Fisher Scientific Inc., FD0573) for subsequent cloning upstream mGFP5 sequence or (NcoI Fast Digest and BstBI Fast Digest , Thermo Fisher Scientific Inc., FD0124 or BstEII, New England Biolabs, R0162S) for subsequent cloning instead mGFP5 sequence. Digested pCAMBIA1302 and PCR products were purified with NucleoSpin ® Gel and PCR Clean-up kit (Macherey nagel). Linear pCAMBIA1302 binary vectors were dephosphorylated using FastAP Thermosensitive Alkaline Phosphatase (Thermo Fisher Scientific Inc., EF0654).

Purified digested PCR products were ligated in dephosphorylated pCAMBIA1302 upstream to mGFP5 and instead of mGFP5 using T4 DNA ligase (Thermo Fisher Scientific Inc.

Introduction

The NOOT-BOP-COCH-LIKE (NBCL) genes, contain BTB/POZ (BROAD COMPLEX, TRAMTRACK and BRICK A BRACK/POXVIRUSES and ZINC FINGER) and ANKYRIN repeat domains and encode co-transcriptional factors involved in the gene regulatory network required for plant boundaries establishment (Aida andTasaka, 2006a, 2006b;[START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF]. These NBCL genes play important roles in stipules, leaves and flowers patterning and identity [START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 encodes a BTB/POZ domain protein required for leaf morphogenesis in Arabidopsis thaliana[END_REF][START_REF] Ha | BLADE-ON-PETIOLE1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]Khan et al., 2012;[START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF][START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF] as well as in abscission process [START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF][START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. NBCL genes were in addition recruited in legume plants to determine the symbiotic organ identity [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. This NBCL gene clade includes genes orthologous to Legume plants from the Fabaceae family belong to the Rosideae, one of the rare clades predisposed to establish symbiosis with nitrogen fixing rhizobia [START_REF] Soltis | Chloroplast gene sequence data suggest a single origin of the predisposition for symbiotic nitrogen fixation in angiosperms[END_REF][START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF]. This symbiosis takes place in a dedicated organ call the nodule. Trifolieae and Fabeae legume tribes form indeterminate nodules characterized by the presence of a persistent apical/central meristematic zone (Nodule Central Meristem, NCM, [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF] and peripheral vasculatures ontologically related to roots [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] possessing root apical meristem-like meristematic zone called nodule vascular meristem (NVM, [START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. Determinate nodules are found in the Phaseoleae and Loteae tribes and display subtle differences in terms of organogenesis and nodule organization, but do not possess a persistent NCM [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. In contrast to the plant-bacteria recognition and infection processes as well as the de novo organogenesis and functioning of the nodule that are well documented [START_REF] Oldroyd | Coordinating Nodule Morphogenesis with Rhizobial Infection in Legumes[END_REF][START_REF] Udvardi | Transport and Metabolism in Legume-Rhizobia Symbioses[END_REF][START_REF] Oldroyd | Speak, friend, and enter: signalling systems that promote beneficial symbiotic associations in plants[END_REF][START_REF] Suzaki | Leguminous Plants: Inventors of Root Nodules to Accommodate Symbiotic Bacteria[END_REF], the processes underlying the acquisition and preservation of the nodule identity as well as the maintenance of different meristematic zones present in this organ remain poorly described. Nodule identity studies were initiated by the discovery of the atypical P.

sativum Pscochleata1 (Pscoch1) and M. truncatula Mtnodule-root1 (Mtnoot1) which are nootbop-coch-like (nbcl) mutants, affected in nodule identity maintenance [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. These mutants are characterized by the emergence of root-like structure from the NVM [START_REF] Vandenbosch | Nodule Initiation Elicited by Noninfective Mutants of Rhizobium phaseoli[END_REF][START_REF] Akasaka | Morphological alterations and root nodule formation in Agrobacterium rhizogenes-mediated transgenic hairy roots of Peanut (Arachis hypogaea L.)[END_REF][START_REF] Voroshilova | A unique pea (Pisum sativum L.) mutant impaired in nodule, leaf and flower development[END_REF][START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Sinharoy | RNA interference highlights the role of CCaMK in dissemination of endosymbionts in the Aeschynomeneae legume Arachis[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]. In indeterminate nodules, the role of these two genes is to repress the root identity of NVM [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF].

To address the role of NBCL genes during symbiosis in the determinate nodule type, we studied the role of the single copy LjNOOT-BOP-COCH-LIKE gene (LjNBCL1), orthologous to MtNOOT1, in Lotus japonicus [START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. Our study revealed that the LjNBCL1 functions are conserved in this species for the maintenance of the L. japonicus determinate nodule identity but also unveiled unexpected and essential roles in L. japonicus vegetative and reproductive organ development.

Results

LjNBCL1 is required for nectary development, leaf proximal/distal patterning and controls the number of axillaries NBCL genes encode highly conserved co-transcriptional factors required for several plant developmental processes and especially for plant boundaries patterning (Reviewed in [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF]. The LjNBCL1 gene belongs to the NBCL clade and more precisely to the legume-specific NBCL1 sub-clade [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF] and was previously described for its role in aerial organs abscission [START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. A second Ljnbcl1 LOTUS RETROELEMENT1 (LORE1) mutant line (line 30119830, Fig. 1a) was isolated for this study. Self-fertilized progeny of this line heterozygous for the Ljnbcl1 mutation segregated as a monogenic recessive trait with seven homozygous wild-type plants, sixteen heterozygous plants and four plants homozygous for the mutant Ljnbcl1 locus (X²: 1,571; df: 2, pvalue: 0,05). The Ljnbcl1 lines 30119830 and 30053558 [START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF] have identical non-abscission and severe sterility phenotypes in agreement with their allelic nature. Data base and Lotus genome analysis reveals that only one NBCL gene is present in this plant in contrast to other legume plants. LjNBCL1 gene expression analysis in the aerial part of wild-type L. japonicus shows that LjNBCL1 transcripts accumulate at low levels in leaves, internodes and in young pods. They were more abundant in nodes and highly present during flower development from stage 10 to 13 (Fig. 2a) according to the floral stages described in [START_REF] Weng | Petal Development in Lotus japonicus[END_REF] These results are in agreement with LjNBCL1 expression data available from the Lotus japonicus Gene Expression Atlas (https://ljgea.noble.org/v2/; Supplementary Fig. 1). To better understand the LjNBCL1 expression kinetic during nodulation, its expression was compared to early symbiotic marker genes expression. LjNODULE INCEPTION and LjEARLY NODULINE40-1 gene expressions are induced early at 2 dpi (Fig. 3b). LjADENYLATE ISOPENTHENYL TRANSFERASE3 (LjIPT3) and LjNBCL1 gene expressions seem to be triggered together and follow a similar kinetic from 2 dpi to 5 dpi. Later, LjIPT3 is strongly induced in nodule while LjNBCL1 gene expression remains lower (Fig. 3b). LjPLETHORA2-LIKE (LjPLT2-LIKE) is the closest ortholog of the M. truncatula MtPLETHORA2 gene [START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]. In L. japonicus, this gene is strongly expressed in RAM and weakly expressed in the root. The LjPLT2-LIKE gene is clearly induced during nodule primordia formation and at the beginning of the L. japonicus determinate nodule development suggesting that LjPLT2-LIKE is necessary for the development of L. japonicus determinate nodule peripheral vasculature (Fig. 3b). From 12 to 20 dpi, LjPLT2-LIKE is no longer expressed. This LjPLT2-LIKE expression pattern may reflect the restriction of the root identity of the determinate nodule vasculature in agreement with the determinate state of NVM in determinate nodules previously suggested in [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF].

LjNBCL1 is required for determinate nodule identity maintenance of L. japonicus

In addition to the study of the two Ljnbcl1:LORE1 insertional mutants (30119830 and 30053558), we performed a RNA interference (RNAi) approach specifically targeting LjNBCL1 transcripts (Ljnbcl1:RNAi; Fig. 1a) via hairy-root transformation [START_REF] Kumagai | Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules[END_REF][START_REF] Okamoto | Hairy root transformation in Lotus japonicus[END_REF]. The efficiency of the Ljnbcl1:RNAi approach was assessed by qRT-PCR on transformed root nodules using a designed-RNAi against the β-GLUCURONIDASE coding gene as control (GUS:RNAi; [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 Cytokinin Receptor Regulates Lateral Root Development and Early Symbiotic Interaction with Sinorhizobium meliloti[END_REF]. This construct significantly reduced the LjNBCL1 mRNA transcripts accumulation by 56% in the transgenic root nodules (Fig. 1b).

Nodulation experiments using Ljnbcl1:LORE1 mutant lines (30053558 and 30119830)

and Ljnbcl1:RNAi hairy-root transformed plants inoculated with M. loti revealed a discrete nodule to root conversion phenotype relative to Mtnoot1 and Pscoch1 mutant nodules meaning that the strength of the mutation on the phenotype is weak (low penetrance). Wild-type L.

japonicus GIFU and GUS:RNAi transformed plants showed nodules with 100% wild-type nodule phenotype (Fig. 4a,c,e). By contrast to the M. truncactula or P. sativum nbcl mutants, the L.

japonicus nodule to root conversion penetrance resulting from Ljnbcl1 inactivation appears very low in both the Ljnbcl1:LORE1 lines and the Ljnbcl1:RNAi transgenic roots with only 4 % and 5

% of the nodules that exhibited ectopic root development, respectively (Fig. 4b,d,e). [START_REF] Pate | Ultrastructure and functioning of the transport system of the leguminous root nodule[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. Intriguingly, in L.

japonicus, it is the opposite. Bundle organization remains collateral but phloem tissues are turned toward the exterior of the nodule and the xylem tissues are turned toward the infection zone (Fig. 5g). In Ljnbcl1 mutant nodules, this atypical organization is conserved but when the NVB that will generate ectopic root starts to dissociate from the tissue containing the infected cells, the xylem and phloem poles delocalize progressively until xylem and phloem poles finally align respectively periclinally and anticlinally to the nodule periphery (Fig. 5h,i). This loss of NVB collateral organization in Ljnbcl1 represents a nodule vascular bundle to root vascular bundle identity switch.

LjNBCL1 gene function is required for floral patterning and development

The first striking phenotype observed in Ljnbcl1:LORE1 mutants was their defect in flower development leading to an almost complete sterility (Fig. 6a,b). A L. japonicus inflorescence presents one to three adaxial bracts and abaxial nectary glands arising at the pedicel base. This pedicel supports the receptacle from which twenty-one floral organs develop, five CHAPTER IV. NOOT-BOP-COCH-LIKE1 is essential for multiple aspects of Lotus japonicus development 169 fused sepals, five petals (one adaxial standard, two lateral wings and two abaxial fused keels), ten

(nine fused and one unfused) anthers and one carpel (Fig. 6c,e,g; [START_REF] Zhang | Proliferating Floral Organs (Pfo), a Lotus japonicus gene required for specifying floral meristem determinacy and organ identity, encodes an F-box protein[END_REF][START_REF] Dong | Floral Patterning in Lotus japonicus[END_REF]. Similarly to mutant leaf phenotype, Ljnbcl1 mutant flowers often display one additional adaxial bract relative to the maximum of three bracts present in wild-type plants (Fig. 6g,h). In addition, Ljnbcl1 mutants are drastically impaired in the formation of the floral organs and develop a range of more or less complex structures (homeosis) instead of a normal flower (see below). One characteristic structure formed instead of a flower is the fused trumpet-like structure shown in Fig. 6c,d. Ljnbcl1 presents an additional homeosis impacting the inflorescence nectaries located in abaxial position at the pedicel axil (Fig. 6c,e,g). By contrast to the nectaries located at leaf axils, that are absent in the mutant, these nectaries seem to always be transformed to leaf-like structures (Fig. 6d,f,h).

Taking into account only the bracts and the leaf-like structures, the simplest organ combination that can be found in Ljnbcl1 is one bract and one leaf-like structure and the most complex structure is four bracts and two leaf-like structures (Fig. 6h). All intermediary combinations exist in the mutant flowers, meaning that based on bract/leaf-like structure, height organ combinations exist in Ljnbcl1.

In addition to these bract/leaf like organs, the structures that develop instead of flowers in Ljnbcl1 can take several aspects (Fig. 7), and were grouped in seven flower-like structures:

absence of any structure (Fig. 7a), prematurely aborted fused trumpet-like structure (Fig. 7b), developed fused trumpet-like structure (Fig. 7c), leaf-like structure (Fig. d), more complex leaflike structure showing organ separations (Fig. 7e), aborted floral-like structure (Fig. 7f) and fullydeveloped but sterile flower (Fig. 7g). All inflorescence organs taken in account, bracts, homeotic leaf-like structures and central floral-like structures, Ljnbcl1 presented at least 56 different combinations of inflorescence organization, highlighting the very crucial role of the LjNBCL1 gene in determining the proper floral developmental frame.

Secondary inflorescence meristems and floral primordia development are affected in the

Ljnbcl1 mutant

In L. japonicus, as in other legumes, after floral transition, the apical meristem of each shoot becomes a primary inflorescence (I1) meristem that at each node, in the axil of the leaf, produces a secondary inflorescence (I2) meristem that generates the floral meristems [START_REF] Benlloch | Genetic control of inflorescence architecture in legumes[END_REF]. In L. japonicus, when floral meristems partitioned from the I2 meristems, they differentiate and get covered by trichomes [START_REF] Feng | Control of petal shape and floral zygomorphy in Lotus japonicus[END_REF]. To understand when the severe defects in floral development of Ljnbcl1 mutants initiate, we studied the ontogeny of the inflorescence of Ljnbcl1:LORE1 mutant plants by scanning electron microscopy.

SEM analysis showed that, as expected, the inflorescence apices of wild-type L. japonicus had normal I2 meristems, formed in the axils of compound leaves, and normal floral primordia, separated by trichomes derived from the differentiated I2 meristems (Fig. 8a-c). In contrast, the structures observed in the inflorescence apices of the Ljnbcl1:LORE1 mutant were very different.

At very early stages of their development, the leaf primordia subtended at their axils a poorly developed dome that was separated from the I1 meristem by a line of developing trichomes similar to the ones observed in the wild-type stage-5 I2 meristem when they differentiate after partition of the floral meristems [START_REF] Feng | Control of petal shape and floral zygomorphy in Lotus japonicus[END_REF]. Structures like wild-type I2 meristems of previous developmental stages were never detected in the mutant apex. The small domes delimited by the trichomes, corresponding to floral meristems, did not progress along with the development of the leaf primordia (Fig. 8d-f). These atypical structures in the Ljnbcl1:LORE1

inflorescence mutant apices suggest that their I2 meristems develop abnormally producing floral meristem-like structures that do not progress normally. This analysis shows that the defects in reproductive structures start very early in Ljnbcl1:LORE1 inflorescence ontogeny. apices, I1s are present, but no normal I2s are found at the leaf axils, and only a line of developing trichomes (I2*, purple), like stage-5 wild-type I2, is observed. This line of developing trichomes delimits a poorly developed dome that would represent a floral primordium-like structure (F*, red). I2 and floral primordia developmental stages are named according to previous descriptions of L. japonicus GIFU floral and inflorescence ontogeny [START_REF] Dong | Floral Patterning in Lotus japonicus[END_REF][START_REF] Feng | Control of petal shape and floral zygomorphy in Lotus japonicus[END_REF]. Scale bars: 50 µm.

Discussion

The strong vegetative and reproductive developmental defects observed in Ljnbcl1 mutant and the absence of an LjNBCL2 sequence in databases suggest that there is only one NBCL (LjNBCL1) in the genome of L. japonicus ecotype GIFU by contrast to other legume plants studied having at least two distinct NBCL genes. In this work we show that in the determinate nodules of L. japonicus, LjNBCL1 gene is constitutively expressed in root, induced during nodulation and thus behaves as the MtNOOT1 gene [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. We demonstrate that as in indeterminate nodule, despite subtle differences existing in organogenesis and nodule organization, LjNBCL1 gene function is conserved for the determinate nodule identity maintenance through NVB root identity repression. A similar phenotype was described in Glycine max determinate nodules inoculated with Bradyrhizobium japonicum mutant strains (ΔphyR, ΔecfG) also exhibiting nodule to root conversion [START_REF] Gourion | The PhyR-sigma (EcfG) signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum[END_REF] but the origin of the nodule ectopic roots was not described in this context. In this study we demonstrate that ectopic roots arising from determinate nodules are initiated from NVB as hypothesized by [START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]. This finding is in agreement with the literature describing that the nodule ectopic roots always initiate from NVM [START_REF] Vandenbosch | Nodule Initiation Elicited by Noninfective Mutants of Rhizobium phaseoli[END_REF][START_REF] Akasaka | Morphological alterations and root nodule formation in Agrobacterium rhizogenes-mediated transgenic hairy roots of Peanut (Arachis hypogaea L.)[END_REF][START_REF] Voroshilova | A unique pea (Pisum sativum L.) mutant impaired in nodule, leaf and flower development[END_REF][START_REF] Ferraioli | Development of Ectopic Roots from Abortive Nodule Primordia[END_REF][START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Sinharoy | RNA interference highlights the role of CCaMK in dissemination of endosymbionts in the Aeschynomeneae legume Arachis[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]. The low penetrance of conversion in Ljnbcl1 compared to Pscoch1 or Mtnoot1 suggest that one of the roles of the NCM in indeterminate nodule is to maintain NVM active to confer them indeterminate growth. In determinate nodules there is no persistent NCM and once vascular strands anastomosis occurred at the nodule apex, NVM become determinate leading to NVB growth arrest [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. This determinate NVM status makes determinate nodule less susceptible to root conversion since this homeosis probably requires constitutively active NVM. Moreover, we also report that L.

japonicus NVB harbour a non-canonical xylem/phloem collateral organization with xylem and phloem turned toward the infected cell and the exterior of the nodule, respectively. We revealed also that this xylem/phloem collateral organization is lost in converted nodule suggesting that Ljnbcl1 nodule vascular bundle identity is lost and acquires a root identity during homeosis.

NBCL genes functions are required for leaf proximal/distal patterning and for the control of shoot branching, these functions are conserved across dicots and grasses [START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF].

Similarly, the present study shows that in L. japonicus the NBCL1 gene fulfilled the same functions. Also, at the base of leaves, NBCL genes must ensure the repression of cell division and the promotion of lateral organ differentiation. In Ljnbcl1, the petiole length increase and the lack of nectary glands may reflect the failure of these two processes. Wild-type L. japonicus exhibits a pair of nectary glands that have been a long time ago proposed to be ontologically related to stipules [START_REF] Irmisch | Noch einige Beobachtungen über die Stipulae bei Lotus, Tetragonolobus und Bonjeania[END_REF][START_REF] Heyn | An old question revived: What are the stipules of Lotus?[END_REF] this study). The complete absence of nectary glands in Ljnbcl1 mutants definitively supports the ontological relationship existing between stipules and nectary glands. LjNBCL1 is thus essential for nectary glands initiation and development in accordance with the role of NBCL as master regulator of the stipule initiation, development and determinacy in P. sativum, M. truncatula and in A. thaliana [START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF][START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF]. Ljnbcl1 inflorescence nectaries undergo an homeosis to leaf-like structure while in the Arabidopsis Atbop1bop2 double mutant, floral nectaries are highly reduced in size [START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF]. The reason why vegetative and floral nectaries are absent or convert in leaf-like structure, respectively, is unclear.

The Ljnbcl1 mutant shows severe defects in floral initiation and patterning characterized by a large range of floral phenotypes from wild-type-like flowers to complete absence of floral organs, suggesting a high instability in cell differenciation/proliferation processes in the floral primordia. Among the floral mutant phenotypes, the fused trumpet-like structure is one of the most impressive one and is reminiscent of the A. thaliana Atcup-shaped cotyledon1/2/3 (Atcuc1/2/3) double and triple mutants, which present fusions of different organs such as cotyledons, sepals, stamens, leaves, stems and pedicels [START_REF] Aida | Genes involved in organ separation in Arabidopsis: An analysis of the cup-shaped cotyledon mutant[END_REF][START_REF] Takada | The CUPSHAPED COTYLEDON1 gene of Arabidopsis regulates shoot apical meristem formation[END_REF][START_REF] Hibara | Arabidopsis CUP-SHAPED COTYLEDON3 regulates postembryonic shoot meristem and organ boundary formation[END_REF]. CUC genes are like NBCL genes involved in organ separation processes and in the establishment of boundary zones, in which they are consistently expressed to repress growth [START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF]. In Arabidopsis, CUC1 promotes the expression of the AtLIGHT-DEPENDENT SHORT HYPOCOTYLS 3 (AtLSH3) and its paralog AtLSH4, which are members of the Arabidopsis LSH1 and Oryza G1 genes family (ALOG; [START_REF] Cho | ORGAN BOUNDARY1 defines a gene expressed at the junction between the shoot apical meristem and lateral organs[END_REF][START_REF] Takeda | CUP-SHAPED COTYLEDON1 transcription factor activates the expression of LSH4 and LSH3, two members of the ALOG gene family, in shoot organ boundary cells[END_REF]; furthermore, in Lycopersicon esculentum it has been recently shown that ALOG proteins are able to interact with NBCL proteins [START_REF] Xu | Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators[END_REF]. Thus, in L. japonicus, boundary zones patterning and regulation might be carried-out through a similar CUC/ALOG/NBCL module in which LjNBCL1 plays a major role.

In Arabidopsis, it has been shown that the NBCL proteins BOP1/2 contribute to floral initiation and patterning promoting the expression of LEAFY (LFY) and APETALA1 (AP1), the main Arabidopsis floral meristem identity genes [START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Blázquez | How floral meristems are built[END_REF][START_REF] Karim | A role for Arabidopsis PUCHI in floral meristem identity and bract suppression[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF]. Therefore, future experiments will indicate if the severe floral defects of the Ljnbcl1 mutants are related to reduced expression of the L. japonicus LFY and AP1 orthologues.

In L. japonicus, floral meristems derive from partitioning of the secondary inflorescence (I2) meristem [START_REF] Feng | Control of petal shape and floral zygomorphy in Lotus japonicus[END_REF]. In the inflorescence apex of the Ljnbcl1 mutant, very early in its development, the only trace of I2 meristem-like structure observed is a line of developing trichomes, identical to the wild-type stage-5 I2 meristem differentiating after forming the floral primordia [START_REF] Feng | Control of petal shape and floral zygomorphy in Lotus japonicus[END_REF]. This line delimited a small dome corresponding to an abnormal floral primordium. The absence of structures like wild-type I2 meristems at earlier developmental stages in the mutant apex suggest that development of these mutant I2 meristems is very premature and fast. This probably negatively affect floral meristem initiation and fits well with the severe floral development defects of the Ljnbcl1 mutant. Acquisition of I2 meristem identity in pea is known to be controlled by VEGETATIVE1 (VEG1), a MADS-box gene from the same clade as AP1 [START_REF] Berbel | VEGETATIVE1 is essential for development of the compound inflorescence in pea[END_REF]. It is likely that VEG1 function is conserved in other legumes, including L. japonicus, where VEG1 orthologs are present [START_REF] Benlloch | Genetic control of inflorescence architecture in legumes[END_REF]. In agreement with this, the phenotype of the soybean Dt2 mutant, with a dominant mutation in the ortholog of VEG1 agrees with the conservation of the VEG1 function [START_REF] Ping | Dt2 is a gain-of-function MADS-domain factor gene that specifies semi determinacy in soybean[END_REF]. In the same way that the Arabidopsis NBCL proteins BOP1/2 promote the expression of the floral meristem identity genes LFY and AP1 [START_REF] Karim | A role for Arabidopsis PUCHI in floral meristem identity and bract suppression[END_REF][START_REF] Xu | Arabidopsis BLADE-ON-PETIOLE1 and 2 promote floral meristem fate and determinacy in a previously undefined pathway targeting APETALA1 and AGAMOUS-LIKE24[END_REF], LjNBCL1 might also be required for the proper expression of the L. japonicus VEG1 ortholog.

Material and methods

Plant material and growth conditions. L. japonicus ecotype GIFU [START_REF] Handberg | Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics[END_REF] and the corresponding Ljnbcl1 LORE1 insertional mutant lines (line 30053558, Couzigou et al., 2016 and line 30119830, this study) obtained from the LORE1 insertion mutant collection (https://lotus.au.dk; [START_REF] Mun | Lotus Base: An integrated information portal for the model legume Lotus japonicus[END_REF]Malolepszy et al., 2016) were used. L. japonicus seeds were scarified using sand paper, surface-sterilized 30 min with sodium hypochlorite, washed three times with sterile water and left overnight in sterile water under agitation at 4°C. Seeds were germinated on agar plates (Kalys Biotech, HP696-5, 7 g.L -1 ) for 2 days at 24°C. For in vitro nodulation, L. japonicus seedling were grown on Buffered Nodulation Media [START_REF] Ehrhardt | Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors[END_REF], solidified with Kalys agar 7 g.L -1 and supplemented with 0,5 µM of 2-aminoethoxyvinyl glycine (Sigma-aldrich). For nodulation in pot, a sand/perlite mixture was used (1/2, v/v). For Rhizobial strain, growth conditions and inoculation. M. loti wild-type strain NZP-2235 [START_REF] Jarvis | Rhizobium loti, a new species of legume root nodule bacteria[END_REF] was used for L. japonicus nodulation. Rhizobia were grown on solid or liquid YEB [START_REF] Krall | Detergent extraction identifies different VirB protein subassemblies of the type IV secretion machinery in the membranes of Agrobacterium tumefaciens[END_REF] for 2 days under darkness at 30°C. For in vitro and sand/perlite nodulation, L.

japonicus seedling or hairy-root transformed plant were inoculated with 1 mL and 25 mL of M. loti suspension at OD: 0,1, respectively.

L. japonicus DNA extraction. L. japonicus DNA were extracted from young leaves using phenol/chloroform procedure. DNA were precipitated using cold sodium acetate 3 M; isopropanol (0,1:1) and washed using ethanol 70 %. DNA samples were dried and resuspended in sterile water. A RNase treatment was finally performed (Roche). A. rhizogenes mediated L. japonicus hairy-root transformation. L. japonicus hairy-root transformations were performed as described in [START_REF] Okamoto | Hairy root transformation in Lotus japonicus[END_REF][START_REF] Kumagai | Gene silencing by expression of hairpin RNA in Lotus japonicus roots and root nodules[END_REF] with minor modifications using A. rhizogenes ARqua1 strain. Hairy-roots were subsequently inoculated with M. loti and nodules were observed at 35 dpi.

Ljnbcl1

qRT-PCR Gene expression analyses. Total RNA extractions were performed from frozen tissues using TRIzol® reagent (Ambion) following manufacturer's recommendations for underground organs and with Na-Borate / TRIzol procedure (adapted from [START_REF] Høgslund | Dissection of symbiosis and organ development by integrated transcriptome analysis of Lotus japonicus mutant and wild-type plants[END_REF] for aboveground organs. RNA samples were treated with the TURBO DNA-free™ Kit (Ambion)

according to the manufacturer's recommendations. Full-length cDNA were synthesized using LjPP2A and LjUBC were used as reference genes for gene expression normalization. Detailed information concerning oligonucleotides used for qRT-PCR are provided in the supplementary Table 3.

Light Microscopy and sample preparation. Sample sections embedded in technovit resine were treated essentially as described in [START_REF] Van De Velde | Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula[END_REF]. Fixed samples were infiltrated 15 min under vacuum (≈500mm Hg) in sodium cacodylate buffer pH: 7 (0,05 M), glutaraldehyde 1 % and formaldehyde 4 % and incubated at 4 °C overnight. Once dehydrated by successive ethyl-alcohol bathes, samples undergone successive 1 h ethyl-alcohol/technovit stock solution bathes (3:1, v/v), (1:1, v/v), (1:3, v/v) and three (1:0, v/v) at 4 °C under agitation.

Samples were included in technovit resin using a teflon Histoform S embedding moulds (Heraeus Kulzer). 5 µm thickness technovit sections were carried out using a microtome (Leica, RM 2155) and tungsten disposable blade (Leica, TC-65). Sections were stained for 10 min with toluidine blue (0,02 %), observed with a microscope (Olympus, BX53) and acquired using cellSens Standard software (Olympus life science).

Introduction

In wild-type plant, fleshy fruit shedding and seed shattering are essential processes that enable offspring propagation and species persistence through the natural detachment of mature fruits from pedicel or seeds from rachis. Shattering and shedding processes are genetically controlled mechanisms that have been amongst the first agronomic traits counterselected by Mankind at the beginning of plant domestication, preventing seed loss and therefore improving yields.

Seed shedding mechanism work through a separation process which occurs at a specialized domain called the abscission zone (AZ, [START_REF] Dong | Seed shattering: from models to crops[END_REF]. The abscission process involves four main steps. First the future abscission zone undergoes a cell differentiation process resulting in few small, densely cytoplasmic and isodiametric cell layers [START_REF] Estornell | Elucidating mechanisms underlying organ abscission[END_REF]. These cell layers will later become able to respond to HEASA/HEASA-LIKE2 receptor-like kinases [START_REF] Cho | Regulation of floral organ abscission in Arabidopsis thaliana[END_REF]Stenvik et al., 2008a, Stenvik et al., 2008b) resulting in a downstream MITOGEN-ACTIVATED PROTEIN KINASE and MITOGEN-ACTIVATED PROTEIN KINASE KINASE activation [START_REF] Cho | Regulation of floral organ abscission in Arabidopsis thaliana[END_REF] that triggers the secretion of cell wall modifying and hydrolyzing enzymes that will degrade the middle lamella between two adjacent cell layers [START_REF] Bleecker | Last Exit: Senescence, abscission, and meristem arrest in Arabidopsis[END_REF][START_REF] Patterson | Cutting loose. Abscission and dehiscence in Arabidopsis[END_REF][START_REF] Roberts | Abscission, dehiscence, and other cell separation processes[END_REF][START_REF] Lewis | Plant separation: 50 ways to leave your mother[END_REF][START_REF] Stenvik | Overexpression of INFLORESCENCE DEFICIENT IN ABSCISSION activates cell separation in vestigial abscission zones in Arabidopsis[END_REF][START_REF] Aalen | IDA: a peptide ligand regulating cell separation processes in Arabidopsis[END_REF].

Finally, the external cell layer on the wound will differentiate in a protective cell fate.

In the abscission process, the correct AZ differentiation and formation is a key step without which the downstream abscission signaling pathway will remain ineffective. In Arabidopsis thaliana, the key regulator genes AtBLADE-ON-PETIOLE1 (AtBOP1) and AtBLADE-ON-PETIOLE2 (AtBOP2) genes are required for both establishment and functioning of the floral abscission zone [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF]. These genes are more generally involved in the regulation of specialized domains called boundaries corresponding to restricted growth area dedicated to separate meristems and lateral organs. These domains have their own genetic program which repress cell proliferation and promote the differentiation of adjacent lateral organs [START_REF] Zadnikova | How boundaries control plant development[END_REF]. The formation of these boundaries controls not only the abscission process but the entire plant development. They have been shown to be involved in many developmental processes, such as leaf differentiation and patterning [START_REF] Ha | BLADE-ON-PETIOLE1 and 2 control Arabidopsis lateral organ fate through regulation of LOB domain and adaxial-abaxial polarity genes[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF], internode elongation, floral meristem identity acquisition, inflorescence initiation [START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF], architecture as well as fruit patterning [START_REF] Ragni | Interaction of KNAT6 and KNAT2 with BREVIPEDICELLUS and PENNYWISE in Arabidopsis inflorescences[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF]Khan et al., 2012). In Legume, NBCL genes have a conserved role in the abscission process of the different aerial organs such as leaves, leaflets, floral pieces and fruits. This conserved role in abscission has been described in different species such as Medicago truncatula, Pisum sativum, Lotus japonicus and Lupinus angustifolius (Clements and Atkins 2001a, b;Frankowski et al., 2015;[START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]. The NBCL gene of Lupinus luteus (LlBOP) has been proposed to have a role in both AZ differentiation and functioning in an ethylen/abscissic acid-dependent manner (Frankowski et al., 2015;[START_REF] Wilmowicz | The influence of abscisic acid on the ethylene biosynthesis pathway in the functioning of the flower abscission zone in Lupinus luteus[END_REF].

AtBOPs
This conserved role has also been described for petal abscission in the Brassicaceae, A.

thaliana and the Solanaceae Solanum lycopersicum and Nicotiana tabacum [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Ichihashi | Evolutionary developmental transcriptomics reveals a gene network module regulating interspecific diversity in plant leaf shape[END_REF][START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF].

In grasses, the NBCL genes are conserved and barley represents the first grass species in which NBCL genes have been characterized. The Hordeum vulgare HvUNICULM4 (HvCUL4) and its closest paralog HvLAXATUM-a (HvLAX-a) have been described as required for tillers formation, ligule and auricles development and for floral organ identity control and rachis development, respectively [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF].

However, since barley has already been selected during domestication for a seed nonshattering phenotype, the involvement of NBCL genes in the AZ establishment cannot be studied in this model plant neither in the other classical domesticated grass model.

In grasses some genes involved in the AZ differentiation have been identified but the studies on rice (Oryza sativa) pave the way on the understanding of the AZ establishment in grasses. In this plant, five genes have been described to be involved in the formation of a functional abscission layer in the pedicel. The BEL1-type homeobox gene SH5 modulates seed shattering by enhancing AZ development and differentiation by inducing two shatteringrelated genes, SHAT1 (SHATTERING ABORTION1) an APETALA2 transcription factor and SH4 (GRAIN SHATTERING QUANTITATIVE TRAIT LOCUS ON CHROMOSOME 4) a trihelix transcription factor [START_REF] Li | Rice domestication by reducing shattering[END_REF][START_REF] Yoon | The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscission-zone development and inhibiting lignin biosynthesis[END_REF][START_REF] Zhou | Genetic control of seed shattering in rice by the APETALA2 transcription factor SHATTERING ABORTION1[END_REF]. SHAT1 and SH4 are required for qSH1 (quantitative trait locus of seed shattering on chromosome 1) expression. qSH1 is a BEL1-type homeobox gene sharing high homology with SH5, that functions downstream of SHAT1 and SH4 [START_REF] Konishi | An SNP caused loss of seed shattering during rice domestication[END_REF] and is probably involved in a positive feedback loop of SHAT1 and SH4 by maintaining the expression of SHAT1 and SH4

in the abscission layer thus promoting AZ formation and differentiation [START_REF] Zhou | Genetic control of seed shattering in rice by the APETALA2 transcription factor SHATTERING ABORTION1[END_REF].

