Théophile Kazmierczak

Li Yang

Eric Boncompagni

Eliane Meilhoc

Florian Frugier

Pierre Frendo
email: pierre.frendo@univ-cotedazur.fr

Claude Bruand

Véronique Gruber

Renaud Brouquisse
email: renaud.brouquisse@inra.fr

Camille Syska

Isabelle Garcia

Maria Jose Soto

Viktor E Tsyganov

Legume nodule senescence: a coordinated death mechanism between bacteria and plant cells

Keywords: Sénescence nodulaire, Glutathion, Cystéine protéase, Régulation transcriptionnelle, Eléments cis-régulateur Nodule senescence, Glutathione, Cysteine protease, Transcriptional regulation, cis-regulatory element Medicago truncatula, Sinorhizobium meliloti SmgshB, glutathione, bacteroid differentiation, senescence Medicago truncatula, nodule senescence, cysteine proteases, cis-regulatory elements, transcriptional regulation

DE THÈSE

Les légumineuses peuvent s'associer aux rhizobia pour développer de novo un organe racinaire, la nodosité racinaire. La nodosité racinaire peut réduire l'azote atmosphérique en azote disponible pour l'hôte végétal. Ainsi, la symbiose fixatrice d'azote joue un rôle important dans l'agriculture avec des apports directs d'azote aux plantes cultivées.

Dans la première partie de la thèse, nous avons caractériser l'impact de la carence bactérienne en glutathion sur la différenciation bactérioïde et le fonctionnement des nodules lors de l'interaction symbiotique entre Medicago truncatula et Sinorhizobium meliloti. Des marqueurs physiologiques, biochimiques, cellulaires et génétiques ont été utilisés pour décrire le nodule fonctionnant dix et vingt jours après l'inoculation de la plante. Nos résultats ont montré que la carence bactérienne en glutathion n'affecte pas la différenciation bactérioïdienne. Cependant, elle induit un processus précoce de sénescence des nodosités chez M. truncatula.

Au cours de la sénescence des nodosités, les activités protéolytiques sont augmentées et se terminent par la dégradation finale des bactéroïdes et des cellules végétales. Par conséquent, les protéases se révèlent être les enzymes caractéristiques de la sénescence des nodosités. Au début de la sénescence nodositaire, une cystéine protéase de la famille des papaïnes, MtCP6 est un bon marqueur moléculaire pour l'initiation de la sénescence des nodules. En ce qui concerne la deuxième partie de la thèse, une analyse consistant en une série de délétion du promoteur de MtCP6 a été réalisée pour identifier les séquences régulatrices présentes sur le promoteur MtCP6 et impliquées dans la sénescence nodulaire.

Afin de comprendre la régulation transcriptionnelle de la sénescence nodositaire chez M. truncatula. Ensuite, la région cis-régulatrice identifiée (67 pb, NS-box) a été validée pour fonctionner dans l'activation de la transcription dans la zone nodulaire III-IV. Nous avons pu montrer que les tétramères de NS-box peuvent induire la transcription dans la zone de sénescence de la nodosité. Afin de déterminer l'importance de NS-box par rapport au promoteur complet (-1720 pb), une analyse fonctionnelle a été réalisée avec suppression de la boîte NS sur le promoteur complet et sur le promoteur minimal (-242 pb). De plus, la validation les rôle potentiel de motif CW localisés dans la séquence NS-box en 5' a été réalisée à l'aide de délétions sites spécifiques. Finalement, la technique de "yeast-one-hybrid" a été utilisée pour identifier des facteurs de transcription interagissant avec le fragment NS-box. Les résultats préliminaires sont présentés.

Dans leur ensemble, les résultats permettent une meilleure compréhension de la régulation et du fonctionnement de la sénescence nodulaire.

Every step I have taken leads the way to the end of my thesis. I am indebted to so many people who have greatly supported me for the last four years. Language here is such a symbolic abbreviation which could not encapsulate all my grateful sensations.

First of all, I would like to thank the members of my thesis jury: Pr. Paola Furla, Dr. Éric Giraud, Dr.

Andreas Niebel and Dr. Florian Frugier, for accepting the invitation of my PhD defence. All the jury members evaluated my thesis with scientific scrutiny, which also allowed a lively discussion in my defence.

I would show my foremost gratitude to my supervisor Prof. Pierre Frendo for accepting me to do PhD research in Symbiose team. He has been very responsible and committed in supervising me throughout my PhD. I am very grateful for the time he has invested in my PhD thesis, my research work and training me to be a scientist. He is also very considerate to include the GSH project in the course my PhD to guarantee enough workload for my graduation. Finally, it gives a great opportunity so that I could develop some knowledge of symbiosis based on observation rather than merely reading. He has also very clear thinking in science that guides me out of many subtleties.

My greatest acknowledgement is undoubtedly to my supervisor Dr. Éric Boncompagni. He has been the one always encouraging and supporting me with his positive thinking and inspiring instructions. He is the first one showing me all the manipulation basics in the lab and always providing me his greatest help. He has been harbouring his warming heart that healing me even during the downs of my PhD.

Life is a long journey, and I am so grateful that I was once here meeting someone like him. He has given me countless opportunities to develop as a scientist as well as to follow my own willingness.

I am so obliged to Dr. Fernanda de Carvalho-Niebel for all the inspiring communications and detailed supervision in Toulouse, as well as the manuscript modifications. It was a great opportunity to collaborate in her lab in Auzeville where I conducted the yeast screening. She is holding the symbol of wisdom in Chinese Zodiac, providing wise ideas in research and efficient communications in science.

She has put forward the project with considerable contributions.

Another one I would thank in Toulouse is Lisa Frances. She is so kind herself and has helped me with many practical considerations. She has facilitated the research with her skills and commitments, which has ensured the experiments (yeast and GUS fluorimetric assay) work out efficiently. I am grateful for all the other members in the ENOD team and have enjoyed the time with a collaborative and motivating environment.

Particular thanks to Dr. Andreas Niebel for all his help and the kind gift (cDNA library for screening). He has also offered me the help in spelling and grammar modifications as a reporter of the thesis jury.

Many thanks for the dedicated language checking and scientific instructions.

I would show my sincere appreciation to Dr. Florian Frugier, who has enlightened the project with expertise suggestions as well as multiple practical considerations following my PhD project and in my PhD defence. I have been greatly inspired by his research ideas.

Figure list

(not including figures in publications/manuscript) Figure 24 The feedforward loop, single-input module (SIM) and dense-overlapping regulons (DOR) found in transcription networks. Figure 30 Yeast-one-hybrid (Y1H) screening strategy to identify NS-box interacting proteins.

Figure 31

Evaluating the specificity of the interaction of the erf candidates to the 4x NS reporter in yeast.

Figure 32 MtERF091 (MtERF1-1) homologues function in leguminous plants.

Figure 33 Analysis of the DNA-binding domain (ERF domain) from the ERF candidates and the homologs in Arabidopsis.

Figure 34 A GCCcore-like motif is present in NS-box.

Figure S2

Heterogenous nodule size appears at 20-and 28dpi following declining nitrogen fixation capacity.

Figure S3

Kinetics of histochemical localization of β-glucuronidase (GUS) activity in nodules carrying promoter-GUS constructions for the MtCP6.

Figure S4

Putative cis-regulatory elements present in the minimal functional MtCP6 promoter (-242bp).

Figure S5 Re-growth and yeast colony PCR of Y1H candidates.

Figure S6

Alignment of ERF candidate sequences (translated protein sequences) to the identical putative ERFs (protein sequence from Mt4.0).

Figure S7

Expression profile of ERF-091, -092, -069 and -073 on MTGEA: M. truncatula gene expression atlas / Noble foundation (https://mtgea.noble.org/v3).

Table list

(not including tables in publications/manuscript)

Table 1 Examples of organisms that can carry out nitrogen fixation.

Table 2 Associations between rhizobia and host plants.

Table 3 Cell types of developmental nodule senescence.

Table 4 Comparison of developmental and dark stress induced nodule senescence.

Table 5 Relative expressions of cysteine protease encoding genes and putative DNA-binding protein encoding genes during root nodule senescence.

Table 6 Phenotypes of GSH deficient rhizobia strains.

Table 7 Cysteine protease genes of M. truncatula involved in nodule senescence with different ID.

Table 8 List of AD in-frame His+ candidates.

Table S1 Strains and plasmids used during the course of this thesis.

Table S2 Examples for BP and three-entry multisite LR reaction mix.

Table S3 List of primers used in yeast-one-hybrid screen strategy Table S4 Web-based resources for plant cis-regulatory sequence analysis used in the course of this thesis.

Smoothest ice A paradise

To him, who is a dancer nice Friedrich Nietzsche

GENERAL INTRODUCTION

I. Nitrogen nutrition

I.1 Anthropogenic changes to the global nitrogen cycle

Nitrogen (N) is one of the primary nutrition elements essential for life, as component of many organic molecules in cell, particularly: amino acids (proteins), nucleic acids (DNA and RNA), and macromolecules such as chlorophyll. In fact, there is an enormous N stock in the ecosphere, but mostly unavailable as in atmospheric dinitrogen (N2), accounting for 78% of Earth's atmosphere [START_REF] Stevens | Nitrogen in the environment[END_REF], or deeply in rocks and sediments. Thus available N for the plant is often limiting in natural conditions, albeit the global N status is changing with increasing rates of N production, N deposition and agricultural N application [START_REF] Reay | Global nitrogen deposition and carbon sinks[END_REF].

The global N production available for use by living organisms is either achieved by natural processes such as lightning and biological N fixation (BNF) or anthropogenic activities like the Haber Bosch process, in a reactive form (Nr) which includes inorganic reduced forms of N (e.g., NH3 and NH4 + ), inorganic oxidized forms (e.g., NOx, HNO3, N2O, and NO3 -), and organic compounds (e.g., urea, amines, and proteins), by contrast to unreactive N2 gas [START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions, and potential solutions[END_REF] (Figure 1). Human activities transform the global N cycle over the past century, bringing about a doubled global N cycling in comparation to natural N production (anthropogenic Nr production: 210 Tg N per year, natural Nr production: 203 Tg N per year) [START_REF] Fowler | The global nitrogen cycle in the twenty-first century[END_REF]. The changes in Nr production are reflecting an increasing combustion of fossil fuels, high N demand in agriculture and industry, and inefficient N utilization [START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions, and potential solutions[END_REF]. Excessive N is largely lost into land, water and air, causing a cascade of environmental problems.

The rising Nr emissions is mostly thought to be deposited on terrestrial ecosystems, in addition to the open ocean and coastal zones [START_REF] Reay | Global nitrogen deposition and carbon sinks[END_REF]. For example, from 1900 to 1980, deposition of oxidized N (such as NOX and N2O) in Europe increased by three to four times, whereas deposition of reduced N (such as ammonia, NH3) doubled [START_REF] Engardt | Deposition of sulphur and nitrogen in Europe 1900-2050. Model calculations and comparison to historical observations[END_REF]. However recently, the N concentrations in plant tissues, often considered an indicator of the amount of N taken up by plants or their N status, have been recently shown declined globally between 1980 and 2017 despite globally increasing availability of N [START_REF] Craine | Isotopic evidence for oligotrophication of terrestrial ecosystems[END_REF], reflecting an inefficient N use. Intermediates, representing nine oxidation states, that donate or accept electrons, thereby contributing to electron flow and conservation of energy in participating microbes. Reactions that comprise the seven major processes of the nitrogen cycle are represented by the numbered circles. Ammonification may be accomplished either by process 1, reduction of dinitrogen (also referred to as 'nitrogen fixation' or 'Nif'), or by process 2, dissimilatory nitrite reduction to ammonium (DNRA). Nitrification is composed of process 3, oxidation of ammonia to nitrite (also referred to as 'nitritation'), and process 4, oxidation of nitrite to nitrate (also referred to as 'nitratation'). Process 5, reduction of nitrate to nitrite, can be coupled to processes 2, 6 or 7 in a population or a community. Denitrification is shown as process 6, which is also referred to as 'nitrogen-oxide gasification'. Anammox is shown as process 7 and is also referred to as coupled 'nitrification-denitrification'. (A) Global fertilizer consumption of primary nutrients (N, phosphate and potash) applied in agriculture. Source: IFASTAT https://www.ifastat.org/;

(B) Global population trends with key dates for the discovery of N as an element in the periodic table and its role in various biogeochemical processes. Also shown is an estimate of the annual production of Nr by the Haber-Bosch process. Adapted from [START_REF] Galloway | Reactive nitrogen and the world: 200 years of change[END_REF]. To feed a growing population which is already over 7 billion, high demand in food production requires intensive agriculture which is largely dependent on the application of inorganic fertilizers (Figure 2A). The agricultural production has been considerable promoted since the industrial revolution (~1840), especially after the invention of Haber Bosch process (Figure 2B) in 1913. As consequences, the change in Nr production is enormous from ~15 Tg N in 1860 to 156 Tg N in 1995 and further increased to 187 Tg N in 2005 [START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions, and potential solutions[END_REF]. The overuse of N fertilizers has led to series of negative impacts as in reduced biodiversity, green-house-gases emissions, polluted air and water quality, soil fertility degeneration and on human health risks.

Even though contemporary focus is on solving environmental problems induced by surplus fertilizers in developed countries, an increasing attention is also drawn on developing countries, wherein small-hold farmers are suffering from the lack of nutrient fertilizers [START_REF] Foley | Solutions for a cultivated planet[END_REF][START_REF] Mueller | Closing yield gaps through nutrient and water management[END_REF]. In these regions, scarce N availability intensifies to the existing food shortage problem mostly caused by failure of affording N fertilizers or due to ineffective N management. While N fertilizers may continue to dominate in intensive agriculture management for some time, a more sustainable agriculture system is ultimately required to keep the N cycle in reasonable balance for the sake of long-term utilization as well as world global conservation. Therein, we may need also in a short-term period of an integrated system using a precise management with high efficiency in N usage, as well as the combination of using biological N fixers such as legumes.

I.2 Plant nitrogen nutrition

As primary producers, plants rely on a large aboveground surface area to collect carbon dioxide and sunlight and a large underground surface area to collect the water and mineral nutrients needed to support their growth and development [START_REF] Oldroyd | A plant's diet, surviving in a variable nutrient environment[END_REF]. Plants optimize their uptake and utilization strategies according to nutrient availability in soil. Typically, roots are well developed in harsh conditions with limited soil nutrients, which keep a limited growth of the upper part resulted in a high root: shoot ratio. Conversely, when soil resources are sufficient, a minimum root system is maintained, and vegetative growth is extended.

A widely accepted view is that the epidermal and cortical cells of the root are the main sites of uptake of ions. A proportion of these ions is subsequently diverted into vacuoles of the cells of these regions, a proportion is consumed on growth of the root, while the remainder is free to diffuse inwards along concentration gradients in the root cytoplasm, through the endodermis, to cells in the stele engaged in loss of ions to the xylem. Under situations where plants are unable to access N and Pi from their immediate environment, they turn to these microorganisms to find new sources of these limiting nutrients. These processes regulate the plants' receptiveness to their microbial communities, promoting symbiotic associations.

Plant nitrogen uptake relies mainly on inorganic forms

Plants mostly uptake N in inorganic forms like nitrate in aerobic soils and ammonium in flooded wetlands or acidic soils (Figure 3) [START_REF] Marschner | Marschner's mineral nutrition of higher plants: Academic press[END_REF]Xu et al., 2012). In return, the resulted acidification or alkalization of the rhizosphere by N uptake changes the soil N availability for plants [START_REF] Marschner | Marschner's mineral nutrition of higher plants: Academic press[END_REF]. Alternatively, plants are proven to be able to utilize organic compounds, such as amides, amino acids. With nitrate and ammonium concentrations in soil solutions ranging from 100µM to 10mM, numerous membrane transporters was composed to cope with different N forms and concentrations (Figure 3) [START_REF] Miller | Nitrate transport and signalling[END_REF]Xu et al., 2012;[START_REF] O O'brien | Nitrate Transport, Sensing, and Responses in Plants[END_REF].

Plants take different routes for nitrogen transportation and storage

Upon absorption of different N sources, biosynthetic activities associated with N assimilation are also found varied in plant roots. Plants exhibit different distribution patterns of the transportation and storage in terms of N forms. In conditions where roots contain a large amount of N beyond its requirements, the remainder will be thereafter transported to shoots for utilization or storage. One extreme of nitrate response spectrum is in woody plants, in which nitrate reduction is being accomplished entirely by their roots, while it is impossible to detect free nitrate in the xylem but high levels of organic solutes of N are present [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF] (Figure 3). The other extremes are in plants such as Borago spp. and Xanthium pennsylvanjcum [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF], such species have roots genetically deficient in the ability to synthesize enzymes of the nitrate reduction pathway (Figure 3). Most herbaceous species like pea, however, localize in the middle of the spectrum, are able to maintain active reductases in both their root and shoot systems [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF].

Analyses of xylem and phloem contents suggest that the xylem supplies target organs with mostly nitrate and a limited range of amino acids, particularly the amide glutamine, whilst the phloem appears to deliver considerably less nitrate and amide but substantial amounts of sucrose and a wide range of amino acids at somewhat higher concentration than in the xylem [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF]. As consequences, xylem nutrition brings in nutrients having on average a C:N ratio of approximately 2:1, whilst in the phloem solutes are dispensed having a C/N ratio of approximately 22:1 [START_REF] Pate | Uptake, assimilation and transport of nitrogen compounds by plants[END_REF]. The leaves are the N target organs during vegetative stage and are turning to source organs during senescence to provide amino acids for seed production (Figure 3). Nitrogen uptake are mainly in the form of ammonium and nitrate by roots. The thicknesses of the arrows schematically represent the relative amounts of nitrogen and sugar inside the plant. Abbreviations: AMT, ammonium transporter; AS, asparagine synthetase; Asn, asparagine; Asp, aspartate; GDH, glutamate dehydrogenase; Gln, glutamine; Glu, glutamate; GOGAT, glutamine-2-oxoglutarate aminotransferase; GS, glutamine synthetase; NAC-TF, certain transcription factors belonging to the NAC family; NiR, nitrite reductase; NR, nitrate reductase; NRT, nitrate transporter.

The absorbed nitrate is partially reduced and assimilated in the roots, and the larger part is transported to the shoot, where it is first reduced to nitrite by nitrate reductase in the cytoplasm and then further to ammonium by nitrite reductase in the plastids and glutamine synthetase (GS) in the plastids and cytoplasm (Xu et al., 2012) (Figure 3). The reduction of nitrate to ammonium requires reducing power which is provided by photosynthesis which produces both energy (ATP) and reducing power (NADPH). Ammonium is further assimilated into amino acids catalysed by the GS/glutamine-2-oxoglutarate aminotransferase (GOGAT) cycle (Figure 3) (Xu et al., 2012). While GS and Asparagine synthetase (AS) are crucial in primary N metabolism, the mitochondrial NADH-glutamate dehydrogenase (GDH) can alternatively incorporate ammonium into Glu in response to high levels of ammonium under stress [START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF].

Nitrogen is reutilized via remobilization during plant senescence

The initiation of leaf senescence is considered as the transition from carbon to nitrogen source. During leaf senescence, internal redistribution of N occurs, and leaf proteins are degraded, particularly from chloroplast, and remobilized to other organs, e.g. seeds in the reproductive stage (Figure 3) [START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF]. In vegetative tissues, Rubisco enzyme is not only a photosynthetic enzyme, but also the repository of recoverable N, in which protein breakdown only starts after the synthesis in young growing leaves [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. The N remobilization is thus regulated sequentially by chloroplastic and vacuolar proteases [START_REF] Desclos | A combined 15N tracing/proteomics study in Brassica napus reveals the chronology of proteomics events associated with N remobilisation during leaf senescence induced by nitrate limitation or starvation[END_REF][START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF]Xu et al., 2012). During N remobilization of leaf senescence, a number of proteases encoding genes are strongly induced [START_REF] Masclaux-Daubresse | Nitrogen uptake, assimilation and remobilization in plants: challenges for sustainable and productive agriculture[END_REF].

Plants response to nitrogen availability

Plant nutrient is systematically regulated by an integrative root and shoot signalling system with a variety of hormones moving between the root and the shoot to both signal nutrient availability and coordinate plant development [START_REF] Oldroyd | A plant's diet, surviving in a variable nutrient environment[END_REF]. There are two scenarios in case of the nitrogen regulation: positive regulation by root N concentration and negative feedback by the shoots. The root response to N involves four signalling processes: local signalling in the root associated with perception of local N; root-shoot-root signalling indicating the presence of roots experiencing low N concentration; root-shoot-root signalling indicating the presence of roots experiencing locally high N concentration; and a systemic inhibitory signal that suppresses root N-foraging activities when shoot N concentration is sufficient [START_REF] Oldroyd | A plant's diet, surviving in a variable nutrient environment[END_REF].

Nitrate response has been well studied in plants. There are two types of nitrate transporters in higher plants: NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters (Tsay et al., 2007). An exception is a plasma membrane transporter NRT1.1 (CHL1), which is a master gene in apical part of root [START_REF] Dechorgnat | From the soil to the seeds: the long journey of nitrate in plants[END_REF], playing roles as nitrate transporter and nitrate sensor of activating the Arabidopsis Nitrate Response 1 (ANR1) to promote nitrate-stimulated lateral root proliferation [START_REF] Zhang | An Arabidopsis MADS box gene that controls nutrient-induced changes in root architecture[END_REF][START_REF] Remans | The Arabidopsis NRT1.1 transporter participates in the signaling pathway triggering root colonization of nitrate-rich patches[END_REF][START_REF] Ho | CHL1 functions as a nitrate sensor in plants[END_REF]. Two low-affinity nitrate transporters in Arabidopsis (AtNRT1.5 and AtNRT1.8) loading and unloading into the root stele or from the shoot vasculature [START_REF] Lin | Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport[END_REF][START_REF] Li | The Arabidopsis nitrate transporter NRT1.8 functions in nitrate removal from the xylem sap and mediates cadmium tolerance[END_REF], and AtNRT1.9 in root companion cells facilitates the loading of nitrate into the root phloem [START_REF] Wang | Arabidopsis nitrate transporter NRT1. 9 is important in phloem nitrate transport[END_REF].

The ammonium transporters (Amt/MEP/Rh family) is widely distributed in nature with high affinity to facilitate the growth on very low concentrations (<5µM) of ammonium salts [START_REF] Khademi | Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å[END_REF]. In high external ammonium concentration, modification of the cytosolic C-terminus of AtAMT1.1 and AtAMT1.2 allows rapid inactivation of the trimer complex to prevent toxic ammonium accumulation [START_REF] Loque | A cytosolic trans-activation domain essential for ammonium uptake[END_REF] [START_REF] Neuhäuser | Regulation of NH4+ transport by essential cross talk between AMT monomers through the carboxyl tails[END_REF].

Ammonium is also shown to trigger lateral root development mediated by AMTs (e.g.,

LjAMT1

;3) [START_REF] Lima | Ammonium triggers lateral root branching in Arabidopsis in an AMMONIUM TRANSPORTER1;3-dependent manner[END_REF]. In Arabidopsis thaliana, the GDP mannose pyrophosphorylase (GMPase) confers a hypersensitivity to NH4 + [START_REF] Qin | GDP-mannose pyrophosphorylase is a genetic determinant of ammonium sensitivity in Arabidopsis thaliana[END_REF].

II. Biological nitrogen fixation (BNF)

II.1 Biological nitrogen fixers

Another major source of N production on earth is from the BNF [START_REF] Burris | Biological nitrogen fixation[END_REF].

N fixation by BNF was estimated to contribute a total of approximately 50-70 Tg N in agricultural systems [START_REF] Herridge | Global inputs of biological nitrogen fixation in agricultural systems[END_REF], and accounting for 60% of the earth N fixation [START_REF] Zahran | Rhizobium-legume symbiosis and nitrogen fixation under severe conditions and in an arid climate[END_REF][START_REF] Canfield | The evolution and future of Earth's nitrogen cycle[END_REF]. BNF is less environmentally problematic as the fixed N are used in situ. The major limitation is that BNF has not found in Eukaryotes, but it is widely distributed in bacteria and Archaea, namely diazotrophic organisms [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF].

They are evolved to use nitrogenase (encoding by nif genes) in fixing dinitrogen to ammonia into biosphere, in phylogenetic groups including all subdivisions of the Proteobacteria, green sulphur bacteria (Chlorbi), Firmibacteria, cyanobacteria and Archaea (Figure 4) [START_REF] Franche | Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants[END_REF]. A larger part of the N fixing Prokaryotes are free-living (nonsymbiotic) in the soil (Table 1), including cyanobacteria, Anabaena and genera such as Azotobacter, Beijerinckia, Clostridium [START_REF] Franche | Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants[END_REF]Taiz et al., 2015). The ability to form a N-fixing symbiosis appears to have evolved relatively recently in land plants, approximately 65 million years ago [START_REF] Doyle | Phylogenetic perspectives on the origins of nodulation[END_REF]. In the N-fixing organisms, diverse physiologies are found (Table 1) (Taiz et al.,

2015):

i) Aerobic: which maintain reduced O2 conditions (microaerobic conditions) through their high levels of respiration (e.g., Azotobacter). Others, such as Gloeothece, evolve to produce O2 photosynthetically during the day and fix nitrogen during the night.

ii) Facultative: which are able to grow under both aerobic and anaerobic conditions, generally fix nitrogen only under anaerobic conditions (e.g., Klebsiella).

iii) Anaerobic: in which O2 does not pose a problem, because it is absent in their habitat.

These anaerobic organisms can be either photosynthetic (e.g., Rhodospirillum), or nonphotosynthetic (e.g., Clostridium).

Alternatively, some other bacteria form host-specific mutualistic associations with higher plants, e.g. Rhizobia & leguminous plants, Frankia & dicotyledonous species (actinorhizal plants) (Table 1) [START_REF] Franche | Nitrogen-fixing bacteria associated with leguminous and non-leguminous plants[END_REF]Taiz et al., 2015). In the case of Gunnera, these organs are existing stem glands that develop independently of the symbiont. In the case of legumes and actinorhizal plants, the N-fixing bacteria induce the plant to form root nodules. (Taiz et al., 2015) In addition, sugarcane and the tiny water fern Azolla can also form associations. with the cyanobacteria Nostoc and Anabaena, respectively (Taiz et al., 2015). N fixing shrub such as gorse, broom, etc. are often pioneers stabilizing soils and increasing N content. Amongst the symbiotic interactions, the actinorhizal symbioses are mutualistic relationships between the actinomycete genus Frankia and a number of dicotyledonous plant genera belonging to eight diverse plant families. The symbiosis occurs in a new root organ called the root nodules. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. The actinorhizal plants share a predilection for marginally fertile soils and the majority are pioneers on nitrogen-poor sites. In addition, many actinorhizal species are able to tolerate environmental stresses such as high salinity, drought, and cold. They inhabit a variety of ecosystems, including coastal dunes, alpine communities, arctic tundra, and forests. Actinorhizal plants are especially important in high latitude regions, such as Scandinavia, Canada, and Alaska, where actinorhizal plants are capable of vigorous growth (Wall, 2000). Much of the new nitrogen entering these ecosystems comes from the actinorhizal symbioses that, on the whole, account for over 15% of the biologically fixed nitrogen worldwide.

II.2 Legume-Rhizobia symbiosis

The most studied nitrogen-fixing symbiosis is between Fabaceae and rhizobia (Azorhizobium, Bradyrhizobium, Photorhizobium, Rhizobium, and Sinorhizobium) (Table 2) [START_REF] Poole | Rhizobia: from saprophytes to endosymbionts[END_REF]. Such symbioses lead to the formation of de novo organ, the root nodule.

Once the primary source of N for agricultural systems, Symbiotic Nitrogen Fixation (SNF) in legumes (approximately 50 million tons per year) contributes to less than half of what is provided by chemical fertilizer N [START_REF] Canfield | The evolution and future of Earth's nitrogen cycle[END_REF]. Legumes have evolved about 60

Million years ago. However, even if several lineages are evolved with N-fixing symbiosis, not all legumes are undergoing N-fixing symbiosis and each legume species has its own cognate rhizobia partners.

Nitrogen fixing symbiosis with leguminous plants (Leguminosae)

Leguminosae (Fabaceae) is the third largest angiosperm family with nearly 770 genera and over 19,500 species [START_REF] Azani | A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny: The Legume Phylogeny Working Group (LPWG)[END_REF]. As for economic importance, the legume family is second after Poaceae, providing nutrition sources to benefit health and livelihoods.

Legumes are long-recognized with three subfamilies: Caesalpinioideae, Mimosoideae, and Papilionoideae. However recently, a new classification of six subfamilies: Cercidoideae, Detarioideae, Duparquetioideae, Dialioideae, Papilionoideae and the recircumscribed Caesalpinioideae, is published on agreement of the legume systematics community based on emerging updating phylogenetic analyses [START_REF] Azani | A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny: The Legume Phylogeny Working Group (LPWG)[END_REF]. Papilionoideae is the largest subfamily with 14 000 species (Figure 5) [START_REF] Azani | A new subfamily classification of the Leguminosae based on a taxonomically comprehensive phylogeny: The Legume Phylogeny Working Group (LPWG)[END_REF], wherein we found many nodulated legume species such as Glycine max (soybean), Phaseolus vulgaris (common bean), Cicer arietinum (chickpea), Vicia faba (broad bean) and Pisum sativum (pea), as well as two well-studied model legumes (Lotus japonicus and Medicago truncatula) (Figure 5). Depicted on a 95% majority-rule Bayesian consensus tree based on analysis of peptide sequences from 81 plastid encoded proteins, subsampling representative taxa from forthcoming phylogenomic analyses.

(Figure 6) [START_REF] Masson-Boivin | Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes?[END_REF]. Most of these bacterial species are in the Rhizobiaceae family in the a-proteobacteria and are in either the Rhizobium, Mesorhizobium, Ensifer (Sinorhizobium), or Bradyrhizobium genera. All a-rhizobial genera belong to the Rhizobiales order, whereas b-rhizobial genera belong to the Burkholderiales order. It is important to note that there are other non-rhizobial species present in these genera. For example, in the Rhizobium genus, there is a well-known non-nitrogen-fixing Agrobacterium tumefaciens.

Figure 6 Unrooted phylogenetic tree of 16S ribosomal DNA sequences from selected a-, b-and g-proteobacteria.

Adapted from (Masson- [START_REF] Masson-Boivin | Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes?[END_REF].

Genera in bold font contain rhizobia. Rhizobial species or strains whose genomes have been fully sequenced are indicated between brackets. Genome sequences have been published for C. taiwanensis, M. loti, S. meliloti, R. leguminosarum, R. etli, A. caulinodans, Sinorhizobium sp. strain NGR234, B. japonicum and Bradyrhizobium sp. BTAi1 and ORS278. Rhizobial genomes with sequencing projects in progress are indicated in square brackets.

III. Medicago truncatula and Sinorhizobium meliloti symbiotic model system

III.1 Medicago truncatula

M. truncatula belongs to the galegoid clade (also known as the inverted repeat-lacking clade, IRLC). M. truncatula is the preeminent model legumes in the study of symbiosis of indeterminate nodules (for determinate: L. japonicus). It is autogamous, with a short generation time and large seed production. It is a diploid specie (2n=16) with a fully sequenced genome of approximately 500 Mb (http://data.kew.org/cvalues/) [START_REF] Arumuganathan | Nuclear DNA content of some important plant species[END_REF], making the genomic and genetic manipulations amendable. A knowledge-based database of this model legume is available at the website LeGOO (Legume Graph-Oriented Organizer) (https://www.legoo.org) (Carri [START_REF] Re | LeGOO: An Expertized Knowledge Database for the Model Legume Medicago truncatula[END_REF].

Medicago genome analysis

The Medicago genome sequencing was initiated in 2003 andpublished in 2011 (Mt3.5) based on BACs (bacterial artificial chromosomes) sequencing [START_REF] Young | The Medicago genome provides insight into the evolution of rhizobial symbioses[END_REF]. In total, there are 62,388 gene loci in Mt3.5, with 3,692 (5.9%) putative TF genes which are significantly nodule-enhanced [START_REF] Young | The Medicago genome provides insight into the evolution of rhizobial symbioses[END_REF]. A refined genome version (Mt4.0; Figure 7A) was released in 2014 using Illumina technology and ALLPATHS-LG assembly method [START_REF] Tang | An improved genome release (version Mt4. 0) for the model legume Medicago truncatula[END_REF]. A 30% additional sequences are encompassed in Mt4.0 (~360 Mb) than

Mt3.5 (246Mb). Mt4.0 includes 50,894 genes and is annotated with a hybrid approach of EST, RNA-seq, protein and gene prediction evidences [START_REF] Tang | An improved genome release (version Mt4. 0) for the model legume Medicago truncatula[END_REF]. The latest version of M. truncatula genome sequences (Mt5.0; Figure 7B) was released in 2018 and developed using PacBio sequencing by Laboratory of Plant-Microbe Interactions (LIPM) [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

Based on that coregulated genes often colocalize, symbiosis-related islands (SRIs) are proposed for the genomic clustering of coregulating genes, provoking SRIs upregulated in nodules or expressed in the nodule differentiation zone (270 and 211, respectively) [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. The nodule enhanced 270 SRIs are consist of 1,960 genes in total, with a mean size of ~46 kb with ~7.3 coregulated genes on average, representing ~87% of the expressed genes in the islands [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. Many nodule-associated genes are present within SRIs, e.g., nodule-specific cysteine-rich peptides (NCRs), glycine-rich proteins (GRPs) or calmodulin-like proteins (CaML), MtENOD11, MtSYMREM1, MtSYMCRK, MtIRE, as well as genes encoding ERF transcription factors [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. The coregulation of neighbouring gene sets are impacted by local chromatin structure determined by epigenetic regulators like DNA methylation status, small non-coding RNAs (ncRNAs) and histone marks [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. CHH (three-nucleotide cytosine context where H can be A, C or T) DMRs (differentially methylated regions) of the differentiation islands were found to be maximal at the gene promoter regions [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF]. The Mt5.0 genome browser integrates various data sources including omics, interactome and mutant collections. respectively) or differentially regulated between laser-dissected nodule zones (specifically expressed in the nodule apex or differentiation zone: yellow and light blue, respectively). [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

Gene expression analysis in the nodulation process

Transcriptome analyses have identified hundreds of plant and bacterial genes that are differentially expressed during nodule development and differentiation. Small-scale transcriptome studies have been carried out on different organs in multiple legume species under a variety of experimental conditions [START_REF] Becker | Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions[END_REF][START_REF] Colebatch | Global changes in transcription orchestrate metabolic differentiation during symbiotic nitrogen fixation in Lotus japonicus[END_REF] El et al., 2004;[START_REF] Mitra | Six nonnodulating plant mutants defective for Nod factor-induced transcriptional changes associated with the legume-rhizobia symbiosis[END_REF]Uchiumi et al., 2004). A more comprehensive Gene Expression Atlas (MtGEA; https://mtgea.noble.org/v3/) is used in transcriptomic analysis based on Affymetrix data of a wide range of biological conditions (Benedito et al., 2008).

A B

Yahyaoui

Organ-specific genes were largely uncovered in the nodules (1,354 genes) and seeds (3,228 genes) (Figure 8A), implying a peculiar function in the specialized organs (Benedito et al., 2008). RNAseq data was coupled with laser-capture-microdissected (LCM) approach (Figure 8B) to shed light on spatial analysis of plant and bacterial gene expression in different nodule zones (SYMbiMICS: https://iant.toulouse.inra.fr/symbimics/) (Roux et al., 2014). Differential and co-expressive analysis of the LCM RNAseq data highlighted 16 expression patterns illustrating successive waves of gene expressions involved from the nodule apical meristem to the N-fixation zone (Figure 8B) [START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

Figure 8 Gene Expression Atlas of M. truncatula.

A) Heat map of organ-specific genes. The colour scale indicates the number of times transcripts for a given gene were detected in the three biological replicates of each organ. Only those genes are shown for which transcripts were detected in all three biological replicates of one organ and no more than once in another organ. Adapted from (Benedito et al., 2008).

B) Hierarchical clustering analysis of genes exhibiting differential expression amongst five laser-dissected nodule zones. Lasermicrodissection of five regions from 15-day-old nodules. ZI: zone I, meristematic region; dZII: distal zone II, (pre)infection region; pZII: proximal zone II, early differentiation region; IZ: interzone II-III, late differentiation zone; ZIII: zone III, N-fixation zone. Source: Symbimics. Adapted from (Roux et al., 2014;[START_REF] Pecrix | Whole-genome landscape of Medicago truncatula symbiotic genes[END_REF].

Mutagenesis of Medicago truncatula

To date, genetics has been revolutionized thanks to the development of genetic tools, such as mutagenesis, gene expression and proteomics [START_REF] Roy | Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation[END_REF] [START_REF] Penmetsa | Production and characterization of diverse developmental mutants of Medicago truncatula[END_REF]Tadege et al., 2008;Pislariu et al., 2012;[START_REF] Roy | Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation[END_REF]. In model species like M. truncatula, forward and reverse genetics have accelerated the understanding of the molecular and cellular processes of the symbiotic relationship. However, reverse genetic approaches are limited to reveal gene functions when gene family is complex or abundant, e.g., the NCRs. T-DNA insertion is largely used mostly in laboratory small scale. M. truncatula mutant population at the Samuel Roberts Noble Foundation is the largest legume mutant collection and were generated using the tobacco retrotransposon Tnt1 (Tadege et al., 2008;Pislariu et al., 2012).

III.2 Establishment of symbiosis

Under N limited conditions, leguminous plants and rhizobia elaborate to develop de novo N fixing root organ, called the root nodule. In this mutualistic relationship, the plant provides the bacteria partner with photosynthates and protective niche, and in return, the symbiotic bacteria fix atmosphere N to supply the plant with ammonium. The lifespan of nodules is a cascade of the initial signal exchange, infection process, development of the functional N fixing nodules and senescence before the final organ death, regulated by specific genes from both the plant and bacteria side with precise timing.

III.2.1 Recognition of legumes and rhizobia

Symbiotic recognition initiates the nodulation process

Establishment of symbiosis requires recognition of both partners with specific signalling.

Under N starvation, plant emit metabolites (flavonoid) into rhizosphere that trigger the expression of bacterial nod genes into lipochitooligosaccharidic nodulation factors (Nod factors). The nod, nif and fix genes are located on large plasmids [START_REF] Kondorosi | Plasmids governing symbiotic nitrogen fixation[END_REF]. The function of nodulation (nod) genes has been extensively investigated by rhizobia mutants. The mutants impaired in the symbiotic interaction are found mostly Nod -(nodulation deficient) or

Fix -(impaired nitrogen fixation) [START_REF] Kondorosi | Plasmids governing symbiotic nitrogen fixation[END_REF]. Four common nod genes (nodABC and D) are conserved in most Rhizobium species and played a role determining root hair curling or nodule initiation, and some other genes for host specificity of nodulation (hsnABC and D; also called nodFEG and H or nodL, nodP and nodQ) differs in among rhizobia strains to determine the host range (Kondorosi & Kondorosi, 1986). The nod D, a LysR-type protein induced by the plant root secreted (iso)flavonoids and betaines [START_REF] Kobayashi | Flavonoids induce temporal shifts in gene-expression of nod-box controlled loci in Rhizobium sp. NGR234[END_REF], is constitutively expressed to regulate the transcription of all the nod gene units. Originally, dual transcription control is found with the activator nodD [START_REF] Rostas | Conservation of extended promoter regions of nodulation genes in Rhizobium[END_REF] binding to a 47bp cis-regulatory element (nod-box), and an occasional expressing repressor protecting a 21bp stretch downstream of the n6 nod-box [START_REF] Rostas | Conservation of extended promoter regions of nodulation genes in Rhizobium[END_REF][START_REF] Kondorosi | Positive and negative control of nod gene expression in Rhizobium meliloti is required for optimal nodulation[END_REF].

Nod factors accumulated in cell walls of root epidermis, perceived by plant receptors (Nod factor perception (NFP), or NFR1/NFR5 in L. japonicus) containing the chitin binding and LysM domain receptor kinases (e.g., LYK3 encoding by HCL gene) [START_REF] Madsen | A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals[END_REF][START_REF] Radutoiu | Plant recognition of symbiotic bacteria requires two LysM receptor-like kinases[END_REF][START_REF] Arrighi | The Medicago truncatula lysine motif-receptor-like kinase gene family includes NFP and new nodule-expressed genes[END_REF][START_REF] Broghammer | Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding[END_REF][START_REF] Moling | Nod factor receptors form heteromeric complexes and are essential for intracellular infection in Medicago nodules[END_REF]. Upon infection of rhizobia, specific plant proteins were found exclusively expressed in nodules [START_REF] Legocki | A nodule-specific plant protein (nodulin-35) from soybean[END_REF][START_REF] Legocki | Identification of "nodule-specific" host proteins (nodulins) involved in the development of Rhizobium-Legume symbiosis[END_REF], thereafter called nodulins, including early nodulins and late nodulins. Plant receptors are crucial for selection/host range determination [START_REF] Radutoiu | LysM domains mediate lipochitin-oligosaccharide recognition and Nfr genes extend the symbiotic host range[END_REF], collaborating with bacterial Nod factors. These receptor genes are also identified with plant mutants with the Nod -and Fix -phenotype, as well as defects in Nod factor responses [START_REF] Roy | Celebrating 20 Years of Genetic Discoveries in Legume Nodulation and Symbiotic Nitrogen Fixation[END_REF].

III.2.2 Bacterial infection is coupled with nodule organogenesis

To allow rhizobial infection, a tubular-like infection thread (IT) is developed by the plant to allow the entry of the entrapped bacterial microcolony present at the surface of the root hair.

The IT grows into root cortex wherein bacteria are released in the plant cell via endocytosis (Figure 9) [START_REF] Brewin | Plant Cell Wall Remodelling in the Rhizobium-Legume Symbiosis[END_REF][START_REF] Oldroyd | The rules of engagement in the legumerhizobial symbiosis[END_REF]. During the infection, cell divisions begin in the inner and subsequently mid-cortex and nodule primordium is thereby formed [START_REF] Schultze | Regulation of symbiotic root nodule development[END_REF]. Bacterial infection and nodule organogenesis are coordinated steps so that multiple genes are required for IT formation and nodulation.

Symbiotic signalling pathway

The plant receptor kinase complexes thus initiate nodule organogenesis in a successive signalling pathway controlling nodulation (Figure 10) [START_REF] Oldroyd | The rules of engagement in the legumerhizobial symbiosis[END_REF]Oldroyd, 2013). An activation of calcium oscillation in form of periodic spiking is believed to be as the centre of the symbiotic pathway [START_REF] Oldroyd | Calcium, kinases and nodulation signalling in legumes[END_REF][START_REF] Oldroyd | Nuclear calcium changes at the core of symbiosis signalling[END_REF][START_REF] Charpentier | Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations[END_REF], with which ion flux changes lead to root hair curling entraps a bacterial microcolony and a subsequent expression of early nodulation genes [START_REF] Charpentier | Nuclear calcium signaling in plants[END_REF]. Nod factors signals transmit to a receptor-like kinase (LjSYMRK), DMI2/MtSYM2 in Medicago, which associates with the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) to the production of mevalonate, which may function as a secondary messenger to the nucleus [START_REF] Kevei | 3-hydroxy-3-methylglutaryl coenzyme a reductase 1 interacts with NORK and is crucial for nodulation in Medicago truncatula[END_REF]. The ion channels, a complex of DMI1 (Lj POLLUX) and CNGC15, collaborate to regulate potassium and calcium fluxes from the nuclear envelope and endoplasmic reticulum [START_REF] Charpentier | Nuclear-localized cyclic nucleotide-gated channels mediate symbiotic calcium oscillations[END_REF]. The calcium spiking signals interpreted inside the nuclear envelop as a calcium-and calmodulin-dependent serine/threonine protein kinase (CCAMK, DMI3/MtSYM13) [START_REF] Singh | Activation of calcium-and calmodulin-dependent protein kinase (CCaMK), the central regulator of plant root endosymbiosis[END_REF], thereafter phosphorylates INTERACTING PROTEIN OF DMI3 (MtIPD3, LjCYCLOPS) inducing symbiotic gene expression [START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF][START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF]. Cell division (indicated with dotted lines) in the inner or mid-cortex and pericycle is initiated early in the interaction between the root and rhizobia and precedes the initiation of infection events. Bacteria are entrapped in a curled root hair, and from this site infection threads (ITs) are initiated. The route of the IT is predicted by pre-infection threads that are densely cytoplasmic subdomains with aligned cytoskeleton. Its progress into the inner cortex where the nodule primordium has formed through a series of cell divisions. From these divided cells, the nodule meristem forms.