The KNOTTED1-LIKE HOMEOBOX gene OSH15 is also involved in the abscission regulation and interacts with both qSH1 and SH5. It has been proposed that OSH15/qSH1 dimer is required for AZ development and that OSH15/SH5 dimer enhance seed shattering through the inhibition of lignin biosynthesis genes at the pedicel AZ [START_REF] Yoon | The BEL1-type homeobox gene SH5 induces seed shattering by enhancing abscission-zone development and inhibiting lignin biosynthesis[END_REF][START_REF] Yoon | KNOX protein OSH15 induces grain shattering by repressing lignin biosynthesis genes[END_REF]. Another gene, qSH3 (grain shattering quantitative trait locus on chromosome 3)

is also important for seed shattering in coordination with qSH1 and SH4 [START_REF] Htun | Effect of quantitative trait loci for seed shattering on abscission layer formation in Asian wild rice Oryza rufipogon[END_REF].

Thus in rice, the pathway of AZ differentiation has been well described, but the involvement of the NBCL genes in the AZ formation has not been studied so far.

In order to unreveal a possible NBCL-dependent regulation of the abscission cell layer development in grass plants, we used Brachypodium distachyon (B. distachyon), a small temperate grass related to the two major small grain cereal species wheat and barley [START_REF] Draper | Brachypodium Distachyon, a new model system for functional genomics in grasses[END_REF][START_REF] Vogel | EST sequencing and phylogenetic analysis of the model grass Brachypodium distachyon[END_REF][START_REF] Opanowicz | Brachypodium distachyon: making hay with a wild grass[END_REF][START_REF] Vogel | Brachypodium distachyon, a new model for the Triticeae. Genetics and genomics of the Triticeae[END_REF], which displays a shattering phenotype. We tested the involvement of the two B. distachyon NBCL genes NPR5-LIKE (BdNPR5-LIKE) and NPR6-LIKE (BdNPR6-LIKE) in the abscission process using Targeted Induced Local Lesions IN Genomes (TILLING) mutants and show that these two genes are involved in different developmental processes but that the single mutants are not altered in the abscission process.

Results

NBCL genes are conserved across dicotyledon and grasses

In order to better understand the organization of this plant specific NBCL gene family, we constructed a maximum likelihood-based phylogenic tree using NOOT-BOP-COCH- 2012;[START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF]. Besides this partial overlapping, the distinct maximal expression patterns of both BdNPR5-LIKE and BdNPR6-LIKE may reflects distinct physiological role in the plant. In the different organs checked, BdNPR6-LIKE is globally more expressed than its paralog BdNPR5-LIKE except in roots and nodes (Fig. 2). BdNPR6-LIKE transcripts are particularly more represented in spikelets compared to BdNPR5-LIKE.

This difference suggests a preponderant role of BdNPR6-LIKE in spikelets development. distachyon ecotype Bd21-3 mutant collection [START_REF] Dalmais | A TILLING platform for functional genomics in Brachypodium distachyon[END_REF]wwwurgv.versailles.inra Among all selected Bdnpr5-like mutant lines, only the two mutants with KO alleles presented phenotypes (especially a glaring liguleless phenotype, see below). Self-fertilized progeny of line Bd7965 heterozygous for the Bdnpr5-like mutation segregated as a monogenic recessive trait with 2 homozygous wild-type plants and 3 heterozygous plants showing wild-type ligule, and 1 plants homozygous for the Bdnpr5-like locus showing liguleless phenotype (Table 1). The family Bd4982 and Bd7965 presented stop codon in the BTB/POZ domain and in the ankyrin repeats domain, respectively (Fig. 3 B; Sup. Fig. 1).

Using a consensus based on 248 NBCL protein sequences (https://www.ncbi.nlm.nih.gov/; https://db.cngb.org/blast4onekp/; [START_REF] Wickett | Phylotranscriptomic analysis of the origin and early diversification of land plants[END_REF][START_REF] Matasci | Data access for the 1,000 Plants (1KP) project[END_REF][START_REF] Xie | SOAP denovo-Trans: de novo transcriptome assembly with short RNA-Seq reads[END_REF][START_REF] Johnson | Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes[END_REF]; Sup. Fig. 1), the absence of phenotype in the family Bd7845, Bd5567, can be explained by an un-conserved amino-acids position. The amino acid modification in family Bd8595 and Bd7526 corresponds to highly conserved positions but their non-polar property remained unchanged (Sup. Fig. 1). For Bdnpr6-like alleles, the mutation in family occurs on a highly conserved position but did not generated any mutant phenotype (Fig. 3 B; Sup. Fig. 1). In the present work the Bdnpr5-like KO mutants and the Bd5998 missense mutant were further studied below. The Bdnpr5-like KO mutant lines present a tillering defect

The first striking phenotype observed in Bdnpr5-like KO mutant lines (families Bd4982 and Bd7965) was a reduced tillering compared to the Bd21-3 wild-type accession (Fig. 4 A-C). In Bdnpr5-like Bd4982 and Bd7965 mutants the tillering defect is visible early during development and seems to be due to a mechanical issue since tiller outgrowth appears to be compromised. Indeed, while in Bd21-3 the culm correctly slide into the sheath to come out from the ligular region, in Bdnpr5-like Bd4982 and Bd7965 mutants this process is altered. The culm failed to slide into the sheath and to properly grow out of it. Instead of coming out the ligular region, the culm starts to twist and to come out from the middle of the sheath (Sup. Fig. 2). Later during Bdnpr5-like Bd4982 and Bd7965 mutant development, tillering is reduced compared to their wild-type relative siblings (Sup. Fig. 3). This tillering defect seems to affect secondary tiller formation because the primary tillers number per plant remains unchanged (Bd21-3: 5,9 ± 1,3; Bdnpr5-like Bd4982: 5,5 ± 1,3; Bdnpr5-like Bd7965: 6,5 ± 1,4), while the number of secondary tillers per plant was significantly reduced in both Bdnpr5-like Bd4982 and Bd7965 mutants compared to the Bd21-3 (Bd21-3: 24 ± 9,1, Bdnpr5-like Bd4982: 8,8 ± 5,4, Bdnpr5-like Bd7965: 11,6 ± 3,7;

Fig. 4 D-E). The same results were obtained when comparing the mutants to their wild-type siblings (Sup. Fig. 4). To check the hypothesis if an eventual mis-organization of the node may avoid the axillary outgrowth, node sections were done. These sections do not reveal any significant differences in Bdnpr5-like mutants relative to Bd21-3 wild-type plant node organization (Fig. 4 F-G).This tillering defect agrees with the presence of the BdNPR5-LIKE transcript in the node (Fig. 2) despite the fact we cannot explain the mechanism involved yet.

As transcripts of BdNPR5-LIKE were present in roots while those of BdNPR6-LIKE are not (Fig. 2) we studied root development in Bdnpr5-like Bd4982, Bdnpr5-like Bd7965, in their corresponding wild-type siblings and in Bd21-3. No significant differences in primary root development, number of secondary roots per plant, secondary root length or mean length of secondary roots were found (Sup. Fig. 5).

Our results show that the B. distachyon BdNPR5-LIKE gene controls tillering of the aerial vegetative part but is dispensable for root development.

phenotype correlates with the BdNPR5-LIKE expression level in the ligular region observed in wild-type plant by RT-qPCR (Fig. 2).

The phenotype observed in the two independent KO alleles shows that in B.

distachyon, the BdNPR5-LIKE gene is involved in the proximal/distal leaf differentiation and more precisely in the formation of the ligule and of the auricles. show that the abscission zone characterized by small differentiated cell layers are correctly established in mutant and wild-type plants (Fig. 7 J, K, L, M).

This suggests that the BdNPR5-LIKE gene alone is not essential for the seed abscission zone establishment and functioning. Only one Bdnpr6-like missense allele isolated in this study gives phenotypes that remember those observed in the corresponding Hvlax-a mutant in barley [START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF].

The similarity of the phenotypes in the two species strongly suggests that Bdnpr6-like Bd5998 represents a true mutant that could be confirmed using a future Bdnpr6-like KO mutant line. The analysis of this mutant line represents the first indication on the involvement of the BdNPR6-LIKE gene in the spikelet architecture and in the control of floral organ number and identity.

Discussion

Seed abscission ability is neither impaired in Bdnpr5-like null mutants nor in Bdnpr6like missense line Bd5998

The original aim of the present study was to explore the role of the NBCL genes in the seed abscission process in a non-domesticated grass species not selected for a non-shattering phenotype or for a particular inflorescence architecture that inhibit florets dispersion.

In domesticated barley the two independent mutations in HvBRITTLE RACHIS1 and HvBRITTLE RACHIS2 genes responsible for the brittle to non-brittle rachis phenotype result in a non-shattering phenotype. These genes encode a couple of signal transducing receptor / protein ligand that control cell wall thickening in the rachis node disarticulation zone [START_REF] Pourkheirandish | Evolution of the grain dispersal system in barley[END_REF]. This pair of receptor / ligand should act upstream the NBCL genes during the abscission process pathway. In addition, in barley, the six-row inflorescence phenotype of barley which appears three times through independent mutations [START_REF] Komatsuda | Sixrowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene[END_REF] resulted in an increased grains production but also generated a seed dispersal difficulty [START_REF] Harlan | Crops and Ancient Man[END_REF][START_REF] Zohary | Domestication of plants in the old world,3 rd edition[END_REF][START_REF] Zohary | Domestication of plants in the old world,3 rd edition[END_REF][START_REF] Fuller | Seed dispersal and crop domestication : Shattering, germination and seasonality in evolution under cultivation[END_REF][START_REF] Fuller | Seed dispersal and crop domestication : Shattering, germination and seasonality in evolution under cultivation[END_REF].

B. distachyon is a small temperate grass related to wheat and barley [START_REF] Draper | Brachypodium Distachyon, a new model system for functional genomics in grasses[END_REF][START_REF] Vogel | EST sequencing and phylogenetic analysis of the model grass Brachypodium distachyon[END_REF][START_REF] Vogel | Brachypodium distachyon, a new model for the Triticeae. Genetics and genomics of the Triticeae[END_REF] and represents a non-domesticated model plant with an effective seed shattering after ripening. The BdNPR5-LIKE gene of B.

distachyon was an excellent candidate to study the seed abscission zone establishment in grasses because contrary to the grass NPR6-LIKE genes, it belongs to an NBCL clade closely related to the dicot NBCL genes known to be involved in AZ formation.

Despite the conserved role of NBCL genes in the process of the AZ formation and functioning in dicot plants [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF][START_REF] Ichihashi | Evolutionary developmental transcriptomics reveals a gene network module regulating interspecific diversity in plant leaf shape[END_REF]Frankowski et al., 2015;[START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]

BdNPR5-LIKE is not essential for the B. distachyon root appartus development

The analysis of the root apparatus development in Bdnpr5-like Bd4982, Bdnpr5-like Bd7965, in their corresponding wild-type siblings and in Bd21-3 did not revealed significant developmental differences. This suggests that BdNPR5-LIKE genes is not essential for primary and secondary root development. In accordance with these findings, in A. thaliana, AtBOP1 and AtBOP2 are both expressed in primary and secondary root vasculatures and are also described as dispensable for normal primary and secondary root development [START_REF] Woerlen | Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDICELLUS is essential for differentiation of secondary xylem in Arabidopsis root[END_REF]. However, in A. thaliana, during radial expansion of the root, AtBOP1 and AtBOP2 are transcriptionally excluded from developing xylems by the class I KNOX genes SHOOT MERISTEMLESS and BREVIPEDILLUS/KNAT1 to allow the differentiation of lignified fibers and vessels [START_REF] Liebsch | Class I KNOX transcription factors promote differentiation of cambial derivatives into xylem fibers in the Arabidopsis hypocotyl[END_REF][START_REF] Woerlen | Repression of BLADE-ON-PETIOLE genes by KNOX homeodomain protein BREVIPEDICELLUS is essential for differentiation of secondary xylem in Arabidopsis root[END_REF]. Excepted for some lineages, monocots have lost the ability to produce vascular cambium and thus to produce secondary xylem and phloem [START_REF] Zinkgraf | Transcript profiling of a novel plant meristem, the monocot cambium[END_REF]. Also, to our knowledge, the function of NBCL genes in monocot roots development has never been described. The use of both

The BdNPR5-LIKE gene is involved in the control of tillering

In barley, HvCUL4 controls the number of axillary buds that develop at a leaf axils, the number of active axillary meristems, the axillary bud outgrowth and thus the tillers formation. The mutant has also been described to produce leafy-side shoot instead of tillers [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF]. The authors suggest that one of the roles of HvCUL4 is to control and guide the development of axillary buds through the definition of main culm/secondary axis boundary presents at the leaf axil [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF].

The

The BdNPR5-LIKE gene promotes proximal/distal leaf differentiation

In barley, HvCUL4 was previously described as essential for the ligule differentiation and outgrowth. The Hvcul4 mutant present a liguleless phenotype coupled with an irregular outgrowth of auricles at the blade-sheath boundary. Furthermore, in the Hvcul4 mutant, ectopic auricle flaps develop on the margins the sheath. Together these observations suggested that HvCUL4 is involved in the proximal-distal patterning of the barley leaf [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF].

In B. distachyon, the two Bdnpr5-like null mutant alleles presented also a liguleless phenotype but in contrast to barley, the development of ectopic auricles from the margins of the sheath was not observed. However, the Bdnpr5-like mutants presented an auricleless phenotype that was not described in barley. We noticed here that the auricles of barley are different from those of B. distachyon in term of shape and that B. distachyon auricles are less expanded.

In the literature, several genes have been shown to be involved in the ligule and auricles formation. In maize, ZmLIGULELESS NARROW (ZmLGN) and its redundant ZmSISTER OF LIGULELESS NARROW (ZmSLN) encode kinases and the Zmlgn corresponding mutants is defective in ligule and auricles formation [START_REF] Moon | The liguleless narrow mutation affects proximal-distal signaling and leaf growth[END_REF].

ZmLIGULELESS1 (ZmLG1), ZmLIGULELESS2 (ZmLG2) and ZmLIGULELESS3 (ZmLG3)
encode respectively a SQUAMOSA PROMOTER-BINDING LIKE protein, a basic LEUCINE ZIPPER (bZIP) transcription factors and a KNOTTED1-LIKE HOMEOBOX protein necessary for the proper ligule and auricles formation [START_REF] Moreno | LIGULELESS1 encodes a nuclear-localized protein required for induction of ligules and auricles during maize leaf organogenesis[END_REF]Walsh et al.,1998;[START_REF] Muehlbauer | Sectors expressing the homeobox gene LIGULELESS3 implicate a time-dependent mechanism for cell fate acquisition along the proximal-distal axis of the maize leaf[END_REF]. The Zmlg3 mutation disrupts the leaf development at the ligular region, the blade, the auricles and the ligule are transformed into sheath-like structure,

ZmLG3 seems to influence cell fate decision and prevent premature differentiation of the leaf [START_REF] Muehlbauer | Sectors expressing the homeobox gene LIGULELESS3 implicate a time-dependent mechanism for cell fate acquisition along the proximal-distal axis of the maize leaf[END_REF][START_REF] Muehlbauer | Ectopic expression of the maize homeobox gene LIGULELESS3 alters cell fates in the leaf[END_REF]. In Zmlg2 mutants, the blade-sheath boundary appears incompletely formed and diffused suggesting a role of ZmLG2 in the bladesheath boundary establishment (Walsh et al., 1998). In Zmlg1 mutant the blade-sheath boundary is formed; ZmLG1 is required later for the pre-ligule cell division [START_REF] Sylvester | Division and differentiation during normal and liguleless-1 maize leaf development[END_REF][START_REF] Moon | The liguleless narrow mutation affects proximal-distal signaling and leaf growth[END_REF]. In Zmlgn mutant, the ZmLIGULELESS1 protein and the auxin transport protein ZmPIN1a are affected [START_REF] Moon | The liguleless narrow mutation affects proximal-distal signaling and leaf growth[END_REF] and it was shown independently that ZmLG1 is regulated directly or indirectly by ZmWAVE AURICLE IN BLADE1 (ZmWAB1) a TEOSINTE BRANCHED 1, CYCLOIDEA, PCF1 (TCP) transcription factor [START_REF] Lewis | Gene regulatory interactions at lateral organ boundaries in maize[END_REF]. On the other hand the ZmLG2 gene regulates the expression of ZmKNOTTED1 (ZmKN1) that itself controls the expression of four ZmPIN1 genes and other auxin-related genes [START_REF] Bolduc | How to Pattern a Leaf[END_REF]. ZmLGN, ZmSLN, ZmLG1and ZmLG2 may thus act in the same pathway in which ZmLGN and ZmSLN seem to function upstream of ZmLG1and

ZmLG2 as signaling components [START_REF] Moon | The liguleless narrow mutation affects proximal-distal signaling and leaf growth[END_REF].

In maize, there are three NBCL genes. ZmNPR5-LIKE (NP_001140651.1 / GRMZM2G060723) is the closest ortholog of BdNPR5-LIKE while ZmNPR6-LIKEa (XP_008669884.1 / GRMZM2G026556) and ZmNPR6-LIKEb (XP_008662362.1 / GRMZM2G022606) are the closest orthologs of BdNPR6-LIKE. Two NBCL genes transcripts have been detected in maize sheath and ligule suggesting that in maize NBCL genes may contribute to the patterning of the blade/sheath boundary, to the ligule initiation and development [START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF]. Investigations using Zmnpr5-like mutant may reveal comparable liguleless and auricleless phenotypes and highlight a relationship with the previously described boundary zone establishment pathway involving ZmLGN, ZmSLN, ZmPINa, ZmWAB1, ZmLG1, ZmKN1 and ZmLG2.

NBCL proteins belong to the NPR1-LIKE protein family that are more related to immunity than development but both plant immune status and development are tightly linked [START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF][START_REF] Smakowska | Organ-specific regulation of growth-defense tradeoffs by plants[END_REF]. Several studies described the interactions between NBCL or NPR1-LIKE proteins and TGACG sequence-specific binding BASIC LEUCINE ZIPPER transcription factors (TGA bZIP TFs). For example, AtNPR1 interacts with TGA 1, TGA2, TGA3, TGA5, TGA6, and TGA7 bZIP TFs to regulate the expression of the salicylic acid induced AtPATHOGENESIS-RELATED1 gene [START_REF] Zhang | Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene[END_REF][START_REF] Després | The Arabidopsis NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors[END_REF][START_REF] Zhou | NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the PR-1 gene required for induction by salicylic acid[END_REF][START_REF] Després | The Arabidopsis NPR1 disease resistance protein is a novel cofactor that confers redox regulation of DNA binding activity to the basic domain/leucine zipper transcription factor TGA1[END_REF][START_REF] Boyle | The BTB/POZ Domain of the Arabidopsis disease resistance protein NPR1 interacts with the repression domain of TGA2 to negate its function[END_REF], AtBOP2 interacts with AtPERIANTHIA in the control of floral patterning [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF] and NtBOP2

is involved in the control of the corolla AZ establishment in tobacco and interact with several bZIP such TGA1a, TGA2.2, TGA2.1, PG13 and RSG [START_REF] Wu | The Tobacco BLADE-ON-PETIOLE2 gene mediates differentiation of the corolla abscission zone by controlling longitudinal cell expansion[END_REF].In B. distachyon, the Bd2g55567 gene which is the closest ortholog (87,3 % of similarity and 78,1 % of identity at protein level) of the bZIP transcription factor ZmLG2 may be a direct interacting partner of BdNPR5-LIKE and participate in the leaf-blade boundary establishment process if these regulatory networks are conserved between plants.

NBCL gene functions were recruited from SAM to pattern grass and dicotyledon leaves

Comparative leaf developmental and morphological studies across grasses and dicotyledons revealed that the monocotyledon leaf blade is not homologous to the dicotyledon blade. Monocot and dicot leaves have different developmental origin and derived from different regions of the leaf primordium. In dicots, the leaves (the petiole and the lamina) are elaborated from the upper part of the leaf primordium "oberblatt" while the basal part of the leaf primordium "unterblatt" is the precursor of the leaf base which is dedicated to generate a sheath around the stem or a leaf base with or without stipule depending on the nodale anatomy [START_REF] Sinnott | Investigations on the Phylogeny of the Angiosperms. 3. Nodal Anatomy and the Morphology of Stipules[END_REF][START_REF] Kaplan | The monocotyledons: Their evolution and comparative biology. VII. The problem of leaf morphology and evolution in the monocotyledons[END_REF][START_REF] Tsiantis | The maize rough sheath2 gene and leaf development programs in monocot and dicot plants[END_REF][START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF]. In monocots, the entire leaves (the sheath, the ligular region and the leaf blade) initiate from the lower part of the leaf primordium while the upper part of the leaf primordium, the precursor of the leaf tip "vorläuferspitze" give rise to a distal leaf rudimentary zone [START_REF] Eichler | Zur Entwickelungsgeschichte des Blattes mit Besonderer Berucksichtigung der Nebenblatt-Bildunger[END_REF][START_REF] Kaplan | The monocotyledons: Their evolution and comparative biology. VII. The problem of leaf morphology and evolution in the monocotyledons[END_REF][START_REF] Tsiantis | The maize rough sheath2 gene and leaf development programs in monocot and dicot plants[END_REF][START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF].

Using laser microdissection coupled with RNA sequencing strategy, trancriptomic analysis in the pre-ligule band (PLB), in adjacent pre-blade and pre-sheath tissues revealed that a set of genes is specifically up-regulated in PBL [START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF]. This gene network involved in the ligule biogenesis is similar to the gene network used in SAM for lateral organ initiation. This suggests that the regulatory network used for ligule initiation and development have been recruited from the pre-existing patterning mechanism of SAM [START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF]. The authors proposed that the positioning and the delimitation of the blade/sheath boundary should be established through the antagonistic signalization module that oppose the proximal KNOX signal to the distal auxin signal along the leaf primordia [START_REF] Johnston | Transcriptomic analyses indicate that maize ligule development recapitulates gene expression patterns that occur during lateral organ initiation[END_REF].

The nbcl mutant studies support and illustrate well the concept developed by [START_REF] Zadnikova | How boundaries control plant development[END_REF][START_REF] Hepworth | Beyond the Divide: Boundaries for Patterning and Stem Cell Regulation in Plants[END_REF][START_REF] Wang | Divide et impera: Boundaries shape the plant body and initiate new meristems[END_REF], they are involved in ligule formation in grasses [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF] this study) and also in stipule formation in dicotyledonous plants. For example in Lupinus angustifolius, Medicago truncatula, Pisum sativum or Arabidopsis thaliana the loss of function of NBCL leads to the simplification, modification or suppression of the stipule [START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF][START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. It is tempting to speculate that grasses ligule and dicot stipules are ontologically related because these organs are both initiated from the lower part of the leaf primordia however it remains unclear if these organs initiate from orthologous boundaries.

Nevertheless stipule and ligule represent lateral organ orthologs since they are modified leaves.

BdNPR5-LIKE gene controls spikelet determinacy

In grasses, flowers are called florets and those arise from a structure called spikelet. A spikelet is defined as a monocot inflorescence unit and its organization define the inflorescence structure.

B. distachyon forms inflorescences named spikes [START_REF] Draper | Brachypodium Distachyon, a new model system for functional genomics in grasses[END_REF] and in this species, as in barley and wheat, these spikes are unbranched [START_REF] Bonnett | The development of the barley spike[END_REF][START_REF] Bonnett | The development of the wheat spike[END_REF].

The establishment of an unbranched spike required several meristem identity transitions. The vegetative meristem first acquires inflorescence meristem (IM) identity, the IM produces axillary meristems/primary branch meristem (AXM/PBM) and the IM ahead and the flower, supplementary organs were found and those show partial to complete stamen-like identity suggesting that both the number and the identity of sexual organ are partially control by BdNPR6-LIKE. Obtaining a null allele for BdNPR6-LIKE will clarify the role of this gene in flower identity establishment.

Conclusion

In this work we studied the roles of two grass NBCL genes orthologs to the [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF].

The subtle differences existing between barley and B. distachyon in terms of inflorescence patterning and determinacy reveal additional phenotypes in B. distachyon compared to barley. In Bdnpr5-like mutants, the number of florets per spikelet is increased.

This suggests that BdNPR5-LIKE is involved in the control of spikelet determinacy. This trait of agronomic interest represents an original target for breeders directly transposable to the B.

distachyon close relative, bread wheat, to improve yield. On the other hand, BdNPR6-like seems to be involved in the spikelet architecture and in the control of flower patterning, more precisely in the lodicule identity and in the number of stamen formed. BdNPR6-LIKE is the closest ortholog of HvLAX-A which is also involved in the floral patterning of the barley inflorescence [START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF].

The aim of the study was to identify the role of the grass NBCL gene in the process of abscission which is a major character in agriculture. By contrast to BdNPR5-LIKE which is not essential for the floret abscission cell layer establishment we cannot affirm that BdNPR6-LIKE is not involved in the abscission process because despite the Bdnpr6-like mutant presented here is not affected in this process, the mutation present in the line Bdnpr6-like Bd5998 may be leaky and may not impact all the developmental processes in which this gene is involved. The characterization of a Bdnpr6-like null mutant will clarify the role of BdNPR6-LIKE in the abscission process. N-Lauroyl sarcosine, 2%; EDTA, 50mM; aqueous phenol, 6,5%, pH: 7,9) were added to the powder. The suspension was vortexed, incubated 5 min at 50°C and shaken 10min at room temperature. 700µL of aqueous phenol pH: 7,9; chloroform; iso-amylalcohol (25:24:1, v:v:v) were added. Suspension was vortexed, centrifuged 10 min at 14000 rpm and aqueous phase was collected. 700µL of chloroform; iso-amylalcohol (24:1, v:v) were add on aqueous phase.

Suspension was vortexed, centrifugated 5min at 14000rpm, aqueous phase was collected and DNA precipitation was carried-out adding cold sodium acetate 3M; isopropanol (0,1:1, v:v).

After 10 min centrifugation at 14000 rpm the supernatant was discarded and the DNA pellet washed with 500 µL of ethanol 70%. After a 5min centrifugation at 14000 rpm the ethanol was discarded, the DNA pellet dried, re-suspended in 100µL of sterile water and heated 5min at 65°C. An RNase treatment is realized using 5 µL of 1 mg.mL -1 RNase (ref.

10.109.134.001, ROCHE). The present thesis works aimed to better understand the mode of action of NBCL in Medicago and pea species forming indeterminate nodules, and in lotus species forming determinate nodules with no persistent apical meristem. We also aimed to discover additional actors of the nodule identity regulatory pathway and find interactions between these partners to improve the understanding of the nodule identity regulation.

Bdnpr5-like and

The legume NBCL1 genes are key regulators of the root nodule symbiosis

The NBCL1 genes expression behaves similarly, independently of the nodule type formed in different legume species. The MtNOOT1, PsCOCH1 and LjNBCL1 gene expressions were constitutive in root and induced in nodule during the nodule formation. Furthermore, a more precise analysis in Medicago revealed that the proMtNOOT1:GUS gene expression is associated in a dynamic manner to the development of the nodule vascular bundles, suggesting a preponderant role for NBCL1 gene in these specific tissues to control their development and their identity (Magne et al., 2017a, b, unpublished). Further investigations should test if NBCL1 pattern of expression always associate to nodule vascular bundles development. Using LORE1 insertional mutant and RNAi approaches in lotus, we were able to demonstrate that LjNBCL1 gene function is conserved for the determinate (lacking NCM) nodule identity maintenance.

Moreover, we determined that ectopic roots arising from lotus determinate nodule also originated from the NVM. Our studies showed that NBCL1 clade is required to maintain the symbiotic organ identity and to repress the NVM root identity in both indeterminate and determinate nodule types. It can be hypothesize that in legume nodules, the NBCL1 clade function is to regulate the meristematic activity of NVM through the activation of boundary gene antagonizing cell divisions.

The role of the legume NBCL1 paralogs, the NBCL2 genes

To find new actor involved in the nodule identity, we looked for the closest Medicago and pea NBCL1 paralog genes. Legumes generally possess a second copy of the NBCL genes. In DISCUSSION

253

Medicago and pea, we identified a second NBCL copy that belongs to a distinct NBCL sub-clade called NBCL2. In Medicago and pea these genes were called MtNOOT2 and PsCOCH2, respectively. The NBCL2 clade has never been studied and the roles of the members of this clade are completely unknown in a context of root nodule symbiosis. To explore the role of the NBCL2 genes we search for KO mutants in both Medicago and pea species. In the laboratory, mutant alleles for MtNOOT2 were already available and we later isolated a mutant KO allele for PsCOCH2 by screening an available pea TILLING collection (Dalmais et al., 2008). We have initiated the characterization of these nbcl2 mutant lines in both Medicago and pea. Our studies revealed that the behavior of these two genes was very similar. The two genes are not expressed in roots and their expression is induced in Medicago and pea indeterminate nodules. Despite their nodule specific behavior, NBCL2 genes are not sensus stricto nodule-specific genes since their transcripts are found in the aerial organs (see below). The presence of NBCL2 transcripts in aerial parts of the plant and in nodules again suggests that the function of this clade has been recruited from pre-existing regulatory functions related to the development of above ground plant organs to serve for nodule development.

The role of NCM on NVM activity and vice versa during nodule development

In Lotus forming determinate nodules, the NCM activity is transient and finally cease.

The functioning of the NVM in determinate nodules is poorly documented but it was suggested that the nodule vascular bundle growth is determinate suggesting that the NVM activity is also transient [START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF]. Relationships between the transient NCM activity and the NVM activities are not well described. Before initiating our studies on the lotus NBCL1 gene (LjNBCL1) we initially thought that in a context of NCM absence, the nodule to root conversion could be significantly exacerbated. Surprisingly, this was not the case and even the opposite. We initially did not observe the nodule to root conversion phenotype in the mutant background and therefore thought that the process of nodule to root conversion did not occur in the Lotus determinate nodules. Later, while checking other developmental processes, we discovered that the nodule to root phenotype exists in the Ljnbcl1 mutant (Magne et al., 2017b, unpublished).

The penetrance of the Ljnbcl1 mutation was very low (≈5%) compared to Mtnoot1 (≈40-50%)

and Pscoch1 (≈90%). We interpreted this very low penetrance by the fact that NVMs and NCM Uncover the role of NBCL2 genes in nodules using nbcl1nbcl2 double mutants

The absence of phenotype in nbcl2 mutants did not allow us to clearly state on the roles of NBCL2 genes in the nodule development and identity. We thus undertook to study the roles of NBCL2 genes using the nbcl1nblc2 double mutant in Medicago and pea. The Mtnoot1noot2 double mutant was already constructed at the beginning of my PhD work and once we discovered the Pscoch2 allele we constructed the Pscoch1coch2 double mutant. The phenotypic characterization of the double nbcl1nbcl2 mutant in both Medicago and pea, clearly indicates that the phenotypes resulting from the nbcl1 mutations were increased by the mutations of nbcl2. In both double mutants the nodulation process was highly impacted with a near complete loss of nodule identity and a strong defect in nitrogen fixation efficiency. The phenotypes of these mutants were roughly similar in terms of consequences but some differences have been revealed.

In Medicago the nodule to root conversion reach 100 % of the nodule leading to a complete fix-phenotype (Magne et al., 2017a, unpublished). The results are slightly different in pea, because the single mutant coch1 showed already 90% of big and pink nodules that are converted. In Pscoch1coch2, the nodule to root conversion penetrance remained similar to the single mutant but the modification of the nodule development was increased. Double mutant nodules were smaller, less pink to completely white, supernumerary and the nitrogen fixation was reduced but not abolished as in Medicago. It is surprising that in pea, the loss of function of these two genes leads to a supernumerary nodule phenotype not observed in Medicago. To explain this supernodulation phenotype in pea we made the hypothesis that the NCM is not functioning correctly and do not produced the CLE peptides regulating the number of nodules formed in the pea root apparatus. To verify if the Pscoch1coch2 supernodulation phenotype is link to the missecretion of CLE peptide, it will be interesting to quantify the CLE12 transcript abundance in nodules or to over-expressed CLE in the double mutant Pscoch1coch2 using the hairy-root technology to determine if the normal nodulation is restored. This experiment can show if the NCM regulation is altered in Pscoch1coch2. In the future, an a priori approach could also be oriented toward the Medicago KNOX and BELL promoter sequences to determine if these genes are targets of TGA bZIP TF interacting with NOOT.

Class II MtKNOX genes are involved in symbiosis [START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF][START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. We highlighted that class II MtKNOX genes expression levels and expression patterns were modified in Mtnoot mutants. We in addition demonstrated a switch of proMtKNOX9:GUS expression pattern from the NCM region to the peripheral vasculature (Magne et al., 2017a, unpublished). proMtKNOX9:GUS is normally expressed in the root cylinder but absent of the nodule peripheral vasculature. This suggest that some elements of the root developmental program have been reused for the development of the nodule vascular bundle but it appears that some regulatory elements such as class II MtKNOX gene have also been recruited for the NCM development. Interestingly, class II KNOX genes are supposed to work antagonistically with the homeotic class I KNOX genes [START_REF] Furumizu | Antagonistic Roles for KNOX1 and KNOX2 Genes in Patterning the Land Plant Body Plan Following an Ancient Gene Duplication[END_REF] and it is known that class I KNOX genes are indirect target of NBCL and are involved in the stem cell niche division, proliferation and maintenance throughout complex phytohormone regulations [START_REF] Zadnikova | How boundaries control plant development[END_REF]. In

Extending the concept of nodule identity-NBCL1 dependent in other types of nodule

This thesis work showed that the symbiotic NBCL1-dependent identity is conserved in different tribes forming nodules with peripheral vasculature such as Trifolieae (M. truncatula), Fabeae (P. sativum) and Loteae (L. japonicus). We can thus ask the question if this NBCL1dependent identity is also conserved in legume forming other type of nodules. In the Phaseoleae tribe, as in Lotus, nodules are determinate and it is probable that nodule identity in this tribe is also NBCL1-dependent. For instance no nbcl1 mutant was described in species forming aeschynomenoïd nodules (determinate nodules). Despite that at least one NBCL gene exists in Aeshchynomene, its involvement in nodule identity is not known. In lupinoïd nodules (several indeterminate meristems) at least one NBCL copy exists but its role in nodulation is also not described. The presence and the implication of NBCL gene(s) in the nodules of the non-legume Parasponia genus (indeterminate meristem/central vasculature) belonging to the Cannabaceae family and in the actinorhizal nodules of actinorhizal plants (indeterminate or determinate meristem/central vasculature) are also not described. The study of NBCL gene(s) in these species could allow to understand if the NBCL genes have played a role during evolution in the acquisition of the ability to form nodules in plant.