The calcium spiking is inducing gene expression of a suit of transcription factors: GRAS domain transcription factors (the DELLA subfamily, nodulation signalling pathway NSP1/NSP2), ERF Required for Nodulation (ERN/ERN2), CCAAT binding Nuclear Transcription Factor Y (NF-YA/YB), as well as Nodule Inception (NIN) [START_REF] Marsh | Medicago truncatula NIN is essential for rhizobial-independent nodule organogenesis induced by autoactive calcium/calmodulin-dependent protein kinase[END_REF]Hirsch & Oldroyd, 2009;Laloum et al., 2013;[START_REF] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF]. DELLAs are recruited by the F-box proteins SLEEPY1 (SLY1) and GA-INSENSITIVE DWARF2 (GID2) to an E3 ubiquitin ligase complex [START_REF] Mcginnis | The Arabidopsis SLEEPY1 gene encodes a putative F-box subunit of an SCF E3 ubiquitin ligase[END_REF][START_REF] Dill | The Arabidopsis F-box protein SLEEPY1 targets gibberellin signaling repressors for gibberellin-induced degradation[END_REF][START_REF] Gomi | GID2, an F-box subunit of the SCF E3 complex, specifically interacts with phosphorylated SLR1 protein and regulates the gibberellin-dependent degradation of SLR1 in rice[END_REF][START_REF] Griffiths | Genetic characterization and functional analysis of the GID1 gibberellin receptors in Arabidopsis[END_REF].

The E3 ubiquitin ligase SKP1-Cullin-F-box (SCFSLY1/GID1) complex polyubiquitinates DELLA proteins and results in their degradation by the 26S proteasome 23, thus stimulating the GA mediated nodulation (Hirsch & Oldroyd, 2009). NSP1 and NSP2 form homo-and heteropolymers interacting with promoters of early nodulins like ENOD11 [START_REF] Journet | Medicago truncatula ENOD11: a novel RPRPencoding early nodulin gene expressed during mycorrhization in arbuscule-containing cells[END_REF]Hirsch et al., 2009;[START_REF] Cerri | Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection[END_REF]. NSP1/NSP2 activates the transcription of ENOD11 as well as the transcription factor ERN1 (Ethylene Response Factor Required for Nodulation1).

ERN1 directly regulates ENOD11 [START_REF] Cerri | Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection[END_REF] via specific binding to a Nod factor responsive cis element, NF-box (Andriankaja et al., 2007), enabling the subsequent bacterial infection process.

The transcription factor NIN is a master gene of nodule organogenesis

During the infection, cells in the inner and subsequently mid-cortex begin dividing and nodule primordium is thereby formed [START_REF] Schultze | Regulation of symbiotic root nodule development[END_REF]. The TF NIN was firstly identified in L. japonicus [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[END_REF], and subsequently evidenced by multiple groups as a master regulatory gene of nodule organogenesis through cytokinin promotion [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] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF]. NIN has been shown to regulate many nodulation related genes such as Rhizobium-induced peroxidase 1 (RIP1) [START_REF] Cook | Transient induction of a peroxidase gene in Medicago truncatula precedes infection by Rhizobium meliloti[END_REF], ENOD11 [START_REF] Cerri | Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection[END_REF], Nodulation Pectate Lyase (NPL) [START_REF] Xie | Legume pectate lyase required for root infection by rhizobia[END_REF], NF-YA1/2 [START_REF] Soyano | Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF] and a number of CLE encoded peptides involved in autoregulation of nodulation (Nakajima et al.) [START_REF] Soyano | Nodule Inception creates a longdistance negative feedback loop involved in homeostatic regulation of nodule organ production[END_REF]. NIN is able to inhibit nodulation in response to nitrate, together with a family of NIN-like proteins (NLPs) through interaction of their carboxy-terminal PB1 domains (Lin et al., 2018). NLPs are found to be nitrate responsive (Lin et al., 2018;Nishida et al., 2018). Furthermore, different cis-regulatory sequences in the NIN promoter are revealed to regulate bacterial infection and nodule organogenesis [START_REF] Yoro | A positive regulator of nodule organogenesis, NODULE INCEPTION, acts as a negative regulator of rhizobial infection in Lotus japonicus[END_REF]. In addition, a remote cis-regulatory region 5kb upstream of the start codon was identified for NIN expression initiate nodule primordium formation, containing putative cytokinin response elements (Liu et al., 2019). Ectopic expression of NIN and the NF-YA1 is able to trigger cortical cell divisions [START_REF] Soyano | Nodule inception directly targets NF-Y subunit genes to regulate essential processes of root nodule development in Lotus japonicus[END_REF].

III.2.1 Bacteroid differentiation

Nodule are developed into determinate and indeterminate nodules in different leguminous plants

When infection thread reaches the primordia, bacteria are released. Bacteria reside in plant cells as an organelle-like vesicles, with a half plant and half bacteria derived double membrane (peribacteroid membrane, PBM), collectively termed symbiosome. In order to perform nitrogen fixation, both plant and bacterial partners undergo differentiation, therein bacteroid differentiation are classified into two types as non-terminal differentiation and terminal differentiation.

1) Non-terminal bacteroid differentiation: In most legume clades like the ones comprising

Lotus japonicus or common bean (P. vulgaris), bacteroids remain similar in size and shape to the free-living rhizobia. They also retain full capacity to resume growth when they are extracted from nodules (Mergaert et al., 2006).

2) Terminal bacteroid differentiation: in galegoid (temperate) legumes like M. truncatula, the endosymbiotic bacteria and the host cells undergo the terminal differentiation.

In consequences, nodules are accordingly developmentally classified in two types:

1) Determinate nodules are round in shape without a persistent meristem, in the model plant L. japonicus and some (Ruvkun et al.)tropical legumes such as Phaseolus, Glycine, and Vigna species.

2) Indeterminate nodules are in cylindric shape caused by continuously generated cells from an apical meristem which was established from nodule primordia, mostly present in temperate legumes, e.g., the model plant M. truncatula. The meristem cells continue to grow into differentiation processes, thus lead to a typical zonation in mature indeterminate nodules.

Zonation in indeterminate nodules are as shown in Figure 11A and At last, a Zone V is also mentioned when cytoplasm of nodule cells is fully degraded, and saprophytic bacteria colonized these senescent nodules. While the development of zonation is gradual without a sharp border, an interzone (zone IZ) is defined between zone II-III corresponding to the bacteria terminal differentiation into bacteroid and characterized by amyloplast accumulation (Vasse et al., 1990). We can also observe the interzone between zone III-IV where the nodule senescence related genes are induced (Perez Guerra et al., 2010;Pierre et al., 2014).

Bacteria grow and divide in the symbiosome, presenting different characteristics in the indeterminate nodule zones [START_REF] Lang | Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules[END_REF]. Seldom bacteria can be found in nodule meristem in which infection thread established (zone I; Figure 11B) [START_REF] Lang | Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules[END_REF]. In nodule zone II, three types of bacteria can be found: small and rod-shaped free-living bacteria, freshly released free bacteria and elongating bacteria (zone II; Figure 11B) [START_REF] Lang | Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules[END_REF]. The bacterial and plant cell elongation mainly occur in the interzone II-III, afterwards bacteroids are fully elongated or in Y-shape occupying the cytosol of host cells in zone III with an organized manner (zone III; Figure 11B) (Kondorosi et al., 2013). The infected cells of nitrogen-fixing nodules are interspersed with uninfected cells that are involved in transport of sugars and nitrogenous compounds in connection with the vascular bundles (zone III; Figure 11B) [START_REF] Peiter | Sugar uptake and proton release by protoplasts from the infected zone of Vicia faba L. nodules: evidence against apoplastic sugar supply of infected cells[END_REF][START_REF] Godiard | MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges[END_REF]. The respiratory activity of living bacteria/bacteroids can be detected by 5-cyano-2,3-di-4-tolyl tetrazolium chloride (CTC) fluorescence (Mergaert et al., 2006). Alternatively, bacterial viability can be detected and microscopically visualized by live/dead staining on fresh nodule sections involving a mixture of the nucleic acid fluorescent dyes SYTO9/PI [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. The bacteria/bacteroids with intact membrane will only stained by SYTO9 (green fluorescence), whilst the damaged ones are stained by PI (red fluorescence) (Figure 11B) [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. (A) The image shows a longitudinal section (10-μm thickness) of a 21-day-old M. truncatula-S. meliloti nodule. IT = infection thread. Adapted from [START_REF] Lang | Transcriptomic Analysis of Sinorhizobium meliloti and Medicago truncatula Symbiosis Using Nitrogen Fixation-Deficient Nodules[END_REF].

(B) The live/dead staining shows nodule zone I-II and cells from zone III and zone IV from a 28-day-old M. truncatula-S. meliloti nodule. The living bacteria/bacteroids are strained with SYTO9 (in green) and the dying bacteroids and plant nuclei are stained with PI (in red). In zone II, infection threads can be observed (Cunningham et al.). In zone III, elongated bacteroid can be observed after terminal differentiation. In early zone IV, senescing bacteroid can be observed with red colour. (Scale bar = 10µm)

Bacteroid differentiation is coupled to genome amplification in indeterminate nodules

During differentiation, bacterial morphological changes like elongation is coupled to genome amplification, membrane modifications, and the loss of reproductive capacity (Van de Velde et al., 2010). Eventually bacteria terminally differentiate into nitrogen fixing bacteroids.

The elongated bacteroids (E-morphotype) are usually 5-to 10-fold longer than the free-living bacteria coupling to the amplification of the bacterial genome (Figure 12A). Cell populations are with 1C/2C content in bacteria, whilst the majority of cells have DNA content >4C and peaked at 24C in bacteroid extractions (Figure 12B) (Mergaert et al., 2006). Due to the extreme differentiation, bacteroids of M. truncatula are uncultivable after the terminal differentiation.

Symbiotic plant cells undergo endoreduplication

Coordinately, the symbiotic plant cells are elongated and coupled with endoreduplication, eventually differentiate into polyploid cells. In Figure 13A, bacteria are released in plant cells in postmitotic stage with 4C DNA content (C being the haploid DNA content) (Kondorosi et al., 2013). Upon activation of NCRs and GRPs, the symbiotic cells further differentiate into 8C, 16C and 32C couple with the endoreduplication (genome replication without mitosis and cytokinesis) and cell enlargement (Figure 13A) (Mergaert et al., 2006;Kondorosi et al., 2013).

Furthermore, in coordination with the host cells, bacterial infection (zone II) also leads to the enlargement of symbiotic cells and endoreduplication cycles resulted in high ploidy levels of 32C and 64C (Figure 13) [START_REF] Cebolla | The mitotic inhibitor ccs52 is required for endoreduplication and ploidy-dependent cell enlargement in plants[END_REF][START_REF] Vinardell | Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules[END_REF]Kondorosi et al., 2013), eventually forming the fully elongated and huge polyploid cells. The symbiotic cells with high ploidy levels are therefore able to undergo extreme cell growth, host thousands of bacteroids and sustain the energy-demanding N fixation (Mergaert et al., 2006). In Figure 13B, a flow cytometry analysis shows proportions of cell ploidy levels at different stages of nodule development [START_REF] Vinardell | Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules[END_REF]. [START_REF] Vinardell | Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules[END_REF].

The plant factors determine terminal bacteroid differentiation with NCR and GRP peptides

Bacteroid differentiation is regulated by several hundreds of plant antimicrobial peptide (AMP)-like nodule-specific symbiotic peptides: nodule-specific cysteine-rich (Brenchley et al.) or glycine-rich protein (GRP) peptides [START_REF] Kevei | Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp[END_REF]Mergaert et al., 2003;Alunni et al., 2007) (Figure 13A). The NCRs are small (30-50aa) contain a signal peptide and a mature peptide, and localized in the membrane or cytosol of bacteroids corresponding to the function

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in bacteroid differentiation (Mergaert et al., 2003;Van de Velde et al., 2010). The corresponding gene expressions are only in infected cells and localized in zones with their presumed function produced in zone II, interzone II-III, and zone III (Mergaert et al., 2003;Van de Velde et al., 2010;Kondorosi et al., 2013).

In a dnf1 mutant of M. truncatula, bacteria can be release in plant cell, but the subsequent differentiation is blocked [START_REF] Wang | Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer[END_REF]. The DNF1 protein is a nodulespecific subunit of the signal peptidase complex (SPC) [START_REF] Wang | Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer[END_REF]. SPC is an early component in the protein secretory pathway in the endoplasmic reticulum (ER) and removes the signal peptide of secretory proteins during their translocation into the ER lumen (Van de Velde et al., 2010;[START_REF] Wang | Multiple regulatory elements in the Arabidopsis NIA1 promoter act synergistically to form a nitrate enhancer[END_REF]. The bacterial terminal differentiation requires NCR trafficking into bacteroid, but the bacteroid differentiation is obstructed in dnf1 mutant when the processing of NCRs blocked at ER (Van de Velde et al., 2006). One of the cationic peptides, NCR247, is expressed in the older zone II and in the interzone II-III [START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF].

NCR247 can penetrate bacteria membranes and form complex with bacterial proteins to affect bacterial cell division, translation, and protein folding [START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF]. In contrast, ectopic expression of NCRs in L. japonicus, for which bacteroids are normally U-morphotype (similar morphology than free-living bacteria), induce bacteroid morphological changes reminiscent of terminally differentiation with elongated bacteroids (Van de Velde et al., 2010).

GRP carry a secretory signal peptide and a glycine rich mature peptide of approximately 100 amino acids [START_REF] Sachetto-Martins | Plant glycine-rich proteins: a family or just proteins with a common motif?[END_REF][START_REF] Kevei | Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp[END_REF][START_REF] Mangeon | Functional diversity of the plant glycine-rich proteins superfamily[END_REF]Kondorosi et al., 2013). Most other GRPs are developmentally regulated by biotic and abiotic factors, e.g., pathogens [START_REF] Molina | Differential expression of pathogenresponsive genes encoding two types of glycine-rich proteins in barley[END_REF], osmotic stress [START_REF] Kim | Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions[END_REF], cold shock [START_REF] Carpenter | Genes encoding glycine-rich Arabidopsis thaliana proteins with RNA-binding motifs are influenced by cold treatment and an endogenous circadian rhythm[END_REF][START_REF] Kim | Functional characterization of a glycine-rich RNA-binding protein 2 in Arabidopsis thaliana under abiotic stress conditions[END_REF] and wounding [START_REF] Showalter | Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding[END_REF]. On the contrary, the nodule specific GRPs requires rhizobia infection but are not responding to abiotic factors [START_REF] Kevei | Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp[END_REF]. MsnodGRP1 and MsnodGRP2 transcripts were found abundant in interzone II-III, suggesting a role in nodule differentiation [START_REF] Kevei | Glycine-rich proteins encoded by a nodule-specific gene family are implicated in different stages of symbiotic nodule development in Medicago spp[END_REF].

Bacterial partnership in bacteroid differentiation: BacA and CtrlA

On the bacterial side, BacA, a membrane protein from the ATP-binding cassette (ABC)

transporter superfamily, plays an important role in bacteroids differentiation [START_REF] Maunoury | Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches[END_REF][START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]Kondorosi et al., 2013). In the S. meliloti mutant bacA, bacteria succeed to release in host cells but is deficient in bacteroid differentiation, resulting in immediate nodule senescence [START_REF] Maunoury | Differentiation of symbiotic cells and endosymbionts in Medicago truncatula nodulation are coupled to two transcriptome-switches[END_REF]. BacA is suggested to protect bacteria against the antimicrobial actions of NCRs which induce membrane permeabilization and bacterial killing in vitro. In contrast, BacA is dispensable for bacterial survival in an M. truncatula dnf1 mutant [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. Therefore, BacA is critical to long-term host infection via allowing the bacterial persistence in symbiotic cells rather than rapid bacterial death [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF].

As a transcriptional regulator, CtrA is considered as the master regulator of cell cycles in some α-proteobacteria such as Caulobacter crescentus as a model system [START_REF] Laub | Global analysis of the genetic network controlling a bacterial cell cycle[END_REF][START_REF] Laub | Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle[END_REF][START_REF] Brilli | The diversity and evolution of cell cycle regulation in alpha-proteobacteria: a comparative genomic analysis[END_REF]. In S. meliloti, CtrA depletion leads to bacterial cell elongation, increase of DNA content and abolished cell division, which are similar to bacteroid differentiation, but the membrane integrity was maintained [START_REF] Pini | Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti[END_REF]. Through microarray-based gene expression analysis, CtrA were found to regulate the transcription of at least 126 genes in S. meliloti, elevating genes such as cell cycle regulators (i.e. ctrA, divJ, divK, sciP), motility genes (i.e. mcp, che, flg, flaAB, fli), cell envelope components and several hypothetical proteins [START_REF] Pini | Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti[END_REF]. CtrA was also shown as a repressor for cell division regulators minC and minD. Therefore, CtrA down-regulation during bacteroid differentiation increases minCD expression, subsequently blocking cell division by inhibiting the FtsZ polymerization and Z-sring formation [START_REF] Cheng | A Sinorhizobium meliloti minE mutant has an altered morphology and exhibits defects in legume symbiosis[END_REF][START_REF] Pini | Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti[END_REF]. CtrA can bind to two consensus sites (TTAA-N7-TTAAC) and (TTAACCAT) to regulate cell cycle genes [START_REF] Laub | Genes directly controlled by CtrA, a master regulator of the Caulobacter cell cycle[END_REF]. In S. meliloti, these binding sites are detected in the promoter regions of mcpZ, pilA1, flaA and divJ via a large-scale Chromatin Immunoprecipitation assay [START_REF] Johnson | Genome-wide mapping of in vivo protein-DNA interactions[END_REF][START_REF] Pini | Cell Cycle Control by the Master Regulator CtrA in Sinorhizobium meliloti[END_REF].

III.2.2 Nitrogen fixation in bacteroids

The bacteroids are able to fix atmosphere nitrogen into ammonia in microaerobic conditions. This BNF process allows for the production of NH3/NH4 + that can be utilized by plants [START_REF] Stein | The nitrogen cycle[END_REF]. Similar as in the Harbor-Bosch process, BNF requires high energy input to break the triple band in N2. Instead of using fossil fuels, the redox reaction of BNF is associated with electron transfer processes with ATP hydrolysis-16 moles of ATP to produce 2 moles of ammonia. The equation of BNF process is as in below:

N2 + 8H + + 8e -+ 16Mg-ATP → 2NH3 + H2 + 16Mg-ADP + 16Pi

Nitrogen fixation is catalysed by the nitrogenase enzyme complexes

This reaction is catalysed by the nitrogenase enzyme complexes [START_REF] Oldroyd | Biotechnological solutions to the nitrogen problem[END_REF], encoding by numerous nif genes, including regulators. The nitrogenase is stringently regulated at transcriptional level (Figure 14A), responding to N resources and oxygen level.

NifA is the master regulator of nif genes, co-dependent with the RNA polymerase sigma factor, σ54 [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF]. The fixLJ genes are positive regulators of the symbiotic expression of the N fixation gens in a cascade: the fixLJ genes activate nifA, which in turn activates nifHDK and fixABCX [START_REF] David | Cascade regulation of nif gene expression in Rhizobium meliloti[END_REF]. The nitrogenase complex consists of a reductase component (Fe protein) and a catalyst component (MoFe protein) (Figure 14B) [START_REF] Burgess | Mechanism of Molybdenum Nitrogenase[END_REF][START_REF] Seefeldt | Mechanism of Mo-dependent nitrogenase[END_REF][START_REF] Oldroyd | Biotechnological solutions to the nitrogen problem[END_REF]. The Fe protein act as electron donors reduced by ferredoxin and flavodoxin, which is dependent on MgATP hydrolysis [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF].

Three metalloclusters, [4Fe-4S], P cluster and FeMo-co [Figure 14B(c)], provide an electron transfer conduit and the catalytic site for N reduction, respectively [START_REF] Oldroyd | Biotechnological solutions to the nitrogen problem[END_REF].

However, O2 can irreversibly damage the Fe protein and the MoFe protein, therefore most of the BNF process must be protected from O2 in various physiological strategies, including anaerobic growth conditions, consumption of excess O2 by respiration, O2 diffusion barriers, or compartmentation of the enzyme spatially or temporally [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF]. In case of legumes nodules, gas permeability is restricted by the nodule cortex as the barrier for O2 diffusion. In addition, an O2-binding protein, leghaemoglobin, which also gives the nodules a pink colour, is present in the cytoplasm of infected nodule cells to facilitate the bacteroid respiration under microoxic conditions. These conditions within nodules are achieved by restricted gaseous diffusion into nodules and high rates of respiration facilitated by oxygenbinding and oxygen-transporting leghaemoglobin in the cytoplasm of plant cells (Ott et al., 2005). Free-oxygen concentrations in nodule cells are as low as 10 -8 M in the fixation zone, permitting nitrogenase to function [START_REF] Tjepkema | Measurement of oxygen partial pressure within soybean nodules by oxygen microelectrodes[END_REF]. Thus, oxygen is maintained in nodules at a level that support respiration but sufficiently low to avoid nitrogenase inactivation (Kuzma et al. 1993). Finally, the released ammonia must be assimilated immediately in root nodules to avoid toxicity. (A) Regulatory cascades controlling nif transcription in S. meliloti. Adapted from [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF].

(B) Nitrogenase structure and genes required for its biosynthesis. Adapted from [START_REF] Oldroyd | Biotechnological solutions to the nitrogen problem[END_REF].

(a) The nitrogen fixation (nif) gene cluster from Klebsiella oxytoca. The nitrogenase structural genes (nifH, nifD and nifK) are coloured according to the crystal structure shown in (b). Remaining genes are colour-coded according to their functions: dark red, Fe protein maturation (nifM); light blue, FeMoco biosynthesis (nifY, nifE, nifN, nifX, nifV, nifB, nifQ); yellow, Fe-S cluster biosynthesis (nifU, nifS, nifZ); green, electron transport (nifJ, nifF); orange, transcriptional regulation (nifL, nifA); grey, unknown function (nifT, nifW).

(b) Structure of the nitrogenase enzyme complex showing the MoFe and Fe protein components, with the three metalloclusters revealed on the left-half of the complex (abbreviated as F, [4Fe4S]; P, P cluster and M, FeMo-co, respectively). ATP hydrolysis by the Fe protein, the route of electron transfer to the catalytic site and the enzyme reaction are also illustrated on the left half of the structure.

(c) Structures of the three metalloclusters in nitrogenase. Genes required for the biosynthesis of each cluster are illustrated below.

IV. Senescence

IV.1 Senescence as a common process

Terminology of senescence

The word "senescence" is originated from the Latin senescere, i.e., to grow old, reflecting its association with senility [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. For semelparous species, the ending timepoint culminates with mating under monocarpic senescence, whereas in iteroparous species, senescence splits in the entities (e.g. whole plant) and the parts (e.g. organs), so that reproduction of parts takes place repeatedly [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. There is a long-term semantic

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controversy about the termination of senescence, aging and death [START_REF] Van Doorn | Senescence and programmed cell death: substance or semantics?[END_REF][START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. Here, we regard aging as the entire process of the lifespan, referring to the changes with time including periods such as growth, differentiation, maturation, senescence and death. Thereof, senescence is the developmental phase that is following the completion of growth; may or may not be succeeded by death; and is antagonistic to the declining of viability (Figure 15) [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. The growth curve is following a typical sigmoidal pattern (Figure 15; S pattern), which is the interplay of the growth potential and limitations, eventually resulted in triggering senescence [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. Senescence is rather a continuous process than a still status, being the later stage of the constitutive aging. The increase in size (S) of a cell mass, tissue, organ, whole plant or population follows a typical sigmoidal pattern. The instantaneous growth rate (G) is maximal atss the inflexion point of the S curve. The relative growth rate (R = G/S) declines progressively over time. V represents viability, which begins to subside at some point following completion of growth. Note that ageing is considered here to refer to change (not deterioration) with time, and includes, but is not limited or defined by, the period of senescence and the terminal phase of decreasing (V).

Senescence is a complex mechanism accompanied with a series of physiological, biochemical and molecular changes. Senescence is characterized by a progressive deterioration of physiological integrity, leading to impaired function and increased vulnerability to death. Death, however, is the terminal period of dying, not belonging to life itself. Death refers to abiotic chemistry while senescence is still related metabolisms with viable tissue.

Recently, senescence is under scientific scrutiny thanks to modern advances in biotechnical approaches. However, the hallmarks of senescence in animals (e.g., somatic mutation, telomere attrition and the costs of repair and maintenance) seem not to be significant in plants, but other factors (e.g. epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, and altered intercellular communication) still matter (Pladys & Vance, 1993;[START_REF] Lopez-Otin | The hallmarks of aging[END_REF][START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF].

Developmental and induced senescence

Senescence and disease may seem to be different process in a distant view.

Senescence can be classified as developmental senescence and induced senescence based on causality. Generally, developmental senescence is believed to be cell programmed.

Comparatively, induced senescence refers to environmental changes. However, they would share a lot of similarities, if we regard time as an abiotic factor in developmental senescence, thereby an accelerated process of senescence would be seen in induced senescence. Multiple physiological processes involved senescence-like and defence-like mechanisms. In this context, defence reactions such as programmed cell death occurring during incompatible interactions share common symptoms with senescence (Lim et al., 2007). The common elements and the specificity of each reactions still need to be elucidated as there are multiple processes depending on the plant species and the environmental constraints. Nevertheless, both senescence and defence reactions involve genetic reprograming which allow the realization of the different physiological responses. Programmed senescence precedes according to a timetable affected by developmental and environmental factors and mediated by genetic programs [START_REF] Jansson | Senescence: developmental program or timetable?[END_REF].

IV.2 Regulation of senescence

IV.2.1 Regulation of plant senescence

Plant senescence is under molecular control such as phytohormone signalling, redox transduction, kinases and transcriptional factors (Lim et al., 2007;Thomas et al., 2009;[START_REF] Kirkwood | On the programmed/non-programmed nature of ageing within the life history[END_REF][START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. Plant hormones including salicylic acid, abscisic acid, jasmonic acid and ethylene advance leaf senescence, whereas others like cytokinin, gibberellins, and auxins delay this process [START_REF] Sarwat | Phytohormones and microRNAs as sensors and regulators of leaf senescence: assigning macro roles to small molecules[END_REF]. Depending on the different plant species and the multiple senescence processes linked to induced senescence or organ specificity, the implication of the different hormones could be different. A striking example is the involvement of ethylene in the fruit ripening with climacteric and non-climacteric plants [START_REF] Gómez | Molecular programme of senescence in dry and fleshy fruits[END_REF][START_REF] Seymour | Fruit development and ripening[END_REF][START_REF] Perez-Llorca | Biosynthesis, Metabolism and Function of Auxin, Salicylic Acid and Melatonin in Climacteric and Non-climacteric Fruits[END_REF].

Downstream of the hormone signalling, numerous TFs has been implicated during senescence (Breeze et al., 2011). For example, an Arabidopsis transcription factor (AtMYB2) is reported as a suppressor for cytokinin mediated axillary bud outgrowth, thus plays a part in the late stage of the whole plant senescence program [START_REF] Guo | AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis[END_REF]). The T-DNA inserted atmyb2 lines have a prolonged life span and are complemented by the AtMYB2 promoterdirected cytokinin oxidase 1 gene (Figure 16) [START_REF] Guo | AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis[END_REF]. Similarly, ethylene response factors are also involved in the regulation of the senescence [START_REF] Koyama | The roles of ethylene and transcription factors in the regulation of onset of leaf senescence[END_REF]. Thus, the genetic regulation of plant senescence occurs through the modification of transcription factor expression and regulation.

Sugar-mediated gene regulation is in the centre of senescence activation

Phytohormones such as cytokinin regulation also mediates leaf senescence by transportation to different organs and changing their status as metabolic sinks (Lara et al., 2004). In this context, energetic metabolism plays a significant regulatory role. Senescence is conceived to be under a sugar-mediated gene regulation [START_REF] Rolland | Sugar sensing and signaling in plants: conserved and novel mechanisms[END_REF][START_REF] Wingler | Metabolic regulation of leaf senescence: interactions of sugar signalling with biotic and abiotic stress responses[END_REF][START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]. Changes in the sucrose to hexose ratio are detected by plant sensors and lead to different transduction pathways and inductive or repressive effects on gene transcription [START_REF] Smeekens | Sugar signals and molecular networks controlling plant growth[END_REF]. A protein kinase SnRK1 is activated by darkness, nutrient starvation and high cellular concentrations of sucrose or low glucose, or both, conditions associated with the induction of senescence [START_REF] Jongebloed | Sequence of morphological and physiological events during natural ageing and senescence of a castor bean leaf: sieve tube occlusion and carbohydrate back-up precede chlorophyll degradation[END_REF][START_REF] Baena-González | Shaping plant development through the SnRK1-TOR metabolic regulators[END_REF][START_REF] Crepin | SnRK1 activation, signaling, and networking for energy homeostasis[END_REF]. The enzyme hexokinases (HXKs) function as glucose sensors and its mutants present senescence-delaying phenotypes, e.g., Arabidopsis HXK1 and rice HXK5 and HXK6 [START_REF] Moore | Role of the Arabidopsis glucose sensor HXK1 in nutrient, light, and hormonal signaling[END_REF][START_REF] Cho | Regulatory functions of nuclear hexokinase1 complex in glucose signaling[END_REF].

Senescence is therefore part of the cell fate in coordination with source-sink communications through the vascular system, during which resources are translocated form the obsolete body parts to new developing structures [START_REF] Guiboileau | Senescence and death of plant organs: nutrient recycling and developmental regulation[END_REF][START_REF] Avila-Ospina | Autophagy, plant senescence, and nutrient recycling[END_REF].

Proteolytic activities are downstream of the senescence process

Proteases are involved in numerous processes in normal cell functioning including germination, development, senescence, immunity, and stress responses [START_REF] Zhou | UBIQUITIN-SPECIFIC PROTEASES function in plant development and stress responses[END_REF][START_REF] Liu | Role of Papain-Like Cysteine Proteases in Plant Development[END_REF]. Proteinases are the central component of executioner in the death machinery which occurs during senescence or cell death (Diaz-Mendoza et al., 2016;[START_REF] Martínez | Activities of vacuolar cysteine proteases in plant senescence[END_REF]Buono et al., 2019;[START_REF] Park | Arabidopsis ubiquitin-specific proteases UBP12 and UBP13 shape ORE1 levels during leaf senescence induced by nitrogen deficiency[END_REF]. They are mostly directly produced at the change of cell structure degradation and most likely localized to the cytosol or in vacuolar compartment.

Some cysteine proteases such as δVPE are expressed in both senescence and nonsenescence Programmed cell death (PCD) contexts (Nakaune et al., 2005), and metacaspases are expressed in both organ senescence and PCD (Breeze et al., 2011;Coll et al., 2014;[START_REF] Olvera-Carrillo | A Conserved Core of Programmed Cell Death Indicator Genes Discriminates Developmentally and Environmentally Induced Programmed Cell Death in Plants[END_REF]. One well known plant PCD mechanism, is found during plant-pathogen interactions, the host cell death is featured with rapid tissue collapse, i.e., the hypersensitive response (HR), regulated transcriptionally and translationally [START_REF] Dangl | Death don't have no mercy: cell death programs in plantmicrobe interactions[END_REF]. In adjacent cells of the HR sites, plant defence genes are transcriptionally elicited to constrain pathogens [START_REF] Somssich | Gene structure and in situ transcript localization of pathogenesis-related protein 1 in parsley[END_REF][START_REF] Heath | Hypersensitive response-related death. Programmed cell death in higher plants[END_REF][START_REF] Balint-Kurti | The plant hypersensitive response: concepts, control and consequences[END_REF].

In leave senescence, autophagy has been also implicated in the incidence of diverse age-related physiological and pathological changes, by degrading and recycling damaged macromolecules and organelles [START_REF] Hanaoka | Leaf senescence and starvation-induced chlorosis are accelerated by the disruption of an Arabidopsis autophagy gene[END_REF][START_REF] Vauclare | Metabolic and structural rearrangement during dark-induced autophagy in soybean (Glycine max L.) nodules: an electron microscopy and 31P and 13C nuclear magnetic resonance study[END_REF]Minina et al., 2013;[START_REF] Avila-Ospina | Autophagy, plant senescence, and nutrient recycling[END_REF]. Macroautophagy involves formation of autophagosomes, which are double membrane-bound structures enclosing macromolecules and organelle residues [START_REF] Kundu | Macroautophagy versus mitochondrial autophagy: a question of fate?[END_REF]. During leaf senescence, several possible routes for internalization of chloroplast components by the central vacuole are proposed, such as autophagosome and senescence-associated vesicle (SAV) trafficking [START_REF] Avila-Ospina | Autophagy, plant senescence, and nutrient recycling[END_REF]. In SAVs, the senescence-associated cysteine-protease encoded by the SAG12 (senescence-associated gene) gene has been detected as a key marker gene (Otegui et al., 2005;[START_REF] Singh | Down-regulation of OsSAG12-1 results in enhanced senescence and pathogen-induced cell death in transgenic rice plants[END_REF].

IV.2.2 Legume-rhizobium symbiotic nodule senescence

The reciprocal symbiosis between legumes and rhizobia are based on mutual benefits of carbohydrates and N exchange. When this nutrient trading is out of the equilibrium, the associated organ, the root nodule, will collapse and turn to general nutrient source instead of carbon sink for nutrient reutilization. Nodule senescence is featured with colour change from to pink to green associated with the degradation of leghaemoglobin (Lb) in the senescence zone present at the base of the nodule (Figure 17) [START_REF] Roponen | The Effect of Darkness on the Leghemoglobin Content and Amino Acid Levels in the Root Nodules of Pea Plants[END_REF]. Inside the indeterminate nodules, bacteroids start to deteriorate in senescence zone IV and ultimately leads to an advanced senescence zone V. Root nodule senescence could happen with natural aging with refined genetic program, with which nodules will progressively enter a senescence stage (developmental senescence, DS). In a more acute way, root nodule senescence can be triggered in a short time by abiotic stresses or recognition failure between the patterners (Induced senescence, IS). Nitrogen-fixing and senescent nodules are from 20-and 28-days post inoculation (dpi) by inoculating S. meliloti with M. truncatula, respectively. Scale bar = 100µm

Symptoms of developmental nodule senescence

In senescent tissues, proteolytic activities are widely triggered with a large scale of protein degradation (Pladys & Vance, 1993), which eventually results in the death of both microsymbionts and plant cells. The cytoplasm becomes less electron dense and vesicles and ghost membranes appear as a result of host and symbiosome membrane disintegration (Timmers et al., 2000). Nodule senescence was observed at 35dpi and 61dpi (Figure 18 A-C) and three types of senescent cells were defined from two senescence stages ( [START_REF] Peiter | Sugar uptake and proton release by protoplasts from the infected zone of Vicia faba L. nodules: evidence against apoplastic sugar supply of infected cells[END_REF]. The conical organization of the senescence front presumably promotes optimal remobilization of breakdown products, because the last cells to die would be located near the vascular bundles at the periphery (Figure 18 A-C). (J) TEM image of a type II senescent cell showing loss of cellular integrity and plant cell wall breakage.

Symptoms of induced nodule senescence

Abiotic factors were evidenced to rapidly trigger nodule senescence, such as continuous dark treatment and extra N provision (Matamoros et al., 1999;[START_REF] Swaraj | Dark-induced changes in legume nodule functioning[END_REF][START_REF] Hernández-Jiménez | Antioxidant defence and damage in senescing lupin nodules[END_REF]Perez Guerra et al., 2010). Induced senescence leads to slightly different symptoms of decaying nodules compared with developmental senescence, with a faster degradation of bacteroid content but remain of the symbiosomal structure (Table 4) (Perez Guerra et al., 2010). The start of the developmental nodule senescence was characterized by numerous vesicles, prominent endoplasmic reticulum, abundant mitochondria and Golgi apparatus, and symbiosome degradation (Figure 19A). During dark induced senescence, the bacteroid content condensed, the peribacteroid space increased (Figure 19B, arrows), and often different symbiosomes fused (Figure 19B,arrowheads). Strikingly, the PBMs remained intact, even when most of the bacteroid content had disappeared, in strong contrast to the developmental senescence, during which eventually the complete symbiosome had dissolved (Figure 19,compare C and D). After vacuolar collapse, the cytoplasm was completely degraded, the mitochondria disappeared, and the only cellular remains were apparently intact PBMs and remnants of bacteroids (Figure 19, E and F). Saprophytic bacteria, derived from infection threads, entered senescing cells at an early stage and multiplied until they occupied most of the space (Figure 19, G and H). In conclusion, dark-induced senescence is a rapid process in which the symbiosome content is degraded in the absence of cellular signs for nutrient remobilization and with a fast colonization by saprophytic bacteria.

Transcriptomics of nodule senescence in M. truncatula

Transcriptomic approaches have been performed to get in sight to the transcriptional regulation of nodule senescence (Van de Velde et al., 2006;Seabra et al., 2012;Cabeza et al., 2014;Serova et al., 2017). Differential gene expression analysis has allowed the identification of the down-regulated genes associated with nodule function such as Lb and NCR peptides. In order to decipher transcriptional regulation mechanism related to nodule senescence, transcriptomic studies (Van de Velde et al., 2006;Seabra et al., 2012;Cabeza et al., 2014) have identified up-regulated genes associated with molecular degradation and nutrient remobilization, such as CPs and PAP (purple acid phosphatase). Moreover, the expression of MtCP1 to MtCP6, except for MtCP4, was found transiently upregulated upon dark treatment, whist the elevation of CP expressions is progressive upon time during developmental senescence (Perez Guerra et al., 2010).

Genetic approaches have revealed consecutive cascades in nodule senescence pathway, corresponding to the development of symptoms (Van de Velde et al., 2006). A transcriptome analysis revealed that developmental nodule senescence in M. truncatula is associated with a carbon sink-to-source transition and with the activation of various genes related to defence and stress. Signal transduction related genes were firstly elevated before the symptoms, which were found in healthy nodules. In addition to the refined transcriptional program, nodule decaying is coupled with a subsequent induction of protease activities associated to ubiquitinoylation reaction. Finally, catabolic events were triggered to transport and reutilize nutrients from macromolecules, such as ATP-binding cassette proteins and phosphate transporters, amino acids, and metal ions. Interestingly, mutations in the STAY-GREEN gene, MtSGR 30 led to aberrant shoot senescence and delayed nodule senescence [START_REF] Zhou | From model to crop: functional analysis of a STAY-GREEN gene in the model legume Medicago truncatula and effective use of the gene for alfalfa improvement[END_REF]. Silencing of two ferritin genes, MtFer2 and MtFer3, increased free iron levels in the nodule and induced early nodule senescence (Dhanushkodi et al., 2018). Nodules of the MtCas31 (cold acclimation specific 31) mutant, which is defective in a dehydrin protein that interacts with the leghaemoglobin MtLB120-1, had elevated transcript levels of senescence genes and reduced nitrogenase activity under drought stress [START_REF] Li | MtCAS31 Aids Symbiotic Nitrogen Fixation by Protecting the Leghemoglobin MtLb120-1 Under Drought Stress in Medicago truncatula[END_REF].

In most monocarpic legumes, pod filling often coincides with nodule senescence accompanied with a declined N fixation capacity in soybeans (Lawn & Brun, 1974), peas and common beans (Bethlenfalvay & Phillips, 1977). Exogenous application of N during pod filling increases both yield and seed protein content [START_REF] Merbach | Effectiveness of symbiotic N2-fixation in leguminous plants, as affected by inoculation with rhizobia, by substrate, n-fertilizing, and 14c-sucrose application (author's transl)[END_REF]. Hence, extending the period of active N fixation by delaying the nodule senescence process might have a beneficial effect on crop yield and seed quality. The existence of soybean (Glycine max)

varieties with a delayed nodule senescence and a longer active N fixation period [START_REF] Espinosa-Victoria | Host variation in traits associated with crown nodule senescence in soybean[END_REF] points to a genetic control of the onset of senescence, suggesting a molecular basis to modify timing and/or progression of developmental nodule senescence.

The coordinated death mechanism of plant-host and rhizobia is discussed in the following review (Kazmierczak et al., 2020).

Abstract

In the legume-rhizobia symbiosis, atmospheric dinitrogen (N 2 ) fixation by rhizobia takes place in a specific root organ, called the nodule. During biological N 2 fixation, the plant provides photosynthetic carbohydrates to bacteria and, in exchange, bacteria feed the plant with reduced nitrogen (N) under ammonia (NH 3 ) form. Like most other organs, the root nodule has a limited lifespan, and eventually enters a senescence process characterized by a decline of N 2 fixation and the coordinated death of both bacteria and plant cells. This senescence process characterized by a metabolic switch from a carbon sink to a nutrient source either results from nodule aging (developmental nodule senescence) or can be triggered prematurely by adverse environmental conditions. Accumulating evidence suggests that redox and hormone signaling contribute to

Introduction

Plant-microbe interactions represent a major aspect of the formation and of the development of ecosystems. Plants often establish symbiotic relationships with specific fungi and bacteria to acquire the nutrients they need to support growth and development [START_REF] Smith | Roles of arbuscular mycorrhizas in plant nutrition and growth: New paradigms from cellular to ecosystem scales[END_REF][START_REF] Van Der Heijden | A widespread plant-fungal-bacterial symbiosis promotes plant biodiversity, plant nutrition and seedling recruitment[END_REF][START_REF] Venkateshwaran | Symbiosis and the social network of higher plants[END_REF]Wang et al., 2017). Amongst the different nutrients, nitrogen (N) has a major importance as it represents a crucial constituent of proteins, nucleic acids and chlorophyll and is often in insufficient amounts in the soil under nitrate and/or ammonia forms. As a result, plants belonging to the legume family have developed a symbiotic association with soil bacteria collectively called rhizobia to increase their N nutrition via the biological fixation of atmospheric dinitrogen (N 2 ) (BNF). In BNF, the plant provides photosynthetic carbohydrates to the bacteria and, in exchange, bacteria feed the plant with reduced N under ammonia (NH 3 ) form. Legumes are a family of dicotyledonous plants of the Fabales order. It is the third most important family of flowering plants after the Orchidaceae and Asteraceae with more than 19,500 species [START_REF] Christenhunsz | The number of known plants species in the world and its annual increase[END_REF]. Economically, Fabaceae is the second largest family after Poaceae and is a valuable source of plant proteins for human food and animal feeding.

The success of the symbiotic interaction relies on the molecular dialogue between the plant and the bacteria (Oldroyd, 2013). The plant root exudates which contain flavonoids and isoflavonoids induce the production by rhizobia of lipochitooligosaccharides, called Nodulation Factors (NFs; Ferguson et al., 2010). NFs are usually essential for the bacterial infection process and the organogenesis of the root nodule, the organ which hosts the N 2 -fixing bacteria. During the infection process, rhizobia attach to root hairs in the susceptible zone of the host roots. The recognition of the NFs by the plant induces the root hair curling and the formation of an infection pocket (Fig. 1). Then, bacteria enter into the root hairs through a membrane invagination and the formation of tubular structures, the infection threads (Fig. 1), which allow the transit of the bacteria from the root surface to the cells of the neo-formed nodule [START_REF] Timmers | Refined analysis of early symbiotic steps of the Rhizobium-Medicago interaction in relationship with microtubular cytoskeleton rearrangements[END_REF]. Later, the bacteria are endocytosed into the plant cell in a new organelle-like structure, called symbiosome. Bacteria inside the symbiosome, called bacteroids, become able to reduce N 2 to NH 3 thanks to the activity of a specific enzymatic complex, the nitrogenase [START_REF] Peters | Nitrogenase structure and function: A biochemical-genetic perspective[END_REF].

Two predominant nodule types exist in legumes (Fig. 1). The nodule type depends on the host plant and is determined by the nature of the nodule meristem which could be either transient or persistent (Oldroyd, 2013). Determinate nodules, with a transient meristem, are spherical since cell divisions occur only during a short period of time. The final growth of the nodule is achieved by cell enlargement. In contrast, indeterminate nodules, with a persistent meristem, are cylindrical as they grow during the whole nodule life by increasing the number and the size of cells. Determinate nodules are generally formed in tropical legumes such as Phaseolus, Vigna and Glycine, whereas indeterminate nodules are found in temperate legumes such as Medicago, Trifolium or Pisum.