NBCL1 genes are involved in the patterning of aerial organs and in abscission

Our study of NBCL genes was initially focused on their roles in the nodule development and identity. However we also reported that theNBCL1 gene transcripts are present in aerial organs in Medicago pea and Lotus (data not presented for Medicago). In Lotus we showed that the Ljnbcl1 mutation impacted the development of several aerial organs and showed impressive flower identity alterations (Magne et al., 2017b, unpublished) in agreement with the published Mtnoot1 and Pscoch1 aerial mutant phenotypes [START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. In all three species, Medicago, pea and lotus, nbcl1 mutations mainly affect the initiation, the development and/or the determinacy of stipules/nectary glands and flowers. It should be noted that the nbcl1 mutant of Lotus showed very strong alterations of flower development compared to pea and M. truncatula suggesting a more important role of this gene in Lotus floral initiation and development. In general, less important effects were observed in leaves. Another important point is that NBCL1

genes in legumes and in other dicots were shown to be involved in aerial organs abscission [START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. We contribute to this work by showing that, similarly, to Medicago, pea and lupinus nbcl1 mutants, the Ljnbcl1 mutant was deficient in abscission.

Uncover the role of NBCL2 genes in the patterning of aerial organs using nbcl1nbcl2 double mutants NBCL2 genes are often co-expressed with NBCL1 genes in aerial organs and both NBCLs transcripts accumulated mainly in nodes where stipules/nectaries arised, in leaves and in flowers.

Moreover, in Medicago, the proNOOT2:GUS expression pattern is associated to the frontiers between the different organs (data not shown) similarly to the published proNOOT1:GUS gene expression pattern [START_REF] Couzigou | The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops[END_REF]. We found that the proNOOT2:GUS expression pattern overlaps with the proNOOT1:GUS at the base of young stipules, at leaf and leaflets abscission zones (pulvini), at the base of floral organs and in pod abscission zones (data not shown in the manuscript).

Extending the concept of the NBCL1-dependent abscission process in grass

The last part of this PhD manuscript aimed at extending the concept of the NBCL1dependent abscission process in a non-domesticated grass species. We used Brachypodium distachyon as model plant since this species has not been selected for a non-abscission character or for spikelet architecture modification that impact the seed shedding. Furthermore, a TILLING mutant collection was available for this model plant [START_REF] Dalmais | A TILLING platform for functional genomics in Brachypodium distachyon[END_REF]. To investigate the role of NBCL genes in the abscission process in grasses, we isolated nbcl mutants for the two Brachypodium NBCL genes. Unfortunately, shortly after obtaining these Brachypodium mutants, [START_REF] Tavakol | The Barley UNICULME4 Gene Encodes a BLADE-ON-PETIOLE-Like Protein That Controls Tillering and Leaf Patterning[END_REF][START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF] published the role of the two first NBCL genes in the development of grass using barley as model plant. However they did not studied the abscission process probably because barley is not a suitable model to study abscission as a result of its domestication.

The Brachypodium study revealed that Bdnpr5-like and Bdnpr6-like single mutants alone were not altered in the process of abscission (Magne et al., 2017c, unpublished). A strong overlap between the two genes expressions was observed in almost all the organs of the plant. Thus, because of their potential redundancies, we were not able to clearly state on the implication of the grass NBCL genes in abscission. The double mutant Bdnpr5-likenpr6-like need to be constructed and characterized to clearly determined if the abscission process is NBCL-dependent in grasses.

NBCL functions in aerial vegetative and reproductive organs patterning are conserved in grasses

Our Brachypodium NBCL study supported well the previous findings and fit with the roles attributed to the barley NCBLs. Indeed, we showed that BdNRP5-LIKE, is involved in the control of tillering and in the promotion of the proximal/distal leaf differentiation and especially for the correct formation of the ligular region. BdNRP5-LIKE is required for the formation of the ligule and auricles as described for its barley ortholog HvUniculme4 (Magne et al., 2017c, unpublished). This work involving several plant models and their corresponding nbcl1 mutants revealed also a common ontology of the organ located at the leaf axil in dicots and grasses.

Mtnoot1, Pscoch1, show modified stipules (asymmetry, simplification or complete homeosis in leaves), Ljnbcl1 lack nectaries that are supposed to be modified stipules [START_REF] Irmisch | Noch einige Beobachtungen über die Stipulae bei Lotus, Tetragonolobus und Bonjeania[END_REF][START_REF] Heyn | An old question revived: What are the stipules of Lotus?[END_REF], Atbop1bop2 lack stipules and the grasses barley and Brachypodium lack the ligule. These organs, dicots stipule and nectaries, and monocot ligules are all located at the leaf axil a region of the leaf that corresponds to a boundary zone whose functioning is regulated by these NBCL boundary genes. The nbcl mutant phenotypes report for all these organs suggest that dicots stipules and nectaries, and monocot ligules have a common origin and should have a common ancestral organ at the leaf axil which has diversified during plant evolution.

Barley and Brachypodium are nonetheless divergent in terms of floral architecture and we found that BdNPR5-like play a role in the inflorescence spikelet determinacy. This character was not reported in barley. We also found similar roles for HvLaxatum-a and BdNPR6-LIKE genes in the control of floral organ identity. Hvlaxatum-a shows complete homeosis of lodicules in stamens and Bdnpr6-like shows a partial homeosis of lodicules in stamen-like structures as well as supplementary stamenoid organs. Finally, we report that BdNPR6-LIKE is also involved in the spikelet architecture relaxation (Magne et al., 2017c, unpublished).

Thus our study is in agreement with other ones and show that the NBCL genes have been conserved in dicots and grasses for inflorescence architecture patterning and the control of floral organs identity.

Conclusions

This PhD work shows that an ancestral function for NBCL genes is conserved for the stipules/nectaries/ligules, leaves and flowers patterning across dicots and grasses.

This work also shows that the role of the NBCL genes in symbiotic organ identity is conserved in different legumes whatever the nodule meristem nature.

It finally indicates that not only the NBCL genes, but complete functional modules involving TGA bZIP TFs and may be ALOG was recruited several times in evolution for plant shaping and inception of new specialized organs, here NBCL/TGA bZIP TF module is reused for the symbiotic organ development. Cet organe symbiotique généré de-novo permet l'hébergement intracellulaire des rhizobia qui, grâce à leurs activités nitrogénase, réduisent l'azote atmosphérique en ammonium, une forme de l'azote directement assimilable par la plante hôte.

Les mécanismes moléculaires sous-jacents à la reconnaissance entre les deux partenaires symbiotiques, au processus d'infection et à l'organogénèse de la nodosité sont bien décrits, cependant l'établissement et la maintenance de l'identité de cet unique organe souterrain restent incompris.

Les Mon travail de thèse a eu pour but d'améliorer la compréhension des rôles des gènes NBCL1, à la fois chez des espèces formant des nodosités indéterminées et déterminées, ainsi que de découvrir de nouveaux acteurs moléculaires impliqués dans l'identité de la nodosité dont la régulation est dépendante des gènes NBCL1, essentiellement par l'utilisation de mutants TILLING, Tnt1 et LORE1 originaux chez trois espèces de légumineuses: la luzerne tronquée, le petit pois et le lotier.

Ce travail rapporte essentiellement l'identification et la caractérisation de nouveaux mutants affectés dans des gènes qui font partie d'un second sous-clade NBCL2 spécifique des légumineuses. Nous avons révélé que les membres de ce sous-clade spécifique des légumineuses NBCL2 jouent d'importants rôles dans le développement de la nodosité, dans l'établissement et la maintenance de l'identité de la nodosité et par conséquence dans le succès et l'efficacité de l'association symbiotique.

Ce travail suggère aussi qu'au cours de l'évolution, le programme de développement de la nodosité a recruté des mécanismes de régulations préexistants afin de réguler le développement de la nodosité et son identité, tel que le module de régulation impliquant des interactions entre des protéines NBCL et des facteurs de transcriptions basic leucine zipper de type TGACG. Nous avons identifié le facteur de transcription MtPERIANTHIA-LIKE, comme un premier partenaire protéique interagissant avec des protéines NBLC dans un contexte de nodosité symbiotique.

Les gènes NBCL sont aussi impliqués dans les réseaux de régulations qui contrôlent le développement et le déterminisme de nombreux organes végétatifs et reproductifs aériens et sont également impliqués dans la capacité d'abscission de ces organes.

Finalement, ce travail thèse a eu pour objectif d'explorer les rôles de ces gènes NBCL très conservés, dans le développement de la graminée non-domestiquée, Brachypodium distachyon.

Title :

The roles of the NOOT-BOP-COCH-LIKE genes in the symbiotic organ identity and in plant development. This de-novo generated symbiotic organ allows the intracellular accommodation of the rhizobia which reduces through their nitrogenase activity the atmospheric nitrogen in ammonium, a nitrogen form usable by the host plant.

The molecular mechanisms underlying the symbiotic partners recognition, the infection process and the nodule organogenesis are well described, however the identity establishment and maintenance of this unique underground organ remain mis-understood.

The Medicago truncatula NODULE-ROOT, the Arabidopsis thaliana BLADE-ON-PETIOLE and the Pisum sativum COCHLEATA genes are members of a highly conserved NOOT-BOP-COCH-LIKE1 (NBCL1) specific clade that belongs to the NON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1-LIKE gene family. In legumes, the members of this NBCL1 clade are known as key regulators of the symbiotic nodule identity.

The present thesis work aims to better understand the roles of the NBCL1 genes, in both indeterminate and determinate nodule forming species and to discover new molecular actors involved in the NBCL1-dependent regulation of the nodule identity essentially using novel TILLING, Tnt1 and LORE1 insertional mutants in three legume species, Medicago, Pisum and Lotus.

This thesis work has allowed the identification and the characterization of new mutants for genes belonging to a secondary legume-specific NBCL2 sub-clade. We revealed that the members of this legume-specific NBCL2 sub-clade play important roles in nodule development, identity establishment and maintenance, and consequently in the success and in the efficiency of the symbiotic association.

This thesis work also shows that during evolution, the nodule developmental program has recruited pre-existing regulatory mechanisms for the nodule development and identity, such as the regulatory module involving interactions between NBCL proteins and TGACG type basic leucine zipper transcription factors. We identified the transcription factor, MtPERIANTHIA-LIKE, as a first interacting partner of NBCL proteins in a context of root nodule symbiosis.

NBCL genes are also involved in the regulatory networks that control the development and the determinacy of many aboveground vegetative and reproductive organs and were also shown as involved in their abscission ability.

In this thesis we also explored the roles of these highly conserved NBCL genes in the development of the non-domesticated grass, Brachypodium distachyon.

Université Paris-Saclay

Espace Technologique / Immeuble Discovery Route de l'Orme aux Merisiers RD 128 / 91190 Saint-Aubin, France

  Legumes are dicot plants of the Fabaceae family. Legume plants belong to the second most important plant family after grasses for mankind and livestock nutrition. Legume plants belong to the first plant species that have been domesticated by mankind in the Fertile

Figure 1 |

 1 Figure 1 | Leguminosae subfamilies organization and phylogeny of Papilionaceae sub groups a, Schematic representation of the six subfamilies classified in the Leguminosae family. This legume phylogeny is based on the matK Bayesian analysis. Clade sizes are proportional to the number of species analyzed. b, Chronogram showing the phylogenetic relationships between the major papilionoid legume groups and their time of evolution. Phylogenetic groups are based on the Legume Phylogeny Working Group data (LPWG, 2013) and the approximate dates of the nodes are taken from Lavin et al. (2005). Nodule characteristics are indicated on the right. IT, infection thread; Ma, million years ago; NPAAA, nonprotein amino acid accumulating, IRLC, Inverted Repeat Lacking Clade. Adapted from LPWG, 2017; Sprent et al., 2017.

  . In the IRLC, legumes such Medicago truncatula (barrel medic), Medicago sativa L. (alfalfa), Melilotus Medik. (white sweetclover), Trifolium repens L. (white clover) and Trifolium pratense L. (red clover), Pisum sativum (pea), Vicia faba L. (faba bean), and Lens culinaris Medik. (lentil) are found.

Figure 2 |

 2 Figure 2 | Overview of nodule structures found in Rosids I clade. a, Simple caesalpinioid indeterminate nodule typical of the Caesalpinioidaceae showing elongated shape similar to the indeterminate nodule of the Papilionoideae subfamily (Cassia quarrei, Corby, 1988). b, Mimosoid indeterminate nodule typical of the Mimosoid clade belonging to the Caesalpinioidaceae showing elongated shape similar to the indeterminate nodule of the Papilionoideae family (Mimosa sp., Elliott et al., 2007). c, Indeterminate nodule typical of the Papilionoideae IRLC showing elongated shape (Medicago trunctula). d, Determinate nodules or desmodiod nodules typical of the Papilionoideae NPAAA clade showing globular shape and lenticels (Desmodium tortuosum, Corby, 1988). e, Aeschynomenoid or Dalbergioid nodules typical of the Papilionoideae showing small and spherical nodules always associated with lateral roots (black arrows) (Aeschynomene americana, Sprent et al., 2017). f, Lupinoid or genistoid nodule typical of the Papilionoideae showing the girdling of the root (Lupinus pilosus, Corby, 1988). g, Crotalarioid nodule showing fan-like multilobed indeterminate structure (Crotalaria monteiroi, Corby, 1988). h, Parasponia nodule with a central vasculature and peripheral infected cells (Parasponia andersonii, Behm et al., 2014). i, Actinorhizal nodules with central vasculature and peripheral infected cortical tissue (Casuarina sp.). m, indicate the presence of a persistent apical nodule meristem. NPAAA, non-protein amino acid-accumulating clade; IRLC, Inverted Repeat Lacking Clade.

(

  soybean), Phaseolus vulgaris L. (common bean), Vigna unguiculata (L.) Walp. (cowpea) and Cajanus cajan L. Huth (pigeon pea) form determinate nodules. In Psoraleeae, Bituminaria bituminosa (L.) form determinate nodules. In the Loteae tribe, species such as Lotus japonicus (lotus), the crops Lotus corniculatus L. (birdsfoot trefoil) and Tetragonolobus purpureus Moench (winged pea) form determinate nodules.

Figure 3 |

 3 Figure 3 | The root nodule symbiosis signalling pathwaySymbiosis signalling is mediated by the recognition of NF by a receptor complex involving the LysM receptor kinases MtNFP/LjNFR5 and MtLYK3/LjNFR1 which associate with the LRR-RLK MtDMI2/LjSYMRK. The recognition of NF activates calcium oscillations in the nucleus. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) associates with MtDMI2/LjSYMRK and is involved in the production of mevalonate. Mevalonate may function as a secondary messenger to the nucleus. Several channels located at the inner nuclear membrane coordinate the release of calcium from the nuclear envelope and endoplasmic reticulum: a complex of MtDMI1/LjPOLLUX and MtCNGC15 regulate counterflows potassium and calcium, allows ions to flow without impinging the membrane polarity; the calcium ATPase MtMCA8 pumps-back calcium into the nuclear envelope. Nuclear calcium oscillations activate MtDMI3/LjCCAMK,which phosphorylates MtIPD3/LjCYCLOPS to promote the induction of symbiosis gene expression. MtIPD3/LjCYCLOPS may form a large complex containing GRAS-domain-containing transcription factors such as NSP1, NSP2 and DELLA that are also necessary for the expression of symbiosis genes like NIN and ERN1. NIN, ERN1 and ERN2 are necessary for rhizobial infection and nodule organogenesis, and NIN also directly activates NF-Y subunit genes which are required for cortical cell division initiation and participate to the rhizobial infection. NIN also induces the expression of MtCRE1/LjLHK1 receptor which perceives and activates the cytokinins pathway and executes a positive regulation on the cortical cell divisions and nodule organogenesis. Adapted from[START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF] Ziepfel and Oldroyd, 2017.

Figure 4 |

 4 Figure 4 | Rhizobial infection and invasion in root epidermis and cortical cells. 1. Rhizobial attachment to root hair and accumulated Nod factor induce initial responses involving root hair deformation and curling. 2. At the tightly curled root hair, cell wall degradation occurs and invagination of the IT membrane in the root hair cell is induced. 3. IT elongation occurs, which is accompanied by cytoplasmic streaming and nuclear movement. 4. During rhizobial infection (1-3) in the root cortical cells, a cytoplasmic bridge or pre-infection thread (PIT) is formed to guide elongating ITs. 5. When an IT reaches the newly divided cortical cell, the IT membrane collapses and rhizobia are released into the cortical cell to form a specialized nitrogen-fixing organelle, the symbiosome. From Suzaki et al., 2015.

Figure 5 |

 5 Figure 5 | Indeterminate root nodule fate map of the nodule primordium to mature nodule. a,b, Origins of the cells in the nodule primordium (a) and in mature nodule (b) are indicated by the same color. The origin of the nodule cortex is not shown. Root pericycle, endodermis and cortical cell layers contribute to the formation of the nodule. The root pericycle and endodermis cells layers give uninfected tissues at the base of the nodules. The inner cortical cell layers C5 and C4 differentiate into more or less 8 infected cell layers at the base of the nodule. The third cortical cell layer (C3) gives rise to the nodule meristem. Once active, the nodule meristem add cells to the nodule. From Xiao et al., 2014.

Figure 7 |

 7 Figure 7 | An indeterminate apical meristematic region including the nodule central meristem and nodule vascular meristem.Above view of a mature Medicago truncatula nodule meristem. The nodule central mersitem (NCM) at the center of the nodule meristem is surrounded by nodule vascular mersitems (NVM). Cytokinin (red) predominate in the NCM with lower auxin response while auxin (blue) is found at high level in NVM. MtWOX5 expression is detected in NCM and extends to the NVM, displaying a star-like pattern. MtPLT1 and MtPLT2 gene expressions (purple stars) were found at high level in NVM but not in the NCM, while MtPLT3 and MtPLT4 expression (red stars) levels were comparable in both NCM and NVM. Scale bar: 50 µm. Based on[START_REF] Osipova | Features of the expression of a meristem-specific WOX5 gene during nodule organogenesis in legumes[END_REF][START_REF] Osipova | WUSCHEL-RELATED HOMEOBOX5 Gene Expression and Interaction of CLE Peptides with Components of the Systemic Control Add Two Pieces to the Puzzle of Autoregulation of Nodulation[END_REF][START_REF] Couzigou | To be or noot to be? Evolutionary tinkering for symbiotic organ identity[END_REF][START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF][START_REF] Franssen | Root developmental programs shape the Medicago truncatula nodule meristem[END_REF]

  genes are key regulators of the symbiotic organ identity In addition to the different genes reported above as potentially involved in the nodule primordia formation and in nodule development, another class of genes was shown to play major roles in the indeterminate nodule development, identity and maintenance. Among the NON-EXPRESSOR OF PATHOGENESIS-RELATED PROTEIN1-LIKE (NPR1-LIKE) genes family, the members of the NOOT-BOP-COCH-LIKE (NBCL) sub-clade are associated to the regulation of plant development. This clade was called NBCL because it groups the M. truncatula MtNODULE-ROOT (MtNOOT), the Arabidopsis thaliana AtBLADE-ON-PETIOLE1 and AtBLADE-ON-PETIOLE 2 (AtBOP1/2), and the P. sativum

  cells which is maintained via the class I KNOTTED-LIKE HOMEODOMAIN (class I KNOX) transcription factors activity inducing a positive regulation on the WUSHEL (WUS)-CLAVATA (CLV) negative feedback loop. In this WUS-CLV regulatory loop, stem cells are maintained in an undifferentiated state through WUS which induces the expression of the CLV3 peptide perceived by the serine/threonine receptor kinase CLV1 that, in return, signals the repression of WUS to control the size of the stem cell domain. In parallel to CLV3 action, the meristem size is positively regulated by cytokinins and BRs. Class I KNOX genes involved in the maintenance of the pool of undifferentiated cells are SHOOTMERISTEM LESS (STM), BREVIPEDILUS/KNOTTED1-LIKE HOMEODOMAIN OF ARABIDOPSIS THALIANA1 (BP/KNAT1) and KNOTTED1-LIKE HOMEODOMAIN OF ARABIDOPSIS THALIANA2 (KNAT2). In SAM, STM represses BOP1/2 to maintain indeterminacy.9.2. The meristem to organ boundary regulationIn the MTOB adjacent to the SAM, a distinct genetic program is executed. The role of this program is to switch-off the meristematic activity and to inhibit the division of cells that are recruited for the formation of a lateral organ. The establishment of the MTOB begins by expressing specific MTOB transcriptional factors such as the CUP-SHAPED COTYLEDON1 (CUC1), CUC2, CUC3 belonging to the NO APICAL MERISTEM (NAM)transcription factors (NAC-TFs) family as well as their direct targets, the ORGAN BOUNDARY1/LIGHT-DEPENDENT SHORT HYPOCOTYLS3 (OBO1/LSH3) and LSH4 that are members of the Arabidopsis LSH1 and Oryza G1 (ALOG)

  BOP1 and BOP2 play crucial roles in the MTOB functioning by participating to the repression of cell division and growth program. BOP1 and BOP2 promote the expression of LBD boundary genes such as AS2 and of the LATERAL ORGAN BOUNDARY (LOB) gene. The boundary-specific TF LOB blocks cell divisions and represses the BRs response via the activation of the BRs-inactivating enzyme PHYB ACTIVATION TAGGED SUPPRESSOR1 (BAS1) which also restricts cell growth and division. LOB expression in turn is regulated by BRs creating a feedback loop. BRs are known to inhibit the CUCs and LOF1 expression, thus the low level BRs content in MTOB allows their expressions resulting in cell division inhibition (Fig. 8).

Figure 8 |

 8 Figure 8 | BOP1/2-mediated brassinosteroids inhibition allows the expression of boundary TFs to block cell division in MTOB. The diagram represents the SAM tissue organization. The central zone is represented in grey, an organ primordium in dark grey and the boundary zone in yellow. LOB expression in the meristem-to-organ boundary is activated by the expression of BOP1 and BOP2 in the primordium and the boundary domain and by the activity of BRZ1 TF, whose expressions depend on brassinosteroids. LOB represses brassinosteroid signaling, especially through the activation of the brassinosteroid-inactivating enzyme BAS1. Low levels of brassinosteroids allow the expression of CUC and LOF, which inhibit growth and allow the MTOB establishment and functioning. Arrows and inhibition lines represent positive (black) and negative interactions (dark red), respectively. The plant hormone brassinosteroid is highlighted in blue. Genes presented with large letters are expressed in the relevant zone of the SAM, while gene names indicated with smaller letters are repressed in the respective area. From Zadnikova et al. (2014).

Figure 9 |

 9 Figure 9 | Exclusion of BOP through the action of PNY-PNF and STM is required for inflorescence meristem integrity and flowering a, PNY, PNF and STM restrict BOP1/2 expression and their downstream effectors ATH1/KNAT6 to boundary domains flanking the IM. This process is essential for inflorescence meristem maintenance, accumulation of FLOWERING LOCUST (FT) and bZIP transcription factor FD and thus for flowering. Atpny-pnf mutant failed to repress the MTOB module activity in the inflorescence meristem. Thus BOP1 acting via an unknown TGA bZIP co-factor directly activates ATH1 and indirectly KNAT6. The MTOB module repress meristem activity, growth and flowering by increasing JA content via the transcriptional promotion of JA biosynthetic genes. FT and FD failed to accumulate and the FT-FD complex does not form. The absence of FT-FD prevents the activation of the floral meristem identity genes such as LEAFY (LFY), APETALA1 (AP1), and CAULIFLOWER (CAL) and thus flowering. b, In floral meristem formation the auxin responsive transcription factor MONOPTEROS (MP) directly activates AINTEGUMENTA (ANT) which triggers proliferation and (LFY) which activate flower development. LFY directly promotes the expression of (AP1) and (CAL) conferring floral fate.Here, BOPs will allow the establishment of floral meristems via promotion of LFY expression, activation of AP1, and repression of IM identity genes. UNUSUAL FLORAL ORGANS (UFO), a LFY co-activator is required for formation of boundaries in the flower. Red letter, inflorescence identity gene. Adapted from[START_REF] Khan | Repression of lateral organ boundary genes by PENNYWISE and POUND-FOOLISH is essential for meristem maintenance and flowering in Arabidopsis thaliana[END_REF] Hepworth and Pautot, 2015 and[START_REF] Khan | BLADE-ON-PETIOLE genes: Setting boundaries in development and defense[END_REF]

  nodule and thus I undertook to investigate the role of NBCL1 in this context using Lotus japonicus. The molecular regulation of the nodule identity is currently not describe, thus to improve our understanding of this regulation, I searched for NBCL1 interacting partners. NBCL1 interacting partners isolation might help us to better understand the molecular environment in which NBCL1 proteins are functioning to drive the development and the identity of the nodule. NBCL1 interacting partner isolation will also allow to explore what are the target genes controlled by NBCL1 for the nodule identity regulation. Also, since NBCL2 paralogs have been discovered, I dedicated a part of my work to investigate their roles in the nodule development and identity by studying the corresponding mutants in Medicago and pea. To deeper understand the role of NBCL genes, nbcl1nbcl2 double mutants were constructed in Medicago and pea and their study was initiated. Another aspect of my work was to investigate if the abscission in Lotus japonicus is also dependent of the NBCL1 function and also to explore if the role of NBCL in abscission is conserved in grasses using Brachypodium distachyon a non-domesticated model species closely related to wheat and barley.

  The first chapter of the manuscript is entitled "Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity". In this part, I have characterized the simple and double Mtnoot mutants available in the laboratory in order to understand the role of MtNOOT2 during nodulation and nodule identity, in Medicago forming indeterminate nodules. This article was submitted for publication to the revue Plant Physiology and we were asked for additional experiments before resubmission. The second chapter of the manuscript is entitled "The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development". In this part, I isolated and characterized TILLING mutants of the PsCOCH2 gene in pea forming indeterminate nodules. I constructed and initiated the characterization of the Pscoch1coch2 double mutant and investigated the roles of PsCOCH2 in nodule development and identity. In this study, I also compared the roles of the pea and Medicago NBCL genes. In addition, I initiated the phenotypic characterization of the Pscoch1coch2 double mutant aerial phenotypes in order to determine if PsCOCH2 plays a role in aerial organ initiation, development and patterning. This part was not submitted for publication. The third part of the manuscript is entitled "The key regulators of the symbiotic identity MtNODULE-ROOT1 and MtNODULE-ROOT2 interact with the TGA bZIP transcriptional factor MtPERIANTHIA-LIKE". The objective of this work was to improve our knowledge concerning the molecular mechanisms underlying the regulation of the Medicago nodule identity mediated by MtNOOT. For this, I initiated an a priori approach to determine if the transcriptional co-factors MtNOOTs interact with the TGA bZIP transcription factor MtPERIANTHIA-LIKE which is specifically expressed in nodules. This part was not submitted for publication. The fourth part of this manuscript is entitled "NOOT-BOP-COCH-LIKE1 is essential for multiple aspects of Lotus japonicus development". In this part, I studied the role of the LjNBCL1 gene in the legume species Lotus japonicus forming determinate nodules without persistent meristem. The objective of this study was to determine if the functions of the NBCL genes are conserved in legume forming determinate nodules. For this, I characterized two mutant alleles for the Lotus LjNBCL1 gene, orthologous to MtNOOT1 and PsCOCH1. Using the Ljnbcl1 mutant alleles, I also investigated the roles of this gene in the development of vegetative and reproductive aerial organs. This article has been submitted for publication in the journal New phytologist but has not been accepted. The fifth part of this manuscript consists in an accepted paper published in the journal New Phytologist entitled "The legume NOOT-BOP-COCH-LIKE genes are conserved regulators of abscission, a major agronomical trait in cultivated crops". This publication describes the function of the legume NBCL gene in abscission using a series of nbcl1 mutants in Medicago, pea, Lotus and Lupinus. This work shows that these genes are essential for the shedding of petals, leaves, leaflets and fruits. I contribute to this work by providing the data concerning the Lotus species. The sixth and last part of the manuscript is entitled "Role of the Brachypodium distachyon NBCL genes BdNPR5-LIKE and BdNPR6-LIKE in development". In this last part of the manuscript, I explored the NBCL gene functions in grasses development and especially studied their role in abscission, a character of interest in agriculture. For this study, I isolated and characterized TILLING mutants for the two NBCL genes in the model plant

CHAPTER I .

 . Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 40 distinct NBCL1 and NBCL2 proteins belonging to the NBCL subclades described above. Legume NBCL2 protein sequences alignment reveals high conservation in both the BTB/POZ and in the ANKYRIN domains (Supplemental Fig. 1). Supplemental Figure S1. Alignment of legume NOOT-BOP-COCH-LIKE 2 (NBCL2) proteins Alignment of legume NBCL2 proteins from Medicago truncatula (MtNOOT2), Cicer arietinum (CaNPR5-likeb), Pisum sativum (PsCOCH2), Phaseolus vulgaris (PvNBCL2), Cajanus cajan (CcNBCL2) and Glycine max (GmNBCL2) was perform using the Multiple Sequence Alignment online software Clustal omega (ebi.ac.uk/Tools/msa/clustalo). The percentage of identity/similarity for shading was setup at 80% using BoxShade Server (ch.embnet.org/software/BOX_form). The BTB/POZ domain and ANKYRIN repeats are underligned by red and blue lines and defined according to Hepworth et al. (2005) and Norberg et al. (2005) respectively. Interestingly, Lupinus, Populus and Salix NBCLs are more related to the legume NBCL1 sub-clade and by contrast Cucurbitaceae NBCLs are more related to the legume NBCL2 subclade (Fig. 1, Supplemental Table

Figure 1 .

 1 Figure 1. Neighbor-joining phylogenetic tree of the plant NBCL proteinsFull-length NBCL protein sequences of different Fabaceae, Brassicaceae, Salicaceae, Solanaceae, Cucurbitaceae and Pooideae species were aligned using ClustalW[START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF]. The phylogenetic tree was built using the neighbor-joining method with the MEGA6 program[START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The tree was drawn according to the evolutionary distances, based on the proportion of amino-acid differences between sequences and was established using pairwise distance analysis on MEGA6 program[START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. Bootstrap method was used and set at 10,000 replicates for the phylogenetic analysis. The tree was rooted using the more distant BTB/POZ-ankyrin repeat AtNPR1 protein sequence. Blue and red open rectangles indicate the legume NBCL1 protein clade and the legume NBCL2 protein clade, respectively. Evolutionary divergence scale bars: 0.05 substitution/site. Medicago truncatula MtNOOT1 and MtNOOT2; Pisum sativum PsCOCH1 and PsCOCH2, Phaseolus vulgaris PvNBCL1 and PvNBCL2; Cicer arietinum CaNPR5-LIKEa and CaNPR5-LIKEb; Lupinus luteus LlBOP1; Cajanus cajan CcNBCL1 and CcNBCL2, Glycine max GmNBCL1 and GmNBCL2; Lotus japonicus LjNBCL1; Solanum lycopersicum SlBOPa, SlBOPb, and SlBOPc; Arabidopsis thaliana AtBOP1, AtBOP2 and AtNPR1; Cucumis sativus CsNPR5-LIKEa and CsNPR5-LIKEb; Cucumis melo CmNPR5-LIKEa and CmNPR5-LIKEb; Populus trichocarpa PtBPL1 and PtBPL2; Salix purpurea SpNPR5-LIKEa and SpNPR5LIKEb; Brachypodium distachyon BdNPR5-like and BdNPR6-like, Hordeum vulgare HvCul4, and HvLAX-A NBCL proteins were used to draw the tree. The above mentioned NBCL protein sequence accession information and origins are listed in the supplemental tableS1.

Figure 2 .

 2 Figure 2. MtNOOT1 and MtNOOT2 gene expression during nodule development MtNOOT1 (A) and MtNOOT2 (B) qRT-PCR gene expression analysis in uninoculated primary root devoid of root apical meristem (2 days post vernalization: PR 0 dpi), in uninoculated root apical meristem (0,5 cm, 2 days post vernalization: RAM 0 dpi), in inoculated primary root (PR 2, 5 dpi), in nodule primordia (Prim 8 dpi) and in nodules (Nod 12, 16 and 21 dpi) inoculated with Sinorhizobium medicae WSM419 strain. The relative expression was normalized against the constitutively expressed MtRNA RECOGNITION MOTIF (MtRRM) and MtACTIN (MtACT) genes and against uninoculated primary root. Results represent means ± SE of three technical replicates and four biological replicates. Prim, primordia; Nod, nodule.

  Figure S2. Comparison of MtNOOT1 and MtNOOT2 gene expression levels during nodulation MtNOOT1 (white bars) and MtNOOT2 (grey bars) gene expressions, in uninoculated primary root devoid of root apical meristem (2 days post vernalization: PR 0 dpi), in uninoculated root apical meristem (0,5 cm, 2 days post vernalization: RAM 0 dpi), in inoculated primary root (PR 2, 5 dpi), in nodule primordia (Prim 8 dpi) and in nodules (Nod 12, 16 and 21 dpi) inoculated with Sinorhizobium medicae WSM419 strain. The gene expression data were normalized against the constitutively expressed MtRRM and MtACT genes. Results represent means ± SE of three technical replicates and four biological repeats. Prim, primordia; Nod, nodule.

Figure 3 .

 3 Figure 3. MtNOOT2 gene expression compared to symbiotic marker gene expression during nodulation MtNOOT2 (diamond) qRT-PCR gene expression analysis compared to different nodulation marker genes, MtPR10 (square), MtNIN (triangle), MtNCR001 (circle) and MtLEGH1 (cross), in uninoculated primary root devoid of root apical meristem (2 days post vernalization: PR 0 dpi), in uninoculated root apical meristem (0,5 cm, 2 days post vernalization: RAM 0 dpi), in inoculated primary root (PR 2, 5 dpi), in nodule primordia (Prim 8 dpi) and in nodules (Nod 12, 16 and 21 dpi) inoculated with Sinorhizobium medicae WSM419 strain. The relative gene expressions were normalized against the constitutively expressed MtRRM and MtACT genes and against uninoculated primary root. Left Y axis represents the relative expression values for MtNOOT2, MtPR10, MtNIN and MtNCR001 genes and right Y axis represent the relative expression values for MtLEGH1 gene. Results represent means ± SEM of three technical replicates and four biological replicates. Prim, primordia; Nod, nodule.

Figure 4 .