Beside the nodule types, the bacteroids possess also three different morphotypes named U, S and E (Kondorosi, Mergaert, & Kereszt, 2013; [START_REF] Oono | Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation[END_REF]. The U-morphotype (for Undifferentiated) corresponds to bacteroids with minor morphological modifications compared to the free-living bacteria and the ability to reverse from the symbiotic lifestyle to the free-living state. The Se and E-morphotypes correspond to large Spherical and large Elongated bacteroids, respectively. These bacteroids cannot return to free-living life and are termed "terminally differentiated". This terminal differentiation is associated with the increase of permeability of the bacteroid membrane and of DNA content compared to free-living bacteria (Mergaert et al., 2006). The bacteroid differentiation is driven by the plant host via the production of a class of peptides called nodule-specific cysteine-rich peptides (NCRs; Van de Velde et al., 2010). To date, NCR peptides have been found to control the terminal differentiation of endosymbionts in inverted repeat-lacking clade (IRLC) legumes and in Aeschynomene spp. (Alunni et al., 2007;[START_REF] Czernic | Convergent evolution of endosymbiont differentiation in Dalbergioid and IRLC legumes mediated by nodule-specific cysteine-rich peptides[END_REF]Mergaert et al., 2003). At the functional level, NCRs induce membrane permeability [START_REF] Lamouche | Symbiotic efficiency of spherical and elongated bacteroids in the aeschynomene-bradyrhizobium symbiosis[END_REF] and have intracellular bacteroid targets such as the cell division protein FtsZ and the chaperone protein GroEL, suggesting that NCRs interact with the bacterial metabolism to induce the terminal differentiation [START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF].

The cellular differentiation of both symbiotic partners is required to allow the symbiotic N 2 fixation. The production of the nitrogenase enzymatic complex in the bacteroid is a prerequisite for the reduction of N 2 to NH 3 . This complex needs high amounts of energy, using sixteen ATP for each N 2 reduced to NH 3 , and as a result demands a high respiration rate in the nodule. However, the nitrogenase activity is extremely sensitive to dioxygen (O 2 ) implying that free O 2 should be present at a very low level in bacteroids [START_REF] Peters | Nitrogenase structure and function: A biochemical-genetic perspective[END_REF]. Multiple strategies allow to deal with this paradox. To decrease the O 2 level in the infected cells, the nodule inner cortex cell layer acts as an oxygen barrier [START_REF] Tjepkema | Measurement of oxygen partial pressure within soybean nodules by oxygen microelectrodes[END_REF]. In parallel, leghemoglobin (Lb), an efficient O 2 -binding protein, present at millimolar concentration in the cytoplasm of infected cells [START_REF] Appleby | The origin and functions of haemoglobin in plants[END_REF], delivers O 2 to the mitochondria and the bacteroids for respiration and decreases free O 2 to the nanomolar range in the N 2 -fixing zone (Ott et al., 2005;[START_REF] Soup Ene | Oxygen as a key developmental regulator of Rhizobium meliloti N2-fixation gene expression within the alfalfa root nodule[END_REF]. Finally, the bacteroid is able to function at a very low oxygen concentration thanks to a specific high affinity cbb3 cytochrome oxidase [START_REF] Preisig | A high-affinity cbb3-type cytochrome oxidase terminates the symbiosis-specific respiratory chain of Bradyrhizobium japonicum[END_REF]. The reduction of N 2 to NH 3 is accompanied by the interuption of NH 3 assimilation into bacteroid amino acids. NH 3 is secreted from the bacteroid into the plant cytosol, for assimilation into the amides glutamine and asparagine, or into ureides [START_REF] Day | Ammonia and amino acid transport across symbiotic membranes in nitrogen-fixing legume nodules[END_REF]. In return, the plant provides carbon and energy sources to bacteroids in the form of dicarboxylic acids, particularly malate and succinate, which are produced from sucrose via sucrose synthase and glycolytic enzymes. Thus, the nodule presents multiple peculiarities, involving a plant-microbe crosstalk associated with a metabolism switch which needs a high energy level under microoxic conditions. These working conditions involve a specific cellular redox control in both symbiotic partners. The contribution of reactive oxygen and N species (ROS and RNS) and the importance of the antioxidant defense in the formation and the functioning of root nodules have been largely demonstrated [START_REF] Alloing | Involvement of glutaredoxin and thioredoxin systems in the nitrogen-fixing symbiosis between legumes and rhizobia[END_REF][START_REF] Damiani | Reactive oxygen species and nitric oxide control early steps of the legume -rhizobium symbiotic interaction[END_REF][START_REF] Frendo | Thiol-based redox signaling in the nitrogen-fixing symbiosis[END_REF]Puppo, Pauly, Boscari, Mandon, & Brouquisse, 2013).

Whereas formation and functioning of the root nodule has been extensively analyzed the rupture of the interaction leading to the nodule senescence has not been much characterized and only few reviews have been dedicated to nodule senescence [START_REF] Bruand | Nitric oxide in plants: Pro-or anti-senescence[END_REF][START_REF] Dupont | The legume root nodule: From symbiotic nitrogen fixation to senescence[END_REF]Puppo et al., 2005;[START_REF] Serova | Symbiotic nodule senescence in legumes: Molecular-genetic and cellular aspects (review)[END_REF]. Some investigators have used the initial decline in nitrogenase activity and BNF efficiency as an indication that nodule senescence has begun [START_REF] Klucas | Studies on soybean nodule senescence[END_REF]. Others authors have defined nodule senescence as the period of rapid nodule decay which occurs at a plant physiological maturity stage, i.e. at leaf yellowing [START_REF] Streeter | Seasonal distribution of carbohydrates in nodules and stem exudate from field-grown soya bean plants[END_REF], after flowering has finished [START_REF] Serova | Symbiotic nodule senescence in legumes: Molecular-genetic and cellular aspects (review)[END_REF], or at pod filling (Bethlenfalvay & Phillips, 1977;Lawn & Brun, 1974). The nodule senescence represents a strong agronomical interest through the possible improvement of N 2 fixation efficiency under adverse environmental conditions to maintain or even increase the yield of legume crops. In this review, we summarize the different modifications of nodule functioning observed during the set up and the progress of nodule senescence, and we focus on the role of antioxidants, ROS/RNS, hormones and proteases in regulating this process.

The regulation of nodule senescence 2.1 Physiological and structural modifications

Although the lifespan of nodules of most legumes is 10e12 weeks, their ability to fix N starts to decline 3e5 weeks post-inoculation (Puppo et al., 2005). Nodule senescence is a highly organized and genetically controlled process that begins with metabolic changes. As nodules and leaves are tightly connected in a reciprocal sink-to-source relationship for carbon and N, any defect in photosynthetic activity will indeed impair the metabolic functions of the nodule associated with N assimilation and consequently provoke nodule senescence (Fig. 2). Developmental senescence (also known as natural senescence) occurs because of organ aging, but exposure to various environmental changes also induces nodule senescence (Fig. 2), e.g. when plants are submitted to drought (Aranjuelo et al., 2013;Gil-Quintana, Lyon, Staudinger, Wienkoop, & Gonzalez, 2015;[START_REF] Gogorcena | N2 fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants[END_REF], cadmium stress [START_REF] Balestrasse | Cadmium-induced senescence in nodules of soybean (Glycine max L.) plants[END_REF][START_REF] Redondo | Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules[END_REF], photosynthesis inhibitors [START_REF] M€ Uller | Trehalose becomes the most abundant non-structural carbohydrate during senescence of soybean nodules[END_REF], prolonged darkness (Hern andez-Jiménez, Mercedes Lucas, & de Felipe, 2002;Matamoros et al., 1999;[START_REF] Perez-Guerra | Comparison of developmental and stress-induced nodule senescence in Medicago truncatula[END_REF]Pierre et al., 2014) et al. al., 1979), or treatments with argon [START_REF] Curioni | Glycolytic flux is adjusted to nitrogenase activity in nodules of detopped and argon-treated alfalfa plants[END_REF], nitrate (Matamoros et al., 1999;Pierre et al., 2014) or abscisic acid (ABA; [START_REF] Gonzalez | Abscisic acid induces a decline in nitrogen fixation that involves leghaemoglobin, but is independent of sucrose synthase activity[END_REF]. Alternately, senescence can result from a deficient recognition between the two partners [START_REF] Banba | Lotus japonicus forms early senescent root nodules with Rhizobium etli[END_REF]. Depending on the type of triggering factor, the senescence process is more or less rapid and implement variable responses (Van de Velde et al., 2006), but a number of common typical features are found in most nodule senescence processes.

During developmental senescence, the first sign of nodule senescence is the drop in measurable N 2 fixation activity that occurs in parallel with a decrease in Lb content (Dalton, Russell, Hanus, Pascoe, & Evans, 1986;[START_REF] Lahiri | Biochemical changes in nodules of Vigna mungo (L.) during vegetative and reproductive stages of plant growth in the field[END_REF]. However, during induced senescence, such as after dark treatment, drought stress, or ABA treatment, the rapid decline in N 2 fixation occurs well before a decrease in Lb transcripts or amounts is observed [START_REF] Escuredo | Involvement of activated oxygen in nitrate-induced senescence of pea root nodules[END_REF][START_REF] Gogorcena | N2 fixation, carbon metabolism, and oxidative damage in nodules of dark-stressed common bean plants[END_REF][START_REF] Gonzalez | Abscisic acid induces a decline in nitrogen fixation that involves leghaemoglobin, but is independent of sucrose synthase activity[END_REF]Matamoros et al., 1999). The most visible sign of senescence is a change of the nodule color, from pink to green, caused by bilirubin and biliverdin accumulation after disruption of the Lb structure [START_REF] Lehtovaara | Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation[END_REF][START_REF] Swaraj | Physiological and biochemical basis of nodule senescence in legumes: A review[END_REF]. In senescent soybean nodules, the green color comes partially from Lb nitration, which highlights the critical role of ROS and RNS in the senescence process [START_REF] Meakin | The contribution of bacteroidal nitrate and nitrite reduction to the formation of nitrosylleghaemoglobin complexes in soybean root nodules[END_REF][START_REF] Navascués | Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules[END_REF][START_REF] Sainz | Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism[END_REF].

In nodules of the determinate type, senescence develops radially (Fig. 1), starting from the center of the nodule and then progressively extending to the peripheral tissues in a few weeks (Puppo et al., 2005). In nodules of indeterminate type, the N 2 fixation zone is constantly renewed and the senescence zone (zone IV) grows as the nodule grows (Fig. 1); it increases from the root proximal zone toward the apical zone of the nodule [START_REF] Montiel | Legume NADPH oxidases have crucial roles at different stages of nodulation[END_REF][START_REF] Perez-Guerra | Comparison of developmental and stress-induced nodule senescence in Medicago truncatula[END_REF]Puppo et al., 2005). Because this zonation is not strictly defined, a senescence inter-zone is sometimes described between zone III and zone IV. Histologically, early nodule senescent cells exhibit a less dense cytoplasm containing numerous vacuoles, altered peroxisomes, mitochondria, starch granules, and late senescent cells ultimately accumulate damaged "ghost" membranes and cell walls (Perez- Guerra et al., 2010;Puppo et al., 2005). In common bean nodule developmental senescence, mitochondria were found to be early targets of oxidative modifications and a potential source of ROS/RNS [START_REF] Matamoros | Mitochondria are an early target of oxidative modifications in senescing legume nodules[END_REF]. A dynamic evolution of both plant and bacteroid cell structures is observed: bacteroids are first degraded whereas plant cells remain alive during the early senescence stage, allowing nutrient remobilization, even though at later stages plant cells are also degraded (Van de Velde et al., 2006;Vasse, de Billy, Camut, & Truchet, 1990). In senescent cells, the degradation of symbiosomes is correlated with the loss of the acidity of the peribacteroid space (Pierre et al., 2013), this acidification being an indicator of the bacteroid functioning in the nodule N fixing zone (Pierre et al., 2013).

The senescence process was analyzed at gene expression level in both leaves and nodules, particularly in Medicago truncatula [START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF]Van de Velde et al., 2006). It appeared that nodule senescence has a significant degree of overlap with leaf senescence, arguing for the recruitment of similar pathways. A strong redox regulation, through antioxidants decrease and ROS/RNS accumulation, as well as hormonal regulations such as abscisic acid, ethylene or jasmonic acid, was evidenced to correlate with catabolic and remobilization processes during nodule senescence (Puppo et al., 2005;Van de Velde et al., 2006).

Redox state, ROS and RNS

Changes in the redox balance, which modify the antioxidant defense and the level of ROS and RNS, is directly associated with the transition between N 2 fixation and senescence in nodules (Fig. 2; Puppo et al., 2005).

Ascorbate (ASC) and glutathione (GSH) are the main reported soluble antioxidants within nodules and their regulation was supposed to control the organ lifespan (Becana, Matamoros, Udvardi, & Dalton, 2010;Loscos, Matamoros, & Becana, 2008). In soybean plants, drought-induced nodule senescence is characterized by a decrease in ASC reduction state and an increase in GSH content (Marquez-Garcia et al., 2015). In bean nodules, the progression of developmental senescence correlates with a decrease in both ASC and GSH concentrations and reduction states (Loscos et al., 2008). Moreover, the decrease in ASC and GSH concentrations in mitochondria correlates to an increase of oxidative modifications of lipids and proteins, supporting the hypothesis that the mitochondria is an early target of senescence in nodules [START_REF] Matamoros | Mitochondria are an early target of oxidative modifications in senescing legume nodules[END_REF]. In contrast, an increased level of GSH in the N 2 -fixing zone is correlated with a higher N 2 fixation efficiency (El Msehli et al., 2011). GSH content is notably involved in the control of glutathione S-transferases (GSTs) activities, which are enzymes involved in the cell protection from an oxidative damage, by catalyzing the conjugation of GSH with oxidative degradation products. The level of the GST9 protein, the most prevalent glutathione-S-transferase in soybean nodules, is accordingly increased during senescence [START_REF] Dalton | Physiological roles of glutathione s-transferases in soybean root nodules[END_REF]. A decrease in N 2 fixation associated with an increase of the oxidatively damaged protein content is observed in GST9-RNAi silenced soybean nodules, illustrating the role of GSTs in nodule senescence through the regulation of antioxidant defences [START_REF] Dalton | Physiological roles of glutathione s-transferases in soybean root nodules[END_REF]. In parallel, the importance of GSH during symbiosis of Sinorhizobium meliloti and Rhizobium tropici has been also demonstrated (Harrison et al., 2005;Muglia, Comai, Spegazzini, Riccillo, & Aguilar, 2008). The S. meliloti and R. tropici strains deficient in gshB, the gene encoding the enzyme catalyzing the second step of GSH synthesis, have a reduced N 2 fixation capacity associated with early nodule senescence. Similarly, mutation in the S. meliloti glutathione reductase gene, gor, impaired N 2 fixation efficiency (Tang et al., 2018).

During the legume-rhizobium symbiosis, both ROS and RNS, and especially nitric oxide (NO), appear to play crucial roles in the establishment and functioning of the interaction (Hichri et al., 2015;Puppo et al., 2013). ROS including superoxide radicals (O 2

.À ) and hydrogen peroxide (H 2 O 2 )

are produced in high amounts in nodules due to elevated rates of respiration in bacteroids and plant mitochondria [START_REF] Matamoros | Mitochondria are an early target of oxidative modifications in senescing legume nodules[END_REF]. Plant NADPH oxidases, known as Respiratory Burst Oxidase Homologues (RBOHs), are plasma membrane enzymes catalyzing the reduction of O 2 to generate O 2 .À using the reducing power of NADPH. Seven Rboh genes have been identified in legume genomes (Marino et al., 2011) and these oxidases are involved in legume-rhizobium symbioses (Arthikala et al., 2014;Marino et al., 2011;[START_REF] Montiel | Legume NADPH oxidases have crucial roles at different stages of nodulation[END_REF]. Other possible sources for H 2 O 2 are cell wall peroxidases, germin-like oxalate oxidases, and diamine oxidases [START_REF] Smirnoff | Hydrogen peroxide metabolism and functions in plants[END_REF]. On the other hand, NO is a reactive diffusible gas known to have important functions as inter-or intra-cellular signaling molecules in plant growth, development and defense against abiotic or biotic stresses (Brouquisse, 2019, and references therein). For a decade, NO has been also identified at all stages of legume-rhizobium symbiotic interactions (Berger, Boscari, Frendo, & Brouquisse, 2019;[START_REF] Hichri | Nitric oxide: Jack-of-All-Trades of the nitrogen-fixing symbiosis[END_REF]Hichri et al., , 2015)). The way NO is produced in legumes is not completely clear. The best characterized sources of NO are the nitrate reductase (NR) and the mitochondrial transfer chain (Horchani et al., 2011). Interestingly, it has been shown in soybean and M. truncatula nodules that the micro-symbiont accounts for 30% up to 90% of NO production thanks to a nitrite reductase (Nir) belonging to the denitrification pathway [START_REF] Calvo-Begueria | Redefining nitric oxide production in legume nodules through complementary insights from electron paramagnetic resonance spectroscopy and specific fluorescent probes[END_REF]Horchani et al., 2011;[START_REF] Gates | Production of nitric oxide and nitrosylleghemoglobin complexes in soybean nodules in response to flooding[END_REF]. Even though ROS and NO are different types of molecules issuing from distinct synthesis pathways, some parallels can be established when looking at their occurrence during symbiosis. Strikingly, NO and ROS have comparable production patterns during symbiosis such as an early production burst in the root in response to rhizobia inoculation, later in the infection thread, then during nodule organogenesis and finally in mature nodules [START_REF] Hichri | Nitric oxide: Jack-of-All-Trades of the nitrogen-fixing symbiosis[END_REF][START_REF] Montiel | Legume NADPH oxidases have crucial roles at different stages of nodulation[END_REF].

Interestingly, NO and ROS play a role in nodule senescence. At a late stage of the nodulation process (e.g. 6 weeks post-inoculation), H 2 O 2 is detected in ultrathin sections of nodules (Puppo et al., 2013). At the ultrastructural level, the senescent nodules of Glycine max and Pisum sativum plants display alterations in the symbiosome structure accompanied by the presence of H 2 O 2 , supporting the idea that ROS have roles also during senescence [START_REF] Montiel | Legume NADPH oxidases have crucial roles at different stages of nodulation[END_REF]. In addition, it has been suggested that NO is present in higher amounts during the late symbiotic stages than at earlier stages [START_REF] Bruand | Nitric oxide in plants: Pro-or anti-senescence[END_REF]. Indeed, NO detected by means of a bacterial biosensor is more abundant in the root-proximal part of M. truncatula nodules, where senescence is known to initiate (Cam et al., 2012). Similarly, NO detected by means of a fluorescent probe accumulates in old Lotus japonicus nodules and Lbs with nitrated heme groups are found in old soybean nodules, indicating the presence of NO in aging nodules [START_REF] Chungopast | Iron-induced nitric oxide leads to an increase in the expression of ferritin during the senescence of Lotus japonicus nodules[END_REF][START_REF] Navascués | Leghemoglobin green derivatives with nitrated hemes evidence production of highly reactive nitrogen species during aging of legume nodules[END_REF][START_REF] Sainz | Leghemoglobin is nitrated in functional legume nodules in a tyrosine residue within the heme cavity by a nitrite/peroxide-dependent mechanism[END_REF]. Remarkably, exogenous addition of a NO donor on mature nodules of M. truncatula lead to an accelerated senescence of the nodules (Cam et al., 2012). Also, several S. meliloti mutant strains, altered on hmp, nnrS1, nnrS2 and norB genes (Table 1) and showing either increased or decreased NO degradation capacity compared to the wildtype, were used to modulate NO levels within nodules and, strikingly, strains deficient in NO degradation induced an early nodule senescence, whereas strains with higher degradation activity led to a delayed nodule senescence (Blanquet et al., 2015;Cam et al., 2012;Meilhoc, Blanquet, Cam, & Bruand, 2013). NO is also involved in stress-induced M. truncatula nodule senescence (Cam et al., 2012). Comparable results were recently obtained in determinate L. japonicus nodules (Fukudome et al., 2019). Indeed, overexpression of a phytoglobin gene LjGlb1-1 (Table 1) decreased NO levels in nodules and delayed nodule senescence.

By analogy, [START_REF] Redondo | Overexpression of flavodoxin in bacteroids induces changes in antioxidant metabolism leading to delayed senescence and starch accumulation in alfalfa root nodules[END_REF][START_REF] Redondo | Alfalfa nodules elicited by a flavodoxin-overexpressing Ensifer meliloti strain display nitrogen-fixing activity with enhanced tolerance to salinity stress[END_REF] reported that overexpression in S. meliloti of a cyanobacterial flavodoxin involved in the response to oxidative stress in bacteria and cyanobacteria improves the oxidative balance, delays developmental nodule senescence in Medicago sativa, and enhances tolerance to cadmium. Overexpression of this flavodoxin also permitted to avoid the decline in nitrogenase activity associated with a salinity stress whether the flavodoxin was expressed in S. meliloti or in M. truncatula (Coba de la Peña, [START_REF] De La Peña | Nitrogen fixation persists under conditions of salt stress in transgenic Medicago truncatula plants expressing a cyanobacterial flavodoxin[END_REF]. In S. meliloti, various ROS scavenging enzymes (katA and katC catalases particularly) are expressed in the N 2 -fixing zone (Roux et al., 2014). The S. meliloti katA katC double mutant displays a reduced N 2 fixation activity and bacteroids underwent faster senescence (Puppo et al., 2013). Rhizobium etli and Bradyrhizobium japonicum possess only one catalase gene and corresponding mutants were not affected in nodule functioning, but ROS scavenging enzymes other than catalases might reduce H 2 O 2 (Puppo et al., 2013). This might also suggest a different role for ROS and antioxidant defense components in the senescence of determinate and indeterminate nodules.

A drastic reduction in N 2 fixation was observed in Phaseolus vulgaris nodules silenced in NADPH oxidase (Rboh) genes probably linked to an early senescence phenotype (Arthikala et al., 2014). By contrast, overexpression of RbohB delays senescence of the nodule. Hence, these elements suggest that ROS produced by RBOHs may have either a promoting, or an inhibiting role during nodule senescence in P. vulgaris. Two Rboh genes (MtRbohA and MtRbohB) were found specifically expressed in the N 2 -fixing zone of M. truncatula nodules (Marino et al., 2011;[START_REF] Montiel | Legume NADPH oxidases have crucial roles at different stages of nodulation[END_REF]Roux et al., 2014). A decrease in MtRbohA expression via a RNA interference (RNAi) approach led to a reduction in the N 2 fixation capacity, showing again the link between MtRBOHA and nodule function (Marino et al., 2011). However, none of these genes displays any detectable promoter activity in the senescence zone of indeterminate nodules, suggesting that they do not directly act in nodule senescence. In addition, RBOHs might have a more prominent role in determinate nodules than in indeterminate nodules, even though the possibility that they participate in nodule senescence should not be eliminated before each gene has been studied in detail in different plants.

Hypotheses on mechanisms by which NO and ROS could trigger nodule senescence are multiple. These molecules can both act at the transcriptional and post-translational levels. Indeed, NO is known to activate the expression of bacterial as well as plant genes (Boscari et al., 2013a;[START_REF] Ferrarini | Expression of Medicago truncatula genes responsive to nitric oxide in pathogenic and symbiotic conditions[END_REF][START_REF] Meilhoc | The response to nitric oxide of the nitrogen-fixing symbiont Sinorhizobium meliloti[END_REF]. However, it is unknown yet which genes or transcription factors (TFs) could be involved at the onset of nodule senescence. One of the main downstream effects of NO is the post-translational modification of proteins such as S-nitrosation of cysteine residues or nitration of tyrosine residues, which might cause conformational changes in the protein with concomitant changes in their activities, turnover or localization. About 80 proteins were found to be S-nitrosated in M. truncatula nodules; among them 20 and 8 plant and bacterial proteins, respectively, were also found to be sulfenylated by ROS (Puppo et al., 2013). These protein targets remain to be characterized to establish a potential link with the nodule senescence. However, the activity of many of them, involved either in the glycolysis, the tricarboxylic acid cycle, the energy regeneration or the N 2 assimilation, were found to be inhibited by NO, suggesting that an increased NO production could directly down-regulate N, carbon and energy metabolism in senescent nodules [START_REF] Boscari | Which role for nitric oxide in symbiotic N2-fixing nodules: Toxic by-product or useful signaling/metabolic intermediate[END_REF]. Interestingly, the enzymatic activity of the plant glutamine synthetases (GS) involved in N assimilation decreases when GS is subjected to NO-mediated tyrosine nitration [START_REF] Melo | Glutamine synthetase is a molecular target of nitric oxide in root nodules of Medicago truncatula and is regulated by tyrosine nitration[END_REF]. Nevertheless, methionine sulfoxidation of GS is also important for its regulation in response to ROS, showing again that both NO and ROS can be involved in the regulation of protein activity [START_REF] Matamoros | Mitochondria are an early target of oxidative modifications in senescing legume nodules[END_REF]. It has also been shown that the nitrogenase activity is inhibited by NO [START_REF] Trinchant | Nitrite and nitric oxide as inhibitors of nitrogenase from soybean bacteroids[END_REF].

NO and ROS are generated in cells at the same time and in response to the same cues, so that it is important to consider their possible interaction and associated consequences. One of the best known interaction between NO and ROS is the production of peroxynitrite (ONOO À ) from NO and superoxide anion (O 2

.À ). This has two potentially important outcomes: first, the reaction removes both O 2

.À and NO from the cell environment, thereby reducing their bioavailability; and second, there is a new compound produced (ONOO À ) which itself can act as a signaling/regulatory molecule, potentially giving different responses than that resulting from ROS or NO signaling alone. As an example, peroxynitrite is more potent than NO alone to trigger tyrosine nitration [START_REF] Astier | Nitric oxide-dependent posttranslational modification in plants: An update[END_REF]. NO/ONOO À -dependent modifications may also alter signaling pathways mediated by other reactive signals. Indeed, Tyr nitration of superoxide dismutase can inhibit its activity and hence alter ROS signaling [START_REF] Holzmeister | Differential inhibition of Arabidopsis superoxide dismutases by peroxynitrite-mediated tyrosine nitration[END_REF]. NO has also been proposed as a negative regulator of RBOH activity in nodulation [START_REF] Damiani | Reactive oxygen species and nitric oxide control early steps of the legume -rhizobium symbiotic interaction[END_REF]. Indeed, it has been reported that the AtRBOHD enzyme is S-nitrosated which thus reduces its ROS generating activity [START_REF] Yun | S-nitrosation of NADPH oxidase regulates cell death in plant immunity[END_REF]. Therefore, NO has a role in controlling ROS levels and hence their associated downstream signaling. In addition, the B. japonicum key regulator of the N 2 -fixing enzymes FixK 2 is reversibly susceptible to oxidation, which might indirectly have an influence on the expression of bacterial genes involved in the NO metabolism regulated by FixK 2 [START_REF] Mesa | Posttranslational control of transcription factor FixK2, a key regulator for the Bradyrhizobium japonicum-soybean symbiosis[END_REF]. Similarly, LsrB, a TF involved in the nodulation and redox regulation, is also regulated by its redox state and might be involved in the senescence process [START_REF] Tang | Regulation of cysteine residues in LsrB proteins from Sinorhizobium meliloti under free-living conditions and symbiotic oxidative stress[END_REF].

Hormones

Plant hormones are other obvious candidate regulatory molecules to control the transition between N 2 fixation and nodule senescence, some of these signals being previously associated with leaf senescence, such as cytokinins that delay leaf senescence (Zwack & Rashotte, 2013). In white lupin, expression of the cytokinin receptor LaHK1, closely related to AHK4/ CRE1 in Arabidopsis thaliana, is increased during nodule senescence (Coba de la [START_REF] De La Peña | A salt stress-responsive cytokinin receptor homologue isolated from Medicago sativa nodules[END_REF]. In L. japonicus, expression of the LjIPT3 (IsoPentenyl-adenine Transferase 3, Table 1) gene encoding a rate-limiting cytokinin biosynthetic enzyme, increases during nodule aging and is associated with vascular bundles of mature nodules. LjIPT3 down-regulation by an RNAi approach triggers a decrease in N 2 fixation, consequently associated with premature nodule senescence (Chen et al., 2014). In addition, a maintained N 2 fixation associated with delayed nodule senescence was observed in drought-stressed nodules of alfalfa inoculated with an S. meliloti strain engineered to overexpress an IPT cytokinin biosynthesis gene and therefore producing increased levels of cytokinins [START_REF] Xu | Effects of engineered Sinorhizobium meliloti on cytokinin synthesis and tolerance of alfalfa to extreme drought stress[END_REF]. Cytokinin may therefore act as a positive regulator of N 2 fixation [START_REF] Boivin | Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula[END_REF][START_REF] Reid | CYTOKININ OXIDASE/DEHYDROGENASE3 maintains cytokinin homeostasis during root and nodule development in Lotus japonicus[END_REF], and possibly as a negative regulator of root nodule senescence as reported during leaf senescence [START_REF] Zwack | Cytokinin response factor 6 negatively regulates leaf senescence and is induced in response to cytokinin and numerous abiotic stresses[END_REF].

Besides cytokinins, several gene expression analyses revealed putative functions for other plant hormones [START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF]Van de Velde et al., 2006). Among those, genes encoding enzymes of the ethylene biosynthesis pathway, such as an 1-aminocyclopropane-1-carboxylate (ACC) oxidase, are induced in senescent nodules (Van de Velde et al., 2006). Interestingly, the lack of an ACC deaminase activity in the Sinorhizobium strain sp. BL3 accelerates nodule senescence in mung bean, suggesting a positive role for ethylene in nodule senescence similarly to what is observed during leaf senescence [START_REF] Tittabutr | Possible role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity of Sinorhizobium sp. BL3 on symbiosis with mung bean and determinate nodule senescence[END_REF]. Lipoxygenase genes associated with Jasmonic Acid (JA) biosynthesis and GA2-oxidase genes associated with Gibberellic Acid (GA) degradation are also up-regulated during nodule senescence, suggesting that JA may positively and GA negatively regulate nodule senescence (Benedito et al., 2008;Van de Velde et al., 2006). Abscisic acid (ABA) could be also involved in nodule senescence since some ABA responsive genes such as ABR17 (ABA Responsive 17) are induced during nitrate-induced senescence [START_REF] Liese | Nitrate application or P deficiency induce a decline in Medicago truncatula N2-fixation by similar changes in the nodule transcriptome[END_REF]. In addition, ABA, which is associated with various environmental stress responses, improves nodule N 2 fixation capacity in alfalfa salt-stressed nodules where antioxidant defenses mediated by the polyamine spermidine and spermine are induced [START_REF] Palma | Involvement of abscisic acid in the response of Medicago sativa plants in symbiosis with Sinorhizobium meliloti to salinity[END_REF], and might therefore regulate senescence progression. Such putative negative effect of ABA on nodule senescence is however not observed in leaves where ABA is a positive regulator of senescence [START_REF] Lee | Age-dependent action of an ABA-inducible receptor kinase, RPK1, as a positive regulator of senescence in Arabidopsis leaves[END_REF].

Considered together, if each of these studies suggests that hormones can intervene in the regulation of senescence, in most cases, functional analyses are lacking to clearly identify how these hormones regulate nodule senescence and more specifically how they may interact with ROS and RNS signaling pathways that are the most extensively linked to nodule senescence. These limited data may nevertheless suggest that common hormonal regulations between leaf and nodule senescence may exist, which could be surprising considering their differences in carbon versus N metabolic status.

Proteases associated with bacteroid and plant cell degradation

A hallmark of cell aging is the progressive decline in proteostasis and the accumulation of misfolded, oxidized and damaged proteins (L opez-Otín, Blasco, Partridge, Serrano, & Kroemer, 2013). Therefore, proteolytic activities are under strict regulation to ensure protease activation and degradation at appropriate time and space. In plants, proteases have been associated with global protein turnover, protein processing, degradation of regulatory proteins, cell growth and differentiation, stress response and developmental senescence, but to date functional evidence of their mode of action remains still limited (Buono, Hudecek, & Nowack, 2019). There are hundreds of protease-encoding genes; for example, more than 800 have been identified in Arabidopsis ( [START_REF] Van Der Hoorn | Plant proteases: From phenotypes to molecular mechanisms[END_REF]. Such a large number of proteases can be grouped into families according to their catalytic residues, aspartic-, cysteine-, serine-, threonine-, glutamic-, asparagine-, or metalloproteases [START_REF] Oda | New families of carboxyl peptidases: Serine-carboxyl peptidases and glutamic peptidases[END_REF]. A description of the protease classification can be retrieved in the MEROPS database (https://www.ebi.ac.uk/merops/; Rawlings, [START_REF] Rawlings | MEROPS: The peptidase database[END_REF].

To date, many reports have elucidated the roles of cysteine proteases (CPs) in nodule senescence and in response to detrimental environments (Cilliers, van Wyk, van Heerden, Kunert, & Vorster, 2018;[START_REF] Perez-Guerra | Comparison of developmental and stress-induced nodule senescence in Medicago truncatula[END_REF]Pierre et al. 2014;Pladys, Dimitrijevic, & Rigaud, 1991;Pladys & Vance, 1993;Van de Velde et al. 2006). The synthesis of CP precursors is orchestrated by a complex transcriptional regulation network. The posttranslational activation of many CPs relies on either self-processing or on the interaction with other enzymes. Their activity is also modulated by inhibitors and co-factors. CPs encoding genes are among the most strongly and specifically induced nodule senescence genes, either developmentally or environmentally triggered (Cabeza et al., 2014;Chungopast et al., 2014;Seabra, Pereira, Becker, & Carvalho, 2012;Van de Velde et al., 2006). Their activity is directly correlated to the protein degradation occurring during senescence, and was documented in various legume plants including soybean [START_REF] Alesandrini | Possible roles for a cysteine protease and hydrogen peroxide in soybean nodule development and senescence[END_REF], Astragalus sinicus (Naito, Fujie, Usami, Murooka, & Yamada, 2000), pea (Kardailsky & Brewin, 1996), and M. truncatula (Pierre et al., 2014).

In soybean, 18 cysteine proteases were identified as actively transcribed in nodules during the developmental senescence (Van Wyk, Du Plessis, Cullis, Kunert, & Vorster, 2014). This expression was increased at specific time points of the senescence process with a majority of CP genes highly expressed in 14 week-old nodules. In Trifolium repens, the identified CP encoding genes were specifically expressed in the senescent zone of the nodule as revealed by a spatio-temporal analysis (Asp, Bowra, Borg, & Holm, 2004). In A. sinicus indeterminate nodules, the papain cysteine protease AsNodF32 expressed in senescent tissues of the nodule was identified from a differential screening of a nodule cDNA library (Naito et al., 2000). An RNAi silencing of AsNodF32 extended the period of the active N 2 -fixation, delaying nodule senescence (Li et al., 2008). In M. truncatula nodules, the Cysteine Protease 6 (MtCP6), (MEROPS family C1A; https://www.ebi.ac.uk/merops/), and a Vacuolar Processing Enzyme (MtVPE; MEROPS family C13), belonging respectively to papain and legumain families, were strongly up-regulated in response to both developmental and environmentally-induced nodule senescence (Van de Velde et al., 2006;Pierre et al., 2014, Table 1). A detailed spatio-temporal analysis of the MtCP6 and MtVPE expression patterns during nodule developmental senescence revealed an expression associated with the transition region between the N 2 -fixing and senescence zones (Fig. 3A andB, Pierre et al., 2014). In nitrate-induced senescent nodules, MtCP6 and MtVPE expression was detected in the whole nodule except in the basal senescence zone (Fig. 3C andD). Subcellular localization of both MtCP6 and MtVPE in senescent nodules was associated with vacuoles of cells non-infected by rhizobia and to symbiosomes of infected cells, suggesting a role of these CPs in symbiosomes and plant cell degradation (Pierre et al., 2014) Silencing of MtCP6 or MtVPE genes delayed nodule senescence and increased N 2 fixation, while their ectopic expression in the N 2 -fixing zone promoted nodule senescence and hindered N 2 fixation (Pierre et al., 2014). In pea, both PsCyp1 and PsCyp15a (CP) genes transcript levels are increased during nodule aging. However, laser capture microdissection of pea nodule zones III and IV revealed a different pattern of expression: the PsCyp1 gene is activated at an earlier stage of nodule senescence as transcripts are found exclusively in zone III, whereas the PsCyp15a gene is expressed in both zone III and senescent zone IV of pea nodule (Serova, Tikhonovich, & Tsyganov, 2017).

CPs were reported to interact with CP inhibitors called cystatins, which act as proteolysis regulators [START_REF] Benchabane | Plant cystatins[END_REF][START_REF] Kunert | Potential use of phytocystatins in crop improvement, with a particular focus on legumes[END_REF][START_REF] Martínez | C1A cysteine-proteases and their inhibitors in plants[END_REF] Makgopa, Cooper, Kunert, & Foyer, 2015;Van Wyk et al., 2014). In soybean developmental senescent nodules, three cystatin genes are co-expressed with CPs (Van Wyk et al., 2014). Remarkably, despite no direct evidence for a role of cystatins during nodule senescence was yet reported, an ectopic overexpression of a rice oryzacystatin in soybean leads to a strong decrease in cathepsin L-like CP activities that are linked to nodule senescence (Quain et al., 2015). The coordinated expression of transcripts encoding cystatins and CPs interacting partners suggests their involvement as a fine-tuning control of proteolysis events associated with nodule senescence. In M. truncatula nodules, serpins (serine protease inhibitors) have also been found to be activated in drought-induced senescence and might play a role as modulators of papain-like CPs (Dhanushkodi, Matthew, McManus, & Dijkwel, 2018).

Increasing data show that the symbiotic establishment relies on the suppression of plant immunity [START_REF] Liang | Nonlegumes respond to rhizobial Nod factors by suppressing the innate immune response[END_REF][START_REF] Yu | Suppression of innate immunity mediated by the CDPK-Rboh complex is required for rhizobial colonization in Medicago truncatula nodules[END_REF]. Transcriptomic studies have revealed transient elicitation of legume plant defense related genes upon rhizobium inoculation [START_REF] Libault | Complete transcriptome of the soybean root hair cell, a single-cell model, and its alteration in response to Bradyrhizobium japonicum infection[END_REF][START_REF] Lohar | Transcript analysis of early nodulation events in Medicago truncatula[END_REF][START_REF] Pucciariello | Homo)glutathione depletion modulates host gene expression during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti[END_REF], followed by their suppressions in the subsequent symbiotic stages [START_REF] Aslam | Bacterial polysaccharides suppress induced innate immunity by calcium chelation[END_REF]. Besides being known as an ending stage of organ development, nodule senescence is also proposed to involve a reversion of the plant innate immunity (Puppo et al. 2005). CPs have also been shown to be involved in salicylic acid induced plant defense to cope with microbial pathogens [START_REF] M€ Ueller | Compatibility in the Ustilago maydisemaize interaction requires inhibition of host cysteine proteases by the fungal effector Pit2[END_REF][START_REF] Shindo | A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14[END_REF][START_REF] Van Der Linde | A maize cystatin suppresses host immunity by inhibiting apoplastic cysteine proteases[END_REF]. Thus, we could hypothesize that activation of CPs during nodule senescence might be linked to increasing plant defense responses linked to the declining nodule N 2 fixation capacity.

Genes encoding components of the SCF (skp1-cullin-F-Box protein) complex, also known as the E3 ubiquitin ligase complex, and genes encoding 26S proteasome AAA-ATPase subunits and the ubiquitin carrier protein 4 are up-regulated during M. truncatula developmental nodule senescence [START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF][START_REF] Perez-Guerra | Comparison of developmental and stress-induced nodule senescence in Medicago truncatula[END_REF]Van de Velde et al., 2006). This suggests that the 26S proteasome pathway may be acting during nodule senescence.

Transcriptomic characterization of nodule senescence

To get insights into transcriptional regulations associated with symbiotic nodule senescence, several transcriptomic approaches were performed initially on M. truncatula nodules. First, a cDNA-AFLP (Amplified Fragment Length Polymorphism) technique was applied on developmental senescent nodules (Van de Velde et al., 2006). A second approach used microarrays on nodules treated for 8h with phosphinotricin, an inhibitor of GS activity and therefore affecting N 2 fixation (Seabra et al., 2012). More recently, an RNAseq was performed on nodules exposed to nitrate for 4 h or 28 h (Cabeza et al., 2014). Similar studies have been also performed in other legumes species than M. truncatula, such as in L. japonicus using microarrays and in soybean using RNAseq, at different time-points during nodule aging (Chungopast et al., 2014;Yuan et al., 2017).

Among the differentially expressed genes identified, the downregulation of specific N 2 fixation associated genes, such as Lb, late nodulins and NCR peptide encoding genes attests the inhibition of the N 2 fixation at the molecular level (Cabeza et al., 2014;Seabra et al., 2012;Van de Velde et al., 2006). Genes related to energy production, such as mitochondrial ATP synthase subunit encoding genes, or amino-acid biogenesis encoding genes, were accordingly repressed (Cabeza et al., 2014;Seabra et al., 2012). An upregulation of genes encoding proteins involved in macromolecule degradation, such as the CPs previously mentioned, and nutrient remobilization (phosphate, amino-acid, ion transporters and ABC [ATP-Binding Cassette] transporters) was observed, whereas ammonium, potassium and most of the sulfate transporters were down-regulated (Cabeza et al., 2014;Van de Velde et al., 2006). Genes associated with redox stress responses such as catalases, oxidoreductases, hydrolases, peroxidases, esterases, glutathione transferases were also upregulated, showing the relevance of redox regulations during nodule senescence (Seabra et al., 2012;Van de Velde et al., 2006), as previously described.

In order to distinguish at the molecular level environmentally inducedsenescence from developmental senescence, selected genes up-regulated during a M. truncatula developmental nodule senescence were tested by qRT-PCR in nodules exposed to darkness for 3e5 days (Perez- Guerra et al., 2010;Van de Velde et al., 2006). Only 50% of the genes selected as up-regulated during developmental senescence were up-regulated during a dark-induced nodule senescence. Genes not regulated in the dark-induced nodule senescence were notably related to the 26S proteasome-mediated protein degradation [START_REF] Perez-Guerra | Comparison of developmental and stress-induced nodule senescence in Medicago truncatula[END_REF], suggesting either a less organized, or more probably an accelerated cellular degradation in dark-induced conditions. To refine the spatio-temporal expression profiles during nodule senescence, M. truncatula nodules were sectioned into apical versus basal zones and pea nodules were microdissected (Serova et al., 2017;Van de Velde et al., 2006). In this way, the M. truncatula or pea ATB2 genes, encoding a bZIP (basic leucine ZIPper) transcription factor (TF), were identified as reaching a peak in fully senescent nodules (Van de Velde et al., 2006;D'haeseleer et al., 2010;Serova et al., 2017).

To evaluate if genes were specifically regulated during the senescence of nodule organs, a transcriptomic analysis was performed using a cDNA-AFLP approach in M. truncatula senescent leaves [START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF]. The overlap between nodule and leaf senescence associated genes was relatively low (23%). When excluding N-metabolism related genes, a higher degree of overlap was nevertheless observed in the early phase, including specific MAP kinases, ubiquitin cycle components, phosphatases, glutathione transferases, and transcription factors; as well as in the late phase, i.e. several CPs and components of the 26S proteasome (Van de Velde et al., 2006;[START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF].

Based on transcriptomic analyses previously described, transcription factors (TF) encoding genes differentially regulated during nodule senescence were identified, mainly from MYB (MYeloBlastosis), bHLH (basic Helix-Loop-Helix), and NAC (NAM ATAF CUC) families (Van de Velde et al., 2006;Seabra et al., 2012;Cabeza et al., 2014;Chungopast et al.;2014;Yuan et al., 2017). Among those, only few TFs were selected for further functional analyses, with the aim of identifying regulatory pathways controlling the transition between N 2 fixation and nodule senescence, either during aging or in response to environmental stress conditions. The NAC TF family was previously largely associated with leaf senescence based on transcriptome and genetic studies performed in Arabidopsis (Breeze et al., 2011), and is also enriched in nodule senescence transcriptomes. Based on RNAseq or microarray experiments, approximately one-third of NAC genes expressed in nodules are induced by nitrate (16/59 in Cabeza et al. 2014;and 21/57 in Benedito et al., 2008) whereas over 30 out of 90 NAC genes are upregulated during Arabidopsis leaf senescence (Breeze et al., 2011). Expression of the M. truncatula TF MtNAC969, closely related to the well-known Arabidopsis leaf senescence associated gene AtNAP (NAC-LIKE ACTIVATED BY AP3/PI), is induced by nitrate and associated with the central region of symbiotic nodules including the N 2 -fixation zone (de Zelicourt et al., 2012). In addition, both AtNAP and MtNAC969 genes are induced during Arabidopsis and M. truncatula leaf senescence, respectively [START_REF] De Michele | Transcriptome analysis of Medicago truncatula leaf senescence: Similarities and differences in metabolic and transcriptional regulations as compared with Arabidopsis, nodule senescence and nitric oxide signalling[END_REF][START_REF] Balazadeh | A gene regulatory network controlled by the NAC transcription factor ANAC092/AtNAC2/ORE1 during salt-promoted senescence[END_REF]. An RNAi silencing of the MtNAC969 TF promotes nodule senescence (Table 1), based on the increased expression of CP genes used as senescence markers, concomitantly to an accumulation of amyloplasts suggestive of an altered nodule metabolism (de Zelicourt et al., 2012). Expression of the M. truncatula MtATB2 bZIP TF is increased during nodule aging senescence, as well as by sucrose but not by a nitrate treatment (Van de Velde et al., 2006;D'haeseleer et al., 2010). Despite this transcriptional regulation pattern, ectopic expression of MtATB2 reduced the number of nodules formed but no senescence phenotype could be observed (D'haeseleer et al., 2010). This highlights that an increased expression in senescent nodules is not a sufficient criteria to identify genes functionally linked to nodule senescence. As MtATB2 is expressed in the peripheral region of nodules containing the vascular bundles and not in the N 2 fixation or senescence central zones, and because of its regulation by sucrose and not by nitrate, this suggests that this TF might sense carbon sources availability and regulate remobilization processes linked to senescence (D'haeseleer et al., 2010). Finally, no TF was yet directly linked to the upregulation of genes encoding CPs or other cell degradation related enzymes, or to the downregulation of Lb or N-assimilation metabolic enzymes encoding genes, or to components of redox pathways. Similarly, a symbiotic bacterial contribution to the regulation of nodule senescence associated genes has not been explored even if some toxin/antitoxin bacterial systems, such as the vapBC-5 (Table 1), have been showed to alter nodule senescence progression (Ol ahet al., 2001;Lipuma et al., 2014).