 4 Figure 4. ProNOOT1:GUS and ProNOOT2:GUS expression patterns during nodule development (A-C) nodule primordia were staged according to Xiao et al. 2014; (A) no detectable ProNOOT1:GUS expression (Blue staining) at stages III-IV. Black arrows indicate anticlinal divisions in C3 and endodermis. Grey arrows indicate periclinal divisions in endodermis and pericycle. (B) longitudinal section of stage V nodule primordium with weak ProNOOT1:GUS expression. Black arrows indicate anticlinal divisions in C2 and C3 derived cells form multiple cell layers. Endodermis and pericycle derived cells form approximately 6 cell layers from the basal part of the nodule primordia. (C) ProNOOT1:GUS expression in stage VI nodule primordium (longitudinal section) in endodermis and pericycle derived cell layers; at this stage, the vascular bundles and the central apical meristem of the nodule are formed. (D) longitudinal section of 8-9 dpi differentiating nodule showing the ProNOOT1:GUS expression associated to the basal part of the nodule and to the cells surrounding the vascular bundles. ProNOOT1:GUS is not expressed in nodule parenchyma tissues or in the nodule fixation zone. Dotted black line separating the Np and Fz from Nv (indicated by asterisk). (E-F) ProNOOT1:GUS expression in the vascular bundles at later stages of nodule development. (G) longitudinal section through a nodule vascular bundle at 8-9 dpi. The ProNOOT1:GUS expression pattern is associated to the nodule vascular bundles. (H) Magnification of (G) focusing

  panel A II-VI). At the young nodule stages, ProNOOT1:GUS expression is associated to the basal part of the nodule derived from the root pericycle/endodermis cell layers and to the young developing nodule vascular bundles (Fig. 4 D and Supplemental Fig. 3 panel A II-III). At later stages of nodule development, ProNOOT1:GUS expression associate essentially to the developing parts of nodule vascular bundles and is reduced in the old and established vascular bundle parts (Fig. 4 E-F and Supplemental Fig. 3 panel A IV-VI). Supplemental Figure S3. ProNOOT1:GUS and ProNOOT2:GUS expression patterns throughout nodule development (Panel A I-VI and i-vi) ProNOOT1:GUS tissue-specific expression pattern in stable transgenic Medicago truncatula R-108 nodules inoculated with S. medicae WSM419. The bright-field pictures of nodules at different developmental stages show the ProNOOT1:GUS expression pattern associated with the nodule vascular bundles. (Panel B I-VI and i-vi) ProNOOT2:GUS tissue-specific expression pattern in stable transgenic Medicago truncatula Jemalong J5 nodule transformed by hairy-root using Agrobacterium Rhizogenes strain A4TC24 and inoculated with S. meliloti Rm41. The bright-field pictures of nodules at different developmental stages show the ProNOOT2:GUS expression pattern associated with both the nodule meristem and the nodule vascular bundles apex. Nodules were collected from 7 to 18 dpi. (Panel A I-VI; panel B I-VI) nodules side views; (Panel B i-vi and panel B i-vi) nodules above views. Scale bars: 500µm.

Figure 5 .

 5 Figure 5. The ProNOOT2:GUS expression pattern is altered in the Mtnoot1 background (A-E) ProNOOT2:GUS expression in 35 dpi nodules. (A) R-108 wild-type nodule expressing the ProNOOT2:GUS fusion in the meristematic zone and along the adjacent vascular bundle extremities. (B) Mtnoot1 young unconverted nodule with ProNOOT2:GUS expression in the apical meristematic zone and ectopic ProNOOT2:GUS expression at the nodule basis. (C) Mtnoot1 nodule showing root identity transition: ProNOOT2:GUS expression is conserved in the nodule meristematic zone but absent in the emerging ectopic root (yellow arrowhead). (D) At advanced stages of Mtnoot1 nodule to root transition, the remaining nodule meristematic region expresses the ProNOOT2:GUS fusion. The ProNOOT2:GUS fusion is also expressed at the nodule basis and in some nodule vasculature. In fully converted structures (white asterisk), the ProNOOT2:GUS apical expression is completely lost. (E) Mature Mtnoot1 fully converted nodule (white asterisk) showing neither apical nor basal ProNOOT2:GUS expression. However, the ProNOOT2:GUS reporter fusion is expressed in nodule primordia (white arrowheads) that developed on the ectopic root of this structure. The nodules and nodule-like structures were formed after infection with S. medicae WSM 419. Yellow arrowheads indicate nodule ectopic roots. Scale bars: 500 µm.

Figure 6 .

 6 Figure 6. Mtnoot1noot2 nodules have lost their identity and their nitrogen fixation ability (A-E) Nodules 35 days after inoculation with S. medicae WSM419 strain; (A) R108 uni-lobed nodule, (B) R108 multi-lobed nodule, (C, D and E) Mtnoot1noot2 nodule-like structures, (F) 60 µm semi-thin section of Mtnoot1noot2 mutant nodule 32 days after S. medicae WSM 419 strain inoculation showing nodule ectopic root vasculature connected to the nodule vascular bundles. (C-F) Yellow arrowheads indicate cryptic roots emerging from Mtnoot1noot2 nodules. Stars indicate pluri-vascularized ectopic root dichotomies; White arrowheads indicate nodule primordia formation on nodule ectopic root; Arrows indicate newly formed nodule-like structures from nodule ectopic root; Asterisks indicate the continuum between nodule vasculature and nodule cryptic root vasculature. Scale bar: 1mm. (G) Characterization of the different nodule phenotypes in R108, Mtnoot1, Mtnoot2 and Mtnoot1noot2 mutants. Wild-type uni-lobed nodules (white bars), wild-type multi-lobed nodules (grey bars), nodule to root conversions (dashed horizontal bars), nodule-like structure (wave bars). (H) Nitrogen fixation in R108, Mtnoot1, Mtnoot2 and Mtnoot1noot2. Asterisks indicate the significant difference compared to the R108 control, *** indicate a p-value < 0,001 (Mann and Whitney test). Results in (G) and (H) represent means ± SE of three independent biological experiments containing nine plants each (27 plants analyzed for each genotype). Total number of nodules analyzed for each genotype: R108, n=205; Mtnoot1, n=141; Mtnoot2, n=153; Mtnoot1noot2, n=94.

Supplemental Figure S4 .

 S4 Nodule occupancy and defense reactions in the mutant backgrounds A-L, Semi-thin and M-P, thin section stained with toluidine blue of M. truncatula nodules in R-108, Mtnoot1, Mtnoot2 and Mtnoot1noot2 backgrounds, at 42 dpi with S. medicae WSM419 eGFP strain (A-L) and at 35 dpi with S. medicae WSM419 strain (M-P). Mtnoot1 and Mtnoot1noot2 mutant nodules are weakly colonized by rhizobia (B, F, J, N and D, H, L P respectively) compared to R-108 and Mtnoot2 in which bacteroids occupate the whole nodositiy (A, E, I, M and C, G, K, O respectively). White arrows indicate the presence of phenolic compounds and suggest defense-like reactions. Number of nodules sectioned and imaged for eGFP rhizobia localization experiment, R-108, n: 26, Mtnoot1: 10, Mtnoot2: 14, Mtnoot1noot2: 28. Semi-thin section thickness: 60 µm; Thin section thickness: 5 m; Scale bar A-L: 500 µm, M-P: 50 µm.

Figure 7 .

 7 Figure 7. Infection of the double Mtnoot1noot2 mutant by rhizobial exo, kps and exo-kps mutants R108 M. truncatula wild-type plant (A-H) and Mtnoot1noot2 mutant (I-P) 35 dpi after inoculation with S. meliloti Rm41 WT (A, E, I and M), S. meliloti Rm41 kps -(B, F, J and N), S. meliloti Rm41 exo -(C, G, K and O) and S. meliloti Rm41 exo -kps -(D, H, L and P). Pictures A-D and I-L represent global view of nodulated roots and pictures E-H and M-P represent magnification of the corresponding symbiotic structures stained with methylene blue. R108 inoculated with S. meliloti Rm41 WT, S. meliloti Rm41 kps -, S. meliloti Rm41 exo -show normal nodulation (A, B, C, E, F and G) while inoculation with S. meliloti Rm41 exo -kps -resulted in the formation of prematurely arrested nodule primordia (D and H). Mtnoot1noot2 inoculated with S. meliloti Rm41 WT, S. meliloti Rm41 kps -, S. meliloti Rm41 exo -show typical nodule to root conversion (I, J, K, M, N and O). Mtnoot1noot2 inoculated with S. meliloti Rm41 exo -kps -shows in addition to small prematurely arrested nodule primordia as observed in R-108, root-like structures presenting central vasculature(s) (L and P). White arrowheads: nodule primordia prematurely arrested; yellow arrowheads: root-like structures presenting central vasculature. Scale bars: A, B, C, D, I, J, K and L, 1 cm; E, F, G, H, M, N, O and P, 200µm.

Figure 8 .

 8 Figure 8. Expression of symbiotic and defense marker genes in R-108, single and double mutant nodules (A-C) Gene expression analysis on mature nodules by qRT-PCR. Wild-type M. truncatula R108 (white bars), Mtnoot1 mutant (downward dashed bars), Mtnoot2 mutant (upward dashed bars), Mtnoot1noot2 (diamond bars). (A) MtNOOT1 and MtNOOT2 gene expression in WT and mutant backgrounds. (B) Expression of defense marker genes MtPAL2, MtPR10 and MtNDR1-LIKE. (C) Expression of symbiotic marker genes MtCRE1, MtNIN, MtNCR001, MtLEGH1, MtDNF2 and MtSYMCRK. Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against R-108 nodules. Results represent means ± SE of two technical replicates and two biological repeats at 29 and 38 dpi with S. medicae WSM419 strain. Asterisks indicate significant difference compared to R-108 nodule (* p-value < 0,01 ; ** p-value < 0,001 ; *** p-value < 0,0001; One-way ANOVA).

  expression of the symbiotic genes MtNCR001 and MtLEGH1 expressed after the primordium stage are significantly reduced in Mtnoot1, not affected in the Mtnoot2 and strongly reduced in Mtnoot1noot2 relative to R-108 (Fig. 8 C). Similarly, the expression levels of the nodule specific genes MtDOES NOT FIX 2 (MtDNF2) and MtSYMBIOTIC CYSTEINE RICH RECEPTOR KINASE LIKE (MtSYMCRK) expressed after rhizobia internalization are also reduced in Mtnoot1, not affected in the Mtnoot2 and strongly reduced in Mtnoot1noot2 relative to R-108 (Fig. 8 C). MtDNF2 and MtSYMCRK are essential to repress immunity in the nodule

  agreement with the MtDNF2 and MtSYMCRK reduced expression in Mtnoot1 and Mtnoot1noot2, the defense-related genes MtPHENYLALANINE AMMONIA LYASE2 (MtPAL2), MtPATHOGENESIS-RELATED PROTEIN10 (MtPR10) and MtNON-DISEASE RESISTANCE1-LIKE (MtNDR1-LIKE), normally repressed in the nodule, are up-regulated (Fig. 8 B; Bourcy

Figure 9 .

 9 Figure 9. Root apical meristem markers, root developmental regulators and plant developmental regulators gene expression analysis (A-C) Gene expression analysis on mature nodules by qRT-PCR. (A) Gene expression analysis of the meristematic marker genes MtWOX5, MtACR4 and MtSHR. (B) Gene expression analysis of members of the MtPLETHORA gene family MtPLT1, MtPLT2, MtPLT3, MtPLT4. (C) Gene expression analysis of members of class II KNOX gene family MtKNOX3, MtKNOX5 and MtKNOX9. Wild-type M. truncatula R108 (white bars), Mtnoot1 mutant (downward dashed bars), Mtnoot2 mutant (upward dashed bars), Mtnoot1noot2 (diamond bars). (A-B) Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against R108 nodules. (C) Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against their respective root organ expression. Results represent means ± SE of two technical replicates and two biological repeats at 29 and 38 dpi with S. medicae WSM419 strain. Asterisks indicate significant difference compared to R-108 nodule (* p-value < 0,01 ; ** p-value < 0,001 ; *** p-value < 0,0001; One-way ANOVA).

  transcripts are almost undetectable by qRT-PCR. No significant differences in MtPLT1, MtPLT3 and MtPLT4 expression levels were observed in Mtnoot2 mutant nodules compared to R-108 nodules (Fig. 9 B). By contrast, MtPLT2 expression level was strongly increased in Mtnoot1 and Mtnoot1noot2 nodules, by 50 and 32 folds, respectively (Fig. 9 B). Interestingly, the MtPLT2 gene expression levels in Mtnoot1 and Mtnoot1noot2 nodules are comparable to the MtPLT2 expression level in non-inoculated R-108 RAM (Supplemental Fig. 5). These results support the identity shift observed in Mtnoot1 and Mtnoot1noot2 mutant organs. Supplemental Figure S5. MtPLETHORA 1, 2, 3 and 4 gene expressions during nodulation MtPLETHORA 1 (Medtr2g098180.1), MtPLETHORA 2 (Medtr4g065370.1), MtPLETHORA 3 (Medtr5g031880.1) and MtPLETHORA 4 (Medtr7g080460.1) gene expression analysis in uninoculated primary root devoid of root apical meristem (2 days post vernalization: PR 0 dpi), in uninoculated root apical meristem (0,5 cm, 2 days post vernalization: RAM 0 dpi), in inoculated primary root (PR 2, 5 dpi), in nodule primordia (Prim 8 dpi) and in nodules (Nod 12, 16 and 21 dpi) inoculated with Sinorhizobium medicae WSM419 strain. The relative expression was normalized against the constitutively expressed MtRNA RECOGNITION MOTIF and MtACTIN genes and against non-inoculated primary root. Results represent means ± SE of three technical replicates and four biological repeats. Prim, primordia; Nod, nodule. The MtNOOT genes regulate class II MtKNOX gene expression in nodules KNOX proteins belong to the THREE AMINO-ACID LOOP EXTENTION (TALE) protein family and encode homeodomain-containing transcription factors. The class I KNOX TFs

MtKNOX3

  and MtKNOX9 gene expression was reduced in Mtnoot1 and Mtnoot1noot2 nodules relative to R-108 nodules. Their transcription levels were reduced by 48% and 25% in the Mtnoot1 and by 76% and 48% in Mtnoot1noot2, respectively (Fig.9 C). By contrast, MtKNOX5 gene expression was slightly induced in Mtnoot1 and Mtnoot1noot2 nodule (Fig.9 C).In wild-type, MtKNOX5 showed similar levels of expression in root and nodules, suggesting that changes in MtKNOX5 gene expression levels do not contribute to a shift from nodule to root identity. No significant differences in gene expression were observed for all three genes in Mtnoot2 nodules relative to R-108 nodules (Fig.9 C).Nodules are highly organized organs with several meristematic domains delimited by boundaries. Although the variation of MtKNOX3 and MtKNOX9 gene expression levels may be linked to the nodule to root conversion phenotype, we hypothesize that their spatial expression patterns could also be MtNOOT-dependent. Using a promoter MtKNOX9:GUS:terminator MtKNOX9 (ProKNOX9:GUS) fusion, the MtKNOX9 expression pattern was investigated in Mtnoot1, Mtnoot1noot2 and R-108 nodules. In R-108 nodules, strong ProKNOX9:GUS expression was observed in the apical meristematic region of the nodule while a weaker signal was observed in the peripheral nodule vascular bundles (Fig.10 A), in accordance with the observations of Di[START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. In Mtnoot1 unconverted multi-lobed nodules we observed an extended ProKNOX9:GUS expression to the entire nodule with a particular strong expression at the base and along the vasculature, suggesting that the restriction of MtKNOX9 CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 64 expression to the apical meristem of nodules is MtNOOT1-dependent (Fig.10 B). By contrast, in the Mtnoot1 converted nodules ProKNOX9:GUS expression in the nodule apical region is lost and a strong expression from the nodule vascular bundles to the ectopic root vasculature is detected (Fig.10 C). Similarly to the expression pattern in the Mtnoot1 mutant, Mtnoot1noot2 wild-type-looking nodules showed ProKNOX9:GUS expression extended to the entire nodule structure with particular strong expression at the base and along the vasculature, and Mtnoot1noot2 converted nodules showed loss of ProKNOX9:GUS expression in the nodule apical region and a strong expression from the nodule vascular bundles toward the ectopic root vasculature (Fig.10 D and E). It is noteworthy, that in the nodule to root conversions events, the ProKNOX9:GUS reporter is expressed in the vasculature of the ectopic root emerging from the mutant nodule, further highlighting the vascular connection between the nodule vascular bundles and the nodule ectopic root vasculatures (Fig.10 C and E).

Figure 10 .

 10 Figure 10. Changes in the expression pattern of the ProKNOX9:GUS reporter fusion in R108, Mtnoot1 and Mtnoot1noot2 mutant nodules (A-E) ProKNOX9:GUS expression in mature nodules 28-35 dpi with S. meliloti. (A) R108 wild-type nodules with ProKNOX9:GUS expression restricted to the apical central meristem zone with weak expression along the vascular bundles of mature R108 nodules. (B) Mtnoot1 unconverted multi-lobed nodules with strong ProKNOX9:GUS expression extended to the entire nodule structure and with particular strong expression in the vasculature. (C) ProKNOX9:GUS expression in Mtnoot1 converted nodules with loss of expression in the apical central meristem region and strong expression in the vascular bundles of nodule to ectopic root conversions. (D) Mtnoot1noot2 wildtype-looking nodule with ProKNOX9:GUS expression with strong ProKNOX9:GUS expression extended to the entire nodule structure and with particular strong expression in the vasculature, similar to the expression pattern in Mtnoot1. (E) Mtnoot1noot2 converted nodule with loss of expression in the apical central meristem region and strong expression in the vascular bundles of nodule to ectopic root conversions, similar to the Mtnoot1 mutant. Scale bars: A, B, D, E: 500 µm; C: 1 mm.

  supporting the hypothesis that nodule-root identity can override the nodule identity. Class II KNOX studies in Mtnoot mutant contexts suggest that the loss of nodule identity observed in Mtnoot1 and in Mtnoot1noot2 nodules is associated to both, a decrease in MtKNOX3 and MtKNOX9 expression levels and to a drastic shift in the MtKNOX9 spatial expression pattern from the apical meristematic region to the vascular tissues.DiscussionLegume plants retained two distinct NBCL genes playing complementary roles for nodule identity maintenanceLegume plants often possess two distinct NBCL genes that were probably present in the legume ancestors. The closely related Cucurbitaceae and Salicaceae families[START_REF] Werner | A single evolutionary innovation drives the deep evolution of symbiotic N2-fixation in angiosperms[END_REF] have retained only one of these two clades with eventual intra-family duplication. A more extensive analysis of the nodulating plants will clarify if this dual NBCL sub-clades model represents a general feature of the symbiotic species or if it is restricted to the species studied here.In this study, we have characterized the MtNOOT1 paralogous gene MtNOOT2. By contrast to MtNOOT1, that belongs to the NBCL1 sub-clade and which is constitutively expressed in roots and induced in nodules, MtNOOT2 gene belongs to the NBCL2 sub-clade, is not expressed in roots and is early induced during nodule formation as PsCOCH2, the NBCL2 member of Pea.During initiation of the nodule organogenesis, MtNOOT1 expression is down-regulated at 2 and 5 dpi, the ProNOOT1:GUS fusion is absent in root stele during the primordia developmental stage III-IV and V and furthermore transcriptomic data indicate that MtNOOT1 is repressed early in roots treated with Nod factors(Herrbach et al., 2017 Table

  , it can be hypothesized that MtNOOT1 and MtNOOT2 proteins interact at the NVM/NCM boundaries to coordinate NVM and NCM development and to repress root identity. This boundary between the two fusions also reflects that another function of MtNOOT1 might be to repress MtNOOT2 expression locally and vice versa. The modifications of the ProNOOT2:GUS pattern in the noot1 background indicates that the maintenance of nodule identity conferred by MtNOOT1 is essential for the correct expression of MtNOOT2. It can also be hypothesised that MtNOOT1 prevents the MtNOOT2 expression from the basal and the peripheral part of the symbiotic wild-type nodule or that MtNOOT2 is ectopically expressed instead of MtNOOT1 in the basal and peripheral part of the Mtnoot1 mutant nodule to compensate for the Mtnoot1 mutation. This would further suggest a partial complementation of the Mtnoot1 mutation by MtNOOT2 and support a partial redundant function between the two proteins.

  in mutant contexts suggests hormonal perturbations in Mtnoot1 and Mtnoot1noot2. It can be hypothesized that the drastic down-regulation of MtKNOX3 observed in Mtnoot1 and Mtnoot1noot2 may cause a reduced MtIPT3 and MtLOG2 accumulation as observed when reducing MtKNOX3 gene expression by RNA interference[START_REF] Azarakhsh | KNOTTED1-LIKE HOMEOBOX 3: a new regulator of symbiotic nodule[END_REF]. Consequently, a decrease in endogenous cytokinin content and/or active form may alter both nodule development and identity. The reduced MtCRE1 expression observed in the Mtnoot1noot2 double mutant may be part of this regulation.Taken together, up-regultion of RAM-related genes and downregulation of MtKNOX3 in Mtnoot1 and Mtnoot1noot2 nodules suggests an increase of auxin level or sensitivity coupled with a decrease of cytokinin content. This perturbation of the auxin/cytokinin hormonal balance may participate to the nodule to root identity conversion observed in Mtnoot1 and Mtnoot1noot2.Moreover, in addition to the down-regulation of the class II MtKNOX3 and MtKNOX9 gene expressions in Mtnoot1 and Mtnoot1noot2 nodules, a drastic rearrangement of the ProKNOX9:GUS expression pattern was observed in the mutant context. Indeed, while the ProKNOX9:GUS expression pattern in the nodule apical meristematic zone is lost, a re-activation of the MtKNOX9 gene expression occurs in nodule vascular tissue recalling the ProKNOX9:GUS pattern in wild-type root. The shift of the ProKNOX9:GUS expression pattern reflects the homeosis event that occurs in Mtnoot1 and Mtnoot1noot2 and support the function of MtKNOX9 in nodule identity previously proposed for the gene belonging to the MtKNAT3/4/5-like genes sub-class (Di[START_REF] Giacomo | KNAT3/4/5-like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ development[END_REF]. It is tempting to speculate that a pre-existing molecular mechanism involving the class II MtKNOX genes in root has been recruited to build the nodule and define its identity in a MtNOOT1-dependent manner.The present study describes MtNOOT2 as an additional molecular actor involved in the nodule identity. This work provides new insights on the molecular basis of the symbiotic organ identity and on our understanding of how Legumes acquired the nodule organogenesis program during evolution. This probably took place by manipulating the pre-existing root developmental program and gene expression domains. The new Mtnoot mutants described here represent interesting genetic tools that will help in future studies to decipher the molecular mechanisms of the symbiotic root nodule identity.

  , Mtnoot2 (NF5464, this study), and Mtnoot1noot2 double mutants (NF2717 crossed with NF5464, this study), R-108 ProNOOT1:GUS (Couzigou et al., 2012) R-108 ProNOOT2:GUS (this study, see below) and Mtnoot1 ProNOOT2:GUS (R-108 ProNOOT2:GUS transgenic crossed with Mtnoot1

  mutants genotyping. M. truncatula noot1 (TNK507) mutant plants homozygous for a Tnt1 insertion 170 bp after AUG codon on the MtNOOT1 gDNA sequence have been genotyped by semi-quantitative PCR. Four oligonucleotides were used. Amplification of MtNOOT1 without insertion using TNK507_D and TNK507_R give a product of 555 bp. Amplification on MtNOOT1 containing the Tnt1 insertion was carried-out using the oligonucleotides TNK507_D and Tnt1 specific oligonucleotide LTR4. The NOOT1-LTR4 PCR product is 179 bp long. A second amplification on MtNOOT1 containing the Tnt1 insertion was carried-out using the Tnt1 specific oligonucleotides LTR6 and TNK507_R. The NOOT1-LTR6 PCR product is 547bp long. M. truncatula Mtnoot2 (NF5464) mutant plants homozygous for a Tnt1 retroelement insertion 438 bp after AUG codon on the MTNOOT2 gDNA sequence have been genotyped by semiquantitative PCR. Four oligonucleotides were used. The amplification of the MtNOOT2 fragment without insertion using NOOT2geno-D and NOOT2geno-R give a 413 bp long PCR product. Amplification on MtNOOT2 containing the Tnt1 insertion was carried-out using the oligonucleotides NOOT2geno-D and LTR4. The NOOT2-LTR4 PCR product is 313 bp long. A second amplification on MtNOOT2 containing the Tnt1 insertion was carried-out using the oligonucleotides LTR6 and NOOT2geno-R. This NOOT2-LTR6 PCR fragment is 274 bp long.

  analysis. Independent experiments carried-out using plants growing in a sand perlite mixture and inoculated with S. meliloti Rm41 gave similar pattern of expression. ProKNOX9:GUS: The A. rhizogenes-mediated plant transformation of R-108, Mtnoot1, and Mtnoot1noot2 seedlings was performed according to[START_REF] Boisson-Dernier | Agrobacterium rhizogenes -Transformed Roots of Medicago truncatula for the Study of Nitrogen-Fixing and Endomycorrhizal Symbiotic Associations[END_REF] using A. rhizogenes strain AR1193. The ProAtUBIQUITIN:DsRed reporter was used for selection of transformed roots using fluorescence microscopy. For the nodulation assay, transformed plants were transferred to Terragreen:Sharp Sand Mix (1:1) (Oil-DriCompany, Wisbech, UK) and inoculated with Sinorhizobium meliloti strain Sm2011 (1,5 ml of culture diluted in water to OD600 = 0.025/plant) and left to grow for 4 weeks. Plants were grown in control environment rooms at 22 °C (80 % humidity, 16/8 h lightdark cycle and 300 μmol m -2 s -1 light intensity).

  CHAPTER I. Medicago truncatula Mtnodule root1/nodule root2 complete loss of symbiotic organ identity 77 incubate at 4 °C overnight. Once dehydrated by successive ethyl-alcohol bathes, samples undergo three successive Technovit stock solution (3:1, v/v), (1:1, v/v) and (1:3, v/v) bathes and three 1 h 100% Technovit stock solution bathes at 4 °C under agitation. Samples were included in technovit resin using a teflon Histoform S embedding moulds (Heraeus Kulzer). Technovit sections were carried out using a microtome RM 2155 (LEICA) and tungsten disposable blade

  stipules in a same node and by the presence of different stipule forms depending on the node position along the stem. iii) Finally, reproductive phase transition stimuli also considerably influences vegetative lateral organs development and results also in modified sink/source relation.[START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF] described the stipule phenotypes associated to the Pscoch1 mutation in the cultivar Torsdag (Hobart line 107). Stipule phenotypes varied acropetally: first nodes[START_REF] Bourgin | UMR 1318[END_REF][START_REF] Vernié | The NIN transcription factor coordinates diverse nodulation programs in different tissues of the Medicago truncatula root[END_REF](5)

Figure 1 |

 1 Figure 1 | PsCOCH1 and PsCOCH2 gene expression in P. sativum a-b, PsCOCH1 (grey bars) and PsCOCH2 (white bars) qRT-PCR relative gene expression analysis in P. sativum cv. Caméor. a, qRT-PCR gene expression analysis in denodulated roots, in nodules (27 days post inoculation, in internodes and in nodes. b, qRT-PCR gene expression analysis in young leaves (2 first leaves from shoot apical meristem), in developing leaves, in young flowers (sepal and petal at same level), in developing flowers (closed flower before aperture), in young seeds (under 2 mm wide) and in developing seeds (4-5 mm wide). All organs were collected on 35 days old plants inoculated with R. leguminosarum wild-type strain P221 (Laguerre et al., 1992). Developing seeds were collected from 42 days old plants. Gene expressions were normalized against the constitutively expressed PsACTIN andPsBETA-TUBULIN3 genes and against denodulated root organs (a) and on PsACTIN and against young seed organs (b). Results represent means ± SEM of three technical replicates and three biological replicates.

Figure 2 |

 2 Figure 2 | PsCOCH1 and PsCOCH2 gene expression profiles from the P. sativum Gene Expression Atlas. PsCOCH1 and PsCOCH2 gene expression profiles were established using the probeset PsCam036654 and PsCam048389 respectively, using the dataset available from the P. sativum Gene Expression Atlas. Gene expression data from plants starting to flower (stage B) and grown under low nitrate condition (LN) which are similar to our growing conditions have been retain and organized by organs. RPKM, Reads Per Kilobase Million; stage C, 20 days after the start of flowering; stage E, 12 days after pollination; N, grown under nitrate condition; dap, days after pollination.

  CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 94 Supplementary figure S4 | NBCL protein consensus The alignment of 248 NBCL proteins was performed using the Multiple Sequence Alignment online software Clustal Omega (ebi.ac.uk/Tools/msa/clustalo) and the protein sequence consensus was obtained using the multiple alignment software Multalin (http://multalin.toulouse.inra.fr/multalin/). High (black box) and low (grey box) consensus levels was set at 90% and 50%, respectively. Consensus symbols: !, is anyone of IV; $, is anyone of LM; %, is anyone of FY; # is anyone of NDQEBZ. Initially, 485 NBCL sequences were obtained from 1KP project BLAST search (https://db.cngb.org/blast4onekp/) and after removing partial or incomplete sequences, 187 NBCL sequences were retained to establish the NBCL protein consensus. In addition, 61 other full-length NBCL protein sequences from NCBI (https://www.ncbi.nlm.nih.gov/) were used. PsCOCH1 (Ps3092, Ps2702) and PsCOCH2 (Ps1178, Ps3340) KO mutations (asterisks) and missense mutations (triangles) located in conserved amino acid positions are indicated above the NBCL consensus sequence. The BTB/POZ domain and ANKYRIN repeats are underlined by red and blue lines and defined according to Hepworth et al. (2005) and Norberg et al. (2005) respectively. Due to the absence of suitable KO lines for PsCOCH1 from the TILLING-NGS screen in Pscaméor background, an existing Pscoch1 full-length deletion mutant (FN3185/1325) in the background JI2822 (a progeny line from JI399 CENNIA and JI15 WBH1458) genetically close to caméor (Ellis, T.H.N, personal communication; Jing et al., 2010), was chosen afterbackcrossing the mutation in caméor, to construct the double mutant Pscoch1coch2 (Table1).For PsCOCH2 alleles, the non-sens mutations (W122* and Q381*) located at the end of the BTB/POZ domain in the family Ps1178 and just after the fourth ANK domain in the family Ps3340 respectively, were recovered (Supplementary figureS4). Unfortunately, due to other unknown deleterious TILLING mutations present in these lines, both lines were drastically impacted in their development and failed to give suitable seeds. Following direct crossfertilization with wild-type Pscaméor, only the mutation in line Ps1178 was saved and the CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 95

Figure 4 |

 4 Figure 4 | Pscaméor and Pscoch2 Ps1178 homozygous mutant shows wild-type phenotype. a, P. sativum var. caméor shows a wild-type indeterminate nodule phenotype. b, Pscoch2 Ps1178 homozygous mutant shows wild-type phenotype. P. Sativum plants were inoculated with R. leguminosarum strain P221 and nodules were collected at 27 days post inoculation from 35 days old plants. Scale bars a,b: 500 µm.

Table 3 |

 3 CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 97 Segregation analysis of double mutants heterozygous for the independent and recessive PsCOCH1 and the PsCOCH2 locus Nodulation experiments on Pscaméor, Pscoch1, Pscoch2 and Pscoch1coch2 mutants showed that Pscaméor and Pscoch2 have a wild-type phenotype (Fig. 5a,c,e), and that Pscoch1 showed pink hybrid nodule-root structures in 90 % of the nodules as described in Fergusson and Reid, 2005 and in Couzigou and Ratet, 2015 (Fig.5b,e). Pscoch1coch2 showed also hybrid nodule-root structure in similar proportion than Pscoch1 (≈90 %, Fig.5d,e) but these hybrid nodule-root structures remain clearly smaller (three times) with faint pink to white coloration indicating a defect in development and in nitrogen fixation efficiency (Fig.5d). Furthermore big pink converted or not nodules as shown in Fig.5a,b,c, were not observed in Pscoch1coch2.Analysis of the nodule number formed in the different Pscoch mutant lines indicates that

Figure 5 |

 5 Figure 5 | Pscaméor, Pscoch1, Pscoch2 and Pscoch1coch2 nodule phenotypes and nitrogen fixation ability. a-d, Nodule from flowering P. Sativum plants inoculated with R. leguminosarum strain P221 at 35 dpi. a, Pscaméor shows a wild-type pink indeterminate nodule phenotype. b, Pscoch1 shows pink nodules converted to root. c, Pscoch2 shows a wild-type pink indeterminate nodule phenotype. d, Pscoch1coch2 double mutant lines from 4 independent crosses show tiny white or pink nodules that convert to roots. Big pink nodules as shown in a,b,c, are not found in Pscoch1coch2. e, Nodule to root conversion penetrance in the Pscoch mutants relative to wild-type. Results represent mean percentage ± SE of wild-type (pink bars) and converted nodule (red bars). Pscaméor present only wild-type nodule and Pscoch1 and Pscoch1coch2 present 90 % of converted nodules, nodule primordia are not represented. f, Number and phenotype of nodules present in Pscoch mutants relative to wild-type. Pscaméor and Pscoch2 show only wild-type nodules and nodule primordia. Pscoch1 show almost only nodules converted to root with few wild-type nodules and few nodule primordia. Pscoch1coch2 shows an increased number of nodules with twice more converted nodules than Pscoch1 and twice more nodule primordia than Pscaméor or Pscoch2 genotypes. g,h, Nitrogen fixation in Pscoch mutants relative to wild-type, expressed per plant (g) and per nodule (without nodule primordia) (h). Asterisks indicate significant differences compared to the Pscaméor control (** indicate a pvalue < 0,01; Mann and Whitney test). Results in (e,f,g,h) represent means ± SE of one biological experiments containing 4 plants for Pscaméor, Pscoch1, Pscoch2 and 16 plant from 4 independent crosses for Pscoch1coch2 representing 612 Pscaméor nodules, 422 Pscoch1 nodules, 511 Pscoch2 nodule and 5172 Pscoch1coch2 nodules. Scale bars a-d, 500 µm.

Figure 6 |Table 4 :

 64 Figure 6 | Pscoch single and double mutants aerial phenotype a-d. Uncompound leaf phenotypes from nodes 3 to 5 of 22 days old plants in Pscoch mutants relative to wild-type. a, Pscaméor shows wild-type leaves with two proximal leaflets and distal tendrils. b, Pscoch1 mutant shows mainly wild-type leaf phenotype but displays occasionally an additional proximal leaflet. c, Pscoch2 mutant shows wildtype leaves, alteration of the leaf patterning were not observed. d, Pscoch1coch2 double mutant shows mainly wildtype leaf phenotype but displays occasionally strong leaf phenotype consisting in an increase of the leaf complexity, with additional proximal leaflets and with distal parts of the leaf reiterating compound leaves or leaflets. e-h. Compound leaf phenotypes from nodes 6 to 11 of 24 days old plants in Pscoch mutants relative to wild-type. e,f,g. Pscaméor, Pscoch1 and Pscoch2 showing wild-type coumpound leaf phenotypes with at least two pairs of proximal leaflets, a pair of distal tendrils and a terminal distal tendril. h, Pscoch1coch2 double mutant shows wild-type coumpound leaf phenotype but displays occasionally leaf morphology alteration. i-l. Stipule phenotypes of 24 days old plants in Pscoch mutants relative to wild-type. i,k. Pscaméor and Pscoch2 show wild-type peltate stipules. j,l. Pscoch1 and Pscoch1coch2 show a range of stipule phenotypes, such absence of stipule (Ab), thread-like stipules (Th), leaflet-like stipules (Lf) or compound leaf-like stipules (Cl). Scale bars a-l: 1 cm.

Figure 7 |

 7 Figure 7 | Pscoch1coch2 floral defects are increased compared to Pscoch1 single mutant a-d. Global views of Pscaméor wild-type pea (a), Pscoch1 single mutant (b) and Pscoch1coch2 double mutant (c,d) flowers. e-h. Dissected views of Pscaméor wild-type pea (e), Pscoch1 single mutant (f) and Pscoch1coch2 double mutant (g,h) floral organs. a,e. Pscaméor shows closed flowers with two asymetric round stipules at the base of the flowers. Wild-type pea flowers are composed by five sepals fused at their bases, five petals, one adaxial large standard, two lateral wings and two short abaxial keels fused on their adjacent edges. b,f. Pscoch1 shows mostly open flowers with a range of floral patterning defects, such as sepals and petals number alteration, reduced number of floral organs, petals identity defects and a loss of the symmetry. Pscoch1 floral stipules were often modified to a lanceolate leaflet shape or absents. c,g. Pscoch1coch2 double mutant showed similar floral patterning defects to Pscoch1 single mutant, with mostly open flowers. Pscoch1coch2 mutant tends to show increased floral defects relative to Pscoch1. d,h. Pscoch1coch2 showed in addition often fusion between adjacent flowers resulting in flowers with supernumerary sepal and pepal organs with petals adaxially fused. St, standard; w, wing; k, keel; s, sepal; stip, stipule; pe, peduncle. Dotted lines represent fusion between petals. Scale bars a-h: 1 cm.

  compared the Pscoch1coch2 double mutant flowers to the Pscoch1 single mutant flowers. Our observations revealed that Pscoch1coch2 and Pscoch1 display similar floral patterning defects leading to an open floral structure and a reduced self-fertility. Again, because of the strong and highly variable floral phenotypes of the Pscoch1 mutant, it was particularly difficult to distinguish if the Pscoch2 mutation accentuated the Pscoch1 mutant phenotypes. A detailed analysis of the floral defects strength needs to be performed between Pscoch1coch2 and Pscoch1 genotypes, as it was done for the stipules morphology alterations, to clearly understand the role of the PsCOCH2 gene in the floral organization and identity. However, we observed a novel and recurrent floral patterning alteration that has not been observed nor described in the Pscoch1 single mutant. This mutant phenotype consists in the fusion of two flowers. The Pscoch1coch2 flowers result sometimes in the fusion of two adjacent flowers forming complex flowers with supernumerary sepals and petals. These flowers conserved a strong open structure that often leads to sterility. We can thus hypothesis a role for PsCOCH2 in the early separation process occurring between the young pea floral primordia, probably in redundancy with its paralog PsCOCH1.Scanning electron microscopy analysis of Pscoch1coch2 inflorescence meristems will allow us to better understand what happens during the early stages of Pscoch1coch2 floral development and will confirm the involvement of PsCOCH2 in the genetic network controlling the floral primordia establishment and separation. Such role in frontiers establishment between organs is well known and described for the Arabidopsis AtBOP1 and AtBOP2 genes which are the closest orthologs of PsCOCH1 and PsCOCH2 genes. Thus a similar role for the pea NBCL genes in organ boundaries establishment and regulation make sense. The absence of floral defects in the Pscoch2 mutant suggests that this gene is not essential for the floral patterning in pea and that PsCOCH1 can perfectly compensate the PsCOCH2 loss of function, indicating also a partial functional redundancy between the two pea NBCL genes. We can hypothesize that PsCOCH2 has evolved from the more ancient and polyvalent PsCOCH1 gene to fulfill new roles in plant development and/or to create additional regulatory steps to better regulate plant developmental processes such as for example the establishment of the nodule root organogenesis and/or a better regulation of the nodule symbiotic performances. The PsCOCH2 gene belongs to the completely unexplored legume-specific NBLC2 subclade. PsCOCH2 and MtNOOT2 represent now the first characterized members of this legume NBCL2 sub-clade and their corresponding mutant alleles represent original genetic tool that can significantly help to increase our knowledge concerning the role of the NBCL2 genes in legume plants. The phenotypic characterization of the Pscoch1coch2 double mutant showed a clear involvement of PsCOCH2 in the regulation of the symbiotic nodule development and identity. We have shown that PsCOCH2 participates to the regulation of the development and the determinacy of the stipules and leaves and we highlight that PsCOCH2 is likely involved in the regulation of the floral patterning in pea. Thus our study provides novel elements significantly improving our understanding of the roles of the NBCL2 clade. Pea NBCL single and double mutants represent important and interesting genetic tools to study the role of legume NBCL genes and their precise functions in the genetic networks underlying nodules, stipules, leaves and flowers development, patterning and identity determinacy in legumes, as it has been the case for the Atbop1bop2 double mutant in Arabidopsis in the past decade. CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 108 Material and methods P.sativum var. caméor TILLING mutant collection screen. The P. sativum var. caméor ethylmethane sulfonate (EMS) TILLING collection represents 4817 individual M2 families (Dalmais et al., 2008). This collection is available at the legume centre for genetic resources at UMR1347-Dijon Institut National de la Recherche Agronomique and was screened by TILLING-NGS technology for putative point mutations in PsCOCHLEATA1 (JN180861.1) and PsCOCHLEATA2 (KX821778, KX821779, KX821780, KX821781) genes. Oligonucleotides sequences used for both TILLING-NGS screening are given in Supplementary Table S5. The TILLING-NGS screen was conducted at the Unité de Recherche en Génomique Végétale, Institut National de la Recherche Agronomique (URGV, INRA). TILLING NGS strategy. Next generation sequencing (NGS) oligonucleotides design. The 20 mers specific sequence of the NGS oligonucleotides were designed and tailed in 5' position with Illumina adaptators, undesirable hairpin formation, 3' complementarity and potential selfdimerization sites of primers were checked using oligocalc: Oligonucleotide PropertiesCalculator online software[START_REF] Kibbe | OligoCalc: an online oligonucleotide properties calculator[END_REF] www.basic.northwestern.edu/biotools/oligocalc.html).Library construction. The DNA region of interest is first amplified using specific NGS oligonucleotides followed by a second PCR using universal Illumina index oligonucleotide couples UiP-F/UiP-R for specific labeling. For each amplicon, the best annealing temperature is determined for the two-step PCR strategy by gradient PCR optimization using TECHNE ELITE/384 thermal cycler (BIBBY SCIENTIFIC LTD.). The PCR mix contains PCR buffer 1X(Tris-HCl pH : 7,9 10mM, MgCl 2 2,5mM, KCl 50mM, Monidet P-40 0,05%), dNTPs 200µM, forward primer 0,1µM, reverse primer 0,1µM, 0,45µM filtered cresol red 500µM, taq polymerase (Tris-HCl pH: 7,9 2mM, KCl 2mM, EDTA 4µM, DTT 40µM, glycerol 2%, Taq 1/2500), DNA matrix 10ng. Gradient program consists in a 4' initial denaturation at 95°C followed by 35 cycles of three steps of 30''denaturation at 95°C, 30'' annealing from 50°C to 60°C and 1' extension at 72°C, the program is ended by a finale 5' extension at 72°C. The best couples in terms of coverage and PCR efficiency, and the best hybridization temperature were selected. The two-step PCR was performed on the TILLING EMS mutated P. sativum DNA collection pooled in two dimensions (X and Y) according to gradient PCR optimizations. Individual plants are identifiable CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 109 thanks to a dual indexing system. The library is pooled, purified using E-gel® Safe Imager™ Real-time Transilluminator G6500 device and e-gel® size select 2% (ref.G661002), (Invitrogen) according to the manufacturer' recommendation, dried using speed vaccum and finally resuspended in 20µL of sterile water. Library quality controls. The purified library quality was assessed using Agilent 2100 Bioanalyzer® device and Agilent DNA 1000 array Kit (ref. 5067-1504). Library quantification was realized using Quant-it™ PicoGreen® dsDNA Assay Kit (ref. P11496) according to manufacturer's recommandations, using an Infinite® M200 PRO micro plate readers (TECAN TRADING AG) at 480 nm and 520 nm for excitation and emission respectively and analyzed using Magellan™ software. Libraries normalization and sequencing. 2 nM of maximum 35 libraries are pooled together and sequenced with Illumina® Miseq® sequencer (Paired-End sequencing, 2 x 300bp) using the Miseq® Reagent Kit V3, 600 Cycle (ref. #MS-102-3003, Illumina Inc.) following manufacturer's instructions. Pooled libraries finale concentration was adjusted to 8-10 pM and the DNA PhiX control represented 10%.Bioinformatics pipeline. For detection of rare single nucleotide variants in a TILLING NGS population, reads provided by Miseq® sequencer were aligned with BowtieII alignment software set at "very-sensitive" on a consensus sequence which serves as reference sequence. Sequences quality was analyzed using CLCbio, filtered using quality score (Phred score; threshold, 25) to remove false-positives and to reduce background noise derived from PCR and sequencing errors.

  acid position and variation, by the type of mutation (intronic, silent, missense, nonsense or splicing), by the individual plate coordinate and the corresponding mutated family. A SIFT (Sorting Intolerant From Tolerant) analysis predicts the deleterious effect of an amino acid change on the protein based on sequence homologies and amino acid physical properties (SIFT-Tool to predict nonsynonmous / missense variant, sift.bii.a-star.edu.sg). CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 110

  Legume plants forming root-nodules are well-studied for their unique ability to establish nitrogen fixing symbiosis with soil rhizobia. Members of the NODULE-ROOT/BLADE-ON-PETIOLE/COCHLEATA-LIKE (NBCL) gene family belong to the NON-EXPRESSOR OF PATHOGENESIS-RELATED PROTEIN1-LIKE (NPR1-LIKE) family and are key regulators of the symbiotic organ identity[START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] Magne et al., 2017a, unpublished).NPR1-LIKEand NBCL genes encode plant-specific co-transcriptional factors containing Broad Complex, Tramtrack, and Bric-a-Brac/POX virus and Zinc finger (BTB/POZ) domain near the N-terminus and a series of four ankyrin repeats near the Cterminus. All the sequenced land plant genomes including primitive mosses possess NPR1-LIKE and NBCL gene homologs

  MtPERIANTHIA-LIKEPhylogenetic analysis of the ten TGA bZIP TFs of A. thaliana and 13 TGA bZIP TFs of M. truncatula revealed a relative conservation of TGA bZIP TFs across the two species. In M. truncatula, two TGA bZIP TFs group with the class V AtTGA8, also known as AtPAN.The closest AtPAN homolog was named MtPAN-LIKE/TGA8-LIKE1 and the second one MtTGA8-LIKE2 (Fig.1a). Three TGA bZIP TFs group with class I AtTGA1 and AtTGA4 and were named MtTGA1/4-LIKE1, 2 and 3. Two TGA bZIP TFs group with class II AtTGA2, AtTGA5 and AtTGA6 and were named MtTGA2/5/6-LIKE1 and 2. Two TGA bZIP TFs group with class III AtTGA3 and AtTGA7 and were named MtTGA3/7-LIKE1 and 2.Four TGA bZIP TFs group with the class IV AtTGA9 and AtTGA10, two of them were relative to AtTGA9 and were thus called MtTGA9-LIKE1 and 2, and the two others were relative to AtTGA10 and were called MtTGA10-LIKE1 and 2.

Figure 1 |

 1 Figure 1 | Phylogenetic tree of M. truncatula and A. thaliana TGA bZIP TF homologs and gene expression analysis of 13 M. truncatula TGA bZIP TF genes in R-108 nodules. a. Maximum likelihood phylogenetic tree of full-length TGA bZIP TFs from A. thaliana (At) and M. truncatula (Mt, highlighted in blue). A. thaliana and M. truncatula TGA bZIP TFs group into five classes (I-V) according to Gatz (2013).The MtPAN-LIKE/TGA8-LIKE1 protein is highlighted in red. The phylogenetic tree was rooted using a distant bZIP transcription factor of M. truncatula. b. qRT-PCR gene expression of 13 M. truncatula TGA bZIP TF genes, MtTGA1/4-LIKE1, MtTGA1/4-LIKE2, MtTGA1/4-LIKE3, MtTGA2/5/6-LIKE1, MtTGA2/5/6-LIKE2 ,MtTGA3/7-LIKE1, MtTGA3/7-LIKE2, MtPAN-LIKE (MtTGA8-LIKE1, red bar), MtTGA8-LIKE2 ,MtTGA9-LIKE1, MtTGA9-LIKE2, MtTGA10-LIKE1 and MtTGA10-LIKE2 were analyzed in R-108 wild-type nodules at 35 dpi with S. medicae WSM419 strain. The 13 M. truncatula TGA bZIP TF gene expressions were ordered from the higher to the lower gene expression. Gene expression values were normalized against the constitutively expressed MtRRM and MtACT reference genes. Results represent means ± SE of three biological repeats and three technical replicates.

  highly expressed genes after MtTGA1/4-LIKE3, MtTGA2/5/6-LIKE2 and MtTGA1/4-LIKE1 just followed by the MtTGA2/5/6-LIKE1 and MtTGA9-LIKE2 that display similar expression levels. The expression level of the other MtTGA bZIP TF genes decreased slowly from the highest to lowest with MtTGA10-LIKE1, MtTGA1/4-LIKE2, MtTGA10-LIKE2, MtTGA9-LIKE1, MtTGA3/7-LIKE1 and MtTGA3/7-LIKE2, respectively (Fig.1b).The probset Mtr.28107.1.S1_at corresponding to the MtPAN-LIKE gene /mtgea.noble.org/v3/) confirmed that MtPAN-LIKE is expressed in nodule and surprisingly also that this gene is strictly nodule-specific (Fig.2a,b).Benedito et al., 2008, expression data also indicate that MtPAN-LIKE gene is induced during nodulation from 4 to 14 days post-inoculation (dpi, Fig.2b).

Figure 2 |

 2 Figure 2 | MtPAN-LIKE gene expression profile from Medicago truncatula Gene Expression Atlas a-b. MtPAN-LIKE gene expression profile established using 11 specific probes from the Affymetrix Medicago truncatula GeneChip. The MtPAN-LIKE associated probeset Mtr.28107.1.S1_at was used. a. Overview of MtPAN-LIKE gene expression profile using the whole MtGEA dataset available on the Medicago truncatula Gene Expression Atlas (MtGEA). b. MtPAN-LIKE gene expression profile using the Benedito et al., 2008 datasets. Gene expression values are expressed in Affimetrix unit and are organized by organs. Dotted line represent noise background; dap, days post pollination; dpi, days post inoculation.

Figure 3 |

 3 Figure 3 | MtPAN-LIKE gene expression compared to symbiotic marker gene during nodulation MtPAN-LIKE (red bars) qRT-PCR gene expression analysis compared to the nodulation marker genes MtNCR001 (yellow bars), MtNOOT2 (blue bars), MtNIN (purple bars), MtNOOT1 (brown bars) and MtPR10 (green curve) in un-inoculated root apical meristem (0,5 cm, 2 days post vernalization: RAM 0 dpi), in uninoculated primary root devoid of root apical meristem (2 days post vernalization: PR 0 dpi), in inoculated primary root (PR 2, 5 dpi), in nodule primordia (Prim 8 dpi) and in nodules (Nod 12, 16 and 21 dpi) inoculated with Sinorhizobium medicae WSM419 strain. The nodulation experiments have been done in vitro. The relative gene expressions were normalized against the constitutively expressed MtRRM and MtACT genes. Left Y axis represents the expression values for MtNCR001, MtNOOT2, MtNIN, MtPAN-LIKE and MtNOOT1 genes and right Y axis represent the expression values for MtPR10 gene. Results represent means ± SEM of three technical replicates and four biological replicates. RAM, root apical meristem; PR, primary root; Prim, primordia; Nod, nodule; dpi, days post inoculation.

Figure 4 |

 4 Figure 4 | Digital in situ localization analysis of MtNOOT1, MtNOOT2 and MtPAN-LIKE gene expression in nodule a-b. MtNOOT1, MtNOOT2 and MtPAN-LIKE gene expression localization based on digital in situ analyses performed by Roux et al. (2014). a. Values represent mean percentage of normalized reads recovered in each laser-capture microdissection (LCM) fraction from 3 repetitions. b, Representation of the different LCM fractions adapted from Roux et al. (2014). FI, Fraction I, meristematic zone; FIId, Fraction II distal region, differentiation and infection zone; FIIp, Fraction II proximal region, differentiation and infection zone; IZ, Inter-Zone, inter-zone between zone II and zone III; ZIII, Zone III, nitrogen-fixation zone. Total LCM reads (DESeq normalized) for MtNOOT1, MtNOOT2 and MtPAN-LIKE were 602.3, 922.2 and 237.1 respectively.

Figure 5 |

 5 Figure 5 | MtNOOT1, MtNOOT2 and MtPAN-LIKE gene structures and Tnt1 insertions localization a-c. Schematic representation of MtNOOT1, MtNOOT2 and MtPAN-LIKE gene structures and their respective Tnt1 retro-element insertions. a, b. MtNOOT1 and MtNOOT2 encode co-transcriptional factors containing brica-brac tramtrack and broad complex/pox virus and zinc finger (BTB/POZ) and ankyrin repeats protein domains. BTB/POZ domain serves as dimerization interface and ankyrin repeats domains serve as protein-protein interaction interface. M. truncatula Tnt1 insertion in lines Tnk507 and NF5464 are localized in the BTB/POZ domains of MtNOOT1 and MtNOOT2, respectively. c. MtPAN-LIKE encode a TGACG sequence specific Basic Leucin Zipper transcription factor (TGA bZIP TF). MtPAN-LIKE contains 11 exons, the sixth, seventh and heigth exons encode for a coiled-coil motif. The sixth exon encodes a DNA-binbing domain, the seventh and heigth exon encode a dimerization interface. Parts of the nineth and the tenth exons encode a protein domain relative to DELAY OF GERMINATION 1 (DOG1). Two glutamine-rich regions (Q-rich I and Q-rich II) are present in the tenth and in the eleventh exon, respectively. M. truncatula Tnt1 insertions in lines NF12413 and NF10706 are localized in the third exon and in the third intron of MtPAN-LIKE, respectively. Exons and introns are represented by ligth-grey rectangles and black lines, respectively. Positions of the M. trunctula Tnt1 insertions are indicated by triangle.

Figure 6 |

 6 Figure 6 | A single MtPAN-LIKE transcript is expressed in R-108 a. In M. truncatula cv. Jemalong A17, four different MtPAN-LIKE alternative transcripts are expressed, using 3 distinct ATG codons and having one alternative splicing site of 12 bp (black triangle). The M. truncatula A17 Medtr5G054900.2, Medtr5G054900.1, Medtr5G054900.3 and Medtr5G054900.4 alternative transcripts present 11, 10, 8 and 8 exons, respectively. Four oligonucleotide couples PANalt1.1, PANalt2.1, PANalt5 and PANalt6 should amplify 1, 2, 4 and 4 MtPAN-LIKE alternative transcripts, respectively. b. qRT-PCR gene expression analysis of theoric MtPAN-LIKE alternative transcripts in R-108 nodules inoculated with S. medicae WSM419 shows no significant difference in abundance between the four theoric MtPAN-LIKE transcripts (On-way ANOVA, alpha level, 0,01). Results represent means ± SE of 8 biological replicates and two technical replicates from nodule cDNA of 16, 21, 29, 38, 51, 55, 57, 64 dpi . c. Schematic representation of the only MtPAN-LIKE transcript of M. truncatula R-108 presenting 11 exons without splicing deletion. a, c. Exons are represented by successive yellow and green rectangles. Positions of the M. trunctula Tnt1 insertion NF12413 is indicated by white triangles. d. qRT-PCR gene expression analysis were performed in Mtpan-like NF12413 single mutant nodules relative to R-108 wild-type nodules at 35 dpi with S. medicae WSM419 strain. Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against R-108 nodules. Results represent means ± SE of three biological repeats and three technical replicates. Asterisks indicate significant differences compared to R-108 nodule (*** p-value < 0,0001; One-way ANOVA).

Figure 7 |

 7 Figure 7 | Mtpan-like mutants nitrogen fixation efficiency is not affected Acetylene reduction assay does not show significant nitrogen fixation difference in Mtpan-like NF10706 and Mtpan-like NF12413 relative to the R-108 wild-type plant control inoculated with S. medicae WSM419 at 35 dpi (Mann and Whitney test). Results represent means ± SEM of three independent biological experiments containing nine plants each (27 plants analyzed for each genotype). Total number of nodules analyzed for each genotype: R108, 599 nodules; Mtpan-like NF10706, 304 nodules; Mtpan-like NF12413 , 492 nodules.

Figure 8 |

 8 Figure 8 | MtPAN-LIKE gene expression analysis in single and double Mtnoot mutant nodules qRT-PCR gene expression analysis on mature nodules. Wild-type M. truncatula R108 (white bar), Mtnoot1 mutant (light blue bar), Mtnoot2 mutant (blue bar), Mtnoot1noot2 (dark blue bar). Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against expression in R-108 nodules. Results represent means ± SE of two technical replicates and two biological repeats at 29 and 38 dpi with S. medicae WSM419 strain. Asterisks indicate significant difference compared to R-108 nodule (** p-value < 0,001 ; *** p-value < 0,0001; One-way ANOVA).

Figure 9 |

 9 Figure 9 | MtNOOT1, MtNOOT2 and MtPAN self-dimerize and interact together in Yeast-two-hybrids a. Y2H analysis of interactions between MtNOOT1, MtNOOT2 and MtPAN-LIKE proteins. All the proteins were used as bait (left) and as prey (top) resulting in 16 different co-transfomants, interactions work in both sens. Y2H shows that MtNOOT1, MtNOOT2 and MtPAN-LIKE are able to form homodimers, MtNOOT1 and MtNOOT2 form hetero-dimers and MtNOOT1 and MtNOOT2 interact both with MtPAN-LIKE. The experiment have been done twice with 2 independent co-transformants for each of the 16 constructs (32 indedendent cotransformants). Co-transformed yeast were dilluted by 100 and grown 6 days on Minimal SD -L-W-H-A, supplemented with 3-AT, 1 mM. b. Beta-galactosidase activities of the 16 constructions were tested and quantitatively measured using o-nitrophenyl-beta-galactopranoside as substrate. Protein/protein interactions tested are represented by blue bars and negative control interactions are represented by white bars.

Figure 10 |

 10 Figure 10 | Subcellular localization of MtNOOT1:GFP, MtNOOT2:GFP and MtPAN-LIKE:GFP fusion proteins. a-g. Confocal images of GFP fluorescence in infiltrated tobacco leaf epidermal cells, images from left to right indicate GFP signal, chlorophyll A signal, white light and merged images, respectively. a. p35S:GFP fusion served as positive control. b,c,d. p35S:NOOT1, p35S:NOOT2, p35S:PAN-LIKE fusions served as negative control. e,f,g. p35S:NOOT1:GFP, p35S:NOOT2:GFP, p35S:PAN-LIKE:GFP fusions subcellular localization. a-g. Constructions were co-inlitrated with HcPRO silencing suppressor. n, nucleus. Bars, 20 µm.

Figure 11 |

 11 Figure 11 | Subcellular delocalization of MtNOOT1:GFP, MtNOOT2:GFP and MtPAN-LIKE:GFP fusion proteins. a-d. Confocal images of GFP fluorescence in infiltrated tobacco leaf epidermal cells, images from left to right indicate GFP signal, chlorophyll A signal, white light and merged images, respectively. a. p35S:NOOT1:GFP fusion subcellular delocalization using p35S:PAN-LIKE. b. p35S:NOOT2:GFP fusion subcellular delocalization using p35S:PAN-LIKE. c. p35S:PAN-LIKE:GFP fusion subcellular delocalization using p35S:NOOT1. d. p35S:PAN-LIKE:GFP fusion subcellular delocalization using p35S:NOOT2. a-d. Constructions were co-inlitrated with HcPro silencing suppressor. n, nucleus. Bars, 20 µm.

  Mtnoot1, Mtnoot2, Mtpan-like single mutants, Mtnoot1pan-like, Mtnoot2pan-like, Mtnoot1noot2 double mutants and R-108 have been characterized for their nodules phenotype 30 days post-inoculation with S. medicae WSM419. R-108, Mtnoot2, Mtpan-like and Mtnoot2pan-like mutants showed only wild-type uni-lobed nodules (≈80 %) and multilobed (≈20 %) nodules in similar proportion (Fig.12a). Mtnoot1 and Mtnoot1pan-like showed nodules converted into roots in similar proportion (≈50 % and ≈40 %, respectively, CHAPTER III. The key regulators of the symbiotic identity MtNODULEROOT1 and MtNODULEROOT2 interact with the TGA bZIP transcriptional factor MtPERIANTHIA-LIKE 134

Figure 12 |

 12 Figure 12 | Mtnoot, Mtpan-like single and double mutant nodule phenotypes and nitrogen fixation efficiencies. a. The proportion of unilobed nodules (ligth green bars), multilobed nodules (dark green bars) and converted nodules (grey bars) was assessed in Mtnoot1, Mtnoot2, Mtpan-like single mutants and in Mtnoot1pan-like, Mtnoot2pan-like and Mtnoot1noot2 double mutants relative to R-108 wild-type plant control. b. The nitrogen fixation efficiencies of these mutant nodule populations were assessed using acetylene reduction assay relative to R-108 wild-type plant control. All mutants were fix + excepted the Mtnoot1noot2 double mutant which was fix -. Statistical analysis was perfomed using a Mann and Whitney test (* p-value < 0,01 ; *** p-value < 0,0001). a,b. Results represent means ± SEM of 3 biological replicates, representing 27 plants for R-108 and Mtnoot1noot2, and 54 plants for Mtnoot1, Mtnoot2, Mtpan-like, Mtnoot1pan-like and Mtnoot2pan-like at 30 dpi with S. medicae WSM419. Total number of nodules analyzed for each genotype: R-108, 342; Mtnoot1, 589;Mtnoot2, 448; 505; 548; 505; Mtnoot1noot2, 336.

  showing nodule to root conversions, the expression level of symbiotic gene markers were slightly reduced compared to the level observed in Mtnoot2, Mtpan-like and Mtnoot2pan-like mutants that showed no symbiotic phenotype however except for Mtnoot1 that show a significant reduction relative to wild-type, expression in Mtnoot1pan-like remains similar to wild-type. MtPR10, MtNON-DISEASE RESISTANCE 1-LIKE (MtNDR1-LIKE) and MtPHENYLALANINE AMMONIA LYASE 2 (MtPAL2) defense gene marker expression analysis showed that only Mtnoot1, Mtnoot1pan-like and Mtnoot1noot2 mutants present upregulation of defense marker genes relative to R-108 (Fig. 13b). Similarly, MtWUSHEL-LIKE HOMEOBOX 5 (MtWOX5), MtCRINKLY4 (MtACR4), MtSHORT ROOT (MtSHR) and MtPLETHORA2-LIKE (MtPLT2-LIKE) root identity marker gene expression were induced in all lines showing nodule to root conversions (Mtnoot1, Mtnoot1pan-like and Mtnoot1noot2 mutants; Fig. 13c). Taken together, nodule phenotype analysis, ARA and qRT-PCR gene expression analysis of symbiotic, defense and root identity marker genes, indicate that the Mtpan-like mutation alone or in association with Mtnoot mutations does not affect symbiosis (Fig. 12,13).

Figure 13 |

 13 Figure 13 | qRT-PCR gene expression analysis of symbiotic, defense and root identity marker genes in Mtnoot and Mtpan-like single and double mutant nodules. a,b,c. qRT-PCR gene expression analysis were performed in Mtnoot1, Mtnoot2, Mtpan-like single mutants and in Mtnoot1pan-like, Mtnoot2pan-like and Mtnoot1noot2 double mutant nodules relative to R-108 wild-type nodules at 35 dpi with S. medicae WSM419 strain. a. qRT-PCR gene expression analysis of the symbiotic marker genes MtNIN (blue bars), MtNCR001 (red bars) and MtLEGH1 (green bars). b. qRT-PCR gene expression analysis of the defense marker genes MtPR10 (blue bars), MtNDR1-LIKE (red bars) and MtPAL2 (green bars). c. qRT-PCR gene expression analysis of the root identity marker genes MtWOX5 (blue bars), MtACR4 (red bars), MtSHR (green bars) and MtPLT2-LIKE (purple bars). a,b,c. Gene expression data represent relative expression normalized against the constitutively expressed MtRRM and MtACT reference genes and against R-108 nodules. Results represent means ± SE of three biological repeats and three technical replicates. Asterisks indicate significant differences compared to R-108 nodule (* p-value < 0,01; ** p-value < 0,001 ; *** p-value < 0,0001; One-way ANOVA).

  /4-LIKE1, MtTGA1/4-LIKE2, MtTGA9-LIKE2, MtTGA10-LIKE1 and MtTGA10-LIKE2 are not significantly affected in the mutant background tested (Fig. 14). No over-expression of other TGA bZIP TF genes can explain the absence of symbiotic phenotype of Mtpan-like mutant, however one of the TGA bZIP TFs studied here can still be functionally redundant with MtPAN-LIKE gene.

Figure 14 |

 14 Figure 14 | qRT-PCR gene expression analysis of M. truncatula TGA bZIP TF genes in R-108, Mtnoot1, Mtnoot2 and Mtpan-like backgrounds qRT-PCR gene expressions of 13 M. truncatula TGA bZIP TF genes, MtPAN-LIKE, MtTGA8-LIKE 2, MtTGA2/5/6-LIKE1, MtTGA2/5/6-LIKE2 , MtTGA3/7-LIKE1, MtTGA3/7-LIKE2, MtTGA1/4-LIKE1, MtTGA1/4-LIKE2, MtTGA1/4-LIKE3, MtTGA9-LIKE1, MtTGA9-LIKE2, MtTGA10-LIKE1 and MtTGA10-LIKE2 were analyzed in Mtnoot1 (red bars), Mtnoot2 (green bars) and Mtpan-like (dark blue bars) single mutant nodules relative to R-108 wild-type (blue bars) nodules at 35 dpi with S. medicae WSM419 strain. The gene expression data were normalized against the constitutively expressed MtRRM and MtACT reference genes. Results represent means ± SE of three biological repeats and three technical replicates. Asterisks indicate significant differences compared to R-108 nodule (* p-value < 0,01; ** p-value < 0,001 ; *** p-value < 0,0001; One-way ANOVA).

Figure 15 |

 15 Figure 15 | Theoritical model of the NOOT/PAN-LIKE interactions in the noduleLongitudinal schematic representation of the half apical part of a M. truncatula indeterminate nodule. The nodule vascular bundle is represented in blue in which the proNOOT1:GUS gene expression is mostly associated to the tip of the nodule vascular bundle represented by the blue color accentuation. The nodule central meristem is represented in red and corresponds to the site of the expression of the proNOOT2:GUS. The region between the nodule vascular meristem (NVM) and the nodule central meristem (NCM) is called the NVM/NCM boundary region. These tissues are located between the outer nodule cortex and the inner apical infection zone. Based on their respective sites of expression and on the different protein/protein interactions previously described, the hypthothetic localization of the proteins MtNOOT1 MtNOOT2 and MtPAN-LIKE in the nodule are represented by blue, red and green ovals, respectively. This scheme represents the hypothetic sites of the different interactions that can occur between these tree protein partners. MtPAN-LIKE localizes mostly in the apical part of the nodule but its exact localization is unknown (question mark), we hypothesized that MtPAN-LIKE proteins are present where the NOOT proteins are, in order to interact with. A potential tripartite interaction between MtNOOT1, MtNOOT2 and MtPAN-LIKE (dotted green ovale) may occur at the NVM/NCM boundary region, the only region in the nodule where both MtNOOT proteins can meet.

  nodules (http://bios.dijon.inra.fr/FATAL/cgi/;Alves-Carvalho el al., 2015). The strong expressions of these three genes, MtTGA1/4-LIKE3, PsCam014568 and bZIP-M43, in M. truncatula, P. sativum and L. japonicus nodules, respectively, suggest an important role for these TGA bZIP TFs during nodulation which is supported by their conservation across different legume species forming different type of nodules. In Mtpan-like mutant, the drastic down-regulation of MtPAN-LIKE and MtTGA1/4-LIKE3 gene expressions did not provoke the loss of nodule identity and the outgrowth of ectopic root on the apical part of the nodule characteristic of the Mtnoot1 mutation. It nethertheless could be interesting to analyze the effect of a complete extinction of the MtTGA1/4-LIKE3 gene on the nodule formation and identity.Despite that the Mtpan-like mutant does not display loss of nodule identity, this study allowed identifying MtPAN-LIKE as a first protein partner able to interact with both MtNOOT proteins in the M. truncatula indeterminate nodule. We thus provide new elements to better understand the completely undescribed regulatory mechanisms involving MtNOOT1 and MtNOOT2 in the nodule identity. We also provide evidences, that in agreement with the existence of the NPR1/TGA and the BOP/TGA protein/protein interaction modules, a conserved regulatory mechanism involving NBCL/TGA bZIP TF module have been recruited for the Medicago-Sinorhizobium symbiotic interaction. The discovery of the direct target(s) of MtPAN-LIKE and the characterization of the corresponding mutant(s) might provide a more complete comprehension of the role of this module in the regulation of the nodule formation and identity.