Concluding remarks -future prospects

Senescence affecting the legume-rhizobia symbiotic nodule organ during aging or in response to environmental conditions remains poorly documented. The best functionally characterized senescence mechanisms are related to the redox dependent inhibition of the N 2 fixation and assimilation. Physiological and molecular approaches show that nodule senescence is followed by a decrease in the level and/or reduction state of the main cellular antioxidants ASC and GSH, and by an increase in the production of ROS and RNS, such as H 2 O 2 and NO. This results in an increase of oxidized/denatured proteins and subsequently in an inhibition of N, carbon and energy metabolisms. In addition from being harmful to cellular processes, both ROS and NO production are also important components of the signaling network used by legumes and their symbionts to communicate at the various stages of the symbiotic interaction. The evolution of efficient ROS/NO degrading systems has most likely enabled plant cells and bacteroids inside nodules to overcome ROS and NO toxicity. Diverse bacterial and plant systems contribute to the control of NO and ROS levels in nodules. Only when these systems are overcome by NO and ROS, these molecules can act as signals to trigger senescence (Puppo et al., 2013). Alternatively, senescence could be a consequence of NO-induced modifications of proteins essential for N 2 fixation. Further studies are needed to link the modifications of these proteins by NO or ROS to nodule senescence.

An increased proteolysis and the induction of plant vacuolar proteases such as CPs and VPEs during senescence are also well documented, and used as a hallmark to follow the senescence progression. Identifying how these nodule specific proteases are regulated and what are their targets remains a challenge to understand better nodule senescence. Both 26S and 20S proteasome proteolytic pathways are additionally induced during nodule senescence, and their contribution should be established. Finally, the nature of bacterial proteases as well as their contribution to the degradation of bacteroid proteins remains to be better documented.

Large-scale transcriptomic analyses from the last decade allowed identifying new candidate genes that may act in the senescence processes. These approaches were however focused on the plant transcriptome, and it would be interesting to analyze in parallel the transcriptional response of symbiotic bacteria to determine if they could contribute to the regulation of nodule senescence progression. In the model legume M. truncatula, partially overlapping plant regulatory pathways were identified between environmental, developmental and leaf senescence. This suggests that a core of common genes is conserved between the senescence of different organs, but also that a subset of genes may specifically regulate senescence in nodules, which may be notably related to the symbiotic and metabolic features of this organ hosting numerous N 2 fixing bacteria. Such putative plant-microbe gene network interactions remain however to be investigated. Only few regulatory genes directly regulating nodule senescence have been yet functionally characterized, such as TFs and hormone-related genes. A future prospect would be to integrate how the diversity of bacteria hosted inside the nodules, beyond rhizobia, could control senescence progression. Indeed, promising recent data [START_REF] Rodrigues | Metabolism of nitrogen and carbon: Optimization of biological nitrogen fixation and cowpea development[END_REF] reported that other soil bacteria than rhizobia might indeed modify the nodule lifespan, as cowpea nodules co-inoculated with Bradyrhizobium sp. and Paenibacillus graminis or Paenibacillus durus plant growth-promoting bacteria present an increased N availability and delayed nodule senescence. Finally, an applied perspective of studying nodule senescence would be, as achieved with "staygreen" leaves, to engineer "staypink" nodules that would have maintained, and even improved, capacities to fix N 2 under environmental stress conditions.

GENERAL INTRODUCTION

94

Proteases play vital roles during nodule senescence in leguminous plants

During nodule senescence, proteolytic activities are strongly increased and associated with the final degradation of bacteroid and plant host cells (Van de Velde et al., 2006;Perez Guerra et al., 2010;Kazmierczak et al., 2020). Hence, proteases are found to be the hallmarks of nodule senescence (Van de Velde et al., 2006;Perez Guerra et al., 2010). Proteases are firstly targeting leghaemoglobin, as consequences of declined N fixation, then rupture of bacteriods, eventually eliminating the microbial partner of the symbiotic associations (Pladys & Vance, 1993). Cysteine proteases have been identified in the cytoplasm of infected nodule cells and their activity appears to increase markedly during senescence (Malik et al., 1981;Pladys et al., 1991). However, interestingly, the knockdown of the CP inhibitor gene MtSERPIN6 accelerated nodule aging (Dhanushkodi et al., 2018).

Transcript-profiling of developmental nodule senescence was revealed in the legume M. truncatula via cDNA-amplified fragment length polymorphism (AFLP) (Van de Velde et al., 2006). Functional clustering of 508 gene tags to corresponding genes demonstrated the transition from carbon sink to general source status of the nodule, as well as the activation of a wide variety of defence and stress responses. Three chronologically upregulating and one downregulating cluster were identified with a nodule senescence-associated differential expression pattern (Van de Velde et al., 2006), indicating consecutive cascades of transcriptional regulation. A selective expression pattern shows this clear transcriptional regulation waves of regulators and CPs in Table 5. The cluster 1 genes were found also upregulating in heathy tissues, indicating an early signal transduction of nodule senescence initiation. It is not surprising that most of the transcriptional regulators are found in this group (Table 5), e.g. MYB, AP2/ERF, bHLH, NAC/NAM and bZIP TFs. Among the TFs, ATB2 is involved in balancing carbohydrate demand and supply in A. thaliana [START_REF] Rook | Sucrose-specific signalling represses translation of the Arabidopsis ATB2 bZIP transcription factor gene[END_REF][START_REF] Wobbes | Control of plant carbohydrate partitioning by the Arabidopsis thaliana ATB2 bZIP transcription factor gene[END_REF]. In M. truncatula, ectopic expression of MtATB2 diminished nodule formation and affected root growth (D 'Haeseleer et al., 2010). MYB4 is suggested to function as repressors in the flavonoid pathway, together with its homologs MYB7 and MYB32 and further interactive with the bHLH transcription factors TT8, GL3 and EGL3 [START_REF] Wang | Arabidopsis MYB4 plays dual roles in flavonoid biosynthesis[END_REF].

Interestingly, in this early cluster, a tag is present that is homologous to genes encoding abscisic acid-insensitive/viviparous related transcription factors and is characterized by an AP2/ERF and B3 DNA-binding domain [START_REF] Kagaya | Regulation by external and developmental signals of the RAV2 gene encoding a DNA binding protein containing AP2 and B3-like domains[END_REF]. The subsequent cluster 2 and 3 are involved in nodule senescence stage I and II (Table 4), corresponding to bacteroid degradation and plant cell death, respectively. Cysteine protease encoding gene expressions are mostly present in these two clusters (Table 5).

Introduction

In plants, as in all other living organisms, proteins are submitted to permanent turnover. The content of a given protein depends on its rate of both synthesis and degradation. The degradation of damaged, misfolded, and potentially harmful proteins is associated with the activity of proteolytic enzymes. Beside this regulatory role in the protein turnover, proteolysis plays numerous roles in the regulation of cellular metabolism, as well as in plant development and defense [START_REF] Thomas | Senescence, ageing and death of the whole plant[END_REF]).

Proteases can be divided into several classes depending on the hydrolysis site of the target protein and on the chemical nature of the catalytic site of the protease. Exopeptidases which act on the termini of (poly)peptide chains can be classified as aminopeptidases when acting on the N-terminus of the protein and as carboxypeptidases when acting on the C-terminus of the protein.

Endopeptidases, also called proteinases, act on the interior of polypeptides. Various amino acids are present in the catalytic site of proteases. In serine, threonine, and cysteine proteases (CPs), the hydroxyl or sulfhydryl group of the active-site amino acid performs a nucleophilic attack on the peptide bond to hydrolyze it. Acidic proteases (aspartic and glutamic proteases) and metalloproteases, which rely on divalent cations for activity such as zinc, cobalt, and manganese, activate a water molecule to act as a nucleophile to attack the peptide carbonyl group. The plant genomes contain around 2% of proteases. According to the MEROPS database (http://merops.sanger.ac.uk; [START_REF] Rawlings | MEROPS: the database of proteolytic enzymes, their substrates and inhibitors[END_REF], the peptidases family contains 847 known and putative members in Arabidopsis thaliana and 799 members in Medicago truncatula of a total of around 40 000 genes per genome. The protease families diverge so far that relationships cannot always be surely demonstrated by comparison of their primary structures. Similarity of the three-dimensional structures allows to classify proteases in clans and families even when primary sequence is not comparable.

The proteases play crucial roles in plant physiology. The selective breakdown of regulatory proteins via the ubiquitin/proteasome pathway controls many aspects of the plant development. As examples, the ubiquitin-proteasome pathway regulates auxin, gibberellin, and jasmonate pathways [START_REF] Wager | Social network: JAZ protein interactions expand our knowledge of jasmonate signaling[END_REF][START_REF] Salehin | SCFTIR1/AFB-based auxin perception: mechanism and role in plant growth and development[END_REF] and light perception via the degradation of regulatory proteins [START_REF] Huang | Beyond repression of photomorphogenesis: role switching of COP/DET/FUS in light signaling[END_REF]. This pathway also controls key aspects of the plant innate immunity and general defense (Marino et al. 2012). Proteases are also responsible for the post-translational modifications of proteins at highly specific sites. This cleavage of proteins is involved in the maturation of enzymes after the subcellular targeting to vacuoles, extracellular space, and organelles such as plastids and mitochondria [START_REF] Kmiec | Shredding the signal: targeting peptide degradation in mitochondria and chloroplasts[END_REF]. Proteolysis is also necessary for protein assembly and to control the activity of proteins and peptides, such as systemin [START_REF] Pearce | Systemin, hydroxyproline-rich systemin and the induction of protease inhibitors[END_REF].

The vacuole is a major compartment involved in protein degradation. This organelle which can represent 90% of the plant cell volume, harbors in addition to other lytic enzymes, the majority of proteolytic activities [START_REF] Matile | Catabolism of carotenoids: involvement of peroxidase?[END_REF]. Numerous proteases have been identified in the vacuole [START_REF] Carter | The vegetative vacuole proteome of Arabidopsis thaliana reveals predicted and unexpected proteins[END_REF]). The vacuolar proteolysis is involved in many cellular processes such as microautophagy and macroautophagy, regulating multiple aspects of plant physiology from mobilization of the storage proteins during seed germination, to initiation of cell death and senescence. The degradation of storage proteins during plant germination allows the remobilization of nitrogen for the formation of new proteins during plantlet development (Tan-Wilson and Wilson 2012).

Protease activity also plays a crucial role in the plant's adaptation to abiotic stresses. In such cases, proteolysis plays a housekeeping role by removing abnormal, inactivated, or denatured proteins, the accumulation of which could be toxic for the cell. In general, the plant adaptation to abiotic stresses goes together with an increased expression of proteases [START_REF] Velasco-Arroyo | HvPap-1 C1A protease actively participates in barley proteolysis mediated by abiotic stresses[END_REF][START_REF] Mishra | Constitutive over-expression of rice ClpD1 protein enhances tolerance to salt and desiccation stresses in transgenic Arabidopsis plants[END_REF]. The stress-induced alteration of photosynthesis also induces a strong modification in cellular metabolism [START_REF] Gururani | Regulation of photosynthesis during abiotic stress-induced photoinhibition[END_REF]. In these stress conditions, the proteases are involved in the redistribution of amino acids for the synthesis of new enzymes and the basis of carbon backbones for energy production through respiration [START_REF] Roberts | Senescence-associated proteases in plants[END_REF]. Proteases are also involved in plant-pathogen interactions. Apoplastic proteases have been shown to be involved in the plant defense against microbial pathogens such as Pseudomonas syringae [START_REF] Xia | An extracellular aspartic protease functions in Arabidopsis disease resistance signaling[END_REF]). Cysteine proteases have also been shown to be required for plant resistance against the necrotrophic fungus Botrytis cinerea (Shindo et al. 2012) and Ralstonia solanacearum (Zhang et al. 2014a). Cathepsins are also involved in the basal immunity of plants against P. syringae [START_REF] Ramirez | An extracellular subtilase switch for immune priming in Arabidopsis[END_REF]. Similarly, a subtilase protease has been shown to be implicated in defense against bacteria and oomycetes [START_REF] Ramirez | An extracellular subtilase switch for immune priming in Arabidopsis[END_REF].

Plant proteases participate to the programmed cell death (PCD) associated with developmental processes and plant defense against abiotic stresses and pathogen invasion (Sueldo and van der Hoorn 2017). In animal systems, the central core execution switch for apoptotic PCD is the activation of caspases (cysteine-containing aspartate-specific proteases). In plants, the existence of caspase-like activity during PCD has been shown to regulate PCD execution [START_REF] Sueldo | Plant life needs cell death, but does plant cell death need Cys proteases?[END_REF]. Proteasome activity also participates in PCD [START_REF] Han | The proteasome is responsible for caspase-3-like activity during xylem development[END_REF]. Proteases are involved in PCD occurring in embryo suspensors [START_REF] Suarez | Metacaspasedependent programmed cell death is essential for plant embryogenesis[END_REF][START_REF] Zhao | A bipartite molecular module controls cell death activation in the basal cell lineage of plant embryos[END_REF], the degeneration of the tapetum during anther development (Zhang et al. 2014b), the inner integument shrinking during seed coat formation (Nakaune et al. 2005), and the development of tracheary elements which constitute the xylem [START_REF] Avci | Cysteine proteases XCP1 and XCP2 aid micro-autolysis within the intact central vacuole during xylogenesis in Arabidopsis roots[END_REF]. Proteases are also involved in PCD occurring during the hypersensitive response induced during plant defense against pathogen microorganisms and viruses [START_REF] Mclellan | Functional redundancy in the Arabidopsis Cathepsin B gene family contributes to basal defence, the hypersensitive response and senescence[END_REF][START_REF] Coll | Arabidopsis type I metacaspases control cell death[END_REF][START_REF] Chichkova | Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity[END_REF]. Likewise, proteases are involved in PCD occurring during abiotic stresses like oxidative and osmotic stresses [START_REF] Chichkova | Phytaspase, a relocalisable cell death promoting plant protease with caspase specificity[END_REF].

Proteolysis is also associated with plant senescence, which is considered as another type of PCD. Senescence is a complex and controlled process, essential for (re)mobilization of nutrients from old and stressed tissues to growing or sink organs [START_REF] Shi | Integration of auxin/indole-3acetic acid 17 and RGA-LIKE3 confers salt stress resistance through stabilization by nitric oxide in Arabidopsis[END_REF][START_REF] Have | Nitrogen remobilization during leaf senescence: lessons from Arabidopsis to crops[END_REF]. It is connected to the degradation of the cellular structures and macromolecules to allow the formation and the relocation of transportable molecules. Leaf senescence has been particularly well studied (Lim et al. 2007;Otegui et al. 2005). It is a developmental process linked to the organ's aging, but it could also be modulated by biotic and abiotic stresses [START_REF] Jajic | Senescence, stress, and reactive oxygen species[END_REF][START_REF] Reinbothe | Plant oxylipins: role of jasmonic acid during programmed cell death, defence and leaf senescence[END_REF]. Proteases are involved in senescence with a major role in nutrient recycling, especially amino acids and nitrogen. Proteases are upregulated during the senescence process and nearly all protease families have been associated with some aspects of plant senescence (Diaz-Mendoza et al. 2016). They are located in multiple cell compartments such as chloroplasts, mitochondria, vacuoles, and senescence associated vesicles (Diaz-Mendoza et al. 2016). In addition to their roles in nutrient mobilization, proteases have been shown to regulate the senescence process. Metacaspases, which are cysteine proteases, are positive regulators of cell death in the context of pathogen-triggered PCD and a negative regulator of the plant senescence (Coll et al. 2014;Minina et al. 2013).

Within leguminous plant nodules, cysteine proteinases are engaged in response to microbial invasion, protein turnover for tissue remodeling, metabolic homeostasis and nutrient reutilization, adaptation to environmental stresses, and leaf and nodule senescence.

Biological nitrogen fixation (BNF) is the biological process by which atmospheric nitrogen (N 2 ) is converted to ammonia by an enzyme called nitrogenase. It is the major source of nitrogen in the biosphere and as such has an important ecological and agronomical role, accounting for 65% of the nitrogen used in agriculture worldwide. The most important source of fixed nitrogen is the symbiotic association between rhizobia and legumes. The nodule is a de novo formed plant organ in which biological nitrogen fixation is achieved by the bacteria (see Chapter 6.1). The bacteria are surrounded by a plant cell membrane, the peribacteroid membrane (PBM), the whole forming an organelle-like structure called the symbiosome where bacteria differentiate into nitrogen-fixing bacteroids. Symbiosomes completely fill the cytoplasm of infected cells. As the bacteria differentiate, infected cells undergo enlargement coupled to repeated endoreduplication cycles -genomic DNA replication without mitosis and cytokinesis -and become large polyploid cells housing thousands of bacteroids [START_REF] Jones | How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model[END_REF][START_REF] Kondorosi | Plant cell-size control: growing by ploidy?[END_REF]; see Section 9.3). The nodule functioning has many particularities, related to the plant-microbe crosstalk and to a metabolism which needs a high energy level under micro-oxic conditions. Whereas leaf senescence has been extensively studied, root nodule senescence has been less characterized. Nodule senescence also has specific features (Puppo et al. 2005). In this context, this review focuses on the roles of proteases in root nodule senescence.

Involvement of plant proteases in the nodule senescence process

Developmental and induced nodule senescence are complex processes causing a strong decrease of N 2 fixation and degradation of leghemoglobin (Lb) showing a typical green color of senescing nodule tissue (Puppo et al. 2005;[START_REF] Lehtovaara | Bile-pigment formation from different leghaemoglobins. Methine-bridge specificity of coupled oxidation[END_REF]. Plants keep in homoeostasis by the continuity of cell degradation and synthesis. However, when the rate of deterioration exceeds that of novel production, plants proceed to whole senescence. In roots, nodule senescence is accompanied with a series of physiological, biochemical, and structural alterations. Nodule senescence has also been regarded as a delayed plant pathogenic response to Rhizobium [START_REF] Mellor | Bacteroids in the rhizobium-legume symbiosis inhabit a plant internal lytic compartment: implications for other microbial endosymbioses[END_REF]). Nodules also individually follow a programmed death for turnover. Stress-induced nodule senescence is a consequence of accumulation of oxidized proteins, loss of thiols content, and general ultrastructural deterioration (Matamoros et al. 1999). Such proteolysis provides substrates to produce new amino acids, hence, contributing to protein turnover. Amino acids required by plants are mainly reused from old amino acids with continuous de novo synthesis.

Microscopic analysis of developmental nodule senescence showed that symbiotic organelle degradation occurs before the plant cell collapses in M. truncatula (Figure 9.4.1.1) (Van de Velde et al. 2006). The nodule degradation process involves a noticeable increase of protease activities leading to a complete degradation of the plant host cell and symbiotic bacteria (Figure 9.4.1.1).

In senescing nodule tissue of soybean, alfalfa, French bean, and pea nodules, numerous proteases activities have been detected (Kardailsky and Brewin 1996;Malik et al. 1981;[START_REF] Pfeiffer | Reversible darkinduced senecence of soybean root nodules[END_REF]Pladys and Vance 1993;[START_REF] Pladys | Senescence in French-bean nodules: occurrence of different proteolytic activities[END_REF]. In determinate nodule, such as soybean, senescence has been shown to be associated with the induction of cysteine protease genes (Alesandrini et al. 2003). Similarly, protease genes have been found to be strongly expressed during developmental senescence in zone IV at the proximal part of the nodule in indeterminate nodule such as M. truncatula or Chinese milk vetch (Fedorova et al. 2002;Naito et al. 2000). A transcriptomic analysis of senescent nodules in M. truncatula using cDNA-AFLP (amplified fragment length polymorphism) confirmed the accumulation of transcripts corresponding to multiple proteases (Table 9.4.1.1) (Van de Velde et al. 2006). At least eight of the identified proteases are cysteine proteases (CPs) that share high homology with the Table 9.4.1.1 Relative expression of various proteases involved in developmental and induced root nodule senescence in M. truncatula cDNA-AFLP transcriptome analysis: N, nitrogen-fixing, indeterminate nodules without senescence zone; ASN2 and PSN2, apical and proximal part of nodules with large green senescence zone, respectively (Van de Velde et al. 2006). RT-qPCR expression patterns of dark induced nodule senescence: N, nodules without signs of senescence; SN3, fully senescent nodules; 1 and 3 ddt (dark-induced nodule senescence compared to young fixing nodules) (Perez Guerra et al. 2010). Lower expression is considered as 1% and higher expression is considered as 100%. nd, stands for no data.

senescence-associated gene 12 (AtSAG12), one of the prominent markers of leaf senescence in A. thaliana (Lohman et al. 1994). AtSAG12 is a typical papain, a CP member of the peptidases (family C1A and clan CA classification from MEROPS database) typically found as lysosomal or secreted (Otegui et al. 2005;[START_REF] Richau | Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics[END_REF]. The overexpression of such CPs suggests an important role of proteases in the regulation of developmental nodule senescence. MtCP6 has been mainly studied as a representative CP from this group of papain in nodule senescence (Van de Velde et al. 2006;Perez Guerra et al. 2010;Pierre et al. 2014). In M. truncatula, another cysteine protease, key marker of nodule senescence was found to be a Vacuolar Processing Enzyme (MtVPE), a protease belonging to legumain families (Table 9.4.1.1) (Sueldo and van der Hoorn 2017). MtVPE, a cysteine protease (member of family C13 from clan CD classification from MEROPS database), is evolutionarily related to caspases and, found to be located in the vacuole (Pierre et al. 2014;[START_REF] Hatsugai | Vacuolar processing enzyme in plant programmed cell death[END_REF]. Both MtCP6 and MtVPE have been found to be strongly upregulated in response to both developmental and environmentally induced nodule senescence such as dark treatment (ddt) (Perez Guerra et al. 2010) (Table 9.4.1.1). Interestingly, only at the onset of nodule senescence, vacuolar SNAREs (soluble N-ethylmaleimide-sensitive factor adaptor protein receptors) proteins (SYP22 and VTI11) that are typical markers of lytic vacuolar membrane identity are found to be present on the peribacteroid membrane [START_REF] Limpens | Medicago N 2fixing symbiosomes acquire the endocytic identity marker Rab7 but delay the acquisition of vacuolar identity[END_REF]. The modification of membrane identity may allow the targeting of vacuolar proteases to the symbiosomes. In this scenario, the low pH present in the peribacteroid space would induce the autoactivation of both CPs and VPEs (Pierre et al. 2013).

In M. truncatula, five cysteine proteases have been identified as specifically expressed in nodules and share a high degree of homology with the MtCP6 family (Pierre et al. 2014). A spatio-temporal analysis of MtCP6 and MtVPE expression pattern in developmental nodule senescence revealed a strong expression associated to the transition region between the N 2 -fixing (III in red) and senescence (IV in green) zones (Figure 9.4.1.1) (Pierre et al. 2014). During senescence induced by three days of ammonitrate treatment, MtCP6 and MtVPE expression was detected in the entire nodule and correlated to the extension of the green senescence zone of the nodule (Figure 9.4.1.1).

Subcellular localization of both MtCP6 and MtVPE in M. truncatula senescent nodules has been associated to vacuoles in non-infected cells and to symbiosomes of infected cells, suggesting a role of these cysteine proteases in symbiosome degradation after the induction of proteases upon developmental or stress-induced senescence (Figure 9.4.1.1) (Pierre et al. 2014). Silencing of MtCP6 or MtVPE genes delayed nodule senescence and extended the zone III ratio relative to zone IV with a concomitant increase of 2.5-to 1.5-fold in nitrogen fixation for both constructions, respectively (Pierre et al. 2014). Conversely, their ectopic expression in the nitrogen-fixing zone promoted an extension of nodule senescence and a reduction of nodule length for both constructions (Pierre et al. 2014), showing the involvement of these two proteases in the nodule senescence process.

Similarly, cysteine protease genes are induced during nodule senescence in other leguminous plants. In soybean, 18 CPs and 6 VPEs were identified as transcriptionally upregulated during developmental nodule senescence (van Wyk et al. 2014). In Trifolium repens, five CP genes TrCP1, TrCP5, TrCP8, TrCP10, and TrCP12 have been identified as having an increased expression mainly in the nodule senescence zone (Asp et al. 2004) (Table 9.4.1.2). In Astragalus sinicus the transcripts are mostly accumulated in senescence zone (IV), as well as in interzone (II-III) under developmental senescence. Interestingly, the expression of a nodule-specific CP, Asnodf32, appears at 14 days post inoculation (dpi) and increases fivefold at 30 dpi (Naito et al. 2000) (Table 9.4.1.2). The important role of Asnodf32 in nodule senescence has been validated by the delayed root nodule senescence correlated to a significant extension of the nitrogen fixation active period obtained by RNAi silencing of the Asnodf32 gene (Li et al. 2008). Furthermore, as in M. truncatula, Asnodf32 RNAi leads to the elongation of nodules suggesting a modification of the nodule development (Li et al. 2008).

Interestingly, using laser microdissection, the analysis of nodule senescence in the mutant line E135F (sym13) of Pisum sativum L. showed the increase of mRNA abundance of two cysteine proteases (PsCyp1 and PsCyp15a) (Table 9.4.1.2) and a thiol protease (PsTPP) (Serova et al. 2017).

Recently, a large collection of M. truncatula mutants has been constructed using ethyl methanesulfonate (EMS) and fast neutron mutagenized or Tnt1-insertion, leading to numerous Nod + Fix -mutants with phenotypes showing signs of early senescence [START_REF] Domonkos | The identification of novel loci required for appropriate nodule development in Medicago truncatula[END_REF]Pislariu et al. 2012). This collection of mutants will represent a huge interest for deciphering the molecular pathways involving proteases in nodule senescence.

Role of cystatin in the nodule senescence process

To date, regulation of the cysteine proteases in legumes is poorly documented. However, recent studies show their interactions in vivo with cystatins, which are cysteine protease inhibitors limiting proteolytic processes in a reversible manner (for review, Benchabane et al. 2010;[START_REF] Martinez | C1A cysteine-proteases and their inhibitors in plants[END_REF]van Wyk et al. 2014;Diaz-Mendoza et al. 2016;[START_REF] Yuan | Search for nodulation and nodule development-related cystatin genes in the genome of soybean (Glycine max)[END_REF]. Cystatins mainly inhibit protease enzymes belonging to peptidase families C1 (papain family) and C13 (legumain family).

In Medicago sativa, expression of the cystatin MsCys1 is developmentally regulated in stems and leaves but not induced by various stress signals. Recombinant MsCys1 shows a strong inhibitory effect on alfalfa leaf CPs activities [START_REF] Rivard | MsCYS1, a developmentally-regulated cystatin from alfalfa[END_REF]). However to date, no study concerning the biological role of such cystatins has been carried out in the nodule under senescence.

In soybean senescent nodules, four cystatin genes are particularly co-induced with GmCYPs during the onset of senescence (van Wyk et al. 2014). Interestingly, two cystatin genes present a downregulation anticorrelated with the induction of GmCYPs expression throughout the transition to nodule senescence (van Wyk et al. 2014). Such coordinated expression of transcripts encoding cystatins and GmCYPs interacting partners suggest their involvement in the tight control of proteolysis regulation associated with nodule senescence. However, no functional analysis aiming to alter their expression has been carried out to determine their relevance in nodule senescence. Interestingly, ectopic overexpression of rice cystatin oryzacystatin-I in soybean leads to a strong decrease in cathepsin L-like cysteine protease activities (Quain et al. 2015). A clear phenotype has been shown with a higher nodulation efficiency of mutant plants in comparison to the wild-type (Quain et al. 2015). Furthermore, overexpression in Lotus japonicus of GmCYS16 gene shows that some cystatin played a positive role in the nodulation process with a significant increase in root nodule numbers [START_REF] Yuan | Search for nodulation and nodule development-related cystatin genes in the genome of soybean (Glycine max)[END_REF].

Proteases from the endosymbiotic bacteria involved in nodule senescence

Similarly to the plant partner, the bacterial partner participates in the establishment and in the functioning of the nitrogenfixing symbiosis. Bacteria may also be tightly involved in the senescence process which leads to the breakdown of the symbiotic partnership.

Bacteria have the capacity to adjust to a wide variety of growth environments. S. meliloti is an interesting model of this adaptability as it has to adapt both to a free-living saprophytic lifestyle and an endosymbiotic environment within the plant cells. This adaptation is provided by many regulatory mechanisms including transcriptional and translational regulation, but also protein degradation. Indeed, in addition to housekeeping functions necessary to remove misfolded or damaged proteins, bacterial proteolysis plays an important role in the regulation of various biological processes including stress response, cell differentiation, sporulation, or pathogenesis (for review, Gottesman 2003; [START_REF] Konovalova | Regulated proteolysis in bacterial development[END_REF]. This regulated proteolysis is mainly realized by energy-dependent proteases belonging to the AAA+ (ATPases associated with different cellular activities) family. Protein degradation starts with the binding of the substrate to the ATPase domain. Then, ATP hydrolysis allows the unfolding of the protein and its transfer to the proteolytic domain. This protease family is divided into four classes (Figure 9.4.1.2): for two of them, proteolytic (ClpP and ClpQ [also called HslV]) and ATPase (ClpA or X and ClpY [HslU]) domains are separated on independent subunits whereas for Lon and FtsH, the two domains are in the same polypeptide.

In the nitrogen-fixing symbiosis, rhizobia undergo important metabolic modifications and adaptations during the transition between the free-living state and the symbiotic association. This transition may include major cellular differentiation with arrest in cell division and genomic endoreduplication and adaptation to an acidic and microoxic environment [START_REF] Jones | How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model[END_REF]Pierre et al. 2013; see Chapter 9.2.2). To date, physiological and molecular mechanisms involved in these processes are still only partially determined. Analysis of symbiotic phenotypes of bacteria deficient in Lon protease has shown the involvement of this protease family in the regulation of nitrogen-fixing symbiosis [START_REF] Summers | The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa[END_REF]Nakajima et al. 2012). Indeed, lon mutants of S. meliloti and Azorhizobium caulinodans in interaction with alfalfa and Sesbania rostrata, respectively, have been shown to induce nodules with little or no nitrogen fixation capacity [START_REF] Summers | The Sinorhizobium meliloti Lon protease is involved in regulating exopolysaccharide synthesis and is required for nodulation of alfalfa[END_REF]Nakajima et al. 2012). Moreover, nodule formation by S. meliloti lon mutants is delayed and abnormal (pseudo-nodules). In both cases, lon mutants also produce and release more extracellular polysaccharides (EPS). Lon is known to be involved in polysaccharides synthesis in bacteria [START_REF] Gottesman | Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes[END_REF]. As EPS were previously associated with early infection steps of the symbiosis such as bacterial attachment to root hairs and invasion [START_REF] Fraysse | Surface polysaccharide involvement in establishing the rhizobium-legume symbiosis[END_REF][START_REF] Laus | Role of cellulose fibrils and exopolysaccharides of Rhizobium leguminosarum in attachment to and infection of Vicia sativa root hairs[END_REF], the authors suggest that at least part of the symbiotic defects obtained with lon mutants may result from abnormal EPS production.

Beside the regulation of EPS production, in many bacteria, Lon proteases are also known to control activation of some toxin-antitoxin (TA) systems by degradation of the unstable antitoxin [START_REF] Christensen | RelE, a global inhibitor of translation, is activated during nutritional stress[END_REF][START_REF] Christensen | Toxin-antitoxin loci as stress-response-elements: ChpAK/MazF and ChpBK cleave translated RNAs and are counteracted by tmRNA[END_REF][START_REF] Brzozowska | Regulation of toxinantitoxin systems by proteolysis[END_REF]. This leaves the toxin, usually acting as an endoribonuclease, free to target specific RNAs thus contributing to a particular biological response. In this context, modification of the symbiotic properties of S. meliloti strains mutated in TA systems has been reported in the interaction with the plant host M. sativa [START_REF] Oláh | Mutation in the ntrR gene, a member of the vap gene family, increases the symbiotic efficiency of Sinorhizobium meliloti[END_REF]Lipuma et al. 2014). Consequently, some symbiotic phenotypes of the S. meliloti lon mutants may also be the consequence of a defect in TA system activation. Another class of energy-dependent proteases, ClpX/AP, seems also involved in nitrogen-fixing symbiosis. In many bacteria, ClpP proteolysis has been associated with regulation of major cellular processes including cell division and differentiation, virulence and stress response [START_REF] Porankiewicz | New insights into the ATP-dependent Clp protease: Escherichia coli and beyond[END_REF]. A critical role of ClpXP proteolysis has been also demonstrated in nitrogen fixation in the plant growth-promoting bacterium Azospirillum brasilense [START_REF] Rodriguez | Pleiotropic physiological effects in the plant growth-promoting bacterium Azospirillum brasilense following chromosomal labeling in the clpX gene[END_REF]). Indeed, an Azospirillum clpX mutant presents enhanced nitrogen fixation associated to higher production of exopolysaccharides. In contrast, clpX disruption is lethal in S. meliloti [START_REF] Kobayashi | Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection[END_REF]). However, mutation of cpdR1, encoding a transcriptional regulator of ClpX, induces small white pseudo-nodules with no nitrogen-fixing capacity [START_REF] Kobayashi | Sinorhizobium meliloti CpdR1 is critical for co-ordinating cell cycle progression and the symbiotic chronic infection[END_REF]. As host plant cells contain numerous bacteria, the authors propose that the bacteroid differentiation step is impaired in the cpdR1 mutant.

Moreover, implication of ClpP proteolysis during nitrogenfixing symbiosis is supported by S. meliloti transcriptomics data. Indeed, the transcript accumulation of some bacterial proteases in the different zones of the nodule can be determined from the plant and bacterial gene expression analysis performed in symbiotic root nodules using laser capture microdissection coupled to RNA-seq (Table 9.4.1.3) (https://iant.toulouse. inra.fr/symbimics/; Roux et al. 2014). This study reveals in particular a significant accumulation of clpP3 proteolytic subunit transcripts in the nodule nitrogen-fixing zone. However, this induction is not correlated with ClpA or ClpX ATPase subunit, which are preferentially expressed in the proximal infection zone. Nevertheless, the transcript accumulation of ATPase subunits seems sufficient to allow the complex formation. Nonetheless, we cannot exclude that ClpP3 could be involved alone in the degradation of short peptides, which can occur with isolated ClpP protease. Moreover, the expression analysis showed The RNA-sequencing was determined in the different zones of S. meliloti 2011 induced nodules (2 wpi) (Roux et al. 2014). Values correspond to the number of reads and colors to their relative abundance (scale in percentage under the table). The transcript relative abundance was estimated using the percentage of normalized counts in each fraction relative to the total number of normalized counts. FI, meristematic zone; FII, infection zone with a distal (FIID) and a proximal fraction (FIIP) zone; IZ, interzone II-III; FIII, fixation zone.

that clpB, a member of the Clp ATPase family, which acts as a chaperone with the DnaJ, DnaK, and GrpE cellular machinery to disaggregate proteins, is also induced in the fixing zone. Therefore, all these data suggest an important role of this protease family in the functioning of the nitrogen-fixing interaction in addition to its probable implication in its establishment. Interestingly, the transcriptomic data analysis also reveals the induction in the nitrogen-fixing zone of a periplasmic carboxyterminal processing serine protease gene, ctpA, known to be critical for viability of Rhizobium leguminosarum when grown on semi-solid medium [START_REF] Gilbert | Mutagenesis of the carboxy terminal protease CtpA decreases desiccation tolerance Chapter 9.4.1: Involvement of proteases during nodule senescence 691 in Rhizobium leguminosarum[END_REF]). The role of the expression of ctpA during symbiosis remains to be determined. Finally, the expression of a S. meliloti metallopeptidase (HrrP) involved in the cleavage of host-encoded nodule-specific cysteine-rich (NCR) peptides may lead to premature bacteroids degeneration [START_REF] Price | Rhizobial peptidase HrrP cleaves host-encoded signaling peptides and mediates symbiotic compatibility[END_REF]. This result suggests a role of this protease in modifying the host-encoded NCR pool. This contributes to the plant-microbe crosstalk in the late development stage and demonstrates the role of bacterial proteolysis to maintain the symbiotic interaction.

The understanding of the implication of bacterium in the nodule senescence is in its infancy. Further analyses have to be performed to acquire a global vision of this critical process.

Conclusion

Many analyses have shown that a large panel of proteases are involved in nodule senescence under developmental or stressinduced senescence. One of the main reasons for the activation of the protease pathways in nodule senescence is linked to nutrient remobilization and programmed degradation of symbiotic partnership, in order to eliminate the useless organ during developmental senescence or to maintain the survival of the plant during stress-induced senescence. Nevertheless, the strong and fast induction of proteases may interfere with an efficient yield production of legumes in agricultural conditions. In a world confronted with global warming and limited access to water, a large investment in plant research attempting to moderate or delay strong responses to abiotic stresses can be seen as an interesting goal in order to produce more resilient plants. In this context, the analysis of the regulation of protease activation during the transition from symbiosis to senescence may allow us to better understand the key mechanisms involved in the senescence initiation. Moreover, such protease genes may also be essential tools for the plant assisted marker selections in order to delay the general plant senescence during adverse climate changes.

The flower that smiles to-day

To-morrow dies;

All that we wish to stay Tempts and then flies.

Percy Bysshe Shelley

OBJECTIVES

To date, it is unclear whether nodule senescence is the consequence of genetic control by plant or attack by microbes or is a result of systematic interplay. Despite intensive studies that have been conducted on the early stages of the rhizobium-legume interaction for last decades, research data are scarce on signal transduction cascades and regulatory functions in terms of the nodule senescence phase. Our to-date very limited understanding on the later stage of nodule senescence necessitates more research. Here in this thesis, we have investigated the roles of a bacterial redox-related gene and a plant protease gene in root nodule senescence with different approaches.

On the bacterial side, the first part of this thesis is intended to characterize the senescence process induced by the bacterial glutathione deficiency in the symbiotic interactions. Time course analysis was performed to analyse the degradation process occurring in the interaction between Medicago truncatula and the SmghsB, a Sinorhizobium meliloti mutant deficient in GSH synthesis. Our scientific question was whether this deficiency induced an early senescence associated to a lack of bacteroid differentiation or an early senescence associated to an early death of bacteroids On the plant side, one plant cysteine protease gene, MtCP6, was proven to play a crucial regulatory role in nodule senescence by delaying senescence with RNAi while accelerating the senescence process with ectopic overexpression (Pierre et al., 2014). Therefore, a comprehensive study on the transcriptional regulation of this cell death executioner is promising to elucidate the nodule senescence regulatory network. The second part of the thesis is the identification of cis/trans factors in the complex transcriptional network involved in the MtCP6 regulation at the onset of nodule senescence.

I. Introduction

I.1 Redox homeostasis in nodulation

ROS production in response to bacterial invasion

Reactive oxygen species (ROS) refer to oxygen-derived molecules, including free radicals (e.g. superoxide, hydroxyl radicals) and non-free radicals (e.g., hydrogen peroxide, singlet oxygen) [START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]. Main sources for ROS are in plants under physiological conditions are respiration, photosynthesis, and N2 fixation [START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]. ROS are highly produced during plant development and in response to stresses. As an example, a burst of ROS is featured in plant hypersensitive reaction responding to pathogen elicitors [START_REF] Lamb | The Oxidative Burst in Plant Disease Resistance[END_REF]).

An oxidative burst is also elicited in the infection of roots by rhizobia (Santos et al., 2001;Puppo et al., 2005). Rhizobia and Nod factor are proven to induce a low level of ROS production which can be effectively blocked by antioxidants such as SOD [START_REF] Santos | Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis[END_REF][START_REF] D'haeze | Role of reactive oxygen species and ethylene in programmed cell death during nodule initiation on Sesbania rostrata. Nitrogen Fixation: Global Perspectives[END_REF][START_REF] Ramu | Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula[END_REF]. A peroxidase encoding gene, rip1, is rapidly triggered by Nod factor and coincides with the superoxide accumulation during early symbiotic processes [START_REF] Ramu | Nod factor induction of reactive oxygen species production is correlated with expression of the early nodulin gene rip1 in Medicago truncatula[END_REF]. Additionally, interfering with the NADPH oxidase-catalysed ROS production by silencing MtRBOHa impairs symbiosis showing that the ROS production is needed to have an efficient nodulation (Marino et al., 2011).

Antioxidants in maintaining nodule function

On the other hand, overproduction of ROS is detrimental for plant development and metabolism. Antioxidant metabolites and enzymes are produced to protect cells from the oxidative damages. The ROS production is kept at a low level after rhizobia infection to maintain a proper function of nodule metabolism including the oxygen sensitive nitrogenase.

However, the nitrogen fixation is high energy-demanding therefore requiring high rates of respiration and thus ROS production. Antioxidants which includes molecules such as glutathione and enzymes such as catalases, superoxide dismutases, and peroxidases are thus required to allow the proper nodule functions [START_REF] Sigaud | Differential regulation of two divergent Sinorhizobium meliloti genes for HPII-like catalases during free-living growth and protective role of both catalases during symbiosis[END_REF][START_REF] Santos | Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis[END_REF][START_REF] Jamet | Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and their crucial role during the infection process[END_REF][START_REF] Dombrecht | Defence of Rhizobium etli bacteroids against oxidative stress involves a complexly regulated atypical 2-Cys peroxiredoxin[END_REF]. Catalases can convert H2O2 to water and oxygen without consuming reductants [START_REF] Scandalios | Catalases in plants: gene structure, properties, regulation, and expression[END_REF]. S. meliloti expresses three genes

CHAPTER I

encoding one bifunctional catalase-peroxidase KatB and two monofunctional catalases KatA and KatC [START_REF] Jamet | Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and their crucial role during the infection process[END_REF]. The KatA is inducible by exogenous H2O2 in free-living rhizobia and is the major catalase gene expressed in bacteroids, whereas KatB and KatC are constitutively expressed in bacteria and in infection threads [START_REF] Jamet | Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and their crucial role during the infection process[END_REF]. An increased catalases activity was associated with bacterial glutathione deficiency in S. meliloti probably through the increase of H2O2 (Harrison et al., 2005). In symbiotic nodules, small antioxidant molecules, such as, glutathione (GSH, γglutamylcysteinyl-glycine), homoglutathione (hGSH) and ascorbate (ASC), are major antioxidants in the protection of nodules from oxidative stresses (Dalton et al., 1986;[START_REF] Frendo | Localisation of glutathione and homoglutathione in Medicago truncatula is correlated to a differential expression of genes involved in their synthesis[END_REF][START_REF] Riccillo | Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance[END_REF][START_REF] Harrison | The synthesis and the roles of glutathione and homoglutathione in legumes[END_REF]Frendo et al., 2005;Harrison et al., 2005;Puppo et al., 2005;Groten et al., 2006;Innocenti et al., 2007;Loscos et al., 2008;Muglia et al., 2008;Tate et al., 2012;Cheng et al., 2017). The ascorbate has been indicated also to have a regulatory role in nodule development as an antioxidant in pea (Groten et al., 2006).