II

  Reverse Transcriptase kit (Invitrogen) in presence of Ribolock RNase Inhibitor (Thermoscientific). Real-time RT-PCR reactions were performed using the LightCycler FastStart DNA Master SYBR Green I kit on a Light Cycler 480 II device according to manufacturer's instructions (Roche). Cycling conditions were as follows: 1 pre-incubation cycle (95°C, 10 min), 40 amplification cycles [(denaturation: 95 °C, 10 s), (hybridization: 60 CHAPTER III. The key regulators of the symbiotic identity MtNODULEROOT1 and MtNODULEROOT2 interact with the TGA bZIP transcriptional factor MtPERIANTHIA-LIKE 148 °C, 15 s), (elongation: 72 °C, 15 s)], 1 melting curve cycle [(denaturation: 95 °C, 15 s), (hybridization: 55 °C, 1 min), (denaturation: 95 °C)], 1 cooling cycle (40 °C, 30 s). Cycle threshold and primer specificities were performed with the LightCycler 480 software release 1.5.0 SP4. Primer efficiencies were calculated with LinReg PCR: Analysis of Real-Time PCR Data, version 11.1. For gene expression normalization, MtACTIN11 and MtRNA RECOGNITION MOTIF were used as reference genes. Reference gene ID, oligonucleotides

  Arabidopsis thaliana (A. thaliana) AtBLADE-ON-PETIOLE1 and AtBLADE-ON-PETIOLE2 (AtBOP1, AtBOP2), to Medicago truncatula (M. truncatula) MtNODULE-ROOT1 (MtNOOT1), to Pisum sativum (P. sativum) PsCOCHLEATA (PsCOCH1) and to Hordeum vulgare (H. vulgare) Uniculme4 (Cul4) and Laxatum-a (Lax-a). Changes in flower development in known nbcl mutants results in supernumerary organs and homeosis of some reproductive organs.

Figure 1 |

 1 Figure 1 | LjNBCL1 gene structure, Ljnbcl1:LORE1 insertions and Ljnbcl1:RNAi probe position and Ljnbcl1:RNAi efficiency a, Scheme representing the LjNBCL1 gene that encodes a bric-a-brac tramtrack and broad complex/pox virus and zinc finger (BTB/POZ) ankyrin protein. Exons are represented by light-grey rectangles. Position of the L. japonicus nbcl1 mutations (LORE1 insertions) are indicated by triangles and the position of the RNAi sequence (263 bp) used for the RNAi approach is indicated by double inverted arrowheads. b, Ljnbcl1:RNAi efficiency was assessed by measuring the LjNBCL1 transcript accumulation by qRT-PCR in 35 dpi nodules inoculated with M. loti strain NZP-2235 coming from hairy-root transformed Lotus japonicus GIFU plants expressing GUS:RNAi or Ljnbcl1:RNAi constructions. Asterisks indicate the significant difference in Ljnbcl1:RNAi transformed nodules compared to the GUS:RNAi transformed nodules control, ** indicates a p-value < 0.01 (one-way ANOVA test). LjNBCL1 gene expression was normalized against the constitutively expressed LjPP2A and LjUBC genes and against the control GUS:RNAi transformed nodules. Results represent means ± SEM of three technical replicates and three biological replicates.

Figure 2 |

 2 Figure 2 | LjNBCL1 gene expression in L. japonicus GIFU aerial part and associated Ljnbcl1 mutant aerial phenotypes a, LjNBCL1 (grey bars) qRT-PCR relative gene expression analysis in wild-type L. japonicus GIFU aerial vegetative organs. Gene expression analysis was performed in inter nodes, nodes containing nectary glands, leaves, flowers at stages 10, 11, 12 and 13 according to floral stages described in Weng et al., 2011 and in developing pods (≈ 1 cm, without petals and stamen). LjNBCL1 gene expression was normalized against the constitutively expressed LjPP2A and LjUBC genes and against expression in leaf organs. Results represent means ± SEM of three technical replicates and three biological replicates. Samples for repeat 1 were collected from 80 days old plant, repeat 2 and 3 samples were collected from 89 days old plants and pods were sampled from 93 days old plants. b, c, d, The Ljnbcl1 mutant presents a supplementary distal leaflet (c, black asterisks) compared to L. japonicus GIFU (b). The penetrance of this phenotype was assessed and 13 % of Ljnbcl1 mutant leaves are hexa-foliated compared to wild-type penta-foliated leaves (d). Results represent percentage means ± SE. L. japonicus GIFU n: 6 plants, 1418 leaves; Ljnbcl1:LORE1 mutant n: 8 plants, 1893 leaves. e, f, Ljnbcl1 mutant presents supplementary axillaries at the leaf axil (f, yellow asterisks) compared to L. japonicus GIFU (e). g, h, in L. japonicus GIFU nectary glands are present at the leaf axil (g, white asterisks), in Ljnbcl1:LORE1 mutant nectary glands are absent (h). i, j, Scanning electron micrograph (SEM) at leaf axil reveals that no trace of nectary gland formation can be detected in the Ljnbcl1:LORE1 mutant (j) compared to L. japonicus GIFU (i , white asterisks). White double headed arrows indicate longer petiole in Ljnbcl1:LORE1 mutant (j) relative to L. japonicus GIFU (i). Scale bars: b, c, e, f, 1 cm; g, h, 200 µm; i, j, 100 µm.

Figure 3 |

 3 Figure 3 | LjNBCL1 gene expression is induced together with symbiotic marker gene expression during nodule development a, LjNBCL1 (grey bars) qRT-PCR relative gene expression analysis. b, LjNBCL1 (grey diamonds) qRT-PCR relative gene expression analysis compared to LjNIN (blue diamonds), LjENOD40-1 (green triangles), LjIPT3 (purple crosses), LjPLT2-LIKE (orange dotted circles) qRT-PCR relative gene expression. a-b, qRT-PCR gene expression analysis in Lotus japonicus GIFU non-inoculated RAM (0,5 cm, 5 days post vernalization: RAM 0 dpi), noninoculated primary roots devoid of RAM (5 days post vernalization: primary root 0 dpi), inoculated primary roots devoid of RAM (2 dpi), inoculated primary roots devoid of RAM with visible nodule primordia (5 dpi) and nodules at 8, 12, 16 and 20 dpi. Plants were inoculated with M. loti strain NZP-2235 and 16 plants were used for each point. Gene expressions were normalized against the constitutively expressed LjSERINE/THREONINE-PROTEIN PHOSPHATASE 2A (LjPP2A) and LjUBIQUITIN-CONJUGATING ENZYME (LjUBC) genes and against noninoculated primary root at 0 dpi. Left Y axis represents the relative gene expression values for LjNBCL1, LjENOD40-1, LjIPT3 and LjPLT2-LIKE genes and right Y axis represents the relative expression values for LjNIN gene. Results represent means ± SEM of three technical replicates and three biological replicates.

Figure 4 |

 4 Figure 4 | Ljnbcl1:LORE1 and Ljnbcl1:RNAi show nodule to root conversion phenotype with low penetrance a, a Wild-type L. japonicus GIFU nodule. b, a Ljnbcl1:LORE1 mutant nodule showing nodule to root identity conversion. c, L. japonicus GIFU transformed by hairy root expressing GUS:RNAi construction showing a wild-type nodule phenotype. d, L. japonicus GIFU transformed by hairy root expressing Ljnbcl1:RNAi showing nodule to root identity conversion. Nodules were obtained following inoculation with M. loti strain NZP-2235. e, Penetrance of the nodule to root identity conversion in Ljnbcl1:LORE1 insertional mutant and in composite plants transformed by hairy root expressing Ljnbcl1:RNAi compared to wild-type L. japonicus GIFU and L. japonicus GIFU plant transformed by hairy root expressing the GUS:RNAi construct. Ljnbcl1:LORE1 insertional mutant and Ljnbcl1:RNAi transformed plant nodules presented 4 % and 5 % of nodules converted to root (dark grey bars), respectively. L. japonicus GIFU and GUS:RNAi transformed nodules showed only wild-type nodules (light grey bars). Results represent percentage means ± SE, error bars for wild-type nodules and nodule to root conversions are indicated in light blue and dark blue, respectively. L. japonicus GIFU nodules (115 dpi), n: 1256; Ljnbcl1:LORE1 insertional mutant nodules (115 dpi), n: 1868; GUS:RNAi transformed plant nodules (35 dpi), n: 1307; Ljnbcl1:RNAi transformed plant nodules (35 dpi), n: 527. Scale bars a-d: 500 µm.

Figure 5 |

 5 Figure 5 | Ljnbcl1 determinate nodule vasculatures connect to ectopic root vasculature and nodule vascular bundle identity is lost a-c, Thin longitudinal sections and d-i, transversal sections of GUS:RNAi transformed nodules (a, d, g), Ljnbcl1:RNAi transformed nodules (b, e, h) and Ljnbcl1:LORE1 insertional mutant nodules (c, f, i) inoculated with M. loti strain NZP-2235 and stained with toluidine blue. a, d, GUS:RNAi transformed nodules display a globular shape typical of wild-type desmodioïd nodules. Peripheral vascular bundles are closed to the rhizobia infected zone. b, c, Ljnbcl1:RNAi transformed nodules and Ljnbcl1:LORE1 insertional mutant nodules show ectopic root emerging from the apical part of the nodule. The vasculatures of these ectopic roots are connected to those of the desmodioïd nodules. e, f, Ljnbcl1:RNAi transformed plant nodules and Ljnbcl1:LORE1 insertional mutant nodules show NVB surrounded by root cortex cell layers that are completely dissociated from the nodule infected zone when compared to those in control GUS:RNAi transformed plant nodules (d). g, h, i, Thin transversal sections of nodule showing vascular bundles. Left-lower and right-upper corners are respectively oriented toward the interior and exterior of the nodule. g, L. japonicus GIFU vasculature localize close to the rhizobia infected cells. Wild-type L. japonicus GIFU vascular bundles are composed of an external vascular endodermis cell layer, pericycle cell layers and the bundle containing xylem tissues and parenchyma cells, and phloem tissues composed of sieve elements, companion and parenchyma cells. The bundle organization is collateral with phloem tissues turned toward the exterior of the nodule and the xylem turned toward the infection zone. h, i, Ljnbcl1:RNAi transformed plant and Ljnbcl1:LORE1 mutant

Figure 6 |

 6 Figure 6 | Ljnbcl1 mutation dramatically affect flower development a, 12 weeks old L. japonicus GIFU wild-type flowering plant. b, 12 weeks old Ljnbcl1:LORE1 mutant without normal flowers. c, Wild-type L. japonicus GIFU flowers present 1 to 3 bracts, a pedicel, inflorescence nectary glands at the base of the pedicel, a pedicel, a receptacle, 5 fused sepals, 5 petals, represented by 1 adaxial standard, 2 lateral wings and 2 abaxial fused keels, 10 stamens and a single carpel (Zhang et al., 2003). d, Ljnbcl1:LORE1 mutant flower presents bracts but instead of floral organs, a unique central trumpet-like organ forms (white arrowhead). In addition, a supplementary leaf-like structure forms at the central organ axil (yellow asterisks). e, f, Magnification of the base of the pedicel region showing an inflorescence nectary in L. japonicus GIFU (e) and the leaf-like structure that develops instead of the nectary in Ljnbcl1:LORE1 mutant (f, yellow asterisks). Flowers in pictures c, d, e and f, were collected from166 days old plants. g-h, Wild-type L. japonicus GIFU floral diagram (according to Dong et al., 2005) (g) and Ljnbcl1:LORE1 mutant floral diagram representing the most complex organ combination that can be found in the mutant, four bracts, two leaf-like structures that develop instead of nectary glands (yellow asterisks) and one central structure (light grey circle, see bellow fig. 7 for central structure) (h). Scale bars: a, b, 5 cm; c, d, 1 mm; e, f, 500 µm. st, stem; in, inflorescence nectary glands; b, bract; pe, pedicel; re, receptacle; s, sepal; sta, standard; w, wing; k, keel; s, stamen; ca, carpel. Black arcs in g represent fused organs.

Figure 7 |

 7 Figure 7 | Gradual Ljnbcl1 mutant flower phenotypes The Ljnbcl1:LORE1 mutants show various floral phenotypes. The flowers can be absent (a, 24 %), replaced by a prematurely aborted fused trumpet-like structure (b, 6 %) or by a more developed fused trumpet-like structure (c, 11 %). Ljnbcl1:LORE1 flowers can be replaced by leaf-like structure (d, 38 %) or by a more complex leaf-like structure showing organ separations (e, 11 %). Finally, some Ljnbcl1:LORE1 plants show prematurely aborted floral-like structure (f, 7 %) or fully-developed but sterile flowers (g, 3 %). Percentages represent the analysis of 1953 flowers from three individual Ljnbcl1:LORE1 plants (166 days). Scale bars: 1 mm.

Figure 8 |

 8 Figure 8 | Ljnbcl1:LORE1 mutation severely affects early development of the L. japonicus GIFU inflorescence apex a-f, Scanning Electron Microscopy of developing L. japonicus GIFU (a-c) and Ljnbcl1:LORE1 (d-f) inflorescence apex. a-c, The L. japonicus GIFU primary inflorescence meristem (I1, blue) produces secondary inflorescence meristems (I2, yellow) at the axil of a compound leaf (L, green). Floral primordia (F, red) are partitioned from the degenerating I2 peripheral regions (I 2 *). Trichome lines mark the formation of floral meristems, as the I2 finally differentiates and become covered by trichomes (I 2 * -see panel b). d-f, In the Ljnbcl1:LORE1 mutant inflorescence

  vegetative development and bloom, a loam/peat/sand mixture was used (70/3,5/7, v/v/v; http://www.puteaux-sa.fr) with expensed clay balls at the bottom of the pot. L. japonicus were grown under 16/8h light-dark cycle, 23/23°C day-night temperature, 60% of relative humidity and 200 µE of light intensity. Plants were watered three times a week (2 times with water / 1 times with nutritive solution in alternance) with N-free nutritive solution (Plant prod, NPK 0-15-40) for nodulation or with N+ nutritive solution (Soluplant, NPK16 6 26) for uninodulated plant.

  SuperScript™ II Reverse Transcriptase kit (Invitrogen) in presence of Ribolock RNase Inhibitor (Thermoscientific). qRT-PCR reactions were performed using the LightCycler FastStart DNA Master SYBR Green I kit on a Light Cycler 480 II device according to manufacturer's instructions (Roche). Cycling conditions were as follows: 1 pre-incubation cycle (95°C, 10min), 40 amplification cycles [(denaturation: 95 °C, 10 s), (hybridization: 60 °C, 15 s), (elongation: 72 °C, 15 s)], 1 melting curve cycle [(denaturation: 95 °C, 15 s), (hybridization: 55 °C, 1 min), (denaturation: 95 °C)], 1 cooling cycle (40 °C, 30 s). Cycle threshold and primer specificity analysis were performed using the LightCycler 480 software release 1.5.0 SP4. Primer efficiencies were calculated with LinReg PCR: Analysis of Real-Time PCR Data, version 11.1.

  hormones signals especially to the jasmonic acid, ethylene and abscissic acid phytohormone signals that promote the abscission signal. In Arabidopsis thaliana floral AZ, following hormonal signalization, the activation of cells separation is carried-out throughout the recognition between INFLORESCENSE DEFICIENT IN ABSCISSION (IDA) peptide and

  LIKE (NBCL) protein sequences from dicotyledon plants and from grasses. The phylogenic tree shows that two to three NBCL proteins are present in most of the plant across grasses and dicots. The B. distachyon BdNPR5-LIKE is the closest ortholog of the barley HvCUL4. These two proteins group with other grass NPR5-LIKE proteins that are present in only one copy in the grasses used in this study. A second NBCL protein of B. distachyon, BdNPR6-LIKE is the closest ortholog of the barley HvLAX-A. These two proteins belong to a second group of protein which contains NPR6-LIKE related proteins. These NPR6-LIKE proteins are found in one copy in Hordeum vulgare, B. distachyon and Aegilops tauschii (Pooideae), but in the other grass genius such Oryza (Ehrhartoideae), Triticum (Pooideae), Setaria, Panicum (Paniceae), Sorghum and Zea (Andropogoneae), two NPR6-LIKE copies were found. The maximum likelihood-based phylogenic tree indicates that the grass NPR5-LIKE protein and the dicot NBCL proteins are closely related while the grass NPR6-LIKE proteins appear more distant. By contrast to the NPR5-LIKE protein group in which only one copy was retained, it appears that during evolution grasses have specifically acquired the NPR6-LIKE genes which have later undergo more recent duplications similarly to the dicots NBCL genes (Fig. 1).

Figure 1 .

 1 Figure 1. Maximum likelihood-based phylogenic tree of monocot and dicot NOOT-BOP-COCH-LIKE (NBCL) proteins Maximum likelihood-based phylogenetic analysis of 45 NBCL proteins. The monocot NBCLs are from B. distachyon (BdNPR5-LIKE, BdNPR6-LIKE), Hordeum vulgare (HvLAX-A, HvCUL4), Oryza sativa japonica (OsjNPR5-LIKE, OsjNPR6-LIKEa, OsjNPR6-LIKEb), Oryza sativa indica (OsiNPR5-LIKE, OsiNPR6-LIKEa, OsiNPR6-LIKEb), Panicum hallii (PhNPR5-LIKE, PhNPR6-LIKEa, PhNPR6-LIKEb), Setaria italica (SiNPR5-LIKE, SiNPR6-LIKEa, SiNPR6-LIKEb), Sorghum bicolor (SbNPR5-LIKE, SbNPR6-LIKEa, SbNPR6-LIKEb), Zea mays (ZmNPR5-LIKE, ZmNPR6-LIKEa, ZmNPR6-LIKEb), Aegilops tauschii (AetNPR5-LIKE, AetNPR6-LIKE), Triticum aestivum (TaNPR5-LIKE, TaNPR6-LIKEa, TaNPR6-LIKEb) and the dicot NBCLs are from Medicago truncatula (MtNOOT1, MtNOOT2), Cicer arietinum (CaNPR5-LIKEa, CaNPR5-LIKEb), Populus trichocarpa (PtBPL1, PtBPL2), Solanum tuberosum (StBOPa, StBOPb, StBOPc), Solanum lycopersicum (SlBOPa, SlBOPb, SlBOPc), Nicotiana tabacum (NtBOP1, NtBOP2, NtBOP3, NtBOP4) and Arabidopsis thaliana (AtBOP1, AtBOP2). The maximum-likelihood tree was rooted using AtNPR1. The Hordeum vulgare and B. distachyon NBCL proteins are highlighted in blue and red respectively.

Figure 2 .

 2 Figure 2. BdNPR5-LIKE and BdNPR6-LIKE gene expression pattern in Bd21-3 wild-type plant The BdNPR5-LIKE (white bars) and the BdNPR6-LIKE (grey bars) transcripts accumulation were measured on 40 days-old wild-type Bd21-3 plants. Gene expression analysis was performed in root, sheath, node, ligular region, leaf blade, rachis node, rachis internode and spikelet (1-1,5 cm length). The BdNPR5-LIKE and BdNPR6-LIKE gene expressions were normalized against the constitutively expressed BdPOLYUBIQUITIN 4 and BdUBIQUITIN-CONJUGATING ENZYME E2 18 reference genes. Results represent means ± SEM of three biological repeats and three technical replicates.

Figure 3 .

 3 Figure 3. Bdnpr5-like and Bdnpr6-like allelic series studied and schematic representation of mutations on the proteins (A) Table summarizing the BdNPR5-LIKE and BdNPR6-LIKE TILLING point mutations studied in this study. a Position of the mutation in mutants from the starting ATG of the coding sequence. b Position of the amino acid substitution in mutants from the starting methionine of the encoded protein. c Values represent predictive deleterious score from the SIFT software. SIFT score ranges from 0 to 1. A score ≤ 0,05 predicts a deleterious effect on protein (http://sift.bii.a-star.edu.sg/). (B) Schematic representation of the BdNPR5-LIKE and BdNPR6-LIKE proteins and position of the amino acids substitutions in the different mutants.

Bd5998

  and Bd3615 resulted in a mutant phenotypes (especially a glaring short-spikelets phenotype, see below). Self-fertilized progeny of line Bd5998 heterozygous for the Bdnpr6like mutation segregated as a monogenic recessive trait with 15 homozygous wild-type plants and 30 heterozygous plants showing wild-type spikelets, and 17 plants homozygous for the Bdnpr6-like locus showing short-spikelet phenotype (Table2). The mutation in family Bd5998 is located in the third ankyrin repeat and corresponds to the replacement of a highly conserved leucine (non-polar) into a phenylalanine (aromatic/hydrophobic) at position 365 (Fig.3 B; Sup. Fig.1). The mutation in family Bd3615 is located in the fourth ankyrin repeat and correspond to the replacement of a highly conserved threonine (polar, uncharged) into an isoleucine (non-polar) at position 381 (Fig.3 B; Sup. Fig.1). The leucine in family Bd5998 and the threonine in family Bd3615 are conserved as far as ancestral land plant genera such in Osmunda, Cyathea, Equisetum, Selaginella, Isoetes, Diphyscium, Andreaea and Megaceros. Mutations in family Bd4087, Bd8706 and Bd4188 did not impact conserved amino-acids and mutant phenotypes were not observed in the corresponding mutants. In family Bd7392, the replacement of an alanine (non-polar) by a threonine (uncharged polar)

Figure 4 .

 4 Figure 4. Bdnpr5-like knock-out lines Bd4982 and Bd7965 present a tillering defect Tillering phenotype of wild-type Bd21-3 (A), Bdnpr5-like Bd4982 (B) and Bdnpr5-like Bd7965 (C) 16 weeks after sowing. Wild-type Bd21-3 plants present several secondary tillers at the base of the plants (A), while in Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965 plants (B, C) few secondary tillers are visible. Numbers of primary (D) and secondary (E) tillers by plant in wild-type B. distachyon, Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965 dry mature plants. Error bars represent standard deviation. Asterisks indicate significant differences (P < 0.001) relative to the wild-type background (Student's t-test). Wild-type B. distachyon, n: 56; Bdnpr5-like Bd4982, n: 36; Bdnpr5-like Bd7965, n: 31. Wild-type B. distachyon represents: Bd21-3, n: 14; BdNPR5-LIKE Bd4982 wild-type sibling, n: 20; BdNPR5-LIKE Bd7965 wild-type sibling, n: 22. Transversal sections of 40 days old Bd21-3 (F) and Bdnpr5-like (G) node stained with toluidine blue showing the absence of detectable differences in tissue organization. Bd21-3 nodes sections n: 17; Bdnpr5-like node sections n: 14. Scale bars: A-C, 10 cm; F-G, 100 µm. Technovit section thickness: 5µm.

Supplementary figure S4 :

 S4 Detailed tillering phenotype of Bdnpr5-like mutants compared to Bd21-3 and wild-type sibling lines Numbers of primary (white bars) and secondary (grey bars) tillers by plant in wild-type B. distachyon Bd21-3, BdNPR5-LIKE Bd4982 wild-type sibling, BdNPR5-LIKE Bd7965 wild-type sibling, Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965 dry mature plants. Error bars represent standard deviation. Asterisks indicate significant differences (*, P<0,05;***,P < 0.001) relative to the wild-type B. distachyon Bd21-3 background (Student's ttest). Wild-type B. distachyon Bd21-3, n: 14; BdNPR5-LIKE Bd4982 wild-type sibling, n: 20; BdNPR5-LIKE Bd7965 wild-type sibling, n: 22; Bdnpr5-like Bd4982, n: 36; Bdnpr5-like Bd7965, n: 31.

Figure 5 :

 5 Figure 5: Bdnpr5-like knock-out mutant lines display liguleless and auriculeless phenotypes at the bladesheath boundary region Blade-sheath boundary region of 40 days-old Bd21-3 (A, D, G); Bdnpr5-like Bd4982 (B, E, H); Bdnpr5-like Bd7965 (C, F, I). A, B, C: side view, D, E, F: adaxial view, G, H, I: abaxial view. In Bd21-3 at the blade-sheath boundary region two auricles and one ligule are present (A, D and G). In the Bdnpr5-like knock-out mutant lines Bd4982 and Bd7965, both auricles and ligule are lacking (B, C, E, F, H and I) and instead of a ligule remains only a fringe of tissues (E, F). For picture D, E and F culms have been removed in order to see the ligule properly. White arrowheads indicate lack of auricles and white asterisks indicates the lack of ligule. Scale bars: 1 mm.

Figure 6 .

 6 Figure 6. Bdnpr5-like knock-out mutants lines display an indeterminate inflorescence phenotype (A) Spikelet phenotype of Bd21-3, (B) Bdnpr5-like Bd4982 and (C) Bdnpr5-like Bd7965 from 16 weeks-old plants. (B) Bdnpr5-like Bd4982 and (C) Bdnpr5-like Bd7965 mutants show an exaggerate spikelet growth and spikelets with an increased number of seeds compared to the wild-type Bd21-3 (A). (D) Seed number per spikelet measurements confirm that Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965 develop more seed per spikelet compared to Bd21-3. Error bars represent standard deviation and asterisks indicate significant differences (P < 0.01) relative to the Bd21-3 genotype (Student's t-test). For Bd21-3, Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965, 473, 510 and 615 spikelets and 3721, 7482 and 7538 seeds were counted respectively. Scale bars: 1 cm.

Figure 7 :

 7 Figure 7: Bdnpr5-like mutant shattering phenotype, seed and seed abscission zone compared to Bd21-3 Shattering phenotype of (A) Bd21-3, (B) Bdnpr5-like Bd4982 and (C) Bdnpr5-like Bd7965 16 weeks-old mature plants. Mature dry seeds of Bd21-3 (D), Bdnpr5-like Bd4982 (E) and Bdnpr5-like Bd7965 (F). Magnification of mature dry seed rachilla and their seed abscission zones in Bd21-3 (G), Bdnpr5-like Bd4982 (H) and Bdnpr5like Bd7965 (I). Longitudinal section of the seed abscission zone from 40 days-old spikelets of Bd21-3 (J-L) and Bdnpr5-like (K-M) stained with toluidine blue. (J-K) Seed abscission zones indicated by black frame are differentiated. (L-M) Seed abscission zones magnification: the abscission cell layers are indicated by black arrows. le, lemma; az, seed abscission zone; pa, palea; ra, rachilla; vb, vascular bundle. Scale bars: A-C, 1 cm; D-I, 1 mm; J, K, 100 µm; L, M, 50 µm. Bd21-3 abscission zone sections n: 13; Bdnpr5-like abscission zone sections n: 12. Tecknovit section thickness: 5 µm.

Figure 8 .

 8 Figure 8. Bdnpr6-like missense mutant line Bd5998 shows a short-spikelet phenotype and floral reproductive organ modifications but its abscission ability is not affected Spikelet phenotype of Bd21-3 (A) and Bdnpr6-like missense mutant line Bd5998 (F) 44 days-old plants. Bdnpr6-like missense mutant line Bd5998 (F) shows a short-spikelet phenotype with packed seeds compared to the wild-type Bd21-3 (A). Reproductive organ organization of wild-type Bd21-3 (B, C) and Bdnpr6-like missense mutant line Bd5998 (G, H) during anthesis of 40 days-old plants. Pictures B and G and pictures C and H represent abaxial view and adaxial view respectively. Wild-type Bd21-3 reproductive organ are usually composed by a feathery stigma, an ovary, two adaxial stamens and two abaxial lodicules (B, C). In Bdnpr6-like missense mutant line Bd5998, reproductive organ organization is affected. In abaxial position, in the picture G, the lodicules of the mutant show a stamen identity characterized by a yellow color indicated by white arrowheads. In adaxial position, in the picture H, two supplementary organs are visible (white asterisks). These two additional organs show a stamen-like identity. sti, stigma; sta, stamen; lo, lodicule; ov, ovary. (D) Penetrance of the mutation Leu365Phe on flower phenotype in Bdnpr6-like missense mutant line Bd5998 compared to Bd21-3, Bdnpr5-like Bd4982 and Bdnpr5-like Bd7965. Wild-type flowers (white bars), flowers with lodicules showing stamen identity (light grey bars) and flowers showing additional stamen-like organs (dark grey bars). Number of flowers disected, n: 206, 224, 211 and 250 for Bd21-3, Bdnpr5-like Bd4982, Bdnpr5-like Bd7965 and Bdnpr6-like Bd5998 respectively. Shattering phenotype of Bd21-3 (E) and Bdnpr6-like missense mutant line Bd5998 (I) from 11 weeks-old plants. The Bdnpr6-like missense mutant line Bd5998 abscission ability is not compromised. Scale bars: A, E, F and I, 1 cm; B, C, G and H: 500 µm.

BdNPR5-

  LIKE over-expressor and KO mutants of B. distachyon class I KNOX homologous to SHOOT MERISTEMLESS and BREVIPEDILLUS/KNAT1 could allow to understand the role of BdNPR5-LIKE gene in B. distachyon roots and more generally in grass roots.

  BdNPR5-LIKE gene is the closest ortholog of HvCUL4 and in this work we show that the corresponding Bdnpr5-like null mutant alleles are also affected in tillers development and more precisely in the secondary tillers formation. Despite that the tillering defect found in B. distachyon agrees with the observations made in barley, two differences between the two species were made: the development of multiple axillary shoots instead of one at a same leaf axil and the presence of leafy side-shoots instead of tillers were not observed in B. distachyon. Taken together, the data on barley and its wild relative species B. distachyon suggest a conserved role of the NPR5-LIKE gene family in the control of tillering among Pooideae. The uncharacterized Bdtillerless1 (Bdtil1) mutant displays a tillering defect and the disruption of the inflorescence development (Derbyshire and Byrne 2013). The causal mutation of this mutant probably not impact BdNPR5-LIKE gene since the Bdnpr5-like null mutants display a distinct spike indeterminacy phenotype. BdTIL1 could represent a new molecular actor involved in tillering control which may help to enhance the understanding of the molecular regulation underlying tillering in grasses. This should also provide new tool for breeding to generate more productive crops since the number of tillers directly impact grain yield.

  Bdnpr6-like mutants genotyping. The first exon of the BdNPR5-LIKE gene (Bd2g60710.1) was amplified using oligonucleotides BdNPR5 Ex1 F1 and BdNPR5 Ex1 R3, allowing the identification of the tilling knockout mutant lines Bdnpr5-like (Bd4982 M3) and the tilling missense mutant lines Bd8037 M2 and M3, Bd8023 M2 and M3, Bd7845 M3 and Bd7526 M3). The first part of the second exon of the BdNPR5-LIKE gene was amplified using oligonucleotides BdNPR5_Ex2.1_F1 and BdNPR5_Ex2.1_R1, allowing the identification of the tilling knockout mutant lines Bdnpr5like (Bd7965 M3). The second part of the second exon of the BdNPR5-LIKE gene wasDISCUSSIONThe root nodule symbiosis Legume plants have the capacity to enter in symbiosis with soil bacteria and to build specific organs called nodules to accommodate nitrogen-fixing rhizobia. This interaction allows the legumes to directly profit of the ammonia produced by the reduction of the atmospheric nitrogen by bacteria. This source of ammonia allows legume plants to grow without addition of nitrogen-containing fertilizers to the soil and thus represent plants of high interest for sustainable agriculture. Due to their particular importance for future sustainable agriculture, it appears necessary to increase our understanding of this interaction to be able to use all the potential of the legume symbiotic interaction.The nodule organogenesis and symbiont accommodation in nodules result from the molecular dialogs involving plant root signals, the flavonoids and specific rhizobia lipochitooligosaccharidic signaling molecules called Nod factors. Many studies described in details the mutual recognition between plant and rhizobia, the bacterial infection process, the downstream Nod factors-dependent signaling pathway leading to nodule organogenesis and formation, and the processes of bacterial differentiation into bacteroïds. However, regulatory mechanisms underlying the correct nodule organogenesis, the acquisition and the regulation of the different meristematic zone identities remains less understood.In temperate legumes, following Nod factors signaling, the nodule primordium formed from the inner cortical root cells that will proliferate and dedifferentiate. At the tip of the growing primordium a new stem cell niche or meristem develops. This nodule central meristem (NCM) allows the indeterminate growth of the nodule. In parallel, peripheral vasculatures develop, driven by RAM-like meristems (nodule vascular meristem, NVM) initiated from the root pericycle and endodermis cells layer. The apical meristem and the vascular bundle initial will then tightly cooperate to control the nodule development. The molecular mechanisms underlying these meristems regulation and coordination are unknown. The nodule represents thus a unique plant organ in which distinct meristematic zone are collaborating to control the growth of this organ.Rare legume mutants show failure of the NVM/NCM coordination. noot-bop-coch-like1 (nbcl1) mutants, such as the Medicago Mtnoot1 and the pea Pscoch1 mutants are altered in nodule identity and are mutated in the highly conserved NBCL1 plant genes. The loss of control of the regulation between NVM/NCM leads to the loss of identity. This phenomenon is characterized by the vascular bundle initial root identity de-repression leading to ectopic root outgrowth.