The ascorbate-GSH pathway involves four enzymes operating in concert to remove H2O2 through ascorbate peroxidase (APX) at the expense, ultimately, of the reducing power of NADH or NADPH (Figure 21) [START_REF] Noctor | ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control[END_REF][START_REF] Wheeler | The biosynthetic pathway of vitamin C in higher plants[END_REF][START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]Foyer & Noctor, 2011). The initial product of APX is monodehydroascorbate (MDHA, CHAPTER I ascorbate free radical), which then disproportionate to ASC and dehydroascorbate (DHA). In the infected cells, APX may participate in the operation of the oxygen diffusion barrier in the nodule parenchyma by adjusting their respiratory activity [START_REF] Dalton | Antioxidant defences in the peripheral cell layers of legume root nodules[END_REF][START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]. MDHA and DHA are reduced back to ASC by specific reductases (Monodehydroascorbate reductase-MR, Dihydroascorbate reductase-DR) using NADH and GSH, respectively. Finally, the oxidized GSH (GSSG) formed by DR is reduced to GSH by glutathione reductase (GR) using NADPH (Figure 21) [START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF].

ROS production resurges during nodule senescence

During nodule senescence, an increase in ROS productions and a diminish in antioxidants oxidize leghaemoglobin and enhance autolytic processes (Matamoros et al., 1999;[START_REF] Becana | Reactive oxygen species and antioxidants in legume nodules[END_REF]Santos et al., 2001;[START_REF] Hérouart | Reactive oxygen species, nitric oxide and glutathione: a key role in the establishment of the legume-Rhizobium symbiosis?[END_REF]Alesandrini et al., 2003;Puppo et al., 2005;[START_REF] Pauly | Reactive oxygen and nitrogen species and glutathione: key players in the legume-Rhizobium symbiosis[END_REF][START_REF] Günther | Metabolism of reactive oxygen species is attenuated in leghaemoglobin-deficient nodules of Lotus japonicus[END_REF]Muglia et al., 2008;Andrio et al., 2013). Eventually, nodule senescence is accompanied with a reduction in nitrogen fixation capacity and an increase in ROS accumulation (Puppo et al., 2005;Muglia et al., 2008).

Therein, oxidative damage of lipids, proteins, and DNA has been observed in nodules during natural and stress-induced senescence (Evans et al., 1999;Matamoros et al., 1999). Large amounts of H2O2 has been shown to accumulate in the cells and apoplast in the central zone of senescing soybean nodules (Alesandrini et al., 2003). These structural and biochemical alterations may result from a switch from a reductive state to an oxidative state, which is a general characteristic of plant senescence (Evans et al., 1999;Puppo et al., 2005;Marquez-Garcia et al., 2015).

I.2 Glutathione in prokaryotes GSH Synthesis involves a two-step ATP-dependent pathway

The tripeptide GSH is a well-known antioxidant and detoxifier existing in many organisms, with a concentration ranging from 0.1 to 10 mM in bacteria [START_REF] Penninckx | Metabolism and functions of glutathione in micro-organisms[END_REF][START_REF] Carmel-Harel | Roles of the glutathione-and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress[END_REF][START_REF] Noctor | Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. GSH synthesis involves a two-step ATP-dependent pathway [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. In the first step, glutamate and cysteine are conjugated by γ-glutamyl cysteine synthetase (γ-ECS; encoding by gshA gene) to form γ-glutamyl cysteine (γ-EC). In the second step, glycine is added to the γ-EC to produce GSH in the reaction catalysed by glutathione synthetase (GSHS; encoding by gshB CHAPTER I gene). Alternatively, hGSH is formed by adding β-alanine to the γ-EC catalysed by homoglutathione synthetase (hGSHS).

i) L-Glu + L-Cys + ATP ↔ γ-Glu-Cys + ADP + Pi ii) γ-Glu-Cys + Gly + ATP ↔ GSH + ADP + Pi
The first step is the rate-limiting and under control of cysteine concentration and the feedback inhibition of the final product GSH [START_REF] Lu | Regulation of glutathione synthesis[END_REF]. Another possible way to control GSH pool is through its degradation by the γ-glutamyl transpeptidase (GGT) to the γ-linkage between the glutamic acid and cysteine [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. However, GGT is localized in the periplasm, which hardly possible to modulate the cytoplasmic GSH level [START_REF] Suzuki | gamma-Glutamyltranspeptidase from Escherichia coli K-12: purification and properties[END_REF].

GSH play multiple roles in prokaryotes

GSH is involved in multiple biological processes including control of cell redox status, progression of the cell cycle, and detoxification and maintaining intracellular K + pools [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]) [START_REF] Ferguson | Importance of glutathione for growth and survival of Escherichia coli cells: detoxification of methylglyoxal and maintenance of intracellular K +[END_REF][START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]. Most of the GSH functions are investigated in Escherichia coli [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. The redox couple GSH:GSSG (reduced and oxidized states of GSH) is carefully controlled in the cytoplasm (Fig 1 .2) [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. Protein glutathionylation, the reversible formation of mixed disulfides between GSH and cysteinyl residues [START_REF] Dalle-Donne | S-glutathionylation in protein redox regulation[END_REF], is thereafter serving as the protection of reactive thiols under oxidative stresses [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. Glutathionylation has been shown to modulate MetE of methionine biosynthesis [START_REF] Hondorp | Oxidative stress inactivates cobalamin-independent methionine synthase (MetE) in Escherichia coli[END_REF], 3'phosphoadenylsulfate (PAPS) reductase of the sulfate assimilation pathway [START_REF] Lillig | Redox regulation of 3'-phosphoadenylylsulfate reductase from Escherichia coli by glutathione and glutaredoxins[END_REF], and the transcription factor OxyR of peroxide oxidative stress [START_REF] Aslund | Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status[END_REF].

During oxidative stress, GSH indirectly reduces the oxyR transcription factor via glutaredoxin 1 [START_REF] Storz | OxyR: a regulator of antioxidant genes[END_REF][START_REF] Zheng | Activation of the OxyR transcription factor by reversible disulfide bond formation[END_REF][START_REF] Aslund | Regulation of the OxyR transcription factor by hydrogen peroxide and the cellular thiol-disulfide status[END_REF]. In E. coli, glutaredoxin is involved in DNA synthesis via providing reduction of Ribonucleotide reductase, which needs at least one of the reducing equivalents NADPH and GSH [START_REF] Ortenberg | Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. Other substrates of the glutaredoxin pathway are PAPS reductase, methionine sulfoxide reductase and arsenate reductase [START_REF] Ritz | Roles of thiol-redox pathways in bacteria[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. GSH is required for bacterial anaerobic growth via reduction of fumarate nitrate reductase regulator (FNR). GSH can be the direct reducing power for FNR, which is necessary to control the switch from aerobic to anaerobic conditions [START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. During anaerobic conditions, GSH is required to activate FNR thus the formed homodimer is with [4Fe-4S] cluster and is able to control bacterial anaerobic growth [START_REF] Ritz | Roles of thiol-redox pathways in bacteria[END_REF].

GSH plays roles in detoxification of xenobiotics. GSH too is known to protect cells from halogens such as chlorine compounds [START_REF] Chesney | Bacterial glutathione: a sacrificial defence against chlorine compounds[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. However, in the glutathione-deficient organisms, resistance can be restored with exogenous GSH caused by chlorine-based oxidants but not by H2O2 [START_REF] Chesney | Bacterial glutathione: a sacrificial defence against chlorine compounds[END_REF]. GSH is required for the enzymes glyoxalase I and II to detoxify methylglyoxal into D-Lactate [START_REF] Masip | The many faces of glutathione in bacteria[END_REF].

GSH can negatively regulate KefB and KefC K+ efflux channels, thus depletion of GSH will lead to K+ leakage and counter cytoplasmatic acidity [START_REF] Ferguson | Importance of glutathione for growth and survival of Escherichia coli cells: detoxification of methylglyoxal and maintenance of intracellular K +[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. The decrease in pH will in turn protect cells against toxicity of the electrophile methylglyoxal in absence of GSH [START_REF] Ferguson | Importance of glutathione for growth and survival of Escherichia coli cells: detoxification of methylglyoxal and maintenance of intracellular K +[END_REF].

I.3 Role of glutathione in nodule functioning.

Glutathione is an important molecule for the growth of S. meliloti. Previous works on the synthesis of GSH during free-living growth and symbiosis of S. meliloti 1021 with M. sativa were examined by Harrison et al. (Harrison et al., 2005). It was found that a S. meliloti mutant strain which was unable to synthesize GSH because of a gene disruption in gshA, was unable to grow under non-stress conditions, precluding any nodulation. This result showed that GSH plays a crucial role in S. meliloti growth. In contrast, a S. meliloti mutant strain with gshB deleted was able to grow, indicating that γ-EC (di-peptide intermediate) could partially substitute for GSH. However, the SmgshB deficient strain showed a delayed nodulation phenotype coupled with a 75% reduction in the nitrogen fixation capacity of Medicago sativa nodules (Harrison et al., 2005).

Preliminary results obtained during the interaction between M. truncatula and S. meliloti gshB mutant strains suggested that the symbiotic interaction was impaired in the bacterial differentiation step [START_REF] El Msehli | Etude de l'interaction symbiotique Légumineuses-Rhizobium [Medicago truncatula-Sinorhizobium meliloti] Sénescence nodositaire et glutathion[END_REF]. The goals of this research project were to analyse in kinetics the symbiotic processes in order to characterize the defective interaction between the S. meliloti 2011 mutant strain (SmgshB) and M. truncatula.

CHAPTER I CHAPTER I

123

II. Results

Yang, L., El Msehli, S., Benyamina, S., Lambert, A., Hopkins, J., Cazareth, J., Under nitrogen-limiting conditions, legumes are able to interact symbiotically with bacteria of the Rhizobiaceae family. This interaction gives rise to a new organ, named a root nodule. Root nodules are characterized by an increased glutathione (GSH) and homoglutathione (hGSH) content compared to roots. These low molecular thiols are very important in the biological nitrogen fixation. In order to characterize the modification of nodule activity induced by the microsymbiont glutathione deficiency, physiological, biochemical, and gene expression modifications were analyzed in nodules after the inoculation of Medicago truncatula with the SmgshB mutant of Sinorhizobium meliloti which is deficient in GSH production. The decline in nitrogen fixation efficiency was correlated to the reduction in plant shoot biomass. Flow cytometry analysis showed that SmgshB bacteroids present a higher DNA content than free living bacteria. Live/dead microscopic analysis showed an early bacteroid degradation in SmgshB nodules compared to control nodules which is correlated to a lower bacteroid content at 20 dpi. Finally, the expression of two marker genes involved in nitrogen fixation metabolism, Leghemoglobin and Nodule Cysteine Rich Peptide 001, decreased significantly in mutant nodules at 20 dpi. In contrast, the expression of two marker genes involved in the nodule senescence, Cysteine Protease 6 and Purple Acid Protease, increased significantly in mutant nodules at 10 dpi strengthening the idea that an early senescence process occurs in SmgshB nodules. In conclusion, our results showed that bacterial GSH deficiency does not impair bacterial differentiation but induces an early nodule senescence.

INTRODUCTION

Nitrogen (N) is the major macronutrient needed for plant growth. However, whereas N is the most abundant element in the atmosphere, it is generally the most limiting plant nutrient in soils. Legume plants are able to interact symbiotically with rhizobia to perform nitrogen fixing symbiosis (NFS). In these mutualistic associations, bacteria deliver nitrogen source to the plants while plants provide bacteria with photosynthates, nutrients required for rhizobium metabolism and a specialized ecological niche that favors their growth. Indeed, the nitrogenfixing symbiosis involves the de novo formation of a new organ, the nodule, associated to a reprogramming of the plant development and metabolism. This allows the intracellular accommodation of several thousands of differentiated bacteria, within nodule cells, called bacteroids which reduce atmospheric nitrogen. Amongst the multiple regulatory processes involved in the setup and the regulation of NFS, a modification of the cellular redox state of the plant partner is observed during the plant infection and nodule functioning (Puppo et al., 2013). Reactive oxygen species production is detected in root hairs in response to nodulation factors [START_REF] Cárdenas | Fast, transient and specific intracellular ROS changes in living root hair cells responding to Nod factors (NFs)[END_REF], the root infection process (Santos et al., 2001;[START_REF] Jamet | H2O2 is required for optimal establishment of the Medicago sativa/Sinorhizobium meliloti symbiosis[END_REF] and in the nodule differentiation zone in Medicago truncatula nodules (Andrio et al., 2013). The implication of the ROS in the symbiotic interaction between legumes and rhizobia has been demonstrated using genetic approaches (Marino et al., 2011;Arthikala et al., 2014). Genetic alteration of the expression of respiratory burst oxidative homologs (Rbohs), proteins involved in the ROS production, significantly modifies the symbiosis between legume plants and the rhizobia (Marino et al., 2011;Marino et al., 2012). Similarly, nitric oxide (NO) production is also detected in the NFS (Hichri et al., 2015). It is required for the optimal establishment of the symbiotic interaction (Hichri et al., 2015;Berger et al., 2019). In mature nodules, NO has a beneficial metabolic function for the maintenance of the energy status under hypoxic conditions, but has also been shown to inhibit nitrogen fixation and to trigger nodule senescence. NO has also been shown to regulate the glutathione (GSH) synthesis in roots (Innocenti et al., 2007). The root nodules contain significantly higher contents of low molecular thiols, GSH and homoglutathione (hGSH), in comparison to roots (Frendo et al., 2013a;[START_REF] Frendo | Thiol-based redox signaling in the nitrogen-fixing symbiosis[END_REF]. GSH is the most abundant low-molecular weight thiol in cells and has several well-known functions. For example, it acts as a redox buffer and as a signaling molecule, and it is also involved in the detoxification of radicals and xenobiotics [START_REF] Rouhier | The role of glutathione in photosynthetic organisms: emerging functions for glutaredoxins and glutathionylation[END_REF]Foyer and Noctor, 2011). GSH is synthesized by a two-step process. In the first step, glutamate and cysteine are conjugated by g-glutamyl cysteine synthetase (g-ECS) to form g-glutamyl cysteine (g-EC). In a second step, glycine is added to g-EC to form GSH in a reaction catalyzed by glutathione synthetase (GSHS). Alternatively, homoglutathione synthetase (hGSHS) catalyzes the addition of balanine to g-EC to form hGSH. Genetic and pharmacological approaches showed that the GSH pool produced by the plant partner is important to allow the nodule development and an efficient biological nitrogen fixation (BNF) (Frendo et al., 2005;El Msehli et al., 2011). On the bacterial side, the use of mutants deficient in the GSHS-encoding gene (gshB) impairs the BNF efficiency during the symbiosis between Medicago sativa/S. meliloti strain 1021, Phaseolus vulgaris/Rhizobium tropici, P. vulgaris/Rhizobium etli, and Pisum sativum/Rhizobium leguminosarum (Harrison et al., 2005;Muglia et al., 2008;[START_REF] Taté | Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness[END_REF]Cheng et al., 2017). The reduced BNF observed with the gshB mutant strain was correlated with nodule and bacteroid early senescence in M. sativa and P. vulgaris (Harrison et al., 2005;Muglia et al., 2008;[START_REF] Taté | Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness[END_REF]. In parallel, recycling of GSH by glutathione reductase was shown to be required to maintain an effective nitrogen fixation (Tang et al., 2018). Finally, a deficiency in bacteroid differentiation was observed for a S. meliloti (strain 2011) mutant for glutaredoxin 1 (Grx1), a glutaredoxin involved in the deglutathionylation of proteins using GSH as a cofactor, in the symbiotic interaction with M. truncatula (Benyamina et al., 2013). Harrison and colleagues (2005) also suggested that glutathione deficiency in S. meliloti strain 1021 interferes with bacteroid differentiation in M. sativa (Harrison et al., 2005) but this hypothesis was not carefully investigated.

In this context, we hypothesized that the genome background of S. meliloti (strain 1021 versus strain 2011) or the genome background of the plant (M. sativa versus M. truncatula) could modify the outcome of the interaction between the two partners. With this in mind, we intended to characterize the impact of the bacterial GSH deficiency on the bacteroid differentiation and the nodule functioning during the symbiotic interaction between M. truncatula and the strain 2011 of S. meliloti. Physiological, biochemical, cellular and genetic markers were used to describe the nodule functioning at 10 and 20 days after plant inoculation, two early time points of nodule functioning. Our results show that bacterial GSH deficiency does not affect bacteroid differentiation. However, bacterial GSH deficiency induces an early nodule senescence process in M. truncatula already detectable at 10-d post-infection.

MATERIALS AND METHODS

Plant Material, Bacterial Strains and Growth Conditions

A mutation in gshB (Smc00419) of S. meliloti 2011 was made by plasmid integration using single-crossover. A 680-bp gshB fragment was PCR-amplified using primers SMc00419-F

(5'-A A G A A T T C C C A T G T T T C G G G C A T C A C C -3 ' ) a n d

SMc00419-R (5'-AACGATC GCTGCAGTGCATGTTGGAGC GAGAATC-3'). The fragment was cloned into EcoRI-PvuI sites of pSUP202, resulting in plasmid pSUP202gshB. Plasmid pSup202gshB was transferred from E. coli to S. meliloti 2011 and recombined into the genomic gshB region via single crossover to give the mutant strain SmgshB in S. meliloti 2011 genetic background. The mutants were screen for resistance to tetracyclin (10 mg per ml) and streptomycin (100 mg per mL). The SmgshB mutant strain in Sm 2011 genetic background was validated by HPLC for the lack of GSH as described previously (Harrison et al., 2005). The delayed growth phenotype observed for the mutant strain was complemented by the addition of GSH (Figure S1). M. truncatula ecotype Jemalong A17, S. meliloti 2011 (wild type), and S. meliloti 2011 gshB (SmgshB) strains were grown as described (Harrison et al., 2005). M. truncatula seeds were then spread on 0.4% agar gel at 4°C for 2 days for vernalization and germinated at 16°C for 1 day. Seedlings were afterwards growing in vitro on Fahraeus medium (Boisson-Dernier et al., 2001). Each seedling was inoculated with 200µl of S. meliloti at an O.D. 600nm of 0.05. All the plants used in this study were grown in a 16-h light (23°C): 8-h dark (20°C) photoperiod. Nodules were harvested at 10-and 20-days postinoculation (dpi). At least three independent biological repetitions were analyzed.

Nitrogenase Activity Assay

Nitrogen fixation activity was determined by C 2 H 2 reduction assay (ARA), using a gas chromatograph. Nodulated roots at 10 and 20 dpi were detached from plants and put inside sealed vials. In each vial, 10% of the atmosphere was substituted with acetylene. After 1 h of incubation at 28°C, gas samples were analyzed with a gas chromatograph system (Agilent GC 6890N; Agilent Technologies, Massy, France). Twelve plants were tested per replicate and at least three different biological replicates were analyzed.

Flow Cytometry

S. meliloti bacteroids were purified as previously described [START_REF] Trinchant | Proline betaine accumulation in alfalfa plants under sodium chloride stress. Exploring its compartmentalization in nodules[END_REF]) using a 320 mOsM extraction buffer. Bacterial cells were fixed by heat treatment (70°C, 15 min) and stained with 10 mg/mL propidium iodide (PI). They were then subjected to flow cytometry analysis with a LSRII Fortessa flow cytometer (BD BioSciences, Rungis, France). Bacterial DNA content was determined from PI fluorescence at 585-625 nm after excitation at 565 nm with a single laser line. Single events were recorded and analyzed with BD FACSDiva v6.1.3 (BD BioSciences) and Kaluza v1.2 software (Beckman Coulter, Villepinte, France), respectively. Experiments were performed with biological triplicates and the recording of more than 200,000 events for each sample to ensure the statistical robustness of each assay [START_REF] Ribeiro | Regulation of differentiation of nitrogen-fixing bacteria by microsymbiont targeting of plant thioredoxin s1[END_REF].

Live/Dead Staining and Microscopic Observations

Nodulated roots and nodules at 10 and 20 dpi, respectively, were harvested and embedded in 4% agarose. Nodule sections of 70µm were prepared with a Vibratome HM650V (Thermo Fisher Scientific, Illkirch-Graffenstaden, France). Afterwards, nodule sections were stained with SYTO ™ 9 and propidium iodide (PI) (LIVE/DEAD BacLight Bacterial Viability Kit, Thermo Fisher scientific) for 20 min as previously described [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. Sections were then mounted on slides and visualized with a laser scanning confocal microscope LSM880 (Carl Zeiss, Marly le Roi, France). The SYTO™ 9 and PI were sequentially excited by 488 nm Argon and 561 nm HeNe laser lines, separately. Both fluorescence signals were recorded on separated detectors. All images were treated with Fiji software (Schindelin et al., 2012). Samples are from at least 30 nodules of 15 plants from three independent replicates.

Quantitative Reverse Transcriptase PCR (RT-qPCR)

Young nodules (10 dpi) and mature nodules (20 dpi) were harvested. Total RNA was extracted from 100 mg of nodules with Trizol (Invitrogen, Illkirch-Graffenstaden, France) and cDNA was synthesized with the Omniscript RT kit (Qiagen, Les Ulis, France) after a DNAse treatment of total RNA. Absence of genomic contamination was ensured by control PCR with intron-spanning primers. Quantitative PCR was performed using AriaMx Real-time PCR system (Agilent, Les Ulis, France). All data were normalized with two constitutively expressed genes, Mtc27 and A38 (M. truncatula) for the M. truncatula genes, and 16S ribosomal RNA and Polyribonucleotide nucleotidyltransferase for the S. meliloti genes. The primer sequences used for PCR are described in Table S1. Each RT-qPCR reaction for each of the three biological experiments was performed in triplicate. The expression fold change was calculated as 2 -ΔΔCt method (Livak and Schmittgen, 2001).

Statistical Analyses

Our data were reported as mean ± standard error. The significance of the results was assessed using the Mann-Whitney nonparametric test, which allows the comparison of small quantitative samples. When the number of samples was enough to define a normal distribution (30 samples), the results were assessed using Student's t-test.

RESULTS

Analysis of Plant Phenotype and Biological Nitrogen Fixation

To test the phenotype of the S. meliloti SmgshB strain, we have analyzed nodule functioning at 10-and 20-dpi which corresponds to young and mature nodules in vitro growth conditions. The phenotypes of the plants and the root nodules were examined, and the nitrogen fixation activity was analyzed by ARA (Figure 1). The nitrogen fixation efficiency was significantly reduced by 53% and 21% in SmgshB inoculated plants compared to control plants at 10 and 20 dpi, respectively (Figure 1A). A significant lower number of nodules was observed for SmgshB inoculated plants compared to the control plants at 10 dpi (Figure S2) and nodule size was not different at 10 or 20 dpi (Figure S2). Phenotypic analysis of root nodules showed that SmgshB nodules presented a less pronounced pink color compared to control ones and present an extended green color on the distal part of nodule at 20 dpi (Figure 1B). A significant diminution of shoot dry weight biomass and a reduced plant development were observed in SmgshB inoculated plants compared to the control plants at 20 dpi (Figures 1C andS3). In conclusion, SmgshB inoculated plants present an altered growth phenotype compared to the control plants.

Analysis of Bacteroid Differentiation Using Flow Cytometry

The bacteroid differentiation is correlated to an increase in size and in DNA content of the bacteroid compared to the bacterium (Mergaert et al., 2006). These criteria could be used to differentiate bacteria from bacteroids using flow cytometry [START_REF] Ribeiro | Regulation of differentiation of nitrogen-fixing bacteria by microsymbiont targeting of plant thioredoxin s1[END_REF]. To test whether GSH deficiency in the bacteria impairs the bacteria differentiation, we have analyzed the bacteroid and bacterial content in nodules from control plants and SmgshB inoculated plants at 10 and 20 dpi using flow cytometry (Figure 2). Bacterial cells showing DNA endoreduplication were observed in both control and SmgshB nodules at 10 and 20 dpi (Figure 2A). The nitrogen-fixing bacteroid (DNA content 4C and more) proportion in control and SmgshB nodules was similar at around 40% at 10 dpi (Figure 2B). In contrast, the proportion of bacteroids was significantly lower in SmgshB nodules than in control ones at 20 dpi, with a reduction of 40% in bacteroid content compared to the control (Figure 2B). Therefore, these results show that bacteroid differentiation occurs in SmgshB nodules in a similar way that in control nodule at 10 dpi. However, the lower content of bacteroids in SmgshB nodules compared to control ones shows that the nodule bacteroid content decreases in mature nodules. This decrease of bacteroid content in SmgshB nodules may be associated to a reduced bacterial differentiation during nodule aging or to a faster bacteroid degradation compared to control nodules.

Analysis of Bacteroid Survival Using Live/ Dead Staining in Nodule

To analyze the survival of bacteroids in the SmgshB nodules, the live/dead staining was used at 10 and 20 dpi (Figure 3). Permeable bacteroid cell membranes that are considered dead or dying and plant nucleus are stained red with propidium iodide (PI), while bacteroids with intact membranes are green due to SYTO ™ 9 staining of nucleic acids [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. No significant difference was observed at 10 dpi between the SmgshB and control bacteroids (Figures 3A,C). At 20 dpi, the nodules are more elongated with a larger zone III in control nodules. No significant bacteroid degradation was observed in the control nodules (Figure 3F). In contrast, SmgshB nodules presented a strong alteration of the nodule cellular structure with numerous dead bacteroids and empty plant cells in zone III (Figure 3H). These results show that the cellular structure of SmgshB nodule is not modified at early time point but present significant degradation at 20 dpi. This suggests that senescence occurs ealier in SmgshB nodules than in control nodules.

Expression of Nodule Marker Genes in SmgshB Nodules

In order to analyze whether the cellular and biochemical alterations observed in the SmgshB nodules could be correlated to modifications in the nodule genetic program, the expression level of plant gene markers was analyzed by RT-qPCR. These genes are involved either in the functioning of nodules or are genes potentially markers of senescence. The expression of eight marker genes was measured to analyze the SmgshB nodule transcriptomic modifications (Figure 4). The expression of marker genes expressed in the nodule differentiation zone (Thioredoxin s1, Trx s1), in the nitrogen fixation zone (Nodule Cysteine Rich Peptide, NCR001 and leghemoglobin, Lb) and during nodule senescence (Cysteine Proteases 6, CP6, Vacuolar Processing Enzyme, VPE, and Purple Acid Protease, PAP) was studied in control and SmgshB nodules. Moreover, the expression of two bacterial genes nifD (nitrogenase molybdenum-iron protein alpha chain) and nifH (nitrogenase iron protein), encoding nitrogenase subunits and expressed in nitrogen-fixing bacteroids, was analyzed in control and SmgshB nodules. Trx s1 is specifically expressed during the symbiotic interaction between M. truncatula and S. meliloti [START_REF] Alkhalfioui | A novel type of thioredoxin dedicated to symbiosis in legumes[END_REF] and its expression is localized in the nodule infection zone [START_REF] Ribeiro | Regulation of differentiation of nitrogen-fixing bacteria by microsymbiont targeting of plant thioredoxin s1[END_REF]. NCR001 is considered as a specific marker of the nitrogen fixation zone (Mergaert et al., 2003) like Leghemoglobin (Mergaert et al., 2003;Ott et al., 2005). The expression of CP6 and VPE, two genes encoding cysteine proteases, is induced during developmental-and stress-induced nodule senescence (Pierre et al., 2014). Finally, the expression of PAP is induced during the nodule senescence process in M. truncatula [START_REF] Xi | Medicago truncatula esn1 defines a genetic locus involved in nodule senescence and symbiotic nitrogen fixation[END_REF]. In silico analysis of this PAP isoform showed that it is significantly more expressed in the nodule than in roots and significantly more expressed in the nitrogen fixation zone than in the nodule meristematic and differentiation zones (Table S2).

Analysis of Trx s1 expression showed that Trx s1 expression was not significantly modified in SmgshB nodules compared to control nodules at 10 and 20 dpi suggesting a normal functioning of the nodule infection zone. The expression levels of NCR001 and Lb are significantly down-regulated in SmgshB nodules in comparison to control ones at 20 dpi. The expression of nifH and nifD was similar in SmgshB and control nodules at 10 dpi and nifH was significantly down-regulated in SmgshB nodules in comparison to control ones at 20 dpi. Regarding the senescence related gene markers, CP6, VPE, and PAP expression are, conversely, significantly up-regulated in SmgshB nodules compared to control nodules. The similar expression of nifH and nifD at 10 dpi strengthens the flow cytometry analysis and shows that the SmgshB bacteria are able to differentiate into bacteroids. Moreover, the down-regulation of the genes involved in BNF efficiency is correlated to the induction of senescence related genes, confirming that an early senescence occurs in SmgshB nodules. The symbiotic interaction established between legumes and bacteria allows the plant to satisfy its needs in nitrogen. The use of a bacterial strain deficient in GSH production alters significantly the biological nitrogen fixation and the growth of M. sativa, P. vulgaris, and P. sativum during NFS (Harrison et al., 2005;Muglia et al., 2008;[START_REF] Taté | Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness[END_REF]Cheng et al., 2017). Moreover, the electronic microscopy analyses suggested that SmgshB mutant strain is partially impaired in the bacteroid differentiation (Harrison et al., 2005). More recently, the glutaredoxin SmGrx1 mutant in S. meliloti strain 2011 showed clearly a deficiency in bacteroid differentiation. Glutaredoxins (Grxs) are small ubiquitous redox enzymes that are involved in the reduction of oxidative modifications using GSH (Benyamina et al., 2013). Thus, our hypothesis is that GSH produced by the strain 2011 is necessary to allow normal bacteroid differentiation in nodules formed on M. truncatula. In this context, we tested the symbiotic interaction between M. truncatula and the GSH deficient-SmgshB mutant constructed in S. meliloti strain 2011 at 10 and 20 dpi, two early time points of nodule nitrogen fixation, using a multidisciplinary approach.

The nitrogen fixation efficiency was significantly impaired in plants inoculated with SmgshB strain (Figure 1). However, this reduction is less noticeable than in plants inoculated with SmGrx1 (Benyamina et al., 2013). Moreover, at 20 dpi, elongated nodules are observed suggesting that the nodule meristem is more active in plants inoculated with SmgshB strain than in plants inoculated with SmGrx1 strain (Benyamina et al., 2013). In M. truncatula, the bacteroids present a terminal differentiation accompanied with a DNA endoreduplication and an arrest in cell division coupled to an increase in cell size [START_REF] Ribeiro | Redox regulation of differentiation in symbiotic nitrogen fixation[END_REF]. The flow cytometry analysis of SmgshB bacteroids shows that DNA endoreduplication occurs in SmgshB bacteroid and no significant difference was observed between SmgshB nodules and control ones at 10 dpi (Figure 2). In contrast, the number of terminally differentiated bacteroids is significantly lower in SmgshB nodules than in control ones at 20 dpi suggesting that there is a faster degradation or a slower formation of bacteroids in SmgshB nodules than in control ones. The live/dead experiments allow us to analyze the viability of the bacteroids in the root nodules. Such live/dead labelling shows an early death of bacteroids in SmgshB nodules compared to control ones at 20 dpi (Figure 3).

Gene expression analysis showed that at 10 dpi, genes expressed in the nitrogen fixing zone, NCR001 and Lb, are similarly expressed in SmgshB and control nodules suggesting that the gene expression in the nitrogen fixing zone is not significantly affected (Figure 4). A similar result is observed for Trx s1, a gene marker for the nodule infection zone, and VPE. Similarly, expression of nifH and nifD genes was similar in SmgshB and control nodules strengthening that SmgshB bacteroid differentiation occurs properly at 10 dpi. In contrast, significant higher expression of CP6 and PAP is already observed in SmgshB nodules compared to control ones at 10 dpi. This result showed that in our plant growth conditions, CP6 and PAP are the earlier senescence marker genes whose expression is significantly modified, suggesting that there is an early development of senescence in SmgshB nodules. At 20 dpi, there is a significant modification in the expression of different genes. NCR001, Lb, and nifH are less expressed in SmgshB nodules compared to control ones showing a correlation between the diminution of the nitrogen fixation efficiency and the reduction of the expression of genes expressed in the nitrogen fixation zone. Despite a strong increase of CP6 expression in 20 dpi mature SmgshB nodule, there is no more a significant difference compared to the control suggesting that a senescence process begins in control nodules in our conditions. In contrast, VPE expression is significantly induced in SmgshB nodule indicating the progression of the senescence at 20 dpi as observed in the live/dead analysis.

Taken together, our data show that, in contrast to SmGrx1 bacterial mutant (strain 2011), SmgshB mutant (strain 2011) is able to differentiate in bacteroids inside M. truncatula cells. The phenotype observed is similar to the one obtained during the interaction of SmgshB mutant (strain 1021) with M. sativa (Harrison et al., 2005). In this context, SmGrx1 should use another reducing power than glutathione to perform its activity. We hypothesize that the Grx1 could use the g-EC accumulated in high quantity in the SmgshB mutant strain as cofactor for the reductive power instead of GSH. Different phenotypes were observed for Arabidopsis thaliana g-ECS (gsh1) and GSHS (gsh2) plant mutants. The ghs1 mutant presents an embryo lethal phenotype whereas the gsh2 mutant has a seedling lethal phenotype suggesting a partial replacement of GSH functions by g-EC [START_REF] Cairns | Maturation of Arabidopsis seeds is dependent on glutathione biosynthesis within the embryo[END_REF][START_REF] Pasternak | Restricting glutathione biosynthesis to the cytosol is sufficient for normal plant development[END_REF]. A similar observation is also true for S. meliloti strain 1021 in which the SmgshA mutant strain cannot divide while the SmgshB mutant strain shows a growth delay under free living conditions (Harrison et al., 2005). g-EC is also used as a cofactor by glutathione peroxidase-1 to detoxify ROS in mice [START_REF] Quintana-Cabrera | g-Glutamylcysteine detoxifies reactive oxygen species by acting as glutathione peroxidase-1 cofactor[END_REF].

The correlation between the GSH content and the BNF in various legumes was the object of several works (Dalton et al., 1986;Evans et al., 1999;Matamoros et al., 1999;[START_REF] Moran | Glutathione and homoglutathione synthetases of legume nodules. cloning, expression, and subcellular localization[END_REF][START_REF] Groten | The roles of redox processes in pea nodule development and senescence[END_REF]. The plant GSH content is crucial for the bacterial infection effectiveness and the nitrogen fixation efficiency. GSH produced in the plant does not compensate the gshB mutation suggesting that it is not able to be transported to the bacteroid [START_REF] Becana | Sulfur transport and metabolism in legume root nodules[END_REF]. In this context, it should be mentioned that sulfate transport from the plant cell to the bacteroids is very active [START_REF] Schneider | Sulfate is transported at significant rates through the symbiosome membrane and is crucial for nitrogenase biosynthesis[END_REF] and that mutation in the sulfate transporter SST1 in Lotus japonicus impairs symbiotic nitrogen fixation [START_REF] Krusell | The sulfate transporter SST1 is crucial for symbiotic nitrogen fixation in Lotus japonicus root nodules[END_REF]. These data suggest that the sulfur metabolism in bacteroid is depending on sulfate transport and that other sulfur containing molecules such as methionine, cysteine or GSH are not transported through the symbiosome membrane. In this sense, the R. etli metZ gene involved in methionine synthesis is required for the efficient interaction with P. vulgaris suggesting that methionine is produced by R. etli bacteroids during symbiosis, and not supplied by the host cells [START_REF] Taté | The Rhizobium etli metZ gene is essential for methionine biosynthesis and nodulation of Phaseolus vulgaris[END_REF]. In contrast, the cysG mutant strain of R. etli, unable to grow on sulfate as the sole sulfur source, was able to induce nitrogen-fixing nodules on P. vulgaris suggesting that cysteine or glutathione is supplied by the plant to bacteria [START_REF] Taté | A cysG mutant strain of Rhizobium etli pleiotropically defective in sulfate and nitrate assimilation[END_REF]. Moreover, the nodules induced by cysteine auxotrophs of S. meliloti Rmd201 were fully effective like those of the parental strain-induced nodules suggesting that the alfalfa host provides cysteine to the bacteroid [START_REF] Abbas | Symbiotic characteristics of cysteine and methionine auxotrophs of Sinorhizobium meliloti[END_REF]. However, even if gshB mutant strains show an impaired symbiotic phenotype, it cannot be excluded that a small fraction of GSH produced in the plant may pass through the bacteroid membrane, partially compensating the bacterial deficiency. The bacterial GSH pool plays also a crucial role for nitrogen fixation efficiency (Figure 5). In the gshB mutant of S. meliloti strain 1021, the GSH deficiency was associated with a higher catalase activity in the bacterial mutant in free-living conditions and a reduction of growth efficiency (Harrison et al., 2005). A decrease in the BNF was observed during the symbiosis with M. sativa correlated to an early bacteroid death. In R. tropici gshB mutant strain, the lower symbiotic efficiency was associated to an early senescence pattern connected to increased levels of superoxide accumulation (Muglia et al., 2008). For Rhizobium etli gshB mutant, the decrease in BNF was correlated to a strong reduction in nodule number and a deficiency of glutamine transport through Aap and Bra transporter [START_REF] Taté | Glutathione is required by Rhizobium etli for glutamine utilization and symbiotic effectiveness[END_REF]. This deficiency in nutrient transport also detected in R. leguminosarum 3841, which fails to efficiently use various carbon sources such as glucose, succinate, glutamine and histidine, may trigger an early nodule senescence. This transport deficiency in R. leguminosarum 3841 gshB mutant strain is also associated to a lower BNF (Cheng et al., 2017). The LysR-type transcriptional regulator LsrB, required for nodulation, also regulates gshA gene expression through modification of cysteine residues [START_REF] Luo | Two new Sinorhizobium meliloti LysR-type transcriptional regulators required for nodulation[END_REF]Tang et al., 2018) and gshB gene expression [START_REF] Lu | The Sinorhizobium meliloti LysR family transcriptional factor LsrB is involved in the regulation of glutathione biosynthesis[END_REF].

In conclusion, the inoculation of M. truncatula with the SmgshB bacterial mutant triggers the development of nodules with a reduced capacity to fix nitrogen. This lower BNF does not seem correlated to a reduced bacteroid differentiation efficiency. However, it is strongly correlated to an early senescence process and the alteration of the expression of multiple nodule gene markers showing the fundamental role of the bacterial GSH pool in nodule functioning.

1

Table S1: List of primers used to quantify gene expression levels by qRT-PCR

Genes

Forward primers

Reverse primers

Mtc27

TGAGGGAGCAACCAAATACC GCCAAAACCAAGCTACCATC

A38

TCGTGGTGGTGGTTATCAAA TTCAGACCTTCCCATTGACA

16S

III. Discussion and perspectives

Bacteroid redox homeostasis ensures symbiotic nitrogen fixation

We have characterized a S. meliloti 2011 mutant strain SmgshB which was generated via integration of a plasmid into the genome by single cross-over recombination in the gene gshB, encoding the enzyme glutathione synthase. The SmgshB strain exhibits a significant reduction in the symbiotic nitrogen fixation capacity, consequently diminishing biomass accumulation in plant aerial parts and accelerating the nodule senescence. Similar evidences have been observed with many other rhizobial strain that the GSH deficiency impairs symbiotic nitrogen fixation (Cheng et al., 2017) (Tate et al., 2012) (Muglia et al., 2008) (Harrison et al., 2005). The symbiotic nitrogen fixation is highly sensitive to the redox status at both transcriptional and post-translational level. The nitrogenase is with high redox potential and prone to be damaged rapidly under exposure to oxygen. Besides, the four regulatory proteins of symbiotic nitrogen fixation, NifA, NifL, FixL and RegB, are also oxygen/redox sensors [START_REF] Dixon | Genetic regulation of biological nitrogen fixation[END_REF]. The anaerobic condition is essential for symbiotic nitrogen fixation, whereas GSH is required to activate FNR transcription regulator to form dimer of [4Fe-4S] cluster subunit to control gene expression under anaerobic conditions [START_REF] Ritz | Roles of thiol-redox pathways in bacteria[END_REF]. It is therefore plausible that the altered redox homeostasis in bacteroid caused by GSH deficiency is detrimental for FNR regulation, thereafter triggering a premature nodule senescence.

GSH deficiency hinders nitrogen-fixing symbiosis

Our study also demonstrates an early senescence phenotype of nodules generated by SmgshB, and we have observed a delayed nodulation phenotype before 10dpi. The symbiotic experiments of the R. tropici CIAT899-13T2 mutant strain show that the mutant strain can induce similar effective nodules albeit outcompeted in co-inoculation with WT strain [START_REF] Riccillo | Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance[END_REF]. The authors later on examined the importance of GSH in symbiosis with the R.

tropici gshB mutant which presented a delayed nodulation, reduced weight in aerial part and early senescence phenotype [START_REF] Muglia | Rhizobium tropici response to acidity involves activation of glutathione synthesis[END_REF]Muglia et al., 2008). The reminiscent phenotype of gshB mutant in R. tropici and S. meliloti implying a conserved functional role of GSH in rhizobia [START_REF] Riccillo | Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance[END_REF] (Harrison et al., 2005).

The expression of SmgshB culminates at the interzone (IZ; Figure 22), also indicating a function of GSH during bacteroid terminal differentiation step which involved DNA endoreduplication. It has been proven in E. coli that the GSH/glutaredoxin system is involved in DNA synthesis ultimately using the reducing power from NADPH [START_REF] Ortenberg | Interactions of glutaredoxins, ribonucleotide reductase, and components of the DNA replication system of Escherichia coli[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF]. The nodule abortion observed in glutaredoxin mutant of S. meliloti (SmGRX1) was associated to an impaired bacteroid differentiation suggesting a crucial role of GSH in this step (Benyamina et al., 2013). Notably, in live/dead staining by a mixture of SYTO 9 and PI, we have observed oversaturated/overlapped florescence in the in the interzone IZ. One possible explanation could be that that, during bacteroid differentiation, PI can penetrate into bacteroids due to the fact that bacteroid membrane permeability is increased by NCRs (Mergaert et al., 2003;Van de Velde et al., 2010;Kondorosi et al., 2013).

Despite PI is nondetectable in free-living bacteria, it could be stained 50% of live bacteroids in addition to all the dead bacteria/bacteroids (Mergaert et al., 2006). Thus, symbiotic cells in interzone IZ could be stained by SYTO 9 and partially by PI with an overlapped florescence. However, the nodules generated by SmgshB presented an early senescence phenotype.

Previous studies have shown that GSH and nitrogen fixation capacity corelate with each other, and that GSH levels decrease in natural or induced senescence in different legumes (Table 6) (Matamoros et al., 1999;Harrison et al., 2005;Muglia et al., 2008;Tate et al., 2012;Cheng et al., 2017) (Dalton et al., 1986;Evans et al., 1999;Matamoros et al., 1999;[START_REF] Moran | Glutathione and homoglutathione synthetases of legume nodules. cloning, expression, and subcellular localization[END_REF][START_REF] Groten | The roles of redox processes in pea nodule development and senescence[END_REF]. In our observation, the bacterial GSH deficiency has affected the nodulation with a delayed nodulation and induced heterogenous size of nodules. We could not detect a significant reduction in bacterial DNA content in early stages of nodulation, indicating that GSH may not be a major component for bacteroid terminal differentiation or that SmGRX1 may use gEC as reducing power. Nevertheless, the premature senescence

CHAPTER I

pattern of the SmgshB infected nodules is proven with enhanced green colour in nodules, reduced nitrogen fixation capacity with less biomass accumulated in plants, early senescent bacteroid in live/dead staining and fast degradation in DNA content, as well as elevated gene expression of nodule senescence markers already seen at 10dpi.

With these gshB mutants showing an early senescence phenotype (Table 6), we therefore conclude that bacterial GSH is important for efficient nitrogen fixation and maintaining nodule function in time. Developmental nodule senescence has been proposed

to result from a decrease in the ascorbate/glutathione antioxidant pool coupled to decreasing carbon-to-nitrogen ratios inside the nodule (Puppo et al., 2005). In this context, analyse of carbon and nitrogen metabolism in nodule obtained with gshB mutant strain could be very interesting to analyse the potential redox regulatory process of carbon and nitrogen metabolism.

GSH endows bacterial stress tolerance

GSH was reported to protect a diversity of bacteria against environmental stresses [START_REF] Chesney | Bacterial glutathione: a sacrificial defence against chlorine compounds[END_REF][START_REF] Crockford | The adaptation of Rhizobium leguminosarum bv. phaseoli to oxidative stress and its overlap with other environmental stress responses[END_REF][START_REF] Masip | The many faces of glutathione in bacteria[END_REF], therefore another evidenced feature of GSH deficient strains is that they are prone to be susceptible to stresses and uncompetitive in nodulation. For example, one R. tropici CIAT899 strain is with an intrinsic tolerance of acidity. One of its mutant strains CIAT899-13T2 is unable to produce GSH and incapable to grow under acidic condition, due to a single Tn5-luxAB insertion in a gene with high similarity of E. coli gshB gene. The mutant presented a sensitivity to low pH levels, high osmolarity, hydrogen peroxide, methylglyoxal toxicity [START_REF] Riccillo | Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance[END_REF]. The acid tolerance can be restored in this mutant by exogenous GSH at 80µM [START_REF] Riccillo | Glutathione is involved in environmental stress responses in Rhizobium tropici, including acid tolerance[END_REF] and by plasmid-based complementation of the gshB gene [START_REF] Muglia | Rhizobium tropici response to acidity involves activation of glutathione synthesis[END_REF]. In terms of the transcription of gshB, it was induced by acidity and UV radiation, but showed no response to environmental constraints, such as osmotic and oxidative stresses, which is known to be mediated by GSH [START_REF] Muglia | Rhizobium tropici response to acidity involves activation of glutathione synthesis[END_REF].

These results suggest that metabolic production of GSH is important in stress tolerance of bacteria under adverse conditions, with peculiar regulation of potassium channels, and involvement in antioxidation and detoxification. In our study, we have also observed an accidental sudden senescence of SmgshB inoculated nodules when growth chamber light was partially off for two days (over the weekend), while the WT type seems more tolerant under weak lumination. However, more experimentations are needed to reveal the role of GSH in stress tolerance of S. meliloti than just a single occurrence.

Transcriptional machinery controls gene expression

Cellular levels of RNA are under control of the complex mixture of transcriptional regulatory mechanisms (Figure 23) [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF][START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. Multiple events are included in transcription in eukaryotes, such as decondensation of the locus, nucleosome remodelling, histone modifications, recruitment of the basal transcription machinery to the core promoter, and binding of transcriptional activators and coactivators to enhancers and promoters [START_REF] Smale | The RNA polymerase II core promoter[END_REF]. The basal transcription is determined with the RNA polymerase binding to the sequence-specific core promoter which is present on all of the genes. The dynamic gene expression is under control of transcriptional regulation network, which is composed of the binding of Transcription Factors (TFs) to the Transcription-Factor Binding Sites (TFBS; the cis-regulatory DNA elements) that are either proximal or distal to a Transcription Start Site (TSS), in addition to co-factors and chromatin structure [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. Sets of numerous TFs proteins are capable to bind to specifically TFBS thus coordinately activating or repressing gene transcription. Therefore, the transcriptional binding is not only determined by the sequence-specificity of TFBS, but also influenced by the adjacent sequences and the proximity of other proteins. TFs have strict spatial requirements in their binding sites that preclude variable spacing. For some TFs, such as a subset of the nuclear receptor family, variable spacing is allowed [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. The bound TFs can interact with cofactors to stabilize the transcription-initiation machinery of gene expression [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]) (Figure 23). The rate of transcription is also highly dependent on epigenetic factors and the three-dimensional structure of chromatin [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. 2) Sets of TFs can operate in functional cis-regulatory modules (CRMs) to achieve specific regulatory properties.

3)

Interactions between bound TFs and cofactors stabilize the transcription-initiation machinery to enable gene expression.

4)

The regulation that is conferred by sequence-specific binding TFs is highly dependent on the three-dimensional structure of chromatin.

Transcriptional regulation network comprises significant simple motifs

The transcriptional regulation network is the interaction between TFs and genes (regulons). TFs themselves are encoded by genes, thereby regulated by other TFs, and so on. These interactions are made of multiple regulatory blocks which are composed of many nodes (genes) and arrows (interactions) (Figure 24) [START_REF] Shen-Orr | Network motifs in the transcriptional regulation network of Escherichia coli[END_REF][START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF].

1) Autoregulation is the autogenous control that a gene is regulated by itself [START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF].

2) The feedforward loop (FFL) is defined by a TF X that regulates a second TF Y, such that both X and Y jointly regulate a regulon Z (Figure 24) [START_REF] Shen-Orr | Network motifs in the transcriptional regulation network of Escherichia coli[END_REF].

3) The single-input module (SIM) is defined by a set of target genes that are controlled by a master TF (Figure 24) [START_REF] Shen-Orr | Network motifs in the transcriptional regulation network of Escherichia coli[END_REF], also known as Multi-Input Motif (MIM) [START_REF] Lee | Transcriptional regulatory networks in Saccharomyces cerevisiae[END_REF]. The genes in SIM always have a common biological function [START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF].

4) The 'Dense Overlapping Regulons (DOR) is a layer of overlapping interactions between regulons and a group of input transcription that is much more dense than corresponding structures in randomized networks (Figure 24) [START_REF] Shen-Orr | Network motifs in the transcriptional regulation network of Escherichia coli[END_REF].

Thereof a complex transcription network is formed by these basic regulatory motifs which are significantly frequent in real transcription. Each network motif has a specific function in governing specific gene expressions (Figure 24) [START_REF] Milo | Network motifs: simple building blocks of complex networks[END_REF][START_REF] Shen-Orr | Network motifs in the transcriptional regulation network of Escherichia coli[END_REF].

Altogether, gene expression is the net effect of the dynamical transcription network. Many genes, as nodes in the network, can be regulated by multiple TFs. Their promoter activity is thus a multi-dimensional input function of the different input transcription factors [START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF].

Additionally, TFs in plants can bind to hundreds or thousands of genomic sequences, but with only a small portion of targets responding [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF]. Each arrow represents for the direct regulation of a TF on its target genes, as activation or repression.

I.1.2 Cis-regulatory elements

Cis-regulatory elements involved in transcriptional regulation in eukaryotes

Non-coding genomic DNA were long-time regarded as junk DNA as without a proteincoding function. For the last decades, due to boosted knowledge on phylogenetic footprinting between orthologous genes, nucleotide composition and the assessment of available transcript data, there are increasing evidences revealing the roles of non-coding sequences in transcriptional regulation and a prominent contribution in evolution [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. The abundant cis-regulatory sequences distribute discretely in cluster in genome. The cis-regulatory elements act across divergent distances in genome to control the spatial and temporal expression of genes. The biological significance of cis-regulatory elements is increasingly uncovered mainly on sequence-conservation-based analyses and application of transgenic approaches in model organisms.

Cis-regulatory elements play different roles in transcriptional control

The cis-elements are recognized with different roles in transcriptional regulation (Figure 25) [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. Transcriptional promoters are in the centre of the basal transcription machinery and located at the 5' ends of genes. Promoters defines the orientation and origin of transcription [START_REF] Maston | Transcriptional regulatory elements in the human genome[END_REF]. Mostly, functional regulatory sequences are more likely to be near the TSS according to the current laboratory annotation [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. But many genes have multiple TSSs. Thus, promoters, that contain one or more TSS(s) are more and more investigated for identification of regulatory sequences.

Promoter regions can be identified with transcript data via recurrent alignment of the 5′ ESTs and/or full-length cDNAs [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF], which is generally accessible in genome browsers.

The cis-regulatory elements have also been revealed with important regulatory functions for gene expression positively (as enhancers) or negatively (as repressors/silencers). The regulatory output will be the net effects of positive and negative inputs, often as consequences of environmental stimuli and developmental cues [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. Some enhancers can also behave like repressors dependent on the differential transcription factor binding [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. Insulators possess two kinds of activities as enhancerblocking or barrier activities (Figure 25, d &e) [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. Enhancer-blocking activity refers to the inhibition of activating effects when placed between enhancers, whereas the barrier activities is the ability to set boundary between active and repressive chromatin [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF].

Regulatory DNA sequences are vital in evolutionary processes

Evolutionary pressures lead to the retention of conserved genetic modules such as the regulatory sequences [START_REF] Rubin | Comparative genomics of the eukaryotes[END_REF]. Genetic mutations on one hand boost genetic diversity, whereas on the other hand mutations with certain functions will be more retained allowing population to adapt and species to evolve. Mutations in functional sequences, which mostly negative effects for fitness, will be lost from the population [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF].

On the contrary, non-functional mutations are free to be fixed by drift as sequences of high genetic variations [START_REF] Kimura | Evolutionary rate at the molecular level[END_REF]. For example, the movement of transposable elements are the major originators to promote mutations and chromosome rearrangements, which is thereafter possible to become heritable after the phenotypic modification and adaptation [START_REF] Biémont | Junk DNA as an evolutionary force[END_REF]. Waves of invasion or loss of transposable elements shape the genome and control the activity of genes during the evolution of populations and species [START_REF] Biémont | Junk DNA as an evolutionary force[END_REF]. The assumption that regulatory mechanisms are conserved in orthologous genes of different species, enables the prediction of conserved regulatory genomic sequences. An establishment of datasets of regulatory sequences is necessary including the protein binding specificity, epigenetic marks, as well as the validated data of regulatory sequences and tissue specificity of characterized promoters.

Increasing evidence enrich the databases of cis-regulatory elements

During the last decades, advances in the identification of the TF binding-sequences have been helpful to decipher the transcriptional regulatory codes [START_REF] Harbison | Transcriptional regulatory code of a eukaryotic genome[END_REF][START_REF] Badis | Diversity and complexity in DNA recognition by transcription factors[END_REF]. The general cis-elements in eukaryotes boxes, such TATA, CCAAT and GC (Mitchell, P. J. & Tjian, R., 1989). In eukaryotes, one of the best described core promoters are the TATA-contained core promoters in RNA polymerase II transcribed genes. TATA box can associate with the basal transcription factors with direct binding with the TATA-binding protein (TBP) [START_REF] Lifton | The organization of the histone genes in Drosophila melanogaster: functional and evolutionary implications[END_REF][START_REF] Smale | The RNA polymerase II core promoter[END_REF][START_REF] Molina | Genome wide analysis of Arabidopsis core promoters[END_REF]. The consensus site of TATA box is TATAWAW, while a wide variety of AT rich sequences can be recognized by TBP [START_REF] Smale | The RNA polymerase II core promoter[END_REF]. It is often located 25-30 bp upstream of a TSS [START_REF] Smale | The RNA polymerase II core promoter[END_REF]. The orientation of the TATA box would have minimal contribution to the direction of transcription [START_REF] Smale | The RNA polymerase II core promoter[END_REF]. TATA box is essential in TATAcontained core promoters, but it is not prevalent in the majority of gene promoters. For example, in Arabidopsis, only about 29% of the promoters are TATA-contained, harbouring the TATA box around -32bp to the TSS, while a large proportion of promoters were identified as TATA-less [START_REF] Molina | Genome wide analysis of Arabidopsis core promoters[END_REF]. The RNA polymerase II, together with other basal TFs, gives only low level of transcription. Other factors, such as regulatory enhancers, are necessary to increase the transcription activity. For example, CAAT box, sometimes also called CCAAT box or CAT box, is the binding sites for the general TFs, i.e., nuclear factor Y (NF-Y; or core binding factors, CBF), with a consensus sequence that is CAAT (Shirsat et al., 1989) or CCAAT (Laloum et al., 2013).

In plant, [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF] et al. have identified DNA-binding specificities of than 80% of these binding sites have a biological relevance based on the analysis of coregulated genes, transcriptomic data, and chromatin hypersensitive regions [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF]. Approximately half of the tested TFs recognized a secondary DNA element differing from its primary motifs. The sequence enrichment is higher in upstream than in downstream regions in terms of enhancers, whereas the spatial restriction is less strict when it comes to the repressors. The sequence frequency is found to be higher in upstream (1kb) in case of positively co-regulated genes, while analogous in negatively co-regulated genes [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF]. These data enriched the datasets for mapping cis-regulatory elements in genome and contributed to decipher transcriptional regulatory, which in return allows the prediction of functions of particular TFs or DNA fragments code.

Several Cis-regulatory elements have been identified in leguminous plants

Functional studies on transcriptional regulators has uncovered numerous cis-regulatory elements in leguminous plants, particularly during the course of symbiosis. In bacterial the infection process, the promoter of MtENOD11 located -411bp upstream of the translation start codon is proven to be sufficient to induce the expression in response to Nod factor and bacterial infection (Boisson-Dernier et al., 2005). Then three TFs ERN1, -2, and -3 were identified to directly regulate the gene expression of MtENOD11 via specific binding to a Nod factor responsive cis-element, the NF-box (between -411bp to -358bp to ATG) (Andriankaja et al., 2007). During rhizobial infection and organogenesis, certain cis-regulatory sequences within ~7kb upstream of NIN are required to regulate bacterial infection but are insufficient for the activation of cortical cell divisions [START_REF] Yoro | A positive regulator of nodule organogenesis, NODULE INCEPTION, acts as a negative regulator of rhizobial infection in Lotus japonicus[END_REF]. More recently, the 5 kb upstream region of NIN is identified to be sufficient for root hair curling and bacterial infection, but not for nodule primordium formation (Liu et al., 2019). Interestingly, a remote cis-regulatory region located 18kb upstream of the start codon is identified for MtNIN expression initiate nodule primordium formation, containing putative cytokinin response elements (Liu et al., 2019).

However, no cis-regulatory element has been found to-date related to nodule senescence regulation.

I.2 Cysteine proteases (CPs) in nodule senescence

There are approximately 140 cysteine proteases (CPs, also cysteine peptidases, cysteine proteinases) among the about 800 proteases encoded by plant genomes [START_REF] Barrett | Handbook of proteolytic enzymes[END_REF]. CPs belong to 15 families in five clans according to the MEROPS classification (http://www.ebi.ac.uk/merops/) [START_REF] Rawlings | The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database[END_REF]. The papain-like proteases C1A (family C1, clan CA) have been described as active proteolytic enzymes induced in plants during multiple physiological processes such as germination, senescence processes and plant defence [START_REF] Sheokand | Cysteine proteases in nodulation and nitrogen fixation[END_REF][START_REF] Gilroy | Involvement of cathepsin B in the plant disease resistance hypersensitive response[END_REF][START_REF] Barrett | Handbook of proteolytic enzymes[END_REF]. Within leguminous plant nodules, CPs are engaged in response to the microbial invasion; protein turnover for tissue remodelling; metabolic homeostasis and nutrient reutilization; adaptation to environmental stresses; leaf and nodule senescence [START_REF] Sheokand | Cysteine proteases in nodulation and nitrogen fixation[END_REF]. In indeterminate nodules, senescence begins from the proximal to the apical part of the nodule.

CP gene expression have been widely associated to nodule senescence of M. truncatula (Fedorova et al., 2002;Van de Velde et al., 2006), soybean (Alesandrini et al., 2003a,b), Chinese milk vetch (Naito et al., 2000), Pisum sativum (Kardailsky & Brewin, 1996) and

Astragalus sinicus (Li et al., 2008).

The upregulation of cysteine protease genes features the onset of nodule senescence

Enhanced activity of proteolytic enzymes is a hallmark of senescence (Pladys & Vance, 1993;[START_REF] Lopez-Otin | The hallmarks of aging[END_REF]. Nodule senescence is featured with lytic-related processes such as rupture of the peribacteroid membranes, lysis of bacteroids, and disorganization of host-cell cytosol (Pladys & Vance, 1993;Van de Velde et al., 2006). Thereof, CPs have been evidenced to activate nodule senescence by multiple groups (Pladys & Vance, 1993;Kardailsky & Brewin, 1996;Naito et al., 2000;Vincent & Brewin, 2000;Alesandrini et al., 2003;Asp et al., 2004;Van de Velde et al., 2006;Li et al., 2008;Perez Guerra et al., 2010;Pierre et al., 2014;van Wyk et al., 2014;Cilliers et al., 2018;Deng et al., 2019). In 2010, a cluster of six M. truncatula CP genes (MtCP1 to MtCP6) were shown to be in relation with senescence process (Perez Guerra et al., 2010). In the same group, other nodule senescence related CPs were found such as TrCP1,5,8,10 (Asp et al., 2004) and AsNODf32 (Naito et al., 2000). The members of this cluster contains an N-terminal signal peptide and a putative tetrapeptide vacuolar targeting signal (LQRD; Chrispeels, 1991), while an HDEL or KDEL endoplasmic reticulum retention signal was absent (Perez Guerra et al., 2010). The protein structure indicates a secretion pathway destined for vacuole-like structures, while later on MtCP6

protease was found to accumulate in the vacuole of uninfected cells or in the symbiosomes of infected cells (Pierre et al., 2014). Considering the discrepancies of gene annotation in genomic and transcriptomic databases, a list of MtCPs involved in nodule senescence are shown in Table 7.

overexpression of MtCP6 leads to a significant increase in N fixation capacity and in size of the zone III in nodule (Pierre et al., 2014).

MtCP6 has been used as a molecular marker for the onset of nodule senescence in numerous studies. Expression of MtCP6 is induced during both developmental senescence and induced nodule senescence, such as dark stress and nitrate treatment (Perez Guerra et al., 2010;Pierre et al., 2014). Using the β-glucuronidase (GUS) reporter gene, the expression of MtCP6 was localized in the basal part of 5wpi nodules, but absent in young nodules (Figure 26) (Perez Guerra et al., 2010). Later on, GUS-promoter analysis of MtCP6 shows a specific expression at the end of N fixation zone (zone III) to the beginning of senescence zone (zone IV), and sharply extended to the whole nodules under abiotic stresses like dark and nitrate treatment (Figure 26) (Pierre et al., 2014). In addition, an accelerated nodule senescence are observed with inefficient symbiosis, with enhanced the development of nodule zone IV and the extension of spatial MtCP6 expression (Cam et al., 2012;Pierre et al., 2014;Yang, L. et al., 2020). The findings of a spatiotemporal expression of MtCP6 at transcriptional level that correlated with proteolytic activities involved in nodule senescence indicate a specific transcriptional regulation of nodule senescence related signals.

To date, functional promoter studies have led to the identification of several nodulespecific cis-elements most in early steps of nodulation, e.g. CYC-box (TGCCATGTGGCA) [START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF], NSP1 (cis-element AATTT) (Hirsch et al., 2009), NFbox (Andriankaja et al., 2007), NIN (AAGMT; a cytokinin-responsive cis-regulatory region) [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] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF]Liu et al., 2019). However, no regulatory sequences have been identified

directly related to nodule senescence. To decipher the early steps of transcriptional regulation of nodule senescence, we looked for in this chapter cis-regulatory elements regulating the transcription of the early senescence marker MtCP6 in transgenic root nodules generated by Agrobacterium rhizogenes.

II. Results (manuscript)

Yang L, Frances L, de Carvalho-Niebel F, Boncompagni E, Frendo P. 2020.

Identification of cis-acting elements involved in the transcriptional regulation of the cysteine protease gene MtCP6 in nodule senescence of Medicago truncatula. In preparation.

1

Identification of cis-regulatory elements involved in the transcriptional regulation of the cysteine protease gene

MtCP6 during nodule senescence of Medicago truncatula

Introduction

Leguminous plants play an important role in sustainable agriculture due to the establishment of a symbiotic association with soil nitrogen-fixing bacteria of the Rhizobiacea family. This symbiosis leads to the development of de novo root organ, the root nodule, in which the bacteria differentiated in bacteroids are able to reduce atmospheric (Ferguson et al., 2010;[START_REF] Syska | Molecular weapons contribute to intracellular rhizobia accommodation within legume host cell[END_REF]. Nodules are developmentally classified into determinate and indeterminate nodules according to the persistence of nodule meristem. In determinate nodules, meristem is nonpersistent, and a radial gradient of senescent cells occurs from the centre and extend to the periphery along with nodule aging (Puppo et al., 2005;Kazmierczak et al., 2020). Conversely, in indeterminate nodules, the presence of a continually active apical meristem (zone I) ensures the continuous growth of the nodule. Cells from the meristematic zone are continuously infected by Sinorhizobium meliloti in the infection zone (zone II). Therein, bacteria reside in plant cells and are surrounded by the plant-originated peribacteroid membrane, collectively known as symbiosome. Thereafter, host plant and bacterial cells initiate a terminal differentiation process in the interzone II-III to form the nitrogen-fixing symbiotic cells (zone III). In zone III, the bacteroids express nitrogenase and reduce atmospheric nitrogen into ammonia. However, the lifespan of root nodule is time-limited and extremely sensitive to abiotic stresses, leading to the development of the senescence zone (zone IV) (Kazmierczak et al., 2020;Puppo et al., 2005). The nodule senescence is characterized with a decline of nitrogen fixation capacity and a coordinated death of both bacteria and plant cells (Puppo et al., 2005;Van de Velde et al., 2006;Perez Guerra et al., 2010;Kazmierczak et al., 2020).

The symbiosis between legumes and rhizobia are mainly based on nutrient exchange of photosynthates and symbiotically fixed nitrogen. Senescence mostly occurs when the nutrient trading is out of equilibrium and the associated organ, the root nodule, will turn to general nutrient sources instead of carbon sink, for nutrient reutilization in other developing organs of the host plants (Puppo et al., 2005;Van de Velde et al., 2006;Perez Guerra et al., 2010;Kazmierczak et al., 2020). Thus, nodule senescence occurs not only during the organ aging (developmental senescence), but it is also triggered by various environmental changes (induced senescence), such as drought (Aranjuelo et al., 2013;Gil-Quintana et al., 2015), dark stress (Perez Guerra et al., 2010;Pierre et al., 2014), and nitrate treatment (Matamoros et al., 1999;Pierre et al., 2014). Accelerated nodule senescence was also observed by interruption of plant genes required for effective symbiosis, e.g. MtDNF2 [START_REF] Bourcy | Medicago truncatula DNF2 is a PI-PLC-XD-containing protein required for bacteroid persistence and prevention of nodule early senescence and defense-like reactions[END_REF]), MtNAC969 (de Zelicourt et al., 2012), MtNPDs [START_REF] Trujillo | Nodule-specific PLAT domain proteins are expanded in the Medicago lineage and required for nodulation[END_REF], or in symbiotic interaction with an inefficient bacteria partner, e.g. S. meliloti gshB (Yang et al., 2020a) and hmp (Cam et al., 2012).

A key feature of nodule senescence in legume is the triggering of proteolytic activities, involved in cellular degradation and nutrient remobilisation (Alesandrini et al., 2003;Puppo et al., 2005;Groten et al., 2006;Kazmierczak et al., 2020;Yang et al., 2020b). Although many factors can affect protease activities, transcriptomic analysis has emphasized the induction of the transcription of a group of cysteine protease encoding genes at the onset of nodule senescence in M. truncatula -S. meliloti symbiosis (Pladys & Vance, 1993;Kardailsky & Brewin, 1996;Fedorova et al., 2002;Alesandrini et al., 2003;Van de Velde et al., 2006;Perez Guerra et al., 2010). In situ hybridizations revealed the tissue-specific expressions of MtCP1 to MtCP6 genes in infected cells located at the periphery of the conical senescence zone starting in the center of the fixation zone (Perez Guerra et al., 2010). A further functional characterization of MtCP6 gene expression has shown its significant role as an early gene marker of developmental and induced nodule senescence (Pierre et al., 2014). Down-regulation of MtCP6 steady state mRNA by RNAi is correlated to a delayed senescence, and conversely, overexpression of MtCP6 leads to a significant increase in nitrogen fixation capacity and in size of the symbiotic zone in nodule (Pierre et al., 2014). Using the β-glucuronidase (GUS) reporter gene, MtCP6 was found to be specifically expressed at the transition between the nitrogen fixing zone (zone III) and the senescence zone (Zone IV) during developmental nodule senescence (Perez Guerra et al., 2010;Pierre et al., 2014). By contrast, under induced nodule senescence conditions of dark stress or nitrate treatment, the spatial expression of MtCP6 changes drastically and extends to the nodule distal part (Pierre et al., 2014). These results suggest a specific spatiotemporal transcriptional regulation of MtCP6 during developmental and induced nodule senescence.

To date, functional promoter studies have led to the identification of several nodule-specific cis-regulatory elements, mostly in early steps of nodule organogenesis [START_REF] Vijn | A 200 bp region of the pea ENOD12 promoter is sufficient for nodule-specific and Nod factor induced expression[END_REF]Andriankaja et al., 2007;Liu et al., 2019). However, no cis-regulatory sequences have been identified directly related to nodule senescence. Nevertheless, a few transcription factors (TFs)

have been identified via differential expression analysis of transcriptomic data, mainly from MYeloBlastosis (MYB), basic Helix-Loop-Helix (bHLH), basic region leucine ZIPper (bZIP), and NAM/ATAF/CUC (NAC) families (Van de Velde et al., 2006;Seabra et al., 2012;Cabeza et al., 2014;Chungopast et al., 2014;Serova et al., 2017;Yuan et al., 2017). For instance, a TF MtATB2 (bZIP family) is regulated by sucrose and is enhanced during nodule senescence (D 'Haeseleer et al., 2010). Recently, the TF MtbHLH2 is reported to be a transcriptional repressor of a cysteine protease encoding gene (MtCP77) and down-regulated accordingly in nodule senescence (Deng et al., 2019).

In this study, in order to decipher transcriptional regulation at the early steps of nodule senescence, we looked for cis-regulatory elements involved in the transcriptional regulation of MtCP6 in transgenic root nodule under developmental and induced nodule senescence. Serial promoter deletion analysis of MtCP6 allowed us to determine the minimal MtCP6 promoter (ProCP6) down to -242bp upstream of the transcription starting site (TSS), which was sufficient to drive a robust tissue-specific expression related to root nodule senescence.

Furthermore, a 67 bases pair sequence of the promoter region, termed as Nodule Senescence (NS) element or NS-box, was defined to drive the tissue-specific expression related to both developmental and nitrate induced nodule senescence using both gain and loss of function experiments. Mutagenesis of putative CAAT/WRKY cis-regulatory elements in the NS-box of MtCP6 promoter impairs the NS sequence regulatory activity. Taken together, these results

showed that we have identified the first cis-regulatory elements involved in the early regulation of root nodule senescence.

Materials and Methods

Plant material, root transformation and growth condition

M. truncatula Jemalong A17 seeds were scarified, surface-sterilized and germinated as described by Boisson-Dernier et al. (Boisson-Dernier et al., 2001). Germinated seedlings were stabbed at hypocotyl with Agrobacterium rhizogenes suspension using a modified protocol from Kereszt et al. [START_REF] Kereszt | Agrobacterium rhizogenes-mediated transformation of soybean to study root biology[END_REF]. Plantlets were thereafter transferred into a mixture substrate of perlite and sand (3:1) with a basic nitrogen supply of 1mM NH4NO3. Plants were grown at 20℃ for one week for optimal transformation and then grown at 23℃ for 2 weeks.

Then transgenic plant roots were selected by cutting off the non-green-fluorescent roots (GFP) under a Leica MZ FLIII fluorescence stereomicroscope (Leica, Wetzlar, Germany). After oneweek-adaptation from the selective cutting, transgenic composite plants were inoculated with 10mL of Sinorhizobium meliloti 2011 pXLGD4 [START_REF] Grefen | A ubiquitin-10 promoterbased vector set for fluorescent protein tagging facilitates temporal stability and native protein distribution in transient and stable expression studies[END_REF] at optical density of OD600nm=0.1. Nodules were harvested at 4 week-post-inoculation (wpi). Nitrogen-treated nodules were from plants applied with 10ml KNO3 (10mM) for 2 days before 4wpi.

Escherichia coli DH5α was cultivated in LB Broth at 37℃ with appropriate antibiotics. S.

meliloti 2011 pXLGD4 was growing in LBMC (LB Broth, added with 2.5mM CaCl2 and 2.5 mM MgSO4) with 100μg/mL streptomycin and 10μg/mL tetracycline at 30℃. Agrobacterium rhizogenes Arqua1 was grown in TY medium (5g/L bacto-tryptone, 3g/L yeast extract, 6mM

CaCl2) with 100μg/ml streptomycin at 28℃ (Quandt, 1993).

Plasmid constructions

Fifteen promoter fragments (-1,720bp, -1,467bp, -1,278bp, -1,088bp, -599bp, -511bp, -356bp, -303bp, -273bp, -242bp, -211bp, -175bp, -141bp, and -80bp) were cloned into entry vector pDONR™ P4-P1r (Invitrogen). Empty entry vector was built by recombination of attB4::empty cassette::attB1 into pDONR™ P4-P1r. Expression vector was constructed by multi-site Gateway recombination of promoter entry vectors with pENTR-GUS and pENTL2L3-T35S into the destination vector pKm43GWD-RolD:eGFP (Cam et al., 2012).

Primer sequences are listed in Table S2.

For gain of function experiments, seamless tetramers of NS region (-242bp to -175bp), NS1

(-242bp to -182bp) and NS2 (-210bp to -175bp) were synthesized into pUC53-Kan (Genewiz corporation, Takeley, United Kingdom). Tetramers were then inserted into EcoRI/HindIII sites upstream of a minimal CaMV 35S promoter (47bp) in a binary vector pLP100 [START_REF] Szabados | New plant promoter and enhancer testing vectors[END_REF]. Binary pLP100 constructs were then introduced into A. rhizogenes Arqua1 by electrotransformation (MicroPulser, Bio-Rad).

Internal and Block Deletion Constructs

Block deletion of the NS region was generated with complementary primers (Table S1) from pDONR™ P4-P1r-ProCP6 (-1,720bp). Similarly, two other promoter deletion constructs (ΔCW, ΔDof) were generated from pDONR™ P4-P1r:ProCP6 (-242bp). Then the deleted promoters (ΔNS, ΔCW and ΔDof) was recombined into pKm43GWD-RolD:eGFP as described in promoter deletion construction.

Histological analyses and microscope observations

Nodules with attached root fragments were harvested at 4wpi, and at 4wpi with a 2d-nitrate treatment. Samples were stained with X-Gluc (5-bromo-4-chloro-3-indolyl-β-D glucuronide, cyclohexylammonium salt; Euromedex) with a modified protocol from Jefferson et al. (Jefferson, 1987). Nodules were incubated at 37°C overnight. Alternatively, short-time staining (4h) were applied to better differentiate staining intensity of promoters less than -303bp. Whole stained nodule images were taken with a Leica MZFLIII stereomicroscope (Leica Microsystems). The nodules were thereafter embedded in 6% (w/v) agarose and sectioned (70µm) with HM650V vibratome (Thermo Scientific). Nodule section images were taken under dark field using Axioplan 2 microscope (Carl Zeiss). From all the histological experiments, more than 100 nodules were analysed from at least 15 independent plants. All experiments were conducted with three biological replicates.

Fluorimetric GUS assays

To quantify enzymatic GUS activity, 20g of nodules were grounded in liquid nitrogen. Total proteins were extracted with GUS buffer (50 mM sodium phosphate, pH 7.5, 10 mM 2mercaptoethanol, 10 mM Na2EDTA, 0.1% Triton X-100, and 0.1% sodium lauryl-sarcosine).

Quantitative GUS activities were measured as previously described (Boisson-Dernier et al., 2005). Total protein (10µg) extraction was measured with 4-methylumbelliferyl-β-Dglucuronide (MUG) as substrate (Biosynth AG). GUS activities were measured using a microtiter fluorimeter (FL600, Bio-Tek). Standard curves were prepared with a range of increasing concentrations of 4-methylumbelliferone (4-MU) (Sigma-Aldrich).

In silico analysis

In silico analysis of MtCP6 promoter was performed in multiple research-based databases (PLACE, PlantPAN and Plantcare) to identify conserved cis-element motifs (Higo et al., 1999;Lescot et al., 2002;Chow et al., 2016). To visualize cis-regulatory element homolog patterns, analysis was carried out on promoters of Cysteine Protease 1 (MtCP1, 1,701bp), MtCP2

(1,700bp), MtCP3 (513bp), MtCP4 (1,700bp), MtCP5 (1,701bp), MtCP6 (1,732bp) which are co-expression genes in nodule senescence with bioinformatic tools [START_REF] De Pater | Characterization of a zinc-dependent transcriptional activator from Arabidopsis[END_REF][START_REF] Bailey | MEME SUITE: tools for motif discovery and searching[END_REF]. Analysis was conducted with MEME algorithm (p-value>0.05) on MEME Suite web server (http://meme-suite.org) with the following parameters: maximum number of motifs

(3), minimum motif width (6), maximum motif width (50), minimum sites per motif (2), maximum sites per motif (600).

RESULTS

The truncated -242bp MtCP6 promoter is sufficient to confer specific spatiotemporal

GUS expression under developmental and nitrate-induced nodule senescence

The MtCP6 promoter-GUS fusion is specifically expressed in the transition between the nodule nitrogen-fixing zone III and the nodule senescence zone IV (Fig. 1A) (Perez Guerra et al., 2010;Pierre et al., 2014). Furthermore, the expression is dramatically upregulated under abiotic stresses like nitrogen treatment and dark stress (Perez Guerra et al., 2010;Pierre et al., 2014).

In order to identify cis-regulatory elements in the promoter region responsive to developmental and induced nodule senescence, fifteen 5' progressive MtCP6 promoter deletions were constructed and fused to a GUS reporter gene (Fig. 1B). The promoter fragments were upstream of TSS, ranging from -1,720bp to -80bp. The position of TSS is determined at the start of 5' UTR (35bp) via the genomic data (ID: Medtr4g079800.1; website:

https://legumeinfo.org/genomes/jbrowse/?data=Mt4.0 ) [START_REF] Tang | An improved genome release (version Mt4. 0) for the model legume Medicago truncatula[END_REF]. The multiple constructions were introduced in M. truncatula via A. rhizogenes transformation. A promoter less construct (Empty vector) was generated for negative control. Transgenic roots and nodules were harvested at 4wpi from composite transgenic M. truncatula plants and stained for detecting GUS activities.

To analyse GUS activity spatiotemporal localization, stained nodules were classified according to the control nodules with a complete MtCP6 promoter expression (Fig. 1B; Fig. S1). Class A corresponded to nodules showing GUS staining tissue at the interzone III-IV. Class B corresponded to those presenting abnormal spatiotemporal expressions. While, Class C corresponded to nodules with no detectable GUS staining. Detectable GUS activity for the full promoter construct (-1,720bp) was present in 87% of the harvested nodules, with 79% of Class A. Detectable Gus activity was present in at least 50% of the harvested nodules, with 40% of Class A for the promoter constructs ranging from -1,467bp to -242 bp. Additional 5′ deletion down to -211bp reduced drastically the percentage of stained nodule to 10%, albeit 9% of nodules were in class A (Fig. 1B). Non-specific GUS staining (Class B) mostly appears in shorter promoters under -211bp, at -175bp, 46% of nodules hold a detectable GUS staining but 43% were from Class B nodules. Interestingly, the -242bp promoter allowed 79% of detectable GUS activity with 97% of Class A whereas the -211bp promoter region allowed 10% of detectable activity with 90% of Class A (Fig. 1B). GUS activity was not detectable in any nodule with the negative control.

To unravel the cis-elements of MtCP6 promoter responding to nitrate, treatment was applied 2 days before harvesting at 4wpi and GUS activity was compared in treated and untreated nodulated roots (Fig. 1C). To evaluate GUS expression intensities, nodule staining was classified into strong (+++), moderate (++), weak (+) and undetectable (-) staining. In total, for all promoter deletion, more than 200 nodules were analyzed from at least 15 independent plants of at least three biological replicates.

Transgenic nodules of composite plants harboring the different MtCP6 promoter deletion constructs were stained for GUS activity overnight and sectioned before microscopic observation, which showed a strong blue precipitation (+++). To obtain a better gradient detection (+; +/-; -), staining period was reduced to 4h in the proximal promoter -303bp upstream of the TSS (Transcriptional Starting Site) (Fig. 1C). Sliced nodules were used to define the different zones of the nodules. Representative images of GUS activity in sliced nodules are presented for each promoter deletion construct (Fig. 1C). Under nitrogen treatment, the GUS staining for the full MtCP6 promoter construct was enhanced in transgenic nodules and the expression zone expanded from proximal to distal part of nodules spreading into the nitrogen fixing zone (zone III). Similarly, to the developmental senescence, a threshold of GUS activity detection was observed between the construction under the control of the promoter constructs at -242bp and -211bp. Whereas the GUS activity was correctly localize in nodule, the promoter deletion at -211bp led to a strong reduction (+/-) of GUS activity under nitrogen treatment (Fig. 1B,1C).

Taken together, these results suggest that a promoter region of 254bp (between -242bp and +12bp) is sufficient to maintain the tissue-specific transcription of MtCP6 during both developmental and nitrate-induced senescence. Specific cis-regulatory elements allowing gene expression during senescence may be present in the promoter region between -242bp and -175bp and bear specific cis-regulatory elements related to this specific regulation. Thus, the MtCP6 promoter region between -242bp and -175bp was termed as the nodule senescence (NS) related box or NS-box.

The NS-box is sufficient to endow nodule senescence expression patterns to GUS reporter gene

Our data suggest that the NS-box corresponding to a 67bp sequence is involved in the regulation of the specific spatiotemporal expression related to nodule senescence pattern. To validate this hypothesis, gain of function analysis was performed with tetramers of complete NS-box (NS; 67bp), of 5'-part of NS-box sequence (NS1, 50bp) and of 3'-part of NS-box

The transcriptional regulation of MtCP6 during nodule senescence may involve multiple cisregulatory sequences. To determine the significance of NS-box regulatory element in the full promoter (-1,710bp), loss of function analysis was conducted with a ΔNS deletion construct (Fig. 3A). Qualitative analysis of GUS activity suggests that transgenic nodules carrying the ΔNS construct expressed GUS similarly to the full promoter (Fig. 3B). The spatial expression localization of GUS was also similar for the construct ΔNS and the complete promoter (Fig. 3C). However, the GUS activity was performed overnight, and the signal seemed to be saturated in these staining conditions. The quantitative analysis of nodule GUS staining performed by fluorometric assay showed that GUS activity in ProCP6 ΔNS nodule (1,879±267

pmol MU/min/mg) was strongly reduced by more than 80% compared to the activity in full promoter control nodule (11,544±965 pmol MU/min/mg) (Fig. 3D).

Taken together, the results showed the significant role of NS-box in MtCP6 promoter activity and suggest the presence of other cis-regulatory sequence(s) allowing the expression of MtCP6 during nodule senescence.

A CW motif is crucial for the NS box activity

The bioinformatic analysis of the full promoter gives abundant putative cis-regulatory elements, which hinders the possible laboratory validation. Hence, we decided to focus on characterization the NS-box which allows the GUS expression in the nodule senescence zone.

To identify the putative cis-regulatory elements present in the minimal sufficient promoter from -242bp to -175bp, we analyze the sequence with Plant cis-regulatory element databases (Higo et al., 1999;Lescot et al., 2002;Chow et al., 2016). Into the NS-box, putative regulatory cis-elements have been found and might be involved in the specific regulation of MtCP6 expression in nodule (Fig. 4). The core promoter element TATA-box (CTATAAATAC) (Shirsat et al., 1989;[START_REF] Grace | Sequence and spacing of TATA box elements are critical for accurate initiation from the β-phaseolin promoter[END_REF]) is located at -32bp upstream of TSS in MtCP6

promoter. Two CAAT-box are found in the NS-box (Shirsat et al., 1989) which are widely recognized as common sequences found in the 5'-non-coding regions of eukaryotic genes were identified. The core sequence of W-box (TGAC) has been found twice within NS-box (Eulgem et al., 1999;Zhang et al., 2004;Xie et al., 2005). The core site required for binding of Dof (DNA binding with one finger) proteins (AAAG) was also observed (Yanagisawa & Schmidt, 1999;Yanagisawa, 2000;[START_REF] Yanagisawa | The Dof family of plant transcription factors[END_REF]. The consensus motif (AATTT) of a GRAS domain transcriptional regulator, Nodulation Signalling Pathway1 (NSP1) (Hirsch et al., 2009), is also present in inside NS-box.

Considering that NS1 was found to be the more important part of the NS-box involved in regulation of MtCP6 spatiotemporal gene expression, selected motifs between -242bp and -211bp, but not found in NS2 (CAAT/WRKY motifs and a tandem of Dof motifs) were validated by loss of function experiments. Thus, we deleted CAAT-WRKY sequence (ΔCW)

and tandem Dof sequence (ΔDof) in the -242pb promoter which allows GUS expression in the nodule senescence zone (Fig. 5A). Constructs were fused to GUS reporter gene, introduced into transgenic roots and qualitative analysis of GUS staining was performed (Fig. 5B).

Histological staining showed a visible but weaker GUS staining in ΔCW nodules in comparison to -242bp MtCP6 promoter. In contrast, no significant difference in GUS activity was observed between the ΔCW nodules and control nodules. 46% and 74% of nodules were found to be from Class A for ΔCW and in ΔDof constructs, respectively (Fig. 5C), while 64% of nodules were stained blue in the -242bp MtCP6 promoter construct. Using fluorometric assay, the lower GUS expression level in nodules was confirmed for ΔCW construct (292±49 pmol MU/min/mg protein) compared to the -242bp MtCP6 promoter construct (as 3,436±378) with the -242bp MtCP6 promoter construct (Fig. 5D).

In conclusion, the CAAT-WRKY motif present in the NS-box, is involved in the transcriptional activity of the MtCP6 promoter (-242bp) during nodule senescence.

DISCUSSION

Promoter activity of MtCP6 is related to the tissue-specific gene expression at the onset of nodule senescence.

Expression of MtCP6 has been used as a specific marker for the breakdown of nodule symbiosis under developmental and induced senescence (Perez Guerra et al., 2010;Cam et al., 2012;de Zelicourt et al., 2012;Pierre et al., 2014;Yang et al., 2020a). Multiple transcriptomic analyses have shown the co-expression of MtCP6 with five other CP genes during nodule senescence (Fig. S2) (Benedito et al., 2008;Perez Guerra et al., 2010). Eight probesets corresponding to CPs (Fig. S2) were highlighted with induction in nodules and with nitrate treatment (Fig. S2) (https://mtgea.noble.org/v3/) (Benedito et al., 2008). Additionally, expression of CPs was triggered in nodules treated with phosphinothricin (PPT), which inhibited the Glutamine synthetase and induced during premature nodule senescence (Fig. S2) (Seabra et al., 2012). These data indicate that CPs are associated to nodule senescence, and they are highly sensitive to plant nitrogen status.

To define cis-regulatory elements involved in the specific spatiotemporal expression at the onset of nodule senescence, we conducted a serial deletion analysis initiated from a 1,732bp

MtCP6 promoter characterized in previous studies (Perez Guerra et al., 2010;Pierre et al., 2014). The spatiotemporal specificity and nitrate response of MtCP6 promoter are preserved in promoter deletions until -242bp to TSS, which is identified as the minimal and sufficient promoter of MtCP6. However, the intensity of the GUS activity decreased gradually concomitantly to the gradual shortening of MtCP6 promoter. Thus, other cis-regulatory motifs, probably functioning as enhancers, might be present upstream the nucleotide -242 in the CP6

promoter. The significant GUS staining obtained with the deletion of the NS-box in the 1,732bp

CP6 promoter supports this idea. We could thus assume that other cis-regulatory elements may be involved in the transcriptional regulation, in addition to the NS-box that controls the tissuespecific transcription in nodule senescence.

NS-box mediates a transcriptional activation at the onset of developmental nodule senescence and nodule response to nitrate

We have determined a specific DNA sequence, the NS-box, that drives a similar transcriptional activity to the -1,720bp CP6 promoter. Interestingly, all MtCPs presenting a correlated expression pattern with MtCP6 (Fig. S2) are sharing large similarities between NS-box element (Fig. S3A&B). To date, no transcriptional regulatory factors (TFs) has been demonstrated to switch on the transcription during nodule senescence. In contrast, few TFs has been evidenced

as repressors for CP gene expressions (de Zelicourt et al., 2012;Deng et al., 2019).

Transcriptomic analysis of the MtbHLH2 mutant enabled the identification of a target gene, MtCP77, and the protein-DNA binding was proved by electrophoretic mobility shift assays and chromatin immunoprecipitation analysis (Deng et al., 2019). Gene expression of MtCP77 is positively related to nodule senescence with a decreased nitrogenase activity, accelerated programmed cell death and reactive oxygen species accumulation (Deng et al., 2019). However, analysis of the MtCP6 promoter sequence down to -1,720bp did not allow the identification of the TF binding sequence known as G-box (5'-CACGTG-3') [START_REF] Menkens | The G-box: a ubiquitous regulatory DNA element in plants bound by the GBF family of bZIP proteins[END_REF].

Nevertheless, it is not found in, however, the less canonical CANNTG motif [START_REF] Kawagoe | Synergism between CACGTG (G-box) and CACCTG ciselements is required for activation of the bean seed storage protein β-phaseolin} gene[END_REF], also known as E-box [START_REF] Hartmann | Differential combinatorial interactions of cis-acting elements recognized by R2R3-MYB, BZIP, and BHLH factors control light-responsive and tissue-specific activation of phenylpropanoid biosynthesis genes[END_REF], can be found multiple times (-1236, -204 and -156 bp) in the MtCP6 promoter. However, no direct evidence has been found concerning the regulation of MtCP6 by the bHLH repressor as the promoter deletion analysis did not show a detectable shift of promoter activity between -175 and -146 bp which harbour one of the Ebox. Finally, the general expression profile of MtCP77 is different from the MtCP1 to MtCP6 suggesting a rather different regulation of expression. The identification of multiple transcriptional repressors strengthens this idea that multiple regulatory elements are involved in the regulation of CP expression in nodule senescence.