  Mtnoot2 and Pscoch2 single mutants did not present any symbiotic defects. Contrary to their nbcl1 mutant paralogs, nbcl2 mutants did not present nodule ectopic root formation and remains fix+. This suggested that the integrity of the nbcl2 mutants nodule identities are not compromised. It appears that NBCL2 genes are not essential for the nodule development and/or symbiotic identity. The detailed studies performed in Medicago revealed that the proNOOT2:GUS gene expression pattern is early associated to the NCM all along the nodule lifespan. Note that late phases of nodule life were not studied. The proNOOT2:GUS gene expression pattern forms a star-like pattern mostly associated to the NCM and extend toward the NVM. Together, proNOOT1:GUS and proNOOT2:GUS expression patterns highlight the existence of boundary domains between the two kinds of meristematic domains. Furthermore, proNOOT2:GUS gene expression pattern in the Mtnoot1 mutant background revealed that the proNOOT2:GUS gene expression pattern dependents of the nodule identity controlled by MtNOOT1. Indeed, in Mtnoot mutant nodules showing loss of identity, the proNOOT2:GUS gene expression pattern is completely changed. This suggests that in Mtnoot1 in addition to the de-repression of the root identity of the NVM, the loss of nodule identity is also associated to the NCM loss. DISCUSSION 254 Also to better understand the function of MtNOOT1, we initiate experiments which aim to determine the proNOOT1:GUS expression pattern in the Mtnoot2 mutant which has no phenotype. If the proNOOT1:GUS is found in the NCM of the Mtnoot2 mutant, it will indicate that MtNOOT1 is able to compensate the loss of function of MtNOOT2 and thus explain why Mtnoot2 has no phenotype. Such results will provide new insights to understand if MtNOOT1 and MtNOOT2 share redundant functions.

  meristematic activities should be transient and cease once nodule vascular bundles are fully developed. This theoretical arrest of NVM activity should prevent the NVM to give rise to ectopic root development. Thus, in my opinion, NCM play an important role in indeterminate nodules development through the maintenance of the NVM activities conferring to the vascular bundle an indeterminate growth. This indeterminate growth allows the vasculature to accompany the development of the nodule and the continuous establishment of the plant nodule vascularization. In nbcl1 indeterminate nodule, the presence of an active NCM thus prolongs the life of the NVM stem cell niche, increases the possibility of NVM to form ectopic root and thus the occurrence of nodule to root conversion. This could explain why indeterminate nodules show a stronger nodule to root penetrance as compared to determinate nodule types. Determinate nodules do not display naturally nodule to root conversion phenotype despite of the non-persistence of the NCM. This indicates that the absence or the loss of the NCM activity does not triggered nodule to root conversion. However, the fact that the NVM might be inactive in determinate nodules occults the consequence of the NCM arrest. Thus, if the loss of NCM activity occurs in presence of active NVM such as in indeterminate nodules, the consequences should be different. To decipher if the NCM controls the NVM activities and if the loss of NCM activity can alone trigger the de-repression of the NVM root derived program necessary for the nodule to root conversion, we can test for example to express a cell cycle inhibitor under the promoter of MtNOOT2 in order to specifically reduce this NCM activity. Also we can try to apply exogenous jasmonic acid or brassinosteroid inhibitors on the NCM to block meristematic activity and determine the impact on NVM meristematic activities. It is tempting to hypothesize that the loss of nodule identity both involves the re-establishment of the root developmental program in NVM but also the inactivation of the NCM. In my opinion, the loss of nodule identity and the associated nodule to root conversion phenotype is a controlled mechanism. This homeosis is the consequence of the plant inability to coordinate properly the developmental programs of both NVM and NCM. I suggest that the mis-establishment of meristematic territories, the mis-regulation of phytohormone contents and the communication failure between meristematic domains triggers this nodule to root homeosis.

  The two nbcl1nbcl2 double mutants have increased phenotypes relative to their corresponding single nbcl1 mutants. This clearly demonstrate that the NBCL2 genes are important for indeterminate nodule development and identity maintenance, and consequently for hosting the bacterial symbiont, the successful symbiotic interaction and thus for the efficiency of the symbiosis. Our work has thus uncovered the existence of new molecular actors, the NBCL2 genes, and their roles in nodule development and identity maintenance. This provides new perspectives to understand the regulatory mechanisms underlying the nodule identity establishment and conservation.As Medicago and pea forming indeterminate nodules, soybean and Phaseolus forming determinate nodules also possess additional NBCL gene paralogs. Phaseolus possesses at least two known NBCL copies and the tetraploid soybean possesses four known NBCL copies.Surprisingly, it appears that only one NBCL (LjNBCL1) paralog has been retain in the Lotus genome. The existence of additional NBCL copies in other legume forming determinate nodule suggests that the Lotus case is maybe atypical. Extending the studies on NBCL2 gene functions in other legumes forming determinate nodules could help to better understand the role of the NBCL2 clade in determinate nodule without persistent meristem.NBCL/TGA regulatory modules have been recruited for nodule developmentIn M. truncatula, we showed that MtNOOT1 and MtNOOT2 are able to form homo and heterodimers together and we identified the nodule specific TGA type bZIP TF, MtPERIANTHIA-LIKE, as a direct interacting partner of the two MtNOOT proteins. This revealed that one NBCL/TGA regulatory module was recruited from pre-existing regulation pathways and re-use as a regulatory element in the nodule developmental pathway. In addition to the discovery of this interaction between NBCL proteins and a TGA bZIP TF in a context of nodule, we showed that at least 11 other MtTGA bZIP TFs are expressed in the Medicago nodules and it could be interesting to test if these transcription factors are also able to interact with the MtNOOT proteins. This study will provide a more complete view of all the TGA bZIP TFs involved with NOOT in the nodule development. In addition, recent publications mentioned that NBCL proteins are also able to interact with members of the Arabidopsis LSH1 and Oryza G1 (ALOG) proteins[START_REF] Xu | Control of inflorescence architecture in tomato by BTB/POZ transcriptional regulators[END_REF]. In Medicago, there are at least 15 genes encoding ALOG proteins and among them, MtTAWAWA-LIKE is particularly interesting because of its nodulespecific behavior. ALOG proteins represent candidates of interest that are susceptible to interact specifically with NBCL proteins in nodules. The identification of the NOOT interacting partner among in TGA bZIP and ALOG protein families will significantly improve our understanding of the gene network involved in the nodule development and identity. In addition, the identification of the MtPERIANTHIA-LIKE TF as an interacting partner of NOOT proteins provides the opportunity to identify the downstream targets of this transcription factor.Investigatethe downstream target of NOOT proteins using their interacting partners NOOT genes encode BTB/POZ and ankyrin repeats domains and do not have DNAbinding elements neither nuclear localization signals. The NBCL genes do not encode transcription factors sensu stricto. The NOOT proteins are co-trancriptional factors and their interacting partners directly interact with downstream target genes promotor sequences and control their activation or repression. Thus, MtPERIANTHIA-LIKE, a transcription factor displaying DNA-binding sequence will help for the characterization of the downstream targets involved in the NOOT regulation pathway in the nodule. Moreover, in Arabidopsis, class I KNOX and BELL promoter sequences are known to be indirect target of the AtBOP1/2 proteins in different developmental pathways.

  addition, the Arabidopsis class I KNOX genes are also involved in the AtBOP regulation. We can thus hypothesized that a regulatory loop involving NBCL, class I KNOX and class II KNOX maybe involved in the identity and the maintenance of the different meristematic zones in the apical part of the nodule. I am convinced that further studies based on the localization of homeotic class I and class II KNOX genes in the different Mtnoot mutant backgrounds could reveal interesting results about the regulation of meristematic domains in the nodule.

  However, nbcl2 mutants never display any aerial development alteration. To understand if NBCL2 genes are involved in the stipules, leaves and flowers development, the Medicago and pea nbcl1nbcl2 double mutants were characterized for their aerial phenotypes and similar observations were made.Couzigou et al., unpublished data, showed that the Mtnoot2 mutation increased the Mtnoot1 mutant aerial phenotypes, such as stipules phenotype amplification and strong flower alteration leading to partial sterility. In pea, preliminary studies tend to show similar exaggeration of the Pscoch1 phenotype in Pscoch1coch2, such as an increase alteration of stipules identity and additional flower defects. Despite that it appears that nbcl2 mutations increased the nbcl1 aerial mutant phenotypes in Medicago and pea, additional studies in Pscoch1coch2 need to be performed to clearly define the NBCL2 role in the development of the pea aerial organ.

Titre:

  Les rôles des gènes NOOT-BOP-COCH-LIKE dans l'identité de l'organe symbiotique et le développement des plantes. Mots clés : Légumineuses, Identité de la nodosité, Symbiose fixatrice d'azote, Développement, gènes NOOT-BOP-COCH-LIKE, Frontière entre organes Résumé : L'association symbiotique entre les légumineuses et les rhizobia aboutit à la formation de la nodosité fixatrice d'azote.

  gènes NODULE-ROOT de Medicago truncatula, BLADE-ON-PETIOLE d'Arabidopsis thaliana et COCHLEATA de Pisum sativum sont membres du clade spécifique très conservé NOOT-BOP-COCH-LIKE1 (NBCL1) qui fait partie de la famille des gènes NON-EXPRESSOR OF PATHOGENESIS RELATED PROTEIN1-LIKE. Chez les légumineuses, les membres de ce clade NBCL1 sont connus comme étant des régulateurs clés de l'identité de l'organe symbiotique.

  Key words : Legume, Nodule identity, Nitrogen fixing symbiosis, Development, NOOT-BOP-COCH-LIKE genes, Organ boundaries Abstract : The symbiotic interaction between legumes and rhizobia results in the formation of a symbiotic nitrogen fixing nodule.

MtNOOT2 gene belongs to a legume specific NBCL2 clade

	Previous studies have shown that MtNOOT1 gene encode a protein containing BTB/POZ
	and ANKYRIN repeat domains required to maintain the nodule identity and belonging to the
	NBCL protein clade of the NPR1-LIKE protein family (Couzigou et al., 2012). Blast analysis
	using MtNOOT1 sequence performed before completion of the M. truncatula genome sequence
	revealed the presence of a new MtNOOT1 paralogous gene in M. truncatula, MtNOOT2. The
	complete sequence of this gene has been isolated using PCR approaches and was later found as
	identical to MTR_1g051025 from the Medicago truncatula genome version Mt4.0 that later
	became available. Using this information we isolated the PsCOCH2 gene sequence from different
	P. sativum accessions (KX821778 for JI2822, KX821779 for JI73, KX821780 for JI128 and
	KX821781 for JI799). The sequence of the MtNOOT1 and MtNOOT2 orthologs are now also
	available from the Phaseolus vulgaris (PvNBCL1-PvNBCL2), Cicer arietinum (CaNPR5-LIKEa-
	CaNPR5-LIKEb), Cajanus cajan (CcNBCL1-CcNBCL2), Lotus japonicus (LjNBCL1) and
	Lupinus luteus (LlBOP1) genome databases (Supplemental Table 1). In order to better understand
	their evolutionary relationships, we compared the legume NBCL protein sequences to non-
	legume NBCL proteins (Brassicaceae, Solanaceae, Salicaceae, Cucurbitaceae) and to more
	distant Pooideae NBCL proteins (Hordeum vulgare HvCUL4 and HvLAX-A and Brachypodium
	distachyon BdNPR5-LIKE and BdNPR6-LIKE) (Supplemental Table

  BTB/POZ-ankyrin repeat AtNPR1 protein sequence. Blue and red open rectangles indicate the legume NBCL1 protein clade and the legume NBCL2 protein clade, respectively. Evolutionary divergence scale bars: 0.05 substitution/site.

	Medicago truncatula
	MtNOOT1 and MtNOOT2; Pisum sativum PsCOCH1 and PsCOCH2, Phaseolus vulgaris PvNBCL1 and PvNBCL2;
	Cicer arietinum CaNPR5-LIKEa and CaNPR5-LIKEb; Lupinus luteus LlBOP1; Cajanus cajan CcNBCL1 and
	CcNBCL2, Glycine max GmNBCL1 and GmNBCL2; Lotus japonicus LjNBCL1; Solanum lycopersicum SlBOPa,
	SlBOPb, and SlBOPc; Arabidopsis thaliana AtBOP1, AtBOP2 and AtNPR1; Cucumis sativus CsNPR5-LIKEa and

CsNPR5-LIKEb; Cucumis melo CmNPR5-LIKEa and CmNPR5-LIKEb; Populus trichocarpa PtBPL1 and PtBPL2; Salix purpurea SpNPR5-LIKEa and SpNPR5LIKEb; Brachypodium distachyon BdNPR5-like and BdNPR6-like, Hordeum vulgare HvCul4, and HvLAX-A NBCL proteins were used to draw the tree. The above mentioned NBCL protein sequence accession information and origins are listed in the supplemental table S1.

  on the junction between the nodule vascular bundle apex and the central nodule meristematic zone, the ProNOOT1:GUS expression is localized to nodule vascular bundle cells, to nodule vascular bundle pericycle cells and the nodule vascular bundle endodermis cells. (I-L) ProNOOT2:GUS expression in nodule primordia and mature nodules of M. truncatula Jemalong J5 hairy roots inoculated with S. medicae WSM419 strain. (I)

	ProNOOT2:GUS
	expression is predominantly located to C3/C4-derived cell layers and weak expression was observed in C2 and in C5
	derived cells layers of stage V of nodule primordia. Black arrows indicate anticlinal divisions in C1 and C2 cell
	layers and grey arrows indicate multiple cell layers derived from periclinal divisions of C3 cell layer. C4/C5 cells
	form about eight cell layers. Pericycle and endodermis form about 6 cell layers to the basal part of the primordium.
	(J) the ProNOOT2:GUS expression in nodules at 12-13 dpi is associated to the nodule apical meristematic zone. (K-
	L) ProNOOT2:GUS expression in the central meristematic region and in the pericycle and endodermis cells of the
	nodule vascular bundles in nodules at 12-13 dpi. (L) magnification of (K) focusing on the junction between the
	nodule vascular bundle apex and the central meristematic zone of the nodule. ep , epidermis; C1, 1 st cortex cell layer;
	C2, 2 nd cortex cell layer; C3, 3 rd cortex cell layer; C4, 4 th cortex cell layer; C5, 5 th cortex cell layer; ed, endodermis;
	pc, pericycle; Nv, nodule vasculature; NCM, nodule central meristem; NVM, nodule vascular meristem; Iz, infection
	zone; Fz, nodule fixation zone; Nc, nodule cortex; Ne, nodule endodermis; Nve, nodule vascular endodermis; Nvp,
	nodule vascular pericycle; Vb, vascular bundle; Np, nodule parenchyma. Red dotted lines indicating the boundary
	between cortex derived cell layers (above) and pericycle/endodermis derived cell layers (below). Sample section
	thickness: 7 µm. Scale bars: 50µm.

  Analyses. Total RNA extractions were performed from frozen tissues with TRIzol® reagent (Ambion). RNA samples were treated with the TURBO DNA-free™ Kit (Ambion) according to the manufacturer's recommendations. Full-length cDNA were

synthesized using SuperScript™ II Reverse Transcriptase kit (Invitrogen) in presence of Ribolock RNase Inhibitor (Thermoscientific). Real-time RT-PCR reactions were performed using the LightCycler FastStart DNA Master SYBR Green I kit on a Light Cycler 480 II device according to manufacturer's instructions (Roche). Cycling conditions were as follows: 1 preincubation cycle (95°C, 10min), 40 amplification cycles [(denaturation: 95 °C, 10 s), (hybridization: 60 °C, 15 s), (elongation: 72 °C, 15 s)], 1 melting curve cycle [(denaturation: 95 °C, 15 s), (hybridization: 55 °C, 1 min), (denaturation: 95 °C)], 1 cooling cycle (40 °C, 30 s). Cycle threshold and primer specificities were performed with the LightCycler 480 software release 1.5.0 SP4. Primer efficiencies were calculated with LinReg PCR: Analysis of Real-Time PCR Data, version 11.1. For gene expression normalization, MtACTIN11 and MtRNA RECOGNITION MOTIF were used as reference genes. Reference gene ID, oligonucleotides

  ProNOOT2:GUS fusion were constructed as described in[START_REF] Cosson | Medicago truncatula transformation using leaf explants[END_REF]. 8 independent transgenic plants were analyzed and 7 showed the pattern of expression described here with different intensities of staining. In order to study the expression of the fusion in the Mtnoot1 mutant background, two independent transgenic plants were crossed to Mtnoot1 mutant plants (altogether four crosses) and the progeny of the crosses screened for the Mtnoot1 phenotype and the expression of the GUS fusion. The pattern of expression described in this study was observed in the four plants.

	https://www.ensa.ac.uk/). The MtKNOX9
	promoter (2,777 bp), the GUS coding sequence (2,016 bp) and the MtKNOX9 terminator (1,369
	bp) were assembled to a level 1 module and combined with the ProAtUBIQUITIN:DsRed

(Discosoma sp. red fluorescent protein) level 1 module into the binary level 2 vector EC15025 (https://www.ensa.ac.uk/).

Agrobacterium tumefaciens mediated plant transformation. M. truncatula transgenic plants expressing the

Agrobacterium rhizogenes mediated root transformation. ProNOOT2:GUS: M. truncatula Jemalong J5 Hairy root transformation was carried-out using A. rhizogenes A4TC24 strain holding ProNOOT2:GUS fusion. The transgenic roots were obtained after kanamycin (25 mg/L) selection on 1,5 % HP696-5 Kalys Agar (Bernin, France; www.kalys.com) plate containing sterilized Fahräeus medium (Fahräeus, 1957) for 2 weeks as described by Boisson-Dernier et al.

The pea COCHLEATA2 gene is a new member of the legume-specific NOOT- BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development.

	Kévin Magne 1,2 , Vladimir Zhukov 3 , Jean-Malo Couzigou 1,2 , Marion Dalmais 1,2 , Abdelhafid
	Bendahmane 1,2 , Christine Le Signor 4 , Richard Thompson 4 and Pascal Ratet 1, 2
	1 Institute of Plant Sciences Paris-Saclay IPS2, CNRS, INRA, Université Paris-Sud, Université
	Evry, Université Paris-Saclay, Bâtiment 630, 91405 Orsay, France.

The matrix indicate high identity between Legume NBLC1 clade member (MtNOOT1, XP_003625012.1; PsCOCH1, AET34790.1; PvNBCL1, XP_007162101.1; CaNPR5-LIKEa, XP_004493408.1; LlBOP1, AGO64649.1; CcNBCL1, KYP46352.1; GmNBCL1, XP_003521136.1; LjNBCL1, AEM62768.1) and between Legume NBCL2 clade member (MtNOOT2, XP 013467409; PsCOCH2, PsCam048389; PvNBCL2, XP_007145550.1; CaNPR5-LIKEb, XP_004514968.1; CcNBCL2, KYP59865.1; GmNBCL2, XP_003518145.1). Maximum identity values for legume NBCL1 and NBCL2 clade members are indicated with black frame. MtNOOT1 and the other member of the legume NBCL1 clade share the following identity percentage: PsCOCH1 93.6%; CaNPR5-LIKEa 89.2%; LjNBCL1 86.3%; PvNBCL1 85.6%; GmNBCL1 84.5%; CcNBCL1 83.9%; LlBOP1 81.6%. MtNOOT2 and the other member of the legume NBCL2 clade share the following identity percentage: PsCOCH2 92.2%; CaNPR5-LIKEb 92.3%; CcNBCL2 83.0%; PvNBCL2 83.0%; GmNBCL2 82.4%. 2 Institute of Plant Sciences Paris-Saclay IPS2, Paris Diderot, Sorbonne Paris-Cité, Bâtiment 630, 91405, Orsay, France. 3 ARRIAM, Laboratory of Genetics of Plant-Microbe Interactions, Podbelsky chaussee 3, 196608, Pushkin, St Petersburg, Russia. 4 Agroécologie, AgroSup Dijon, Institut National de la Recherche Agronomique (INRA), Université Bourgogne Franche-Comté, 21000, Dijon, France. * Present address: ETH Zurich -Institute of Microbiology HCI F 437, CH-8093 Zurich, Switzerland. CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 82

Results PsCOCH1 and PsCOCH2 gene expressions are induced in the pea indeterminate nodules

  required for indeterminate nodule development. Mtnoot2 single mutant does not show any particular mutant phenotype neither in underground nor in aboveground plant organs but the double mutant Mtnoot1noot2 nodules display 100 % of nodule to root conversion, a complete loss of identity and a fix minus phenotype(Magne et al., 2017a, unpublished). MtNOOT2 belongs to the distinct legume specific NBCL2 sub-clade that has never been investigated and the function of this gene clade remained poorly understood. To better characterize the NBCL2 gene II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 88 http://bios.dijon.inra.fr/FATAL/cgi/pscam.cgi;[START_REF] Alves-Carvalho | Full-length de novo assembly of RNA-seq data in pea (Pisum sativum L.) provides a gene expression atlas and gives insights into root nodulation in this species[END_REF] and to the previous results obtained in M. truncatula indeterminate nodules (Fig.1a; Fig.2;Magne et al., 2017a, unpublished). Both PsGEA and qRT-PCR transcripts accumulation analysis, reveal that PsCOCH2 is not expressed in root and thus appears nodule specific as its putative ortholog

family functions in legume plants, we have undertaken the study of the MtNOOT2 orthologous gene in P. sativum, PsCOCHLEATA2 (PsCOCH2) in a similar context of legume forming indeterminate nodules.

In this work we report for the first time the isolation and the characterization of a pea Pscoch2 mutant obtained by Targeted Induced Local Lesions IN Genomes coupled with Next Generation Sequencing (TILLING-NGS) and with the aim to decipher the role of this gene in leaf, stipule and flower patterning as well as to discover if its role during the nodule formation is similar to the MtNOOT2 gene.

The P. sativum PsCOCH1 and PsCOCH2 genes are orthologs to the M. truncatula MtNOOT1 and MtNOOT2, respectively

[START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] Magne et al., 2017a, unpublished)

. In M. truncatula, MtNOOT1 is constitutively expressed in root and induced during nodule development while MtNOOT2 is not expressed in root but early induced in nodule.

PsCOCH1 and PsCOCH2 gene expression analysis by qRT-PCR in wild-type Pscaméor show a strong expression of both genes in nodule at 27 dpi and a lower expression was observed for PsCOCH2 compared to PsCOCH1, in accordance with the PsCOCH1 and PsCOCH2 gene expression data available from the P. sativum Gene Expression Atlas (PsGEA; CHAPTER MtNOOT2. However, while in PsGEA data, PsCOCH1 seems to be constitutively expressed in root as MtNOOT1 in M. truncatula, we were not able to detect PsCOCH1 gene expression in root by qRT-PCR in our conditions (Fig.

1a

; Fig.

2

.;

Magne et al., 2017a, unpublished)

. In M. truncatula and P. sativum indeterminate nodules NBCL1 and NBCL2 genes are well induced for nodulation.

Table 1 | Pscoch1 allelic serie and Pscoch2 mutant lines studied

 1 Table summarizing thePsCOCH1 and PsCOCH2 TILLING point mutations studied in this study. a Position of the mutation in mutants from the starting ATG of the coding sequence. b Position of the amino acid substitution in mutants from the starting methionine of the encoded protein (* indicate stop mutation). c Values represent predictive deleterious score from the SIFT software. SIFT score ranges from 0 to 1. A score ≤ 0,05 predicts a deleterious effect on protein (http://sift.bii.a-star.edu.sg/).

Among all the analyzed PsCOCH1 alleles, no mutant lines presented the expected coch1 phenotypes which consist in nodule to root conversion phenotype and aerial developmental defects. Using a NBCL proteins consensus based on 248 NBCL protein sequences (https://www.ncbi.nlm.nih.gov/; https://db.cngb.org/blast4onekp/;

[START_REF] Wickett | Phylotranscriptomic analysis of the origin and early diversification of land plants[END_REF] Matasci et al., 2014;[START_REF] Xie | SOAP denovo-Trans: de novo transcriptome assembly with short RNA-Seq reads[END_REF][START_REF] Johnson | Evaluating methods for isolating total RNA and predicting the success of sequencing phylogenetically diverse plant transcriptomes[END_REF] Supplementary figure

Table 2 | Segregation analysis of the PsCOCH2 Ps1178 locus

 2

CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development 96

  CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development

	times with nutritive solution in alternance) with HY N-free nutritive solution for nodulation (For
	5 liters of HY N-free solution: 50 mL of stock solutions (KH 2 PO 4 , 0,1 M; MgSO 4 , 0,1 M); 12,5
	mL of stock solutions (K 2 SO 4 , 0,1 M; CaCl 2 , 2H 2 O, 0,1 M); 5 mL of stock solutions (KCl, 50
	mM; H 3 BO 3 , 23,8 mM; MnSO 4 , H 2 0, 5 mM; ZnSO 4 , 7H 2 0, 1 mM; CuSO 4 , 1 mM; MoNa 2 O 4 ,
	2H 2 0, 0,7 mM; Na-Fe-EDTA, 98mM); pH: 7) or with N+ nutritive solution (Soluplant, NPK16 6
	26) for non-nodulated plant.
	Rhizobial strain, growth conditions and inoculation. Rhizobium leguminosarum bv. viciae
	, wild-type strain P221 (Laguerre et al., 1992) was used for P. sativum nodulation. Rhizobia were
	purified on G-50 sephadex column, mixed with formamide and concentrated using speed grown on solid or liquid Yeast Extract Mannitol (YEM; Bergersen,1961) agar plate (Na 2 HPO 4 ,
	vacuum. Samples and fluorescent markers (50-1500 bp DNA sizing standard IR® DYE 700 and 460 mg.L -1 ; MgSO 4 , 100 mg.L -1 ; FeCl 3 , 4 mg.L -1 ; CaCl 2 , 40 mg.L -1 ; sodium hydrogeno
	800) were denaturated and loaded on 0,2 mm thickness LI-COR acrylamide gel (20 mL KB + LI-glutamate, 1.1g.L -1 ; mannitol 10 g.L -1 ; yeast extract, 200 mg.L -1 ; bactoagar, 15 g.L -1 ; pH 6,8) for
	crossing Pscoch1 FN3185/1325 (back-cross1) with Pscoch2 Ps1178 (back-cross 2). P. sativum
	were surface-sterilized 20 min with sodium hypochlorite and washed three times with sterile
	water. Seeds were vernalized between two filter papers on agar plates (Kalys Biotech, HP696-5,
	7 g.L -1 ) for 4 days at 4°C under darkness. Seeds were germinated 48h at 24°C under darkness for
	acclimatization before sowing. For nodulation assays in pot, a sand/perlite mixture was used (1/2,
	v/v). For vegetative development and bloom, a loam/peat/sand mixture was used (70/3,5/7, v/v/v;
	http://www.puteaux-sa.fr) with expensed clay balls at the bottom of the pot. P. sativum were
	111

COR, 196 µL APS (10%), 19,6 µL TEMED). Samples were analyzed using LI-COR DNA sequencer (http://www.licor.com) between 680 and 800 nm. Oligonucleotides used for SNP detection assays by are given in supplementary table

S5

.

Plant material and growth conditions. Wild-type P. sativum var. caméor, the corresponding ethyl methanesulfonate tilling mutants lines Pscoch1 (Ps3092, Ps2702, Ps2240, Ps2058, Ps547) and Pscoch2 (Ps1178, Ps3340,

Dalmais et al., 2008

, http://urgv.evry.inra.fr/UTILLdb), and wildtype P. sativum var. JI2822 and the corresponding fast neutron bombardment full-length deletion mutants lines Pscoch1 (FN3185/1325,

[START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] John Innes Centre germplasm; www.jic.ac

.uk/ GERMPLASM/) were used. Double mutants Pscoch1coch2 were obtained by grown under 16/8h light-dark cycle, 24/24°C day-night temperature, 60% of relative humidity and 200 µE of light intensity. Plants were watered three times a week (2 times with water / 1 2 days under darkness at 28°C. For nodulation in sand/perlite substrate, P. sativum seedling were inoculated with 25 mL of R. leguminosarum suspension at OD 600 : 0,1.

  CHAPTER II. The pea COCHLEATA2 gene is a new member of the legume-specific NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development

	Pscoch1 and Pscoch2 mutants genotyping strategy. The tilling point mutation in lines Pscoch1
	(Ps3092, Ps2240, Ps2702, Ps2058, Ps547) and Pscoch2 (Ps1178, Ps3340) were checked by PCR
	amplification and sequencing. PCR amplifications were carried out using High Fidelity Taq DNA
	polymerase (EUROBIO GAETHF004D). Detailed information about mutation location are given
	supplementary table S2 and S3 and information concerning oligonucleotides couple used for PCR
	amplification are provide in supplementary table S5. PCR products were sequenced by Sanger
	(www.eurofinsgenomics.eu) and analyzed with A plasmid Editor 2.0.49 sofware. For the
	Pscoch1 line FN3185/1325, the PsCOCH1 full-length deletion borders are not known and
	homozygous mutant were selected by phenotyping for the coch1 phenotypes.
	112

Gene expression analyses.

Total RNA extractions were performed from frozen tissues using TRIzol® reagent (Ambion) following manufacturer's recommendations. RNA samples were treated with the TURBO DNA-free™ Kit (Ambion) according to the manufacturer's recommendations. Full-length cDNA were synthesized using SuperScript™ II Reverse Transcriptase kit (Invitrogen) in presence of Ribolock RNase Inhibitor (Thermoscientific). qRT-PCR reactions were performed using the LightCycler FastStart DNA Master SYBR Green I kit on a Light Cycler 480 II device according to manufacturer's instructions (Roche). Cycling conditions were as follows: 1 pre-incubation cycle (95°C, 10min), 40 amplification cycles [(denaturation: 95 °C, 10 s), (hybridization: 60 °C, 15 s), (elongation: 72 °C, 15 s)], 1 melting curve cycle [(denaturation: 95 °C, 15 s), (hybridization: 55 °C, 1 min), (denaturation: 95 °C)], 1 cooling cycle (40 °C, 30 s). Cycle threshold and primer specificity analysis were performed using the LightCycler 480 software release 1.5.0 SP4. Primer efficiencies were calculated with LinReg PCR: Analysis of Real-Time PCR Data, version 11.1. Relative transcripts accumulations were normalized on PsACTIN and PsBETA-TUBULIN3 reference genes. Denodulated root, nodule, internode and node PsCOCH1 and PsCOCH2 expression levels were normalized on denodulated root reference organ. Young leaf, developing leaf, young flower, developing flower, young seed and developing seed PsCOCH1 and PsCOCH2 expression levels were normalized on young seed reference organ. The specificity of PsCOCH1 and PsCOCH2 qRT-PCR products amplified from nodule cDNA were sequenced by Sanger (www.eurofinsgenomics.eu). Detailed information concerning oligonucleotides used for qRT-PCR analysis are provided in the supplementary Table

  NBCL genes are undeniably key regulators of nodule identity, however, the molecular mechanism underlying the regulation of the symbiotic organ identity mediated by NBCL genes is completely unknown.

	Based on the NPR1-like/TGA and NBCL/TGA interactions described for plant
	immunity and development, the present study aims to investigate if a similar regulatory
	mechanism involving NBCL/TGA interaction exists in M. truncatula nodules to allow nodule
	development and/or maintain the symbiotic nodule identity. Here we report the identification
	of the AtPAN closest orthologous gene in M. truncatula, MtPERIANTHIA-LIKE (MtPAN-
	LIKE), as a nodule specific gene and the characterization of the corresponding Mtpan-like
	Tnt1 insertional mutant. We suggest that NBCL/TGA interactions might exist in nodule and
	demonstrate that MtNOOT1, MtNOOT2 and MtPAN-LIKE are able to interact with

. Since only nbcl mutant plants exhibit such nodule to root homeosis,

  MtPAN-LIKE gene expression induction during nodulation was confirmed by qRT-PCR analysis. In our experiments MtPAN-LIKE transcripts were detected from 5 to 21 dpi (Fig.3). MtPAN-LIKE gene can be consider as an early symbiotic gene since it expression is induced simultaneously with MtNOOT2 and MtNODULE INCEPTION (MtNIN) expression that are detected from height to twenty-one dpi. Later from 12 to 21 dpi the late induced

	MtNODULE SPECIFIC CYSTEINE RICH PEPTIDE 001 (MtNCR001) is detected. We can
	note that all these symbiotic genes are triggered in parallel with the downregulation of the
	MtPATHOGENESIS RELATED PROTEIN 10 gene (MtPR10) which is induced at the
	beginning of the nodulation process and then repressed during nodule development in
	agreement with Bourcy et al. (2013). MtNOOT1 is constitutively expressed in the root,

slightly down-regulated at the beginning of the nodule primordia formation and induced from 8 to 21 dpi as MtNOOT2, MtNIN and MtPAN-LIKE. Our gene expression analysis also reveals that during the nodulation MtPAN-LIKE is more expressed than MtNIN, MtNOOT1

Table 1 | Segregation analysis of double mutants heterozygous for the independent MtNOOT1 and MtPAN-LIKE loci.

 1

Table 2 | Segregation analysis of double mutants heterozygous for the independent MtNOOT2 and MtPAN-LIKE loci.

 2

  We show that MtPAN-LIKE gene is a nodule-specific gene which behaves as an early symbiotic gene. Furthermore, the MtPAN-LIKE gene symbiotic nature was confirmed by testing the expression of the MtPAN-LIKE gene in the Mtnoot mutant backgrounds. It was previously shown that symbiotic genes are downregulated in the Mtnoot1 and Mtnoot1noot2 mutant backgrounds(Magne et al., 2017a, unpublished) and we obtained similar results for MtPAN-LIKE suggesting that MtPAN-LIKE expression is dependent of the nodule identity. Based on the work of[START_REF] Roux | An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing[END_REF] we highlight that GUS and the proNOOT2:GUS expression patterns in the nodule vascular bundle apex and in the nodule apical meristem, respectively[START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF] Magne et al., 2017a, unpublished). However, we did not yet confirmed the gene expression pattern of MtPAN-LIKE gene in nodule using GUS reporter or in situ hybridization and the exact localization of the expression of this gene is still unknown. The precise localization of the MtPAN-LIKE gene expression could significantly add to the comprehension the role of this gene. In addition we showed by qRT-PCR that MtNOOT1, MtNOOT2 and MtPAN-LIKE genes were simultaneously expressed. Thus, the MtNOOT1, MtNOOT2 and MtPAN-LIKE spatial and temporal common gene expression patterns supported well our hyphothesis of a conserved NBCL/TGA regulation module in the nodule of M. truncatula involving MtNOOT1/2 and MtPAN-LIKE.

Thus, MtPAN-LIKE gene was designated as a suitable candidate gene potentially involved in M. truncatula nodule identity regulation. MtNOOT1, MtNOOT2 and MtPAN-LIKE shared a similar spatial expression pattern and that their transcripts were mostly found in the apical part of the nodule. The apical localization of MtNOOT1 and MtNOOT2 found in Roux et al. (2014) is coherent with the previously described proNOOT1:

  We further demonstrated that MtNOOT1, MtNOOT2 and MtPAN-LIKE proteins were able to interact with themselves and with each other using yeast-two-hybrid technology and beta-galactosidase assay. It means that MtNOOT1 and MtNOOT2 are able to form homodimers and heterodimers and that MtPAN-LIKE is also able to form homodimer and to interact with both MtNOOT proteins. Also we confirmed the interactions between MtPAN-LIKE and the two MtNOOT proteins independently using an original transient subcellular protein-GFP delocalization assay. In this experiment we were able to delocalize the MtNOOT nucleo-cytoplasmic GFP signal to the nucleus by co-expressing MtPAN-LIKE proteins and to delocalize the MtPAN-LIKE nuclear GFP signal to the cytoplasm by co-expressing MtNOOT proteins. Transient delocalization experiments indicate that MtPAN-LIKE is able to bind MtNOOTs proteins and to address them to the nucleus. Thus we demonstrate that MtNOOT1 and MtNOOT2 interact in vivo with MtPAN-LIKE in planta and in yeast.

Although this kind of interactions have already been described in different defense and developmental processes in Solanaceae and in Brassicaeae families, to our knowledge, it is the first time that such an interaction between NBCL and TGA bZIP TF is described in a legume plant and in a such original context of nodule. Based on these results and on the literature dealing about BTB/POZ and ankyrin repeat domains protein/protein interaction, it can be hypothesize that MtNOOT1, MtNOOT2 and MtPAN-LIKE execute dynamic interactions involving inactive form of MtNOOT1 and MtNOOT2 oligomers and active form of MtNOOT1 and MtNOOT2 momomers that should be able to interact with the MtPAN-LIKE TF to target genes involved in nodule identity. It can also be hypothesize that MtNOOT1, MtNOOT2 and MtPAN-LIKE form a complex as previously described in the perianth patterning regulation involving BOP1/BOP2/PAN in Arabidopsis. It is also possible that in the nodule the MtNOOT1 and/or the MtNOOT2 regulate the turnover of the MtPAN-LIKE by playing a role of CULLIN E3 adaptor mediating its degradation by proteasome

[START_REF] Zhang | BLADE-ON-PETIOLE proteins act in an E3 ubiquitin ligase complex to regulate PHYTOCHROME INTERACTING FACTOR 4 abundance[END_REF]

. In the absence of both Mtnoot2 and Mtpan-like phenotypes, we currently aim to over-express MtNOOT2 and MtPAN-LIKE, independently, to see if they alter the nodule identity.

:LORE1 insertional mutants genotyping strategy.

  In line 30053558, the LORE1 retroelement is inserted 2077 bp after ATG codon on gDNA sequence, 1134 bp after AUG codon on mRNA sequence (NCBI Acc. No.: JN408495) and between amino acids isoleucine 378 and The RNAi_LjNBCL1#1 (263 pb) amplicon was designed against the second exon of the LjNBCL1 ARNm sequences (1452 pb) from the 474 th to the 736 th nucleotide from the AUG codon. Amplicon specificity was determined through BLAST analysis (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastn) against L. japonicus genome.

	glutamic acid 379 of the 483 amino acids LjNBCL1 protein (NCBI Acc. No.: AEM62768). In
	line 30119830, the LORE1 retro-element is inserted 1803 bp after ATG codon on gDNA
	sequence, 860 bp after AUG codon on mRNA sequence and between amino acids leucine 286
	and alanine 287 of the 483 amino acids LjNBCL1 protein. The presence of the 5406 bp length
	LORE1 TY3-gypsy type retrotransposon (NCBI Acc. No.: AJ966990) at the LjNBCL1 locus was
	checked by PCR in lines 30053558 and 30119830 using primer described in the supplementary
	table 1.
	RNA interference. RNAi_LjNBCL1#1 amplicon have been amplified from L. japonicus ecotype GIFU cDNA using
	Phusion® High-Fidelity DNA Polymerase (Neb) following the manufacturer's recommendation,
	cloned in pENTR/D-TOPO vector (Life technologies) and introduced by Gateway LR
	recombination (Invitrogen) in both anti-sens and sens in pFRN-RNAi binary vector (Gonzalez-
	Rizzo et al., 2006; derived from binary pFGC5941 vector; NCBI Acc. No.: AY310901).
	Construct of interest were introduced in Agrobacterium rhizogenese (A. rhizogenese) ARqua1
	(streptomycin R -derivative strain of A4T; Quandt et al., 1993) by electroporation (Bio-rad, E. coli
	Pulser). The LjNBCL1:RNAi construction transcript structure has been predicted using the online
	software RNAfold web server and the « minimum free energy » pairing algorithm
	(rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). Control ARqua1, expressing GUS:RNAi construction
	was gently provided by Dr. Mathias Brault. Oligonucleotides used for the RNA interference
	strategy are described in the supplementary table 2.

BdNPR5-LIKE and BdNPR6-LIKE gene are expressed in many organs in B. distachyon

  As few expression data for BdNPR5-LIKE and BdNPR6-LIKE existed in the literature and in databases (https://phytozome.jgi.doe.gov/pz/portall;[START_REF] Goodstein | Phytozome: a comparative platform for green plant genomics[END_REF], we analyzed the BdNPR5-LIKE and BdNPR6-LIKE expression using RT-qPCR in the wild-type B. distachyon ecotype Bd21-3. RT-qPCR expression data reveals that both genes are expressed in many organs (Fig.2). Our expression data are also coherent with the expression data available on the recently released B. distachyon gene expression atlas (www.gene2function.de; http://www.bar.utoronto.ca;[START_REF] Sibout | Expression atlas and comparative coexpression network analyses reveal important genes involved in the formation of lignified cell wall in Brachypodium distachyon[END_REF]. BdNPR5-LIKE maximum transcript accumulation was detected in nodes. Transcription was also detected in sheaths, ligular regions, rachis nodes and spikelets at a lower level. BdNPR5-LIKE transcripts were also detected at low level in roots and in leaf blades but were below the detection level in rachis internodes (Fig.2). BdNPR6-LIKE maximum transcript accumulations were detected in nodes and in spikelets at similar level. BdNPR6-LIKE transcripts were also detected in sheaths, in ligular regions and rachis nodes and are more abundant in these organs compared to BdNPR5-LIKE transcripts. BdNPR6-LIKE accumulate at a low level in leaf blades and in rachis internodes. By contrast to BdNPR5-LIKE, BdNPR6-LIKE transcripts do not accumulate in roots (Fig.2). BdNPR5-LIKE and BdNPR6-LIKE gene transcripts accumulate in different organs across the B. distachyon plant body, implying that these two genes may be involved in several developmental processes. Furthermore, the expression pattern of BdNPR5-LIKE and BdNPR6-LIKE partially overlap like their closest paralogs HvCUL4 and HvLAX-A in barley (RNAseq data, http://ics.hutton.ac.uk/morexGenes, IBSC,

table S1: Overview of the mutations identified in BdNPR5-LIKE and BdNPR6-LIKE genes by TILLING-NGS

  .fr/tilling/brachypodium.htm). 99 % and 65 % of the coding sequence were screened for BdNPR5-LIKE and BdNPR6-LIKE genes, respectively. 55 and 37 TILLING mutants were identified for BdNPR5-LIKE and BdNPR6-LIKE respectively. A summary of the tilling results and a detailed list of the tilling mutations obtained for BdNPR5-LIKE and BdNPR6-LIKE are given in Sup. Table 1 and Sup. Table 2, respectively.

	Supplementary

Table 1 | Correlation between the segregation of the BdNPR5-LIKE Bd7965 locus and the liguleless mutant phenotype Table 2 | Correlation between the segregation of the BdNPR6-LIKE Bd5998 locus and the short-spikelet mutant phenotype

 1

  our results indicate that in B. distachyon the KO Bdnpr5-like mutants is not affected in the formation of the seed abscission zone nor in the abscission ability. The Bdnpr6-like missense mutant Bd5998 was also not affected in seed abscission ability but because the single amino-acid change (Leu365Phe) may not affect all the BdNPR6-LIKE functions and or might represent a weak mutation, we cannot conclude that this gene is not involved in abscission. Obtaining a null allele for BdNPR6-LIKE will clarify the role of this gene alone or in a Bdnpr5-like context, in the seed abscission process.BdNPR5-LIKE and BdNPR6-LIKE genes share 73,9 % of similarity and 59,5 % of identity at the protein level and are both expressed in rachis nodes and in spikelets containing seed AZs. This may indicates that the two genes are functionally redundant and thus explains the absence of a loss of shattering ability in simple mutants. A potential NBCL-dependent regulation of AZ formation between rachises and seed bases could allow the discovery of new molecular actors involved in the abscission zone establishment in grasses that may facilitate new species domestication and provide new tools for breeding strategies.

  Johnston et al., 2014 in which a pre-existing genetic network used to initiate lateral organ in SAM is reused for the development of other organ at other boundaries. NBCL genes are involved in boundaries formation and promote lateral organ formation in SAM

  Arabidopsis thaliana BOP1/BOP2 encoding BTB/POZ and ankyrin proteins, in the nondomesticated model plant B. distachyon, BdNPR5-LIKE and BdNPR6-LIKE. BdNPR5-LIKE is involved in the tillering control representing an important trait directly linked to grain yield and in the promotion of proximal/distal leaf differentiation, more precisely in the ligule and the auricles formation. These results are in agreement and confirm the role of the HvCUL4 gene in barley which is the closest ortholog of BdNPR5-LIKE and represents the first monocot NBCL gene characterized

  To our knowledge, in barley, the double mutant incubate at 4 °C overnight. Once dehydrated by successive ethyl-alcohol bathes, samples

	undergo three successive Technovit stock solution (3:1, v/v), (1:1, v/v) and (1:3, v/v) bathes
	and three 1 h 100 % Technovit stock solution bathes at 4 °C under agitation. Samples were
	included in technovit resin thanks to a teflon Histoform S embedding moulds (Heraeus
	Kulzer). Technovit sections were carried out using a microtome RM 2155 (LEICA) and
	tungsten disposable blade (TC-65, LEICA). 5 µm thickness sections were stained 10 min by
	immersion in toluidine blue (0,02 %) and observed with a microscope BX53 (OLYMPUS)
	and acquired using cellSens Standard software.
	B. distachyon genomic DNA extraction. Young leaves were frozen in liquid nitrogen and
	grinded with a tissue lyser MM400 (RETSCH) two times 30 seconds at a 25.s -1 frequency.
	600µL of fresh DNA extraction buffer (NaCl, 350mM; Tris-HCl,10mM, pH: 7,6; urea, 7M;

Acknowledgments

Je souhaite exprimer ma profonde gratitude à Pascal Ratet, chef et directeur de thèse.

Acknowledgements

This work was supported by the CNRS and by the grants ANR-14-CE19-0003 (NOOT) from the Agence National de la Recherche to PR. The authors thank Dr. Mathias Brault for providing pFRN:RNAi plasmid, A. rhizogenes strain ARqua1 and control GUS:RNAi construction. We gratefully thank Cristina Ferrandiz for the help to interpret the identity of the meristems in the SEM pictures and Frederique Guinel for help to interpret the identity of vascular tissue organization in nodule vascular bundle of L. japonicus. We thank Dr. Simona Radutoiu for providing the Na-Borate/TRIzol RNA extraction prorocol. This work has benefited from the facilities and expertise of the Servicio de Microscopía Electrónica Universitat politècnica de Valencia (http://www.upv.es/entidades/SME/) and of the IMAGIF Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil Général de l'Essonne.

Acknowledgments

We thank Sébastien Antelme who generated the mutagenized Brachypodium distachyon ecotype Bd21-3 population. We thank Christine Saffray and Raphaël Costa who helped to develop the TILLING-NGS strategy, Guillaume Beaumont and Brahim Mania who developed the TILLING-NGS bioinformatic pipeline. This work has benefited from the facilities and expertise of the IMAGIF Cell Biology Unit of the Gif campus (www.imagif.cnrs.fr) which is supported by the Conseil Général de l'Essonne. We also thank Gane Ka-Shu Wong, Michael Deyholos, Yong Zhang and the other people involved in the oneKP initiative to give access to their large-scale gene sequencing data through a BLAST search portal. We gratefully thank Pr. Marie Dufresnes for scientific discussions and careful reading of the manuscript.

Supplemental table

 S3. List of primers used for qRT-PCR analysis and genotyping

CHAPTER II.

The pea COCHLEATA2 gene is a new member of the legume-specific

NOOT-BOP-COCH-LIKE2 clade involved in root nodule symbiosis and aerial organ development

Identification of TILLING mutations in PsCOCH1 and PsCOCH2 genes

The PsCOCH1 gene is involved in nodule identity maintenance through the repression of root identity inside the indeterminate nodule of P. sativum, in stipule development, in leaf size and patterning, in inflorescence architecture and development, and in floral organ identity [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Sharma | COCHLEATA controls leaf size and secondary inflorescence architecture via negative regulation of UNIFOLIATA (LEAFY ortholog) gene in garden pea Pisum sativum[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. In P.

sativum, the role of the second NBCL gene PsCOCH2 is completely unknown and PsCOCH2 knockout (KO) mutants are not available.

To study the role of PsCOCH2 using Pscoch2 single mutant and to later construct the double mutant Pscoch1coch2 in a similar P. sativum background, TILLING mutations in the PsCOCH1 and PsCOCH2 genes were screened by TILLING coupled with Next Generation Sequencing technology (TILLING-NGS) using the P. sativum var. caméor TILLING mutant collection (Dalmais et al., 2008). 97 % and 99 % of the coding sequence were screened for PsCOCH1 and PsCOCH2 genes, respectively. 66 and 75 TILLING mutants were identified for PsCOCH1 and PsCOCH2 respectively. A summary of the tilling results and detailed lists of the TILLING mutations obtained for PsCOCH1 and PsCOCH2 are given in Supplementary Table S1, S2, S3, respectively. Based on SIFT score (http://sift.bii.a-star.edu.sg/) which represents the predictive deleterious effect of a mutation on the protein function, one KO and 4 missense mutants (SIFT score: 0), were chosen for PsCOCH1. Similarly, two KO lines (SIFT score: 0), were chosen for PsCOCH2 (Fig. 3; Table 1). A schematic representation of the mutations position along their respective protein is presented Fig. 3. Acetylene reduction assay. Acetylene reduction assays (ARA) were performed on individual plants inoculated with R. leguminosarum strain P221 at 35dpi with a protocol modified from [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. Basically, the nodulated root system of one plant was placed in 21 mL glass vial sealed with rubber septa in presence of 200 µL of water. Acetylene gas (500 µL) was injected into each vial and 2 h incubation was performed. Ethylene production was measured using a Gas Chromatograph (Agilent Technologies, 7820A) equipped with a GS-Alumina column (50 m x 0,53 mm) with hydrogen as carrier gas. Column temperature and gas flow were adjusted at 120 °C and at 7,5 mL.min -1 , respectively. For each sample analysis, 1 mL of gas was injected in the gas chromatograph. expressing the viral protein helper component-proteinase (HC-Pro) RNA silencing inhibitor [START_REF] Mallory | A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco[END_REF] at OD HCPro = ∑OD constructions tested . Agroinfiltration. Agroinfiltrations were performed as described in [START_REF] Sparkes | Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[END_REF] Briefly, 1 h before infiltration, tobacco plants are placed under white light and large third and fourth leaves from apex were chosen for agroinfiltration. Leaves abaxial surface were gently pierced, the A. tumefaciens mixture was infiltrated using a 1 mL syringue and plant were replace under normal growth conditions.

Supplementary table S1 | Overview of the mutations identified in

Light microscopy and samples preparation. In Ljnbcl1:LORE1 homozygous mutants, 13 % of the leaves presented an additional leaflet relative to L. japonicus GIFU suggesting that LjNBCL1 is required for the proximal/distal patterning of the leaf (Fig. 2b,c,d). In addition, while L. japonicus wild-type plants produce one axillary at each leaf axil, Ljnbcl1:LORE1 homozygous mutants often produced multiple axillaries at a single leaf axil suggesting that LjNBCL1 is required to control the number and the growth of these axillaries (Fig. 2e,f).

Together with Tetragonolobus and Bonjeania, Lotus possesses a pair of original organs at each node called nectary glands which were proposed to be modified stipules by [START_REF] Irmisch | Noch einige Beobachtungen über die Stipulae bei Lotus, Tetragonolobus und Bonjeania[END_REF] and Heyn (1976) (Fig. 2g,i). These nectary glands are completely absent in Ljnbcl1 and scanning electron microscopy (SEM) analysis indicated that no trace of these nectary gland appendages remain at the leaf axil (Fig. 1h,j). In contrast, a significant increase of the petiole length was observed in Ljnbcl1 relative to the wild-type (Fig. 2i,j; Supplementary Fig. 2). These results support the general role of the NBCL genes in both stipule and nectary developments and define CHAPTER IV. NOOT-BOP-COCH-LIKE1 is essential for multiple aspects of Lotus japonicus development 161 the leaf proximal region identity, as proposed before in A. thaliana, M. truncatula and P. sativum [START_REF] Gourlay | Pea compound leaf architecture is regulated by interactions among the genes UNIFOLIATA, COCHLEATA, AFILA and TENDRIL-LESS[END_REF][START_REF] Yaxley | Leaf and flower development in pea (Pisum sativum L.): Mutants cochleata and unifoliata[END_REF][START_REF] Ha | The BLADE-ON-PETIOLE 1 gene controls leaf pattern formation through the modulation of meristematic activity in Arabidopsis[END_REF][START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF][START_REF] Norberg | The BLADE ON PETIOLE genes act redundantly to control the growth and development of lateral organs[END_REF][START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF]. Thus, in Lotus the LjNBCL1 gene is essential for the nectary gland formation.

LjNBCL1 is constitutively expressed in root and induced during nodulation

To uncover if LjNBCL1 participates to the L. japonicus ecotype GIFU nodule development, its expression was measured in root apical meristem (RAM), in root and in determinate nodule throughout a nodulation kinetic using Mesorhizobium loti (M. loti) strain NZP-2235 [START_REF] Jarvis | Rhizobium loti, a new species of legume root nodule bacteria[END_REF] as symbiotic partner.

LjNBCL1 is constitutively expressed in non-inoculated L. japonicus root and its expression in RAM was detected at very low level (Fig. 3a). 2 days post inoculation (dpi),

LjNBCL1 gene expression level remains similar to non-inoculated root. At 5 dpi, when nodule primordia are visible, the LjNBCL1 expression increased. At 8 dpi, its expression in nodules reached its maximum (Fig. 3a) and started to decrease at 12 dpi. From 16 dpi to 20 dpi LjNBCL1 gene expression seems to stabilize (Fig. 3a). This kinetic is compatible with the LjNBCL1 gene expression profile obtained from the Lotus japonicus Gene Expression Atlas (https://ljgea.noble.org/v2/; Supplementary Fig. 1).

As described for the M. truncatula and P. sativum nbcl1 mutants [START_REF] Ferguson | Cochleata: Getting to the root of legume nodules[END_REF][START_REF] Couzigou | NODULE ROOT and COCHLEATA maintain nodule development and are legume orthologs of Arabidopsis BLADE-ON-PETIOLE genes[END_REF], Ljnbcl1:LORE1 and Ljnbcl1:RNAi mutant nodules had a wild type level of nitrogen fixation (Supplementary Fig. 3) when assessed using acetylene reduction assay (ARA; [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF]. Detailed histological analysis of the GUS:RNAi transformed root nodules shows the classic wild-type globular shape of determinate nodule type (Fig. 5a,d;[START_REF] Corby | Types of rhizobial nodules and their distribution among the Leguminosae[END_REF][START_REF] Walsh | Vascular transport and soybean nodule function. 1: Nodule xylem is a blind alley, not a throughway[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]. In contrast, in Ljnbcl1:RNAi and Ljnbcl1:LORE1 mutant nodules, an apical nodule-root structure is clearly present (Fig. 5b,c). Longitudinal sections of the Ljnbcl1 nodules reveal the origin of these nodule ectopic roots and show the vascular bundle continuum between nodule vascular bundles (NVB) and nodule ectopic root vasculatures. This demonstrates that nodule ectopic roots originate from NVM as in indeterminate nodule (Fig. 5a-c In agreement with the literature describing wild-type determinate nodule organization, transversal sections of the GUS:RNAi nodules show peripheral vascular bundles deeply incrusted in nodule parenchyma and close to the rhizobia infected cells. In determinate nodules, 2 to 3 boundary cell layers separate the NVB from the first infected cells [START_REF] Brown | Symplastic transport in soybean root nodules[END_REF][START_REF] Guinel | Getting around the legume nodule: I. The structure of the peripheral zone in four nodule types[END_REF]Fig. 5d,g; Supplementary Fig. 4). By contrast, in Ljnbcl1 converted nodule, NVB are surrounded by additional root cortex-like cell layers and tend to be isolated from the nodule infected zone (Fig. 5e,f,h,i). In Ljnbcl1 converted nodules the number of cells separating NVB from infected cells is significantly increased and reaches 5 to 6 cortex cell layers (Fig. 5e,f; Supplementary Fig. 4). Comparison of completely dissociated Ljnbcl1 nodule root structures and L. japonicus wild-type roots revealed that the number of cortex cell layers surrounding the vasculature (4 to 5 cortex cell layers; [START_REF] Wopereis | Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype[END_REF] were not significantly different (Supplementary Fig. 5), highlighting the shift to root identity. Acetylene reduction assay. Acetylene reduction assays (ARA) were performed on individual plants inoculated with M. loti at 35dpi with a protocol modified from [START_REF] Koch | Reduction of acetylene to ethylene by soybean root nodules[END_REF].

Basically, the nodulated root system of one plant was placed in 21 mL glass vial sealed with rubber septa in presence of 200 µL of water. Acetylene gas (500 µL) was injected into each vial and 2 h incubation was performed. For each sample, 1 mL of gas was injected. Ethylene production was measured using a Gas Chromatograph (Agilent Technologies, 7820A) equipped with a GS-Alumina column (50 m x 0,53 mm) with hydrogen as carrier gas. Column temperature and gas flow were adjusted at 120 °C and at 7,5 mL.min -1 , respectively. were chosen for BdNPR6-LIKE (Fig. 3 A). Following BdNPR5-LIKE gene mutations genotyping, homozygous mutants were found in families Bd8595, Bd7845, Bd4982, Bd7526, Bd7965 and Bd5567 (Fig. 3 A). For the BdNPR6-LIKE gene, homozygous mutants were found in families Bd7392, Bd5998, Bd4087, Bd8706 and Bd4188 (Fig. 3A). The brief description of the different mutations is given Fig. 3 A and a schematic representation of these mutations along their respective protein is presented Fig. 3 B.

Scanning

Supplementary figure S1: NBCL protein consensus

The alignment of 248 NBCL proteins was performed using the Multiple Sequence Alignment online software Clustal Omega (ebi.ac.uk/Tools/msa/clustalo) and the protein sequence consensus was obtained using the multiple alignment software Multalin (http://multalin.toulouse.inra.fr/multalin/). High (black box) and low (grey box) consensus levels was set at 90% and 50%, respectively. Consensus symbols: !, is anyone of IV; $, is anyone of LM; %, is anyone of FY; # is anyone of NDQEBZ. Initially, 485 NBCL sequences were obtained from 1KP project BLAST search (https://db.cngb.org/blast4onekp/) and after removing partial or incomplete sequences, 187 NBCL sequences were retained to establish the NBCL protein consensus. In addition, 61 other full-length NBCL protein sequences from NCBI (https://www.ncbi.nlm.nih.gov/) were used. BdNPR5-LIKE (Bd8595, Bd7526, BdBd4982, Bd7965) and BdNPR6-LIKE (Bd7392, Bd5998) KO mutations (asterisks) and missense mutations (triangles) located in conserved amino acid positions are indicated above the NBCL consensus sequence. The BTB/POZ domain and ANKYRIN repeats are underligned by red and blue lines and defined according to [START_REF] Hepworth | BLADE-ON-PETIOLE -Dependent Signaling Controls Leaf and Floral Patterning in Arabidopsis[END_REF]

The BdNPR5-LIKE gene is involved in the ligule and the auricles formations

In grass, the junction between the basal sheath and the distal leaf blade is called the blade-sheath boundary region or ligular region. This region is located between the top of the sheath and the base of the leaf blade. These two different organs are separated and articulated by two wedge-like structures called auricles. The ligule is another organ which extends from the adaxial surface of the blade-sheath boundary and wraps the culm in Bd21-3 wild-type plant [START_REF] Sylvester | Division and differentiation during normal and liguleless-1 maize leaf development[END_REF]) (Fig. 5 A,D,G).

The second striking phenotype of the two Bdnpr5-like KO mutant lines (families Bd4982 and Bd7965) is the complete lack of both the ligule and auricles tissues. As described above, in Bd21-3, auricles and ligules are well differentiated (Fig. 5 A,D,G) but in both Bdnpr5-like KO mutant lines the ligule does not develop and only a fringe of differentiated tissue is visible (Fig. 5 B,C,E,F). The auricles are also absent resulting in an almost continuous structure between the sheath and the leaf blade (Fig. 5 B,C,E,F,H,I). This Despite that this spikelet indeterminacy resulted in an increase in florets number per spikelet, the seed yield per plant was not significantly increased in the Bdnpr5-like KO mutant lines probably as the consequence of the reduced number of secondary tillers (Sup.

Bdnpr5-like mutants are not impaired in seed abscission ability

NBCL genes are involved in the abscission process of different organs in various plant species such as Brassicaceae, Solanaceae and Papillonoidaceae [START_REF] Mckim | The BLADE-ON-PETIOLE genes are essential for abscission zone formation in Arabidopsis[END_REF]Wu et al., 2012;[START_REF] Couzigou | NOOT-dependent control of nodule identity: nodule homeosis and meristem perturbation[END_REF]. In B. distachyon, the KO mutations in BdNPR5-like gene do not alter seed shattering (Fig. 7 A,B,C). The seeds of Bdnpr5-like KO mutants do not show any morphological difference (Fig. 7 D,E,F) and the seed abscission zone does not present any sign of mis-formation. The abscission zones of both Bd21-3 and Bdnpr5-like KO mutants look-like identical, are clearly separated and a sharp scars remains after seed shatering (Fig. 7 G, H, I). Thin sections of Bd21-3 and Bdnpr5-like mutants seed abscission zones also clearly Supplementary figure S7: Bdnpr6-like missense mutant line Bd5998 floral reproductive organ modifications Reproductive organ organisation of wild-type Bd21-3 (A, F) and Bdnpr6-like missense mutant line Bd5998 (B, C, D, E, G, H, I, J) during anthesis of 40 days-old plants. Pictures from A to E and picture from F to J represent adaxial and abaxial views respectively. Wild-type Bd21-3 reproductive organ are usually composed by a feathery stigma, an ovary, two adaxial stamens and two abaxial lodicules (A, F). In Bdnpr6-like missense mutant line Bd5998 (carrying Leu365Phe mutation), reproductive organ organization is affected. In the pictures B, G, the lodicules of the mutant show a stamen identity characterized by a yellow color indicated by white arrowheads. In adaxial position, one or two supplementary organs are visible (white asterisks), in C, H. These organs do not seem to have a defined identity. In D, I one of the two supplemental organs displays a stamen-like identity and in E, J the two additional organs show a stamen-like identity. sti, stigma; ov, ovary; sta, stamen; lo, lodicule. Scale bars: 500µm. AXM/PBM later differentiate into terminal spikelet meristem (TS) and spikelet meristems (SM) respectively. TS and SM finally produce flower meristems [START_REF] Kellogg | Early inflorescence development in the grasses (Poaceae)[END_REF]. The B. distachyon unbranched spike is composed by a terminal spikelet and several lateral spikelets which hold in average 10-12 florets organized in distichous phyllotaxy along the central rachis [START_REF] Draper | Brachypodium Distachyon, a new model system for functional genomics in grasses[END_REF][START_REF] Kellogg | Early inflorescence development in the grasses (Poaceae)[END_REF][START_REF] Derbyshire | MORE SPIKELETS1 is required for spikelet fate in the inflorescence of Brachypodium distachyon[END_REF].

In the present study, the Bdnpr5-like null mutant alleles show a particular phenotype that was not described in the barley Hvcul4 mutant. It consists in an exaggerated and a continuous production of florets on both terminal and lateral spikelets. This floret overproliferation on spikelets present in B. distachyon but absent in barley, might be explained by subtle differences in organogenesis existing among the unbranched spike-making species.

The barley IM is indeterminate and no terminal spikelet formed, each AXM or primary branch gives three determinate flowered-spikelets that give one or three florets in two-rowed and six-rowed barley, respectively [START_REF] Bonnett | The development of the barley spike[END_REF][START_REF] Moncur | Floral initiation in field crops: An atlas of scanning electron micrographs[END_REF][START_REF] Babb | Genetic and morphological characterization of the barley uniculm2 (cul2) mutant[END_REF][START_REF] Kellogg | Early inflorescence development in the grasses (Poaceae)[END_REF][START_REF] Koppolu | Six-rowed spike4 (Vrs4) controls spikelet determinacy and row-type in barley[END_REF]. In contrast to barley, the wheat IM is of the determinate type and shifts to a spikelet meristem identity. This differentiation fixes the number of spikelets that will be formed. Each AXM or primary branch gives a single indeterminate spikelet producing 6 or more florets [START_REF] Bonnett | The development of the wheat spike[END_REF][START_REF] Moncur | Floral initiation in field crops: An atlas of scanning electron micrographs[END_REF]. B. distachyon is more similar to wheat, since the IM is also determinate and presents a spikelet meristem differentiation. Each AXM or primary branch gives a single indeterminate spikelet producing 10-12 florets [START_REF] Kellogg | Early inflorescence development in the grasses (Poaceae)[END_REF][START_REF] Derbyshire | MORE SPIKELETS1 is required for spikelet fate in the inflorescence of Brachypodium distachyon[END_REF]. [START_REF] Dobrovolskaya | FRIZZY PANICLE Drives Supernumerary Spikelets in Bread Wheat[END_REF], the Triticum eastivum TaFRIZZY PANICLE (TaFZP, [START_REF] Dobrovolskaya | FRIZZY PANICLE Drives Supernumerary Spikelets in Bread Wheat[END_REF], the Zea mays ZmBRANCHED SILKLESS1 (ZmBD1, [START_REF] Chuck | The control of spikelet meristem identity by the branched silkless1 gene in maize[END_REF][START_REF] Komatsu | FRIZZY PANICLE is required to prevent the formation of axillary meristems and to establish floral meristem identity in rice spikelets[END_REF] and the Oryza sativa OsFRIZZY PANICLE (OsFZP, Komatsu et al., 2003) are orthologous genes encoding APETALA2/Ethylene Response Factor transcription factors (AP2/ERF TF) that are important regulator of the spike architecture conserved among grasses. These AP2/ERF TFs are involved in the maintenance of the spike architecture through the control of spikelet number and development and the control of the floret meristem identity. Basically, these mutants show a spikelet to branch identity conversion. These branches look-like wild-type spikes, forming multiple lateral spikelets and one terminal spikelet. In B. distachyon, Bdmos1 mutation finally results in a ramified B. distachyon spike that recalls rice inflorescence architecture. Since Bdmos1 is described as producing supernumerary spikelets [START_REF] Derbyshire | MORE SPIKELETS1 is required for spikelet fate in the inflorescence of Brachypodium distachyon[END_REF][START_REF] Dobrovolskaya | FRIZZY PANICLE Drives Supernumerary Spikelets in Bread Wheat[END_REF], it would be interesting to generate the double mutant Bdmos1/npr5-like, that should result in a more productive B. distachyon in which both spikelets and florets will be supernumerary. The barley Hvlax-a mutant presents also a homeotic conversion of lodicules into stamen-like structures [START_REF] Jost | A homolog of BLADE-ON-PETIOLE 1 and 2 (BOP1/2) controls internode length and homeotic changes of the barley inflorescence[END_REF]. Bdnpr6-like mutant shows a less severe but similar phenotype. Indeed, while Bd21-3 lodicules are normally completely flat and translucent, the Bdnpr6-like lodicules are thicker, narrower and present a yellow coloration suggesting a partial stamen-like identity. By contrast to Hvlax-a, Bdnpr6-like does not exhibit a complete conversion of lodicule into stamen structures. On the other hand, in adaxial position of the Imaging, light microscopy and sample preparation. Pictures of whole organs were acquired using a Nikon AZ100 Macroscope (http://www.nikoninstruments.com). Sample sections embedded in Technovit resine were treated essentially as described in [START_REF] Van De Velde | Aging in legume symbiosis. A molecular view on nodule senescence in Medicago truncatula[END_REF]. In brief, fixed samples were infiltrated 15 min under vacuum (≈500mm Hg) in sodium cacodylate buffer pH: 7 (0,05 M), glutaraldehyde 1 % and formaldehyde 4% and amplified using oligonucleotides BdNPR5_Ex2.2_F2 and BdNPR5_Ex2.2_R2 allowing the identification of the tilling missense mutant lines Bdnpr5-like (Bd5567 M3).

The

Material and methods

B. distachyon ecotype

The first part of the second exon of the BdNPR6-LIKE gene (Bd4g43150.1) was amplified using oligonucleotides BdNPR6 Ex2.1 F2 and BdNPR6 Ex2.1 R1, allowing the identification of the tilling missense mutant lines Bdnpr6-like (Bd7392 M3, Bd8485 M3). The second part of the second exon of the BdNPR6-LIKE was amplified using oligonucleotides BdNPR6_Ex2.2_F1 and BdNPR6_Ex2.2_R1, allowing the identification of the tilling missense mutant lines Bdnpr6-like (Bd4087 M3, Bd4188 M3, Bd5998 M3 and Bd8706 M3).

PCR were carried-out using High Fidelity Taq DNA polymerase (GAETHF004D, EUROBIO) and PCR products were sequenced by Sanger (http://www.eurofinsgenomics.eu) and analyzed with A plasmid Editor 2.0.49 software. Oligonucleotide sequences and PCR product length are described in Sup. Table 3.

Phylogenetic tree construction. Full-length NBCL protein sequences from dicotyledon plants and grasses were obtained by BLAST search on NCBI and Phytozome v12.1 (https://blast.ncbi.nlm.nih.gov/; https://phytozome.jgi.doe.gov/). NBCL protein sequences were aligned using ClustalW [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice[END_REF] and the phylogenetic tree was built using maximum likelihood-based method using the Jones-Taylor-Thornton (JTT) substitution model with the MEGA6 program [START_REF] Tamura | MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0[END_REF]. The maximum-likelihood tree was evaluated using 1000 bootstrap replicates.

Gene Expression

Analyses. The BdNPR5-LIKE (Bd2g60710.1) and BdNPR6-LIKE (Bd4g43150.1) transcripts accumulation were assessed in different organs of 40 days old wild type B. distachyon cv. Bd21-3. Total RNA were isolated using Trizol © reagent, (http://www.invitrogen.com) from three independent collects of root, sheath, node, ligular region, leaf blade, rachis node, rachis internode and spikelet (1-1,5 cm). RNA samples were treated with the TURBO DNA-free™ Kit (Ambion) according to the manufacturer's recommendations. Full-length cDNA were synthesized from 1 µg of RNA template using Superscript II reverse transcriptase (Invitrogen) in presence of Ribolock RNase Inhibitor (Thermoscientific). Real-time RT-PCR reactions were performed using the LightCycler 3.

Protein sequence pairwise levels of similarity/identity. Similarity/identity matrices of protein sequences were generated using Matrix Global Alignment Tool (MatGAT; [START_REF] Campanella | MatGAT: An application that generates similarity / identity matrices using protein or DNA sequences[END_REF]; available at www.angelfire.com/nj2/arabidopsis/MatGAT.html).

Supplementary table S3: Primers used for TILLING-NGS screen, genotyping and gene expression

Underlined sequences represent the 5' 26 bp illumina adaptators.