The synthetic promoter of NS-box-based tetramers restores partially the transcriptional activity of the -242 bp promoter (Figure 2D). This result showed that positive transcriptional regulators may be involved in MtCP6 transcriptional regulation in nodule senescence. The partial restoration of the transcriptional regulation is quite common as for NF-box that was proven to be sufficient to mediate the nod factor-elicited gene activation, for which the corresponding synthetic promoter constituted of NF-box tetramers recovered less than half of the GUS activity in fluorometric assays (Boisson-Dernier et al., 2005;Andriankaja et al., 2007). Moreover, repetitive number of the same element within a synthetic promoter could have a silencing effect on transcriptional activation [START_REF] Müller | Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis[END_REF][START_REF] Zürcher | Medtr7g066240.1 DNA-directed RNA polymerase I, II Medtr1g108640.1 DUF4228 domain protein Medtr7g092580.1 transmembrane protein, putative Medtr4g075290.1 peptidyl-prolyl cis-trans isomerase Medtr7g056557.1 leguminosin group 567 LEED...PEED secreted peptide Medtr4g029600.1 extensin-like repeat protein Medtr5g084260.1 Nodule-specific Glycine Rich (NGR) Peptide Medtr4g103920.1 glyceraldehyde-3-phosphate dehydrogenase Medtr4g021725.1 metallothionein Medtr2g042510.1 Nodule-specific Glycine Rich Peptide MtNodGRP2B Medtr5g464340.1 phosphate-responsive 1 family protein Medtr8g010280.1 Defensin related Medtr4g065960.1 Nodule Cysteine-Rich secreted peptide Medtr0240s0040.1 Nodule Cysteine-Rich secreted peptide Medtr6g090485.1 Nodule Cysteine-Rich secreted peptide 1 Candidates encoding proteins with DNA regulatory functions are shaded with grey colour. Testing the specificity of the interaction of the ERF candidates in yeast To validate the specificity of interaction of the ERF factors with the NS-box regulatory sequence, we used purified plasmid preparations (see Methods) of ERF091 (group IX ERF), ERF092[END_REF].

The gain of function experiments using the synthetic NS-box promoter showed that the NSbox activity is sensitive to the nodule nitrate treatment. This suggests that the NS-box plays a regulatory role during developmental and nitrate induced senescence. Henceforth a potential shared transcriptional regulation between developmental and nitrate induced nodule senescence is plausible. However, transcriptional regulators for nitrate response are generally specific. In Arabidopsis, a Nitrate-Responsive cis-regulatory Element (NRE) of 43bp, containing conserved sequence 5'-tGACcCTTN10AAGagtcc-3', was identified and conserved in nitrite reductase (NIR) gene promoters [START_REF] Konishi | Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cisregulatory elements for nitrogen response[END_REF][START_REF] Konishi | Roles of the transcriptional regulation mediated by the nitrateresponsive cis-element in higher plants[END_REF]. NRE is binding to NIN-Like Proteins (NLPs) (Konishi & Yanagisawa, 2013) and activate NRE-dependent transcription in plant response to nitrate (Konishi & Yanagisawa, 2013;Lin et al., 2018;Nishida et al., 2018). In M. truncatula, in the NAC family TF, MtNAC969 was reported to be up-regulated with nitrate treatment, albeit negatively regulate developmental nodule senescence (de Zelicourt et al., 2012).

Putative CAAT and WRKY cis-regulatory elements orchestrate the specific transcriptional role of NS-box

Gene transcription is the net effect of subtle equilibrium conferred by multiple regulators, so that multiple cis-regulatory elements are generally required to determine promoter activity. In this work, site specific deletion have been carried out to validate the potential TF binding sites, onto the CAAT-box and the core sequence of consensus WRKY binding site together (ΔCW), as well as a tandem Dof binding site (ΔDof) (Fig. 4; Fig. 5) Whereas, no significant effect of Dof deletion was observed in the promoter activity, a decrease of GUS activity with the ΔCW promoter construct strongly suggests an activating transcriptional role for CAAT/WRKY sequence (Fig. 5C). In plants, CAAT-box are binding the transcription factor family (NF-Y) (Laloum et al., 2013). NF-Y regulatory element is composed of three subunits: NF-YA, NF-YB and NF-YC, where NF-YA is the subunit that directly interact with the CAAT-box.

Interestingly, MtNF-YA1 gene is specifically expressed in nodule meristematic zone and in distal infection, and gene silencing leads to strong alterations in nodule meristem function and poor nodule development (Combier et al., 2006). MtNF-YA1 belongs to a subgroup of group I where gene are mostly expressed in root and nodule tissues and others MtNF-YA are indeed expected to play key roles in nodule development processes (Laloum et al., 2013). W-box is the consensus sequence for binding sites of WRKY TFs (Eulgem et al., 1999;Zhang et al., 2004;Xie et al., 2005). WRKY TFs have been identified as predominantly involved in transcriptional modulators during abiotic stresses. In M. truncatula, WRKY genes Medtr8g005750 and Medtr3g093830 are responding to nod factor and S. meliloti. Interestingly, in tobacco plants, an overexpression of chickpea CaWRKY50 lead to an early plant senescence [START_REF] Kumar | WRKY domainencoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s)[END_REF] and in Arabidopsis thaliana, WRKY genes (AtWRKY53, 54, and 80) are found to be either positive or negative regulators of plant senescence [START_REF] Ülker | The WRKY70 transcription factor of Arabidopsis influences both the plant senescence and defense signaling pathways[END_REF][START_REF] Potschin | Senescence networking: WRKY18 is an upstream regulator, a downstream target gene, and a protein interaction partner of WRKY53[END_REF][START_REF] Kumar | WRKY domainencoding genes of a crop legume chickpea (Cicer arietinum): comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s)[END_REF]. The WRKY binding-site has also been detected in the Nitrate Responsive Element (NRE) in the Arabidopsis NIR1 promoter [START_REF] Konishi | Identification of a nitrate-responsive cis-element in the Arabidopsis NIR1 promoter defines the presence of multiple cisregulatory elements for nitrogen response[END_REF].

Overall, this study identified a specific DNA sequence (NS-box) positively associated to nodule senescence from the MtCP6 promoter. The fact that NS-box responds to both developmental and induced nodule senescence strongly suggest that nodule senescence and nodule response to environmental changes share, at least partially, similar transcriptional regulatory processes. In the future, transactivation activities between NS-box and some transcription proteins could be identified with yeast-one-hybrid approach. Alternatively, a targeted approach using NF-Y and WRKY to analyse NS-box-TFs interaction could be also considered.

FUNDING

LY is supported by a doctoral fellowship from the China Scholarship Council (CSC). This work was supported by the "Institut National de la Recherche Agronomique", the "Université Nice Côte d'azur", and the French Government (National Research Agency, ANR) through the "STAYPINK" project reference # ANR-15-CE20-0005) and the "Investments for the Future" LABEX SIGNALIFE: program reference # ANR-11-LABX-0028-01.

ACKNOWLEDGEMENTS

We are largely indebted to the symbiosis team members for numerous nodule harvests. Previously identified cis-regulatory elements area indicated as: TATA-box with double underline (CTATAAATAC) (Shirsat et al., 1989;[START_REF] Grace | Sequence and spacing of TATA box elements are critical for accurate initiation from the β-phaseolin promoter[END_REF], ARR1-binding-site (NGATT) in dashed line, NSP1 consensus motif (consensus motif AATTT) underlined (Hirsh et al., 2009); or annotated with name: CAAT-box (Shirsat et al., 1989), CCAAT-box [START_REF] Rieping | Synergistic effect of upstream sequences, CCAAT box elements, and HSE sequences for enhanced expression of chimaeric heat shock genes in transgenic tobacco[END_REF][START_REF] Haralampidis | Combinatorial interaction of cis elements specifies the expression of the Arabidopsis AtHsp90-1gene[END_REF][START_REF] Wenkel | CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis[END_REF]Laloum et al., 2013), WRKY (W-box) (Eulgem et al., 1999;Zhang et al., 2004;Xie et al., 2005); core binding site of Dof proteins (AAAG) (Yanagisawa & Schmidt, 1999;Yanagisawa, 2000). Table S1: Primer sequence list

Primers

Sequences (5' -3')

-1720 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCCGCCATCTATCTTCATTCGTCA -1467 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCTGAGAGAGATGAATGGTACTAT -1278 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCATGAGATGAGATGAGATGAGATGA -1088 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCGATAGTGATTGAGGACCGCT -599 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCTGAAATAAAGAGCTATTTGTTG -511 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCTTGTAAAAAATAATCAAAATTATCTA -356 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCCCATAGAATTTGCCATCACAAT -303 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCCATCACTTAGTTACGTGAAGCA -273 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCACACGTTAGTTAACGTGAAACA -242 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCTTGATTGTGACATACTTTCTTT -211 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCATGCGTTCACTTGACAAAGAAT -175 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCGAAAATTCTCACATTCTCACAG -141 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCGATTCCTACATTGGGTCTCATT -80 F (attB4) GGGGACAACTTTGTATAGAAAAGTTGTCTTAGCACAAATTATTAACATAATA attB1R-ProCP6 GGGGACTGCTTTTTTGTACAAACTTGGAAGAAGTGCTGTGCTTTTACCC empty vecoter F (attB4-attB3) GGGGACAACTTTGTATAGAAAAGTTGTCCAAGTTTGTACAAAAAAGCAGTCCCC empty vecoter R (attB4-attB3) GGGGACTGCTTTTTTGTACAAACTTGGACAACTTTTCTATACAAAGTTGTCCCC M13F GTAAAACGACGGCCAG M13R CAGGAAACAGCTATGAC pLP100-MSC F TGCCACCTGACGTCTAAGAAAC GUS R AAGACTTCGCGCTGATACC ΔNS 5' R CTGTGAGAATGTGAGAATTTTCTAACTCAGTTTGTTTCACGTTAAC ΔNS 3' F GTTAACGTGAAACAAACTGAGTTAGAAAATTCTCACATTCTCACAG ΔCW F CTTTGTATAGAAAAGTTGTCTTGTACTTTCTTTGTAAGAAAAATGC ΔCW R GCATTTTTCTTACAAAGAAAGTACAAGACAACTTTTCTATACAAAG ΔDOF F GAAAAGTTGTCTTGATTGTGACATAGTAAGAAAAATGCGTTCACTTGACA ΔDOF

III. Discussion

Tissue-specific expression of MtCP6 reflects its biological function

Transcriptomic analyses (Van de Velde et al., 2006;Perez Guerra et al., 2010) reveal a peculiar nodule senescence related transcriptional regulation with the specific expression pattern of MtCP6 at the transition between the nitrogen fixing zone and the nodule senescence zone (Perez Guerra et al., 2010;Pierre et al., 2014). CP genes are found mostly expressed starting from zone III via the RNA-seq approach coupled with laser-microdissection on different nodule zones (Figure 27) (Roux et al., 2014). In addition to rhizobial infection, CP genes are also upregulated with arbuscular mycorrhizal infection (Gomez et al., 2009;Hogekamp et al., 2011). Similar with rhizobial infection (Gourion et al., 2015), the initial stages of mycorrhizal colonization have also been reported with a fast activation of plant defence response before suppression during symbiosis (Harrison & Dixon, 1993). As good molecular markers for nodule senescence, the transcriptional activation of CP genes during mycorrhizal infection supports the hypothesis that nodule senescence might be a delayed plant defence response. Indeed, CP genes seemingly have a common biological function with highly correlated expression patterns. The corresponding regulatory motifs are more likely Multi-Input Motif or Dense Overlapping Regulons under control of one or several TFs, rather than Feedforward Loop or Autoregulation [START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF]. Thus, the NS-box cis-regulatory elements that we have identified could be representative for the biological function of MtCP6 in root nodule senescence and early response to plant nitrogen status. Expression of MtCPs based on RNA-seq coupled to laser micro-dissection within spatial zonation corresponds to successive developmental stages. Source: Symbimics (https://iant.toulouse.inra.fr/symbimics/) (Roux et al., 2014).

In this study, serial promoter deletion analysis of MtCP6 allowed us to decipher the transcriptional regulation of the tissue specific expression manner of MtCP6.

IV. Perspectives

NS-box-based promoters could serve for tissue-specific and transactivation studies at the onset of nodule senescence

The NS-box contains the minimal regulatory elements required for the transcriptional activation of MtCP6 which is specifically expressed at the onset of nodule senescence. For example, it would be possible to apply the synthetic promoter of the NS tetramer in nodule senescence-related studies. For instance, the tissue-specific feature might be used for regulation studies using a GAL4-VP16/UAS transactivation system [START_REF] Sevin-Pujol | Development of a GAL4-VP16/UAS trans-activation system for tissue specific expression in Medicago truncatula[END_REF].

Alternatively, the NS-box synthetic promoter might also be used as sensor to monitor plant nitrogen status if we consider its high sensitivity to nitrate treatment. However, the transcriptional output of the NS-box is not restricted in nitrate response, as it exhibits a dynamic spatiotemporal pattern. It is more likely that the NS-box would be able to be used as a more general sensor of the nodule fitness status. In this context, more evidences are necessary to prove a multi-response of the NS-synthetic promoter to biotic and abiotic factors.

Additionally, the activity of a synthetic promoter is determined by the sequence conservation, number of binding sites, identity of flanking, orientation and distance between the core motifs [START_REF] Zurcher | A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta[END_REF][START_REF] Wu | The 6xABRE Synthetic Promoter Enables the Spatiotemporal Analysis of ABA-Mediated Transcriptional Regulation[END_REF]. An improvement on NS-based promoter might be done to robustly monitor the fitness status of the nodules and the transition from symbiotic interaction to senescence. et al., 2007). TF proteins are generally recognized by the presence of a conserved DNA-binding domain (DB) (Mitchell, Pamela J & Tjian, Robert, 1989;[START_REF] Ptashne | Transcriptional activation by recruitment[END_REF] which may be conserved across eukaryotic organisms or plant specific [START_REF] Libault | Legume transcription factor genes: what makes legumes so special?[END_REF]. TFs can act as activators (enhancers) or repressors to change mRNA production of RNA polymerase II (Figure 28) [START_REF] Alon | An introduction to systems biology: design principles of biological circuits[END_REF]. Expression of transcriptional activators are expected to have overlapped expression patterns to the corresponding target genes, while the repressors might display opposite expression patterns [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF].

A novel cis-regulatory

A B

TF-coding genes accounts for a large number of genes in plant genomes, ranging from 6% to 10%, with 2000 TF encoding genes in Arabidopsis [START_REF] Riechmann | Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes[END_REF][START_REF] Riechmann | Transcription factors of Arabidopsis and rice: a genomic perspective[END_REF]. In M. truncatula, 1298 putative TF genes, mostly (1169) falling in 45 known plant TF families, were identified on the Affymetrix Medicago GeneChip [START_REF] Günther | Metabolism of reactive oxygen species is attenuated in leghaemoglobin-deficient nodules of Lotus japonicus[END_REF]. In the gene expression atlas of M. truncatula, key regulators such as TFs are revealed to orchestrate genetic reprograming during development and differentiation among organs (Benedito et al., 2008). Among these putative TF genes, 532 are differentially expressed during nodule development and may therefore play important roles in symbiotic nitrogen fixation (Benedito et al., 2008).

I.2 TFs associated with nodule senescence processes in legume plants

To date, many TFs have been functionally characterized to play key roles during nodulation, for example, GRAS family proteins [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]Hirsch et al., 2009), CYCLOPS [START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF][START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF], DELLA, (Fonouni-Farde et al., 2016b;[START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF][START_REF] Fonouni-Farde | DELLA1-gibberellin signaling regulates cytokinin-dependent symbiotic nodulation[END_REF], ERN (Andriankaja et al., 2007;[START_REF] Cerri | Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection[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] and NF-Y (Combier et al., 2006;[START_REF] Laloum | Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis[END_REF][START_REF] Laporte | The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection[END_REF][START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF]. The first TF found in legume plants, NIN, has been intensively studied in both cis-and trans-regulation with prominent roles in rhizobial infection and nodule organogenesis [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[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] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF]Liu et al., 2019).

ZPT2-1 [START_REF] Frugier | A Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis[END_REF] and EFD [START_REF] Vernié | EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula[END_REF] were related to nodule differentiation process.

During nodule senescence stage, only a few of TFs have been identified and essentially only by correlative transcriptomic analyses, e.g. MYB (MYeloBlastosis), bHLH (basic Helix-Loop-Helix), and NAC (NAM ATAF CUC) families (Kazmierczak et al., 2020;Van de Veldeet al., 2006;Seabra et al., 2012;Cabeza et al., 2014;Chungopast et al., 2014;Yuan et al., 2017).

Nevertheless, a few of them have been functional characterized and directly linked to the transition between N-fixation to nodule senescence. MtATB2 (bZIP family) is regulated by sucrose and is enhanced during nodule senescence (D 'Haeseleer et al., 2010), while the TF MtbHLH2 was recently reported to be a transcriptional repressor of a cysteine protease encoding gene (MtCP77) and down-regulated accordingly in nodule senescence (Deng et al., 2019). Regarding to TFs regulating induced nodule senescence, MtNAC969 was evidenced to be upregulated during nitrate treatment, but it was negatively related to nodule developmental senescence (de Zelicourt et al., 2012).

I.3 Search for trans-regulatory elements interacting with NS-box

To search for interactive trans-proteins that might interact with the cis-regulatory NSbox sequence identified in this study , we chose to use the DNA-centered Yeast-One-Hybrid (Y1H) screening approach [START_REF] Li | Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system[END_REF][START_REF] Bass | Gene-Centered Yeast One-Hybrid Assays[END_REF], derived from the yeast-two-hybrid approach used to identify protein-protein interactors [START_REF] Fields | A novel genetic system to detect protein-protein interactions[END_REF]. The Y1H strategy, firstly developed to detect yeast proteins that can bind to specific DNA sequences [START_REF] Li | Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system[END_REF][START_REF] Wang | Molecular cloning of the olfactory neuronal transcription factor Olf-1 by genetic selection in yeast[END_REF]. The approach is based on the generation of a 'bait' DNA sequence that is inserted in front of a reporter gene that is then integrated by recombination into yeast genome. Auxotrophic gene reporters (HIS3, URA3, TRP1, and LEU2), which allow yeast growth on corresponding amino acid deficient selective medium are generally used here. While the prey protein (DNA-BP) is fused to an activation domain (AD) of yeast TF Gal4 in a 'prey' hybrid vector (thus named as one-hybrid). High affinity interaction of the cis-element and the trans-factor would bring the AD close to the transcription initiation site of the reporter gene, thereby enabling yeast growth in auxotrophic conditions and selection of a positive yeast clone. However, false-positive yeast candidates can grow because of a basal level of transcription of the reporter gene (e.g. histidine) by non-specific interaction of endogenous yeast proteins to the cis-binding sites. Thus, a proper concentration of histidine biosynthesis inhibitor 3-amino-1,2,4-trizol (3-AT) is often used in these screens in order to limit the background.

One hybrid screen has been successfully used to identify several plant TFs, e.g. during nodulation (Andriankaja et al., 2007), lipid transfer during arbuscular mycorrhiza symbiosis [START_REF] Jiang | Medicago AP2-domain transcription factor WRI5a is a master regulator of lipid biosynthesis and transfer during mycorrhizal symbiosis[END_REF] and plant cold responses [START_REF] Zhang | Opposing Control by Transcription Factors MYB61 and MYB3 Increases Freezing Tolerance by Relieving C-Repeat Binding Factor Suppression[END_REF]. Indeed, this approach has led to the identification of ERF transcription factors (ERNs) that bind to the NF-responsive box.

We therefore decided to use a similar approach to identify TF-interacting factors of the NS- (Andrinakaja), extracted from E. coli, were then used for sequencing and/or retransformation of yeast bait strains 4x 4NS-box (this study) and 4x NF-box (Andriankaja et al., 2007). Dilution series (DO= 1, 0,1 and 0,01) of the yeast transformants was spotted on SD-L, SD-H-L, and SD-H-L + 15 mM and their growth was then monitored for 3-4 days, for evaluating the specificity of the interaction towards the 4x NS-box or 4x NF-box regulatory sequences.

III. Results

Yeast-one-hybrid (Y1H) screening for NS-box interactive proteins

Before initiating the one-hybrid screen, it was important to first (i) generate the yeast YM4271 bait strain carrying the 4x NS-box-HIS3 reporter and to (ii) select the best yeast bait candidate showing low HIS3 expression levels that can be inhibited by 3-AT (Histidine enzyme inhibitor) for performing the screen. The generation and selection of such strain, named YM4271-4xNF-box-His3 was done before at the LIPM as described in the Method sections. When I arrived in Toulouse, we have then used this strain to test the optimal concentration of 3-AT to be added to the media for inhibiting the HIS3 background levels. The YM4271-4xNS-His3 strain was grown on SD/-His plates with increasing concentrations of 3-AT (0, 1, 2, 4, 6, 8 and 10mM). At 3 days post-transformation, the concentration of 10mM 3-AT was effective to reduce the background levels of the bait yeast strain. We therefore decided to perform the one-hybrid screen on SD/-Leu/-His media supplemented with 10 mM 3-AT. The Y1H screening strategy used for the identification of NS-box-interacting TFs is schematically represented in Figure 29. The AD fusion cDNA library from nodules was generated in a pGAD-HA vector (Dualsystems Biotech AG, Switzerland) (Baudin et al.) and used to transform the yeast YM4271-4xNS-box-HIS3 bait reporter strain. The potential interaction of a DNA-binding protein to the NS bait sequences (the tetramer of the NS-box) should result in the transcription activation of the HIS3 gene reporter, thereby enabling the His + candidate yeast clones to grow on selective histidine-deficient SD media supplemented with 3-AT. The Leucine (LEU2) marker on the cDNA library vector enables the yeast to grow in -Leu auxotrophic conditions, and thereby to select the yeast clones with positive transformation of pGAD plasmids.

The Y1H screening was conducted using 20 µg pf DNA from a M. truncatula nodule cDNA library, prepared from RNA samples extracted from isolated nodule samples collected 4-and 19-days post-inoculation with S. meliloti [START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF]. After evaluating the efficiency of transformation (see Methods in ANNEX III) we calculated that approximately 1.11

x 10 5 independent yeast clones were screened here (Figure 30A). After 3 to 4 days posttransformation, yeast colonies growing above the background were individually collected in water and classified according to their colony size and time of appearance in three main groups a, b and c (Figure 30B). The a-type (colony size > 1mm) and b-type (colony size < 1mm) were collected 3-days post-transformation while the c-type were new colonies (~ 1mm) that appeared at 4-days post-transformation. (A) Schematic view of the different steps of the one-hybrid strategy used to screen a nodule cDNA library using a yeast YM4271-4xNS-His3 reporter strain. At 3 days-post-transformation, the bait yeast does not significantly grow in SD-Leu-His media with 10mM 3-AT. When transformed with the nodule cDNA library, the yeast colonies grow in SD-Leu media with 10mM 3-AT (due to the Leu reporter in the pGAD library vector) and in the SD-Leu-His media with 10mM 3-AT if specific TF-NS box interaction takes place or in case of non-specific interactions. (C) sequencing analysis allowed to obtain information on candidates having their ORF in frame with the AD domain (potential true interactors) their homology to known Medicago proteins. Selected candidates were then used for DNA plasmid preparation and re-transformation of bait yeast strains for testing interaction specificity. In total, 88 clones were selected as primary candidates (His + clones) with distinguished colony size above the background. These 88 clones were confirmed to grow on selective conditions after re-dropping on SD/-Leu/-His plates with 10mM 3-AT. Among the 88 PCRamplified clones that were analysed by sequencing most of them (52) contained either artefactual vector sequences or were not in frame with the AD activation domain. Despite this large proportion of apparent false positives, 29 clones were in frame with the AD domain in the gene fusion, and this included 15 clones encoding proteins with DNA regulatory functions (in grey shade, Table 8). Interestingly, seven of them code for ERF-type transcription factors belonging to the APETALA2/ethylene responsive factor (AP2/ERF) superfamily (Shu et al., 2015;[START_REF] Phukan | Regulation of Apetala2/Ethylene Response Factors in Plants[END_REF]. These positive clones, which are termed here ERF091 (MtERF1- [START_REF] Penmetsa | Production and characterization of diverse developmental mutants of Medicago truncatula[END_REF][START_REF] Varma Penmetsa | The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations[END_REF][START_REF] Anderson | The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[END_REF]), (ii) to be induced by abiotic stresses conditions (group VIII, MtERF073) (Shu et al., 2015) or (iii) to be involved in the regulation of gene expression in anaerobic conditions, including during nodulation [START_REF] Rovere | ERF-VII family as key players in hypoxic signaling and adaptation in Medicago truncatula[END_REF] (group VII, MtERF069/MtERFB2.3). The overrepresentation of members of the ERF group IX (Five out of the seven clones), expected for "true positives in this type of approach, is encouraging and suggest a potential regulatory role of ERFs of this group for driving CP6 expression in nodules.

Additionally, another positive clone codes for a TCP (TEOSINTE BRANCHED / CYCLOIDEA / PROLIFERATING CELL FACTORS) family TF MtTCP20 (Medtr7g028160.1) was identified before by an Y1H screens as a nitrate-mediated regulator of AtNIA1 (Wang et al., 2010). MtTCP20 is homologous to AtTCP20 (At3g27010) of A. thaliana., which is recently reported to interact with the NIN-like protein (NLP) transcription factors AtNLP6&7 to orchestrate plant response to nitrate availability [START_REF] Guan | Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway[END_REF][START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF].

Additionally, the mutant tcp20 showed an accelerated leaf senescence, indicating a repressive role in transcriptional regulation [START_REF] Danisman | Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically[END_REF]. In M. truncatula, 21 putative TCPs were identified and subjected into class I (also known as PCF or TCP-P) and class II (also known as TCP-C, with subclasses: CIN and CYC/TB1) [START_REF] Wang | Genome-Wide Identification of TCP Family Transcription Factors in Medicago truncatula Reveals Significant Roles of miR319-Targeted TCPs in Nodule Development[END_REF]. But there is not so much functional characterization of TCP proteins in symbiosis. Although it is tempting to speculate that this candidate might have a regulatory function in MtCP6-associated nodule expression, caution should be taken before further analysis since only one TFC candidate was found following this screening. transform the yeast bait strain YM4271-4x NF-box strain as a control. NFbox was the cisregulatory elements identified preciously to be the binding site for the TF ERN1 (Andriankaja et al., 2007). The specific interaction of the positive control ERN1 to the NF-box results in prominent yeast growth in selective SD-Leu-His+ 3-AT conditions at 2.5-days after transformation (Figure 31). In contrast, no significant yeast growth is observed when the same strain was transformed with the ERF091, -092, -073 or-069 plasmids. This indicate that these other ERF factors are unable to specifically interact with the NF-box. On the other hand, these ERF were able to interact with the NS-box resulting in growth of the YM4271-4X NS-box strain in selective conditions (Figure 31). Although ERN1 can to a certain extent interact nonspecifically to the NS-box, this interaction is significantly less strong compared to the group IX ERF factors. In conclusion, these results validate the specific interaction of the ERF candidates (ERF091, -092, -073 and -069) to the NS-box regulatory sequences in yeast.

IV. Discussion

ERF regulators as new possible regulators of nodule senescence

We have conducted a yeast-one-hybrid screening using the NS-box tetramer as a bait sequence and identified among 29 positive candidates, 7 clones encoding ERF TFs. These clones code for four ERF TFs belonging to three ERF family groups (IX, VII and VIII). The most represented clones (five out of the seven ERFs) belong to the ERF group IX. Members of this group have been widely reported to participate in defence responses or in hormone signalling in plants. MtERF091 (MtERF1-1) was identified previously in the ethylene-sensitive sickle mutant [START_REF] Penmetsa | Production and characterization of diverse developmental mutants of Medicago truncatula[END_REF][START_REF] Varma Penmetsa | The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations[END_REF]. The defence response to R. solani was also proven with an early induction of promoter activity containing the 4×GCC::luciferase construct in transgenic hairy roots of the skl mutant [START_REF] Anderson | The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[END_REF].

MtSkl encodes the ortholog of the Arabidopsis ethylene signalling protein Ethylene-Insensitive2 (EIN2) [START_REF] Varma Penmetsa | The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations[END_REF]. The skl mutant is presenting a hypernodulation phenotype [START_REF] Penmetsa | Production and characterization of diverse developmental mutants of Medicago truncatula[END_REF][START_REF] Varma Penmetsa | The Medicago truncatula ortholog of Arabidopsis EIN2, sickle, is a negative regulator of symbiotic and pathogenic microbial associations[END_REF].

Previous work demonstrated that ectopic overexpression of MtERF091 driven by the CaMV 35S promoter did not interfere with root nodule symbiosis (Figure 32A) [START_REF] Anderson | The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[END_REF]. However, if MtERF091 plays a major role during later stages of nodule senescence, a phenotype would not be observed with a CaMV 35S promoter which is known to be non-fully functional in conditions of low oxygen pressure, as expected to occur in mature nodules. The homologue of MtERF091 in L. japonicus, LjERF1, has been shown up-regulated in roots upon ethylene and jasmonic acid and activating downstream defence genes (Figure 32B) [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], which is similar to the Arabidopsis homologue AtERF1 [START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defence[END_REF].

Interestingly, LjERF1 was induced as early as 3h after inoculation with M. loti, whereas repressed at a later stage of nodulation (4 dpi) [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], implying a transient plant defence response upon rhizobia infection followed by genetic suppression in the later nodulation process. . The shoots and roots of hormone-treated seedlings were collected at 3, 6, and 24 h after treatment. Adapted from [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].

However, the expression pattern of these ERF candidates appears not to be analogous to MtCP6 expression according to Medicago gene expression atlas (Figure S 7). It might be therefore necessary to test if these ERFs (or ERFs from the same group) are upregulated under developmental and stress induced senescence conditions, e.g. via RNAseq analysis of senescing nodules. Nevertheless, TF proteins might still function at low protein/transcript levels. It remains to be clarified the regulation activities of a TF protein and the potential feedback regulation on its own gene expression. Another possibility is that a TF could have distinct target genes in different developmental stages or is dynamically regulated under environmental conditions. For example, the oxygen sensitive TF RAP2.12 (group VII ERF) has been shown to migrate from the plasma membrane to the nucleus upon hypoxia, thereby activating expression of genes adaptative to low-oxygen conditions [START_REF] Kosmacz | The stability and nuclear localization of the transcription factor RAP 2.12 are dynamically regulated by oxygen concentration[END_REF].

Thus, it would be hard to predict its function according to the expression pattern of a single TF protein, considering multiple transcriptional actors may coordinate together for the transcriptional outputs in situ.

B A

Our findings suggest a potential role of this ERF factor in the regulation of senescence genes in nodules. Provided with a validated interaction of these ERF candidates with NS-box, our results could support the hypothesis that nodule senescence is a recovery of plant defence responses. Upon nodule senescence, the symbiosis between legume and rhizobia is lost because of perturbed nutrient trading, wherein the beneficial bacteria could be regarded by the host plant similarly to a detrimental pathogen. Consequently, the activation of plant defence related regulators (e.g. MtERF091/MtERF1-1) might mark the initiation of a signal transduction pathway for the loss of the legume-rhizobia mutualism. ABA and ethylene have been suggested to play roles in nodule senescence process based on the transcriptomic profiles of senescing nodules, which demonstrates the up-regulation of ethylene-related genes (Van de Velde et al., 2006). Interestingly, homologs of the ERF factors characterized here were shown to be under the control of ethylene, JA or ABA signalling pathways [START_REF] Muller | Ethylene Response Factors: A Key Regulatory Hub in Hormone and Stress Signaling[END_REF]Shu et al., 2015;[START_REF] Phukan | Regulation of Apetala2/Ethylene Response Factors in Plants[END_REF][START_REF] Shu | APETALA 2-domain-containing transcription factors: focusing on abscisic acid and gibberellins antagonism[END_REF][START_REF] Xie | AP2/ERF Transcription Factor Regulatory Networks in Hormone and Abiotic Stress Responses in Arabidopsis[END_REF].

It is therefore plausible that these ERF regulators might represent downstream responses of these hormone regulated pathways to promote nodule senescence.

DNA-binding domain are conserved among the ERF candidates

ERF TFs comprise a DB domain of about 60aa, typically binding to the canonical GCCbox with high affinity in its target gene promoters [START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF]. All the TF candidates ERF091, ERF092, ERF069 and ERF073 are found to be conserved in this DB domain (Figure 33A), and homologous to the Arabidopsis ERFs, AtERF1A [START_REF] Lorenzo | ETHYLENE RESPONSE FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defence[END_REF], AtERF5 [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF], AtRAP2.3 (Related To APETALA2 PROTEIN3) [START_REF] León | RAP2. 3 negatively regulates nitric oxide biosynthesis and related responses through a rheostat-like mechanism in Arabidopsis[END_REF] and AtERF9 [START_REF] Maruyama | The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi[END_REF], respectively.

The phylogenetic analysis of the homologs is shown in Figure 33B. Moreover, the ERF domains are highly conserved in ERF candidates (ERF091, ERF092, ERF069 and ERF073) and their Arabidopsis homologs (Figure 33C). However, these ERF candidates (ERF091, ERF092, ERF069 and ERF073) do not show similar amino acid residues outside the ERF domain. ERF domain of AtERF1A consists of a three-stranded anti-parallel b-sheet and an ahelix packed approximately parallel to the b-sheet (Figure 33D) [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF]. This ERF domain binds to the target DNA which bends slightly at the central CG step (Figure 33D) [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF]. and AtERF9) [START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Maruyama | The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi[END_REF][START_REF] León | RAP2. 3 negatively regulates nitric oxide biosynthesis and related responses through a rheostat-like mechanism in Arabidopsis[END_REF]. The arrangement of the secondary structural elements is annotated as in AtERF1A [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF].

Sequences of the two consensus motifs are at the top. Amino acid residues interacting with nucleotides of GCC-box are underlined [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF] [START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF][START_REF] Cerri | Régulation et fonctions de facteurs de transcription ERF ERN au cours de la symbiose entre Medicago truncatula et Sinorhizobium meliloti[END_REF]. The structure of ERF domain is indicated with a three-stranded anti-parallel b-sheet and an a-helix. et al., 2009); or annotated with name: GCC-like motif (Andriankaja et al., 2007), CAAT-box (Shirsat et al., 1989), WRKY (W-box) (Eulgem et al., 1999;Zhang et al., 2004;Xie et al., 2005); core binding site of Dof proteins (AAAG) (Yanagisawa & Schmidt, 1999;Yanagisawa, 2000).

(B) Oligonucleotides of GCC-related boxes and C-repeat/DRE. Nucleotide sequences of GCC-box (Ohme-Takagi & Shinshi, 1990;[START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF], GCC-like motif (Andriankaja et al., 2007), C-repeat/DRE [START_REF] Stockinger | Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[END_REF] protein toxicity in yeast, 2) the TF may be absent from the prey source, 3) the TF might only bind to DNA as a heterodimer (and the current Y1H system expresses only one TF at a time, 4) the TF needs posttranslational modifications (Fuxman [START_REF] Bass | Gene-Centered Yeast One-Hybrid Assays[END_REF], 5) the TF may have an efficient DNA binding domain but not be expressed in the senescence conditions.

Thus, complementary approaches are necessary to validate the interactions observed in Y1H.

The use of transient expression in heterologous system such as Nicotiana bentamiana and in vitro analysis of protein/DNA interaction could be used to validate the interactions.

In Arabidopsis, TCP20 has been reported to activate nitrate assimilatory genes via interacting with NIN-Like Proteins (NLPs) [START_REF] Guan | Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway[END_REF][START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF]. TCP20 is also negatively involved in jasmonic acid (JA) biosynthesis and in this context it repress its target gene LIPOXYGENASE2 (AtLOX2) [START_REF] Danisman | Arabidopsis class I and class II TCP transcription factors regulate jasmonic acid metabolism and leaf development antagonistically[END_REF]. JA has been involved in plant responses to environmental stresses and leaf senescence [START_REF] Creelman | Biosynthesis and Action of Jasmonates in Plants[END_REF][START_REF] He | Evidence supporting a role of jasmonic acid in Arabidopsis leaf senescence[END_REF]. The role of JA in nodule senescence has been suggested based on transcriptomes of senescing nodules, wherein gene targets of JA were shown to be upregulated (Van de Velde et al., 2006). The stress-related role of JA was tested on nodules of common bean, particularly in the ascorbate-glutathione pathway, which contributes to antioxidative protection in nodules (Dalton et al., 1986;Becana et al., 2010). The authors found that JA treatment had some major effects on nodules, with an accumulation of hGSH, inhibition of g-glutamylcysteine synthetase (gECS) activity, transcriptional induction of ascorbate oxidase (AO) and posttranslational suppression in dehydroascorbate reductase (DR) activity (Loscos et al., 2008). TCP20 is revealed to response nitrate and interact with NLPs [START_REF] Guan | Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway[END_REF][START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF]. Nevertheless, TCP20 is not expressed in an organ-specific manner neither in A. thaliana nor in M. truncatula. It is thus hardly predictable that TCP20 could interact alone with the NS-box, which directs tissue-specific expression in root nodules.

Other His + clones not encoding typical TFs with recognized DNA binding domains

With regard to other positive clones in addition to TF candidates, two histone H2B, Medtr1g068600.1 and Medtr7g099960, are found 4 and 1 times, respectively (Table 8). H2B

is one of the fundamental histone proteins (H3, H4, H2A, H2B and H1) that wrap the genomic DNA to form nucleosomes of chromatin in eukaryotic cells. Histone modifications on aminotermini, including methylation, acetylation, phosphorylation and ubiquitination are thought to carry epigenetic information regulating chromatin dynamics and gene expressions [START_REF] Strahl | The language of covalent histone modifications[END_REF][START_REF] Jenuwein | Translating the histone code[END_REF][START_REF] Zhang | Transcriptional regulation by histone ubiquitination and deubiquitination[END_REF]. However, histones are widely distributed in eukaryotic cells, it is not very well-understood if histone modifications are related to genomic DNA sequence specificity.

Apart from that, the output of Y1H screen also resulted in three NCR peptides (Table 8).

NCRs are defensin-like peptides possessing antimicrobial activity and therefore are classified as AMPs (Van de Velde et al., 2010;[START_REF] Maróti | Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules[END_REF]. NCRs are abundant and with more than 500 members in M. truncatula [START_REF] Maróti | Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules[END_REF], which are essential for bacteroid terminal differentiation (Mergaert et al., 2003;Mergaert et al., 2006;Van de Velde et al., 2010;[START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF]. During terminal differentiation of bacteria, NCRs were shown to suppress bacterial reproduction and increase bacterial membrane permeability, also indicating their positive roles during nodule senescence, when the fragilized bacteroid membranes can be efficiently degraded by the plant cell proteases (Vasse et al., 1990;Van de Velde et al., 2006;Van de Velde et al., 2010). The NCR MtNFS2 was shown to functions as a negative regulator of symbiotic persistence, suggesting a host control on the success of the symbiotic partnership (Wang et al., 2017). However, there are currently no evidence of NCRs in transcriptional regulation or relationship to CP gene expressions.

V. Summary of potential transcriptional actors for MtCP6 expression

Overall, the 67-bp-long cis-regulatory elements (NS) is sufficient to drive transcriptional gene activation during developmental nodule senescence and in response to nitrate. A summary of the potential regulation for MtCP6 gene expression is in Figure 35. In the Y1H screening, the mostly represented NS-interacting TFs belong to ERFs involved in plant defence responses. ERF091 (group IX) has been found to be pathogen related and involved in ethylene/jasmonate signalling pathway in M. truncatula [START_REF] Anderson | The B-3 ethylene response factor MtERF1-1 mediates resistance to a subset of root pathogens in Medicago truncatula without adversely affecting symbiosis with rhizobia[END_REF]. Gene expression of ERF073 (group VIII) appears to be sensitive to many environmental stimuli (Shu et al., 2015). Arabidopsis ERF genes in groups VIII and IX have often been implicated with plant response to disease and stresses in ethylene and/or JA signalling pathways [START_REF] Nakano | Genome-Wide Analysis of the ERF Gene Family in Arabidopsis and Rice[END_REF]. The group VII ERF members, including ERF069, might be regulators under hypoxia/anaerobic conditions via perception of gaseous signals (nitric oxide: NO) [START_REF] Gibbs | Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors[END_REF][START_REF] Rovere | ERF-VII family as key players in hypoxic signaling and adaptation in Medicago truncatula[END_REF]. NO was shown to be produced at interzone III-IV by using a NObiosensor S. meliloti strain, thus proposed to be a signal in developmental and induced nodule senescence. (Cam et al., 2012). In M. truncatula nodules, NO3-(nitrate) is reduced to NO2-, which is subsequently reduced to NO via the mitochondrial and bacteroid electron-transport chains (Horchani et al., 2011;Hichri et al., 2015). In presence of NO, the group VII ERFs were shown to be degraded in a N-end rule pathway of targeted proteolysis [START_REF] Gibbs | Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors[END_REF].

ERF069 is thus more likely to play a role under the anaerobic conditions inside nodules.

Another potential nitrate response candidate is TCP20, which has been proven to interact with NLPs in nitrate response of Arabidopsis [START_REF] Guan | Nitrate foraging by Arabidopsis roots is mediated by the transcription factor TCP20 through the systemic signaling pathway[END_REF][START_REF] Guan | Interacting TCP and NLP transcription factors control plant responses to nitrate availability[END_REF].

VI. Perspectives

Based on the NS-box from Chapter II, we searched the interacting TFs via Y1H.

However, this preliminary result requires further validation or potentially characterization to prove roles of the candidates in nodule senescence (Figure 36). Firstly, to validate the interaction of TF candidate and the NS-box, another spot assay experiment in yeast is necessary to be done with the yeast strains transformed with corresponding plasmids. In this context, a higher concentration (up to 45mM according to the Clontech protocol) of 3-AT may be considered to validate the interaction of TF candidates and NS-box. Moreover, considering that the TFs are fused to the GAL4-AD (activation domain) which activates in situ the PminHIS3 for HIS3 expression, the transactivation in yeast hybrid may only provide evidence for the function of the TF BDs (DNA-binding domains). We therefore require further characterization to determine if the TF candidates act as activators or repressors. Although the ERF factors characterized in this study are supposed to act as activators, we need anyhow to show it experimentally. For that we propose to use a N. benthamiana (Andriankaja et al., 2007) transactivation system that allow us to examine the regulatory activity of a TF towards NS-containing promoter-GUS gene fusions.

In this system co-infiltration of A. tumefaciens strains containing respective TF constructs (under the 35S promoter) and the 4xNS-GUS constructs will allow us to evaluate by histochemical and fluorimetric (quantitative assays) the relative transcription activity of these transcription factors. In preliminary experiments with L. Frances (LIPM, Toulouse), we demonstrated the low basal of a 4x NS-box-GUS fusion in this system and we now aim to use it in the presence of the ERF TFs and other important controls. This includes the use of mutated NS versions at the GCC-rich motif, mutated versions of the ERFs without the DNAbinding domain and other unrelated ERFs (such as ERN1) to test specificity in the transcription assays.. If they act as activators, we can also subsequently evaluate if their overexpression is sufficient to activate the transcription of their target (CP6) in M. truncatula in non-inoculated plants, as previously done for other symbiotic regulators [START_REF] Laloum | Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis[END_REF][START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[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]. Complementary analyses of GFP-protein fusions will provide information on the subcellular localization of these factors.

If the candidates are validated by yeast drop test and transactivation in N. benthamiana, it will be important to validate the TF-DNA interaction by complementary approaches such as DNA pull-down [START_REF] Wu | Analysis of protein-DNA binding by streptavidin-agarose pulldown[END_REF][START_REF] Kliszczak | DNA mediated chromatin pull-down for the study of chromatin replication[END_REF], EMSA (electrophoretic mobility shift assay) [START_REF] Hellman | Electrophoretic mobility shift assay (EMSA) for detecting protein-nucleic acid interactions[END_REF] or in vitro translation DNA-binding assay [START_REF] Nomoto | In vitro Protein-DNA Binding Assay (AlphaScreen® Technology)[END_REF]. To identify other targets of TF proteins in the genome, it might also be performed with an and CHIP-PCR [START_REF] Ren | Genome-wide location and function of DNA binding proteins[END_REF][START_REF] Nelson | Protocol for the fast chromatin immunoprecipitation (ChIP) method[END_REF] to determine a genome-wide binding positions in vivo. The CHIP-PCR strategy has been successfully used both in N. benthamiana and M. truncatula to study the DNA-protein binding activity of other interaction as previously done for the ERN ERF regulators (Andriankaja et al., 2007;[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]. If ERF and TCP candidates are validated to bind to the NS-box, it might be interesting to carry out functional studies on its mutants via reverse genetic approaches. From the Nobel Medicago mutant bank, several mutants are available for MtCERF092 (e.g. Solexa-NF5874-6), MtERF069 (e.g. NF9447_high_9, NF7237-high-3, and NF19360_high_33) and MtERF073 (e.g. NF1441_high_58, NF5110_high_25). However, functional redundancy can be an issue here, we can then envisage to use alternative strategies to knockout (CRISPR-Cas) or knockdown (RNAi) closely related ERF members and complementary ectopic expression of TF encoding genes to understand their roles in the regulation of CPs expression. With regards to TCP20, we can envisage testing its potential roles in developmental nodule senescence and in nitrate signalling. The mutant line (NF0759_high_45) is available from the Nobel Medicago mutant bank. From the gene expression atlas, an up-regulation of MtTCP20 can be seen in 16-day-old nodules treated with nitrate (10mM, 2 day) (Benedito et al., 2008).

To search effective phenotypes of TFs in terms of nodule senescence, an example can be found in Chapter III, wherein a premature nodule senescence phenotype has been found to be induced by ineffective bacterial partner as a glutathione deficient S. meliloti mutant On the plant side, a cysteine protease encoding gene MtCP6 was firstly identified from a group of CP genes up-regulated during nodule senescence (Van de Velde et al., 2006;Perez Guerra et al., 2010). MtCP6 has been proven to modulate nodule senescence positively and negatively via ectopic overexpression and RNA interference approaches, respectively (Pierre et al., 2014). Expression of MtCP6 in nodules is specifically at interzone III-IV and sensitively response to environmental stresses such as dark-and nitrate-treatments (Perez Guerra et al., 2010;Pierre et al., 2014). In this study, a 67bp-long nodule-senescence related cisregulatory element (NS-box) is therein identified from the 1.7kb-long 5' flanking sequence of MtCP6. A tetramer of the NS-box was able to drive expression pattern at the onset of nodule senescence and was also responsive to nitrate, which is reminiscent of the native promoter activity of MtCP6. Using the NS-box, numerous transcription factors have been trapped from nodule cDNA library and were found to be identical to plant defence related genes. Our study has uncovered several actors of the root nodule senescence regulation mechanism. We have also provided a vision to decipher a complex 3D transcriptional network, which could be triggered by multiple extrinsic and intrinsic stimulus while terminating at activating the executors of the coordinated bacteroid and plant cell death.

In addition to the plant host, the bacterial partner is also involved in the participatory decision-making process in the departure of symbiotic friendship. Inside nodules, rhizobia reside in plant cells to fix dinitrogen necessitating an anaerobic condition mostly due to the extreme oxygen-sensitivity of nitrogenases. We have shown that a S. meliloti 2011 mutant SmgshB, in which the glutathione synthetase encoding gene gshB is disrupted, induced a premature nodule senescence phenotype. In association with such a rhizobium strain deficient with the antioxidant GSH, a series of genetic and phenotypic changes have occurred in nodules probably caused by accumulated oxidative stresses inside bacteroids. The ascorbate-GSH or "Foyer-Halliwell-Asada" pathway is recognized as a key player in H2O2 metabolism [START_REF] Noctor | ASCORBATE AND GLUTATHIONE: Keeping Active Oxygen Under Control[END_REF][START_REF] Matamoros | Biochemistry and molecular biology of antioxidants in the rhizobia-legume symbiosis[END_REF]Foyer & Noctor, 2011). H2O2 accumulation, together with CP gene expressions, have been observed in all of the central infected tissue at the onset of the nodule senescence process (Alesandrini et al., 2003). Notably, expression of MtCP6 was the most elevated gene in RT-qPCR gene expression analysis of WT S. meliloti 2011 and SmgshB inoculated nodules. This result indicated an early and sensitive transcriptional response to induce nodule senescence in case of the ineffective bacteria with impaired nitrogen fixation capacity.

In the future, analysis of plant and bacterial actors regulating the developmental and stress senescence will be an interesting scientific topic to develop. The expression of NCR peptides has been already shown to play a major regulatory role in the interaction between the two partners (Mergaert et al., 2003;Alunni et al., 2007;Van de Velde et al., 2010;[START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF][START_REF] Farkas | Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms[END_REF]. However, the NCR peptides are not present in legumes producing determined nodules suggesting that these peptides are only a fraction of the plant proteins regulating the interaction with the bacteria. In developmental senescence, microscopy analysis suggests that the symbiosome is firstly degraded in the symbiotic plant cells. We could predict that this degraded organelle could produce molecular signals which could induce senescence of the plant cell. However, this scenario is maybe not identical in stress induced senescence in which a faster degradation of bacteroid content undergoes in the absence of cellular signs for nutrient remobilization. In this context, the identification of communicating signals from the host and the bacteria involved in the senescence processes represent a major interest to decipher the similar but distinct nodule senescence processes triggered by intrinsic cellular signals or by environmental perturbations.

Is nodule senescence a delayed plant defence response?

Upon symbiotic recognition, symbiotic microorganisms like rhizobia can accommodate into plant cells with the host-specific Nod factors binding to the plant kinase-like receptors.

Nod factors are actually derivatives of chitin oligosaccharides which are potent inducers of plant immune responses, but the lipid modification on its chitin-oligosaccharide backbone may enable a compatible recognition by host plants [START_REF] Zipfel | Plant signalling in symbiosis and immunity[END_REF]. Nod factor-dependent nodulation is not the only strategy to enter symbiosis. Some Bradyrhizobium strains were found to be able to elicit root and stem nodules even lacking nod genes [START_REF] Giraud | Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia[END_REF][START_REF] Masson-Boivin | Establishing nitrogen-fixing symbiosis with legumes: how many rhizobium recipes?[END_REF][START_REF] Okazaki | Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system[END_REF]. Therein, rhizobia type III (T3SS), or type IV (T4SS) secretion systems are required for nodule organogenesis [START_REF] Deakin | Symbiotic use of pathogenic strategies: rhizobial protein secretion systems[END_REF][START_REF] Okazaki | Hijacking of leguminous nodulation signaling by the rhizobial type III secretion system[END_REF]. Evidence is emerging that symbiotic colonization needs a suppression in plant immunity, for reviews (Gourion et al., 2015;Tóth & Stacey, 2015). Transcriptomic analysis has also shed light on the suppression of plant defence genes during nodulation (Van de Velde et al., 2006). The DNF2 and SymCRK plant genes, encoding a phosphoinositide phospholipase C (PI-PLC)-like protein and a cysteine-rich, non RD receptor-like kinase, respectively, can suppress defence reactions by an unknown mechanism (Oldroyd, 2013).

It was proposed in 2005 that nodule senescence may be a delayed response on the part of the plant to rhizobium as a potential pathogen (Puppo et al., 2005). Upon rhizobial infection, a plant defence response is transiently activated with an oxidative burst, similar to that observed in plant pathogen response (Santos et al., 2001;Puppo et al., 2005). While accepting infection by the bacterial partner, the plant keeps reactive oxygen species (ROS) low, which is indeed necessary for the proper functioning of the rhizobial nitrogenase enzyme complex (Puppo et al., 2005;Muglia et al., 2008). Finally, during nodule senescence, a decrease in N fixation levels and an increase in ROS occur (Puppo et al., 2005;Muglia et al., 2008). The transcriptomics of nodule senescence (Van de Velde et al., 2006) revealed a wide variety of activation in plant defence (e.g., glutathione transferase) and stress responses (e.g.

wound-inducible P450 hydroxylase). We have seen that CP genes are elevated during nodule and multiple plant defence relate proteins were trapped in the Y1H screening using the nodule senescence related element we identified. Indeed, papain-like cysteine proteases (PLCPs)

have been considered as central hubs in plant immunity, which are targeted by secreted pathogen effectors to suppress immune responses [START_REF] Misas-Villamil | Papain-like cysteine proteases as hubs in plant immunity[END_REF]. Arabidopsis null mutants for the PLCP RD21 are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinereal (Shindo et al., 2012). The study in this thesis has been dedicated

to deciphering the transcriptional regulation signalling upstream in activation of the PLCP-like enzymes (CPs) in symbiotic organ. In comparison, the investigations on plant immunity in symbiosis and pathogenesis, including the roles of CPs, might shed light on the distinct evolution of host-rhizobia or host-pathogen relationships. In the future, it might be interesting to compare symbiosis and pathogenesis in parallel to uncover the similar but distinct signalling pathways.

II. Deciphering nodule senescence mechanism at transcriptional level

Transcriptional actors are involved in determination of nodule success

To date, many researches have contributed to decipher the transcriptional cascades during early stages of nodulation. Numerous identified TFs have been revealed to be vital in symbiosis establishment between legume and rhizobia, e.g. IPD3 (known as CYCLOPS in L.

japonicus) [START_REF] Messinese | A novel nuclear protein interacts with the symbiotic DMI3 calciumand calmodulin-dependent protein kinase of Medicago truncatula[END_REF][START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF][START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF], a gibberellin (GA) induced repressor DELLA (Fonouni-Farde et al., 2016a;Fonouni-Farde et al., 2016b;[START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF][START_REF] Fonouni-Farde | DELLA1-gibberellin signaling regulates cytokinin-dependent symbiotic nodulation[END_REF], the GRAS protein NSP1,2 [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]Hirsch et al., 2009;Hirsch & Oldroyd, 2009;[START_REF] Jin | DELLA proteins are common components of symbiotic rhizobial and mycorrhizal signalling pathways[END_REF][START_REF] Hartmann | Functional analysis of arbuscular mycorrhiza-related GRAS transcription factor genes of Medicago truncatula[END_REF], ERNs (ERF group V) (Andriankaja et al., 2007;[START_REF] Cerri | Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection[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], NIN [START_REF] Schauser | A plant regulator controlling development of symbiotic root nodules[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] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF], cytokinin Response Regulators RRs [START_REF] Gonzalez-Rizzo | The Medicago truncatula CRE1 cytokinin receptor regulates lateral root development and early symbiotic interaction with Sinorhizobium meliloti[END_REF], and the CCAAT-binding TF NF-Ys [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] Laloum | Two CCAAT-box-binding transcription factors redundantly regulate early steps of the legume-rhizobia endosymbiosis[END_REF][START_REF] Laporte | The CCAAT box-binding transcription factor NF-YA1 controls rhizobial infection[END_REF][START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF][START_REF] Zanetti | Plant NF-Y transcription factors: Key players in plant-microbe interactions, root development and adaptation to stress[END_REF]. Some of the interacting cis-regulatory DNA elements have therefrom been uncovered, e.g. CYC-box (TGCCATGTGGCA) [START_REF] Singh | CYCLOPS, a DNA-binding transcriptional activator, orchestrates symbiotic root nodule development[END_REF], NSP1 (cis-element AATTT) (Hirsch et al., 2009), NFbox (Andriankaja et al., 2007), NIN (AAGMT; a cytokinin-responsive cis-regulatory region) [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] Vernié | The NIN Transcription Factor Coordinates Diverse Nodulation Programs in Different Tissues of the Medicago truncatula Root[END_REF]Liu et al., 2019), cytokinin sensor TCS (or TCSn) [START_REF] Muller | Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis[END_REF][START_REF] Zurcher | A robust and sensitive synthetic sensor to monitor the transcriptional output of the cytokinin signaling network in planta[END_REF][START_REF] Liu | Imaging TCSn:: GFP, a synthetic cytokinin reporter, in Arabidopsis thaliana[END_REF]. Several TFs have been related to maintain nodule function, e.g. EFD (Ethylene response factor required For nodule Differentiation) (ERF group V) [START_REF] Vernié | EFD is an ERF transcription factor involved in the control of nodule number and differentiation in Medicago truncatula[END_REF], ZPT2-1 [START_REF] Frugier | A Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis[END_REF], Regulator of Symbiosome Differentiation (RSD) [START_REF] Sinharoy | The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula[END_REF].

However, it is not well understood about the transcriptional regulation of nodule senescence process. The TF MtATB2 (bZIP family), which was identified from transcriptomic profile of senescent nodules (Van de Velde et al., 2006), is shown later by the authors under the regulation of sucrose (D 'Haeseleer et al., 2010). Cysteine proteinase gene CP77

(GenBank: QFO38998.1; in M. truncatula A17: Medtr4g065077.1) was identified as one of the highly upregulated gene of the transcriptional repressor bHLH2 (Medtr5g030430.1) from the transcriptome of bhlh mutants and was related to nodule senescence (Deng et al., 2019).

bHLH2 was shown to be expressed antagonistically to the nodule senescence processing, while CP77 is dramatically upregulated at 28dpi in the bhlh2 mutants. The transcriptional binding of bHLH2 to G-box on the promoter of CP77 was confirmed by Electrophoretic Mobility Shift Assay (EMSA) and chromatin immunoprecipitation [START_REF] Korkuc | Characterization and identification of cis-regulatory elements in Arabidopsis based on single-nucleotide polymorphism information[END_REF] [START_REF] Dejardin | Purification of proteins associated with specific genomic Loci[END_REF]. More recently, an in situ approach has been developed to capture the locus-specific chromatin events with biotinylated nuclease-deficient Cas9 and sequence-specific guide RNAs (Liu et al., 2017).

Nevertheless, we have already identified the NS-box and multiple TFs that may be involved in the nodule senescence regulation. Concerning the NS-box, we did not find TF that putatively bind to the CW motif. As these sequences seem to play an important role in the NSbox activity, it will be important to test whether the TF candidates bind to DNA using this sequence or if other candidates have been missed. To answer to this question, the screening of a senescence specific cDNA library by Y1H could be performed to increase the number of putative senescence related TFs. We could also construct two different cDNA library deriving from RNAs extracted from developmentally senescent nodules or stress induced senescent nodules. However, a short-term perspective might still depend on the validation of the transcription factor candidates from the Y1H experiments. Multiple approaches could be used validate the target genes. As an example, ChIP is a powerful tool to search for the target DNA locus distributed in chromatin [START_REF] Nelson | Protocol for the fast chromatin immunoprecipitation (ChIP) method[END_REF]. Identification of transcriptional regulators

Forward and reverse approaches can be used to decipher a transcription network

In our study, we intended to decipher to some extent the transcriptional regulation of nodule senescence in a DNA-centered strategy. Generally, gene transcriptional networks can be explored following two approaches: (1) given a TF, one could identify the target genes subjected to the transcriptional control with binding activity (forward approach), or (2) given a gene of interest (GOI), one could search for the upstream TFs (reverse approach). In both conditions, it would be more efficient to initiate from a central player (protein/gene) with evidenced biological functions.

The TF-centered strategy includes a priori characterization of a TF and the identification of the molecular targets. Firstly, functional characterizations are required with phenotyping via mutagenesis, subcellular localization, in addition to interaction with other proteins. An evidenced functional TF could be thereby used to identify its target genes and the specific binding sites. However, many years of research may be spent to screen a functional TF considering genomic abundance, functional compensation and technical limitations. The following identification of target genes can also be challenging. In this context, chromatin immunoprecipitation [START_REF] Korkuc | Characterization and identification of cis-regulatory elements in Arabidopsis based on single-nucleotide polymorphism information[END_REF] is a powerful assay which provided crucial insights into the protein-DNA interactions distributed in chromatin [START_REF] Nelson | Protocol for the fast chromatin immunoprecipitation (ChIP) method[END_REF]. Sometimes, the targets have been screened/selected from genes with perturbated expression based on the transcriptomic profile of a TF mutant, following by transactivation validations. Nevertheless, the screening approach is easy to be flooded with too many candidates, for the reason that hundreds of genes can be modified in the mutant. Furthermore, the transcriptional binding of certain TF family is conserved to specific sequences with marginal variations, which is abundant in non-coding genomic sequences. Thus, a transactivation analysis in vivo is more likely to reflect a biological context spatially and temporally than in vitro assays.

In contrast, promoter of the GOI can be used for fishing the interactive proteins, as we have conducted. In this case, it is often necessary to conduct the classical promoter deletion analysis, which is often labour-intensive particular in planta/vivo. Moreover, the cis-regulatory elements of GOI are not necessarily to be binding sites of certain TFs. The function might be also conferred via other transcriptional factors such as DNA methylation and chromatin conformation. In this context, it would be complicated to fish the interactive TFs, as well as the subsequent validation in terms of transcriptional binding and biological function of the TF candidates. In addition to Y1H [START_REF] Li | Isolation of ORC6, a component of the yeast origin recognition complex by a one-hybrid system[END_REF], numerous approaches have been developed to capture interacting proteins with a priori knowledge of the target loci. Using a bait nucleotide probe, its associated proteins could be isolated and identified via a protocol called Proteomics of Isolated CHromatin segments (PICh) [START_REF] Dejardin | Purification of proteins associated with specific genomic Loci[END_REF]. More recently, an in situ approach has been developed to capture the locus-specific chromatin events with biotinylated nuclease-deficient Cas9 and sequence-specific guide RNAs (Liu et al., 2017).

To date, many bioinformatician are also dedicated to decoding the genome sequences via continuously updating algorithms. Even though to master the comprehensive transcription networks remains to be a distant aspiration and ambitious hope, it has been developed to refine bioinformatic prediction of the regulatory sequences based on flooded data from genome sequencing and large-scale expression data [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. Even so, current bioinformatic predication of conserved sequences alone is of limited usage which could hardly represent the highly dynamic chromatin status in situ. Nonetheless, it still provides valuable guides and previous evidences in prior to experimental validations. In return, laboratory data can be used to refine the prediction models, which are generally generated from promoter deletion mapping, and mutagenesis of regulatory sequences [START_REF] Wasserman | Applied bioinformatics for the identification of regulatory elements[END_REF]. Overall, despite of the current protein-coding system, we still lack sufficient knowledge to understand how the regulatory information is encoded in genomic DNA sequences. A systematic combination of multiple transcriptional analyses from different perspectives would contribute to the decoding of regulatory sequences.

III. The future of symbiotic nitrogen fixation

Is it beneficial to delay nodule senescence in agricultural production?

For the sake of sustainable agriculture and global equity, an increasing attention has been paid on the substitution of industrial fertilizer with biological N fixers. In the nitrogen-fixing symbiotic nodules, plant and bacterial partners associate together with a nutrient trading agreement, therein nodules are kept in an appropriate number, size and nitrogen-fixing efficiency. A prominent feature of nodule senescence is the declining symbiotic nitrogenfixation capacity, which shuts down the nutrient exchange. A strict genetic control is therein involved to determine the fate of a nitrogen-fixing symbiosis. Several genes have been related

nodule senescence, mostly due to the early senescence phenotype observed in its mutant, such as LjIPT3, MtNAC969, MtCP6 and MtVPE in plants (de Zelicourt et al., 2012;Chen et al., 2014;Pierre et al., 2014), as well as nnrS, norB and and vapBC in bacteria (Meilhoc et al., 2013;Lipuma et al., 2014;Blanquet et al., 2015). Nevertheless, many of such studies showed early senescence phenotype with reduction in N fixation capacity, via gene knock-down. Thus, the essential role of these genes to maintain N-fixing instead of directly triggering nodule senescence is deductive. Nonetheless, a delayed senescence phenotype has been found by overexpression of two genes involved in NO detoxification, LjGlb1-1 and hmp (Cam et al., 2012;Fukudome et al., 2019), and a vapB toxin mutant (Lipuma et al., 2014). Accumulated toxic molecules thereof have a direct impact on nodule senescence. In terms of the degradation stage, the delayed nodule senescence phenotype has been observed via silencing two cysteine protease genes MtCP6 (Pierre et al., 2014) and MtCP77 (Deng et al., 2019). It appears feasible to keep the pink nodules fixing nitrogen beyond the timing -'StayPink', which entitled the ANR funding project. Thus, it might be considered to apply in agriculture the senescence-delay strategies to promote plant growth or the protein content of seed production.

Nevertheless, it is still not sure if a prolonged nitrogen fixation period would benefit plant growth. To verify a positive role on plant production by hindering nodule senescence, it necessitates more related experiments or evidences in field conditions. There is still a long way to go from the basic scientific research to industrial/agronomic implementation, let alone the disputes on genetically modified organisms. From an evolutionary point of view, all plastids, such as chloroplast and mitochondria, are derived from the uptake of a prokaryotic ancestor that gradually evolved into a plant organelle [START_REF] Mcfadden | Evolution: red algal genome affirms a common origin of all plastids[END_REF]. Symbiosome may also be considered as an N-fixing organelle and might represent an intermediate phase in an analogous bacteria-to-plant organelle evolution (Van de Velde et al., 2006). It might be possible in the future to repress plant defence genes that function in nodule senescence so as to accelerate the evolution of the nitrogen-fixing symbiosome evolve to organelle in root cells. Ultimately, it could probably facilitate the engineering of symbiosis into cereal crops to increase yield and protein content. Additionally, a possible solution might be still hidden in the natural plant and rhizobium populations, in which efficient plant-rhizobium partners are trained in response to diverse environments. Substitutivity, compatible and efficient symbiotic strains might be selected from the training of repeated inoculation processes.

I. Plant and bacteria materials

I.1 Medicago truncatula

I.1.1 Seed sterilization and germination

The M. truncatula seed coat, and especially its hydrophobic, waxy outer tegument, must be scarified in order to allow the penetration of water and O2 that trigger germination [START_REF] Garcia | Seed storage and germination[END_REF]. Seed coat can be scarified mechanically by sandpaper or chemically by sulfuric acid. Here in this thesis we used only the chemical method. M. truncatula Jemalong A17 seeds (0.4g ≈ 100 seeds) were immersed in Sulphuric acid (H2SO4, 96%) for 6 minutes and sufficiently rinsed with cold sterile water. Then the seeds are sterilized with bleach 6° for 3min and rinsed thoroughly with sufficient sterile water. The seeds were afterwards soaked in water for more than 3h. The scarified seeds were thereafter spread on 0.4% agar and the plates were put inverted 2d at 4°C for vernalization and 1d at 16°C for gemination.

Plant growth conditions on soil

Germinated seedlings were transferred into pots containing a mixture of sand with perlite

(1:3). Each pot was planted with 2 planets and covered for 3 days to keep humidity. Plants were regularly watered with nitrogen-free fertilizer (Plant-Prod: 0-15-40, Boulogne Billancourt, France) or reverse osmosis water. Plants were growing in growth chamber with a photoperiod of 16/8h and thermoperiod of 23/20°C respectively for day and night.

In vitro plant growth conditions

Seedlings were afterwards transferred on Fahraëus medium [START_REF] Barker | Growing M. truncatula: choice of substrates and growth conditions[END_REF], with 6 plants for each petri dish. Top part of the gel (approx. 1/3) was removed to allow the growth of the plant aerial parts, whilst plant roots were protected from light by black plastic paper.

Plants were growing in growth chamber with a photoperiod of 16/8h and thermoperiod of 23/20°C, respectively for day and night. To observe nodule phenotypes (nodule number, size, colour, etc.) in comparison to the wild-type nodules (WT). However, it may need to bear in mind that plant may not be grown more than 20 days in vitro, as this culture condition is not a good choice for mature nodules undergoing nodule senescence. In addition, exposure of the root system to light had a detrimental effect on nodule development, which necessities an addition of the ethylene biosynthesis inhibitor AVG to agar plates [START_REF] Gourion | Protocols for growing plant symbioses; rhizobia[END_REF].

antibiotics at 30°C. Escherichia coli DH5α was cultivated in LB Broth (Miller)

II. Molecular biology methods

II.1 Cloning

The polymerase chain reaction (PCR)

To generate promoter fragments, a 50µl PCR reaction was done with the Q5 ® High-Fidelity DNA Polymerase (ref. M0491, NEB) according to the manufacture's guide. In primer design, site-specific sequences could be directly flanked by attB sites. In this case, a higher annealing temperature might be considered, but still depending on the primers. Alternatively, in order to avoid the long primers at around 40bp, attB adapters can be add with a two-PCR procedure: 1) 1 st PCR using primers with site-specific sequences flanked by partial attB sequences; 2) PCR purification to remove the first step primers and primer-dimers; 3) 2 nd PCR with attB adapters using 10µl of the purified 1 st step PCR products (annealing: 55 ℃). An efficient amplification of DNA fragments is necessary to be observed with a gel electrophoresis before continuing the procedure. Finally, it is recommended to purify the PCR products with the Wizard SV Gel and PCR Clean up system (ref. A9282, Promega) in order to remove attB primers and primer-dimers, which can recombine efficiently with the donor vector in BP reaction.

BP/LR reaction for Multisite Gateway cloning

Gateway cloning was conducted according to the manufacturer's instructions (Invitrogen) with a BP reaction step and a LR reaction step. A Gateway reaction mix of 5µl for BP reaction an 8µl for LR-plus reaction was used. For multisite Gateway cloning, LR Clonase TM plus enzyme mix would be more efficient than the normal LR Clonase TM , while the later could still be used. BP/LR Clonase TM enzyme mix are sensitive to repeated freeze-thaws, therefore is necessary to be aliquoted and better stored at -80°C until immediately before use. To using an equimolar number of plasmids/DNA-products would enhance the BP/LR reaction efficiency (Table S 2). Examples for generate promoter:GUS constructs for MtCP6 promoter deletions of Y1 (-1467bp) and C187 (-175bp), as well as an empty vector for promoter:GUS negative control. 1µl BP/LR Clonase TM was used in each reaction.

In BP reaction, PCR amplified DNA fragments were recombined with pDONR P4-P1r, respectively. In BP reaction, the entry vectors obtained from BP reaction were separately recombined with two other entry vectors (pENTR-GUS and P2-P3-T35S) and the destination vector pKM43-RolD::EGFP. The reaction mix could be incubated at 25°C for 1h-overnight.

The reaction was later stopped by adding 1µl Proteinase K (37°C, 10min) prior to transform into E. coli competent cells. For mutagenesis, PCR amplification were performed on the entry vectors with forward and reverse primers containing mutations. The PCR products were transformed into E. coli DH5a, and confirmed with subsequent colony PCR, plasmids isolation and sanger sequencing (Genewiz). The entry vectors carrying the wanted DNA mutations were recombined into destination vector by the multisite LR reactions.

Restriction cloning

As for restriction cloning, PCR products and vector plasmids were digested with corresponding restriction endonucleases (NEB) to the manufacture's guide in a final volume of 50µl, respectively. The PCR-DNA/vector-backbone was gel purified and ligated with a molar ratio 6:1 with T4 DNA Ligase (ref M1801, Promega) in a final volume of 20µl (22°C, ~16h).

The reaction was inactivated at 70°C for 5min. Considering the presence of salts, the ligation mix is transformed into E. coli DH5a using the chemical transformation method (42°C, 30s) [START_REF] Inoue | High efficiency transformation of Escherichia coli with plasmids[END_REF].

II.2 Transforming bacteria competent cells

E. coli transformation

Electro-competent cells of E. coli were prepared for plasmid transformation.

II.3 Composite transgenic plants

A. rhizogenes ARqua1 strain is growth from glycerol -80°C stock on TY/CaCl2 plates with appropriate antibiotics at 28°C one week in advance. An overnight preculture (1.5ml) was then prepared 2d in advance. The preculture (400µl) was used to inoculate the TY/CaCl2 liquid medium (10ml) the day before plant transformation. On the day of transformation, bacteria were harvested (5000rpm, 10min) and rinsed before used. The bacteria pellet was finally resuspended in PS medium (peptone and sucrose, 1%) for stab-injecting into the hypocotyl part of plantlets.

After germination, seedlings of M. truncatula ecotype A17 were under limited growth at 4°C for 5 days in dark, in order to obtain thick shoots at hypocotyl region. Chimeric M. truncatula roots were produced by stabbing ARqua1 suspension at hypocotyl region. Plantlets were then settled in a mixture of perlite and sand (3:1). For an optimal plant growth, NH4NO3

(~5mM) of is recommended to provide at this step. However, after the 4-week transgenic root growth period, nitrogen nutrient should be washed away to avoid any nitrogen-mediated inhibition of the following inoculation. Plants were under growth condition with a photoperiod The two images show before and after cutting off the non-transgenic root parts from the chimeric plant. The non-transgenic roots were cut and discarded, while the transgenic were confirmed with green florescence (EGFP marker) and used for further studies.

The plant was 5 weeks old, including one-week limited growth under 4°C, one-week transformation under 20/20°C and threeweek limited growth under 23/20°C.

Transient Expression in N. benthamiana leaves

The A. tumefaciens GV3101/GV3103 strains harbouring the constructs PAM-PAT35S-3xHA-GTW were growing in LB medium under antibiotic selection at 28℃. The overnight cultures were harvested and resuspended in Agro-buffer [10 mM MgCl2, 10mM MES, with 100 mM acetosyringone (No. D13440-6; Sigma-Aldrich), pH=5.6], subsequently incubated at room temperature for more than 3h. To check the interactions of TF candidates and promoter fragments, the corresponding resuspensions were adjusted to O.D.=0.5 and mixed prior to coinfiltration into leaves of 3-week-old N. benthamiana. The leaf discs were collected 36h after infiltration for histochemical GUS staining or frozen in liquid nitrogen and stored at -80℃ for further processes.

Before After

Non-transgenic roots Transgenic roots

Plant total RNA extraction

Total RNA was extracted from 100mg plant materials harvested and grounded in liquid GoScript™ Reverse Transcriptase). A control PCR was carried out on cDNA with a pair of intron-spanning primers of Leghaemoglobin 1 gene [START_REF] Hopkins | MtZR1, a PRAF protein, is involved in the development of roots and symbiotic root nodules in Medicago truncatula[END_REF] to ensure the absence of genomic DNA contamination.

Quantitative PCR (qPCR)

Then cDNA was then diluted 40 times and 5µl cDNA 40X was firstly deposited in the 96well qPCR plate. Then 10µl reaction mix (0.45µl Specific forward and reverse primer 10µM, 7.5µl master Mix, 1.9µl Nuclease-Free H2O qPCR, 0.15µl CXR Reference Dye) was added as described in the user manual of Go-Taq qPCR master Mix (ref A6002, Promega). The reaction was carried out in triplicates with AriaMx Real-time PCR system (Agilent) with qPCR cycle as:

an initial denaturation (95°C, 3min), 40 thermal cycles (95°C 3s, 60°C 30s) and a melting cycle (95°C 30s, 65°C 30s, 95°C 30s). Housekeeping genes Mtc27 [START_REF] Favery | The endosymbiosis-induced genes ENOD40 and CCS52a are involved in endoparasitic-nematode interactions in Medicago truncatula[END_REF]MtA38 (del Giudice et al., 2011) were used as endogenous control. Stability of the reference genes were evaluated with an R package, RqPCRBase (Hilliou & Tran). Relative expressions were analysed with 2 -ΔΔCt methods (Livak & Schmittgen, 2001) and confirmed with the output analysed with the R package.

II.4

Yeast-One-Hybrid screening (Y1H)

The Yeast-One-Hybrid screening of the tetramer of NS-box sequence for interacting TF candidates was done at the LIPM in Toulouse, under the supervision of F. de Carvalho-Niebel (DR CNRS) and the practical assistance of Lisa Frances (TR INRA). For performing such screen, we need first to generate and validate the yeast strain carrying the bait sequence (4x NS-box) before performing the screen of the cDNA library for NS-box interacting factors. We used the yeast reporter YM4271 strain (Clontech, Palo Alto, CA) for generating the bait 4x NS-box yeast and a nodule cDNA library (kindly provided by A. Niebel, LIPM) for performing the One-Hybrid screen following protocols adapted in F. de Carvalho-Niebel' team (Andriankaja et al., 2007) on the basis of the "Clontech Matchmaker One-Hybrid system.

II.4.1

Yeast strain, culture conditions and transformation.

The yeast YM4271 strain (MATa,112,ade5::hisG) [START_REF] Liu | Identifying DNA-binding sites and analyzing DNAbinding domains using a yeast selection system[END_REF] Testing the ability of the 4x NS-box YM4271 reporter strain to grow in selective screen conditions

The non-specific activation of the HIS3 reporter can results in yeast growth in the absence of Histidine. It is therefore important, before any cDNA library screen, to select the growth conditions that minimize this non-specific background level. The 3-aminotriazole (3-AT), a competitive inhibitor of the His3 enzyme, can be used to inhibit growth driven by background HIS3 expression. In this study, we first randomly selected 5 bait 4xNS-box strains and tested their ability to grow (in SD/-His media supplemented with increased concentrations of 3-AT (0mM, 1mM, 2mM, 4mM, 6mM, 8mM, 10mM and 15 mM). A relative inhibition of growth was obtained at 10 to 15mM of 3-AT, we therefore selected one of these yeast 4xNSbox strains for testing their ability to grow in selective 3-AT conditions after DNA transformation.

To this end, we transformed the 4xNS-box (this study) and the 4x NF-box (Andriankaja et al., 2007) bait yeast strains with respective water control and a prey plasmid for expressing the NF-box-interactor ERN1 ERF transcription factor (Andriankaja et al., 2007). Serial dilutions (O.D.600nm = 1, 0.1, 0.01) of the transformed strains were generated in 96-well plates and spotted (3-5μl) on selective media (SD/Leu-, SD/His-/Leu-and SD/His-/Leu-+ 10mM or 15 mM 3-AT). It is very important here to well homogenize the yeast solution before pipetting and make sure that the volume pipetted inside the tips are the same. Wait until the plates are dry and incubate at 28-30℃ for 2 days. Scan the plates when a differential growth is observed.

The 4x NS-box yeast showed a higher growth background level then the 4xNF-box strain, nevertheless we could control it by the addition of 3-AT to the media at a given time frame.

II.4.3 Yeast-one hybrid screen of a nodule cDNA library

For performing the yeast-one hybrid screen using our 4xNS-box yeast bait strain, we decided to use a nodule cDNA library prepared in pGAD-HA vectors (Dualsystems Biotech AG, Switzerland, (http://www.dualsystems.com/) from RNA samples extracted from 4 and 19 days-old nodules [START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF]. The screening of the library was done under 10 mM 3-AT selective conditions.

For the Y1H screening, the yeast YM4271 4x NS-box strain was transformed with 20μg of DNA of the nodule cDNA library and the transformed yeast were selected on SD/-Leu/-His with 10 mM 3-AT. Transformation efficiency of the library was evaluated by plating 1µl (1µl/200µl) of the transformed yeast on SD/-Leu. The appearance of the yeast colonies was monitored over time (at 28°C) and yeast clones were classified according with their time of appearance and their colony sizes (that measured from 1 to >2mm in diameter at 3 to 4 days after transformation).

Selection and validation of His+ yeast candidates

Candidate yeast clones grown in selective conditions (His+) were recovered from the screening plates in 10μl sterile H2O with a toothpick. To recover clones and confirm their growth in selective conditions, 2-5μl of the yeast suspension was plated on SD/-Leu/-His, SD/-Leu, or SD/-Leu/-His + 15mM 3-AT plates. After growth, yeast colony PCR was performed using 2μl of the yeast suspension and using the primer pairs listed in Table S 3. Each PCR reaction was performed with 2μl yeast suspension, with 0.5mM of each primer in a final volume of 20µl. The cycling conditions were as follows: 95°C for 10min followed by 35 cycles of denaturation at 95°C for 30s, annealing at 55°C for 30s, and extension at 72°C for 3min. 15μl

of the PCR reaction were analysed on a 1.5% agarose gel. Positive clones comprising amplified bands were analysed by sequencing using 5μl of the PCR reaction. If the colony PCR resulted in more than one bands, DNA bands were PCR purified (Promega) and sent for sequencing.

After carefully analysing the DNA sequencing chromatograms, the candidate sequences

were analysed for open reading frames that were in frame with the N-terminal Activation Domain (AD). Sequences and translated proteins of His+ candidates were compared by blastn, blastx and blastp algorithms [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] 8) and of ERN1 (Andriankaja et al., 2007) were then used to respectively transform 4x NF-box and 4x NS yeast bait strains. The interaction of respective TF candidates and bait tetramer sequences were evaluated by spot inoculation of 1, 0,1 and 0,01 dilution series of yeast in SD/-Leu; SD/-Leu-His; and SD/-Leu-His + 15 mM 3-AT.

Bioinformatic methods in identification of regulatory elements

In silico analysis of MtCP6 promoter was performed in multiple research-based databases (PLACE, PlantPAN and Plantcare) to identify conserved cis-element motifs (Higo et al., 1999;Lescot et al., 2002;Chow et al., 2016). The used datasets of plant regulatory binding sites are in Table S2.

Table S 4 Web-based resources for plant cis-regulatory sequence analysis used in the course of this thesis.

Resource name URL

III.2 Histology and Microscopic observation

GUS staining

Transgenic roots were harvested via the EGFP selection at 4-wpi, 6-wpi, as well as 4wpi with ammonium nitrate stress (3d). Nodules with root fragments were stained with X-Gluc

(5-bromo-4-chloro-3-indolyl-β-Dglucuronide, cyclohexylammonium salt) (Euromedex) as described (Jefferson, 1987). Nodules with attached root fragments were pre-fixed in cold acetone 90% for 1h at -20°C. After three times rinse with phosphate buffer (pH=7.4, 77.4% NaH2PO4 100mM, 22.6% Na2HPO4 100mM), nodules were submerged in the GUS-staining solution (X-Gluc 0.5mg/ml, potassium ferricyanide 0.5mM, Potassium Ferrocyanide 0.5mM, prepared solution with phosphate buffer) after 20min of vacuum. Nodules were then incubated at 37°C either overnight or 4h in order to see a different staining intensity. (Note: overnight staining nodules would be over-stained for the native promoter of MtCP6, whereas it is necessary for the synthetic promoters containing 4xNS. Samples might be kept at 4°C in phosphate buffer and the staining time could be under strict control in the next morning to be stopped at a visible blue colour). Stained nodules were embedded in 4-6% (w/v) agarose and sections (70µm) were prepared with HM650V vibratome (Thermo Scientific). Images were taken in dark-field using Axioplan 2 microscope (Carl Zeiss). From all the experiments, more than 100 nodules were analysed from at least 16 plants. The experiments were conducted with three biological replicates.

GUS fluorimetric assay

In order to obtain a better estimation of population (nodule GUS activities) means, we have increased the sample size by harvesting transgenic nodules of every 5 plants into one tube. As results, we measured the GUS activity with three independent samples (15 plants)

for each construction each biological replicate. In total, 9 samples were analysed with three biological replicates conducted for each construction, which should have increased the power of experiment. The harvested nodule samples were immediately frozen in liquid nitrogen and stored at -80°C. The florimetric assays was performed under the practical assistance of Lisa Frances (TR, INRA).

To quantify enzymatic GUS activity, each nodule sample were grounded independently in liquid nitrogen with mortar and pestle. Nodule powder (50mg) was mixed with 400µl GUS buffer [sodium phosphate 50 mM, pH 7.5; Na2EDTA 10mM, Triton X-100 0.1% (ref T8787, Sigma-Aldrich); 2-mercaptoethanol 10 mM (ref 63689, FLUKA), and sodium lauryl-sarcosine 0.1% (ref 61747, Sigma-Aldrich)], and vortexed until dissolved. The supernatant was taken after two times centrifugation (maximum speed at 14 000rpm, 15min, 4°C). Protein concentration of each supernatant was determined via the Bradford protein assay (Bradford reagent, Bio-Rad) [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Samples were either directly used for the following fluorometric assay or stored at -70°C until use.

As previously described (Boisson-Dernier et al., 2005) This method was then applied to test bacteria/bacteroid viability on fresh nodule sections [START_REF] Haag | Protection of Sinorhizobium against host cysteine-rich antimicrobial peptides is critical for symbiosis[END_REF]. Nodules were harvested and kept in phosphate buffer (pH=7.2, 100mM) on ice, then embedded in 4-6% agarose and sectioned longitudinally with Vibratome HM650V (Thermo Scientific) into 70μm slices. Afterwards nodule sections were stained with 1.5µl/ml Component A (SYTO 9 and PI) and 1.5µl/ml Component B (SYTO 9 and PI) for 20 min. It would improve the staining with gently shaking if possible, and the staining can be stopped via rinse with phosphate buffer. Sample sections were then mounted on slides and observed with a laser scanning confocal microscope LSM880 (Carl Zeiss, Oberkochen, Germany). The excitation/emission maxima used for SYTO 9 stain is 488nm (Argon laser) and 506-545nm, respectively. As for PI, the fluorescence is excited at 561nm (DPSS laser) and its emission was detected at 650-684nm. 5' flanking sequences of MtCP6 (-242bp to the TSS) was analysed using plant cis-regulatory element databases PLACE, PlantCare and PlantPAN 2.0 (Higo et al., 1999;Lescot et al., 2002;Chow et al., 2016).

ERF91 ERF92 ERF69 ERF73

Figure

  Figure Nitrogen-cycle intermediates and major processes of the nitrogen cycle.

Figure

  Figure Historical changes rely on nitrogen fertilizer in intensive agriculture.

Figure

  Figure Schematic routes of nitrogen uptake from the rhizosphere, transportation and assimilation, and remobilization inside the plant.

FigureFigure

  Figure Phylogenetic 16S tree in prokaryotes carrying nif genes.Figure Phylogeny of the Papilionoideae subfamily.

Figure 6 FigureFigureFigure

 6 Figure 6 Unrooted phylogenetic tree of 16S ribosomal DNA sequences from selected a-, band gproteobacteria. Figure Medicago mt4.0 and mt5.0. Figure Gene expression atlas of M. truncatula. Figure Schematic representation the coordinated bacterial infection and nodule organogenesis.

FigureFigure

  Figure Plant symbiotic intracellular signalling pathways.Figure Overview of nodule zones and bacteroid differentiation stages in indeterminate nodules.

Figure 12 FigureFigureFigureFigureFigureFigureFigureFigure

 12 Figure 12 Size, shape, and DNA content of free-living, cultured S. meliloti bacteria and bacteroids isolated from nitrogen-fixing M. truncatula nodules. Figure Endoreduplication of the host plant cells is coupled with bacteroid differentiation in nodules. Figure Nitrogenase regulation, gene and structure. Figure Idealized growth and viability curves terminated at activating senescence. Figure Restoration of the atmyb2-1 plants to the wild type (WT), morphologically and in terms of levels of endogenous cytokinin, by intact AtMYB2 or by PAtMYB2-AtCKX1. Figure The nitrogen-fixing and senescent nodules of M. truncatula. Figure Microscopic analysis of nodule senescence in M. truncatula. Figure Microscopic analysis of the dark-induced nodule senescence in M. truncatula. Figure The cDNA-AFLP samples for transcriptome analysis nodule senescence in M. truncatula.

Figure

  Figure Antioxidant enzymes of legume nodules.

Figure

  Figure Expression of gshB in laser-dissected zones of 15-day-old nodules.

Figure

  Figure Model of the transcriptional machinery.

Figure

  Figure Classes of known regulatory elements.

Figure 26

 26 Figure 26 Histochemical analysis of MtCP6 expression throughout nodule development or induced nodule senescence.

Figure 27

 27 Figure 27 Transcriptional activation of CP genes at the onset of nodule senescence.

Figure 28

 28 Figure 28 Schematic representation of the transcriptional machinery under control of activator (A) and repressor (B) TFs, respectively.

Figure 29

 29 Figure 29 Schematic overview of the yeast one-hybrid screening procedure.

Figure 35

 35 Figure 35 Summary of transcriptional elements presumably involved in the expression of MtCP6 during nodule senescence.

Figure 36

 36 Figure 36Flow chart of perspectives to characterize TF candidates.

Figure S1

 S1 Figure S1Example of transgenic roots of M. truncatula generated by A. rhizogenes Arqua1.

Figure 1

 1 Figure 1 Nitrogen-cycle intermediates and major processes of the nitrogen cycle. Adapted from (Stein & Klotz, 2016).

Figure 2

 2 Figure 2 Historical changes rely on nitrogen fertilizer in intensive agriculture.

Figure 3

 3 Figure 3 Schematic routes of N uptake from the rhizosphere, transportation and assimilation, and remobilization inside the plant. Adapted from (Xu et al., 2012)

Figure 4

 4 Figure 4 Phylogenetic 16S tree with prokaryotes carrying nif genes. Adapted from (Franche et al., 2009).

Figure 5

 5 Figure 5 Phylogeny of the Papilionoideae subfamily. Adapted from (Azani et al., 2017).

Figure

  Figure 7 Medicago Mt4.0 and Mt5.0. (A) Increased number of chromosome-anchored sequences in Medicago Mt4.0 compared to Mt3.5. Red-coloured portion of the chromosomes represent BAC sequences used in Mt3.5, while the white regions on the chromosomes represent newly anchored sequences in Mt4.0. (Tang et al., 2014). (B) Medicago Mt5.0 indicated with symbiosis-island. Symbiosis-related genomic islands represent physical clusters of genes strongly up-or downregulated when comparing whole nodules versus root systems (blue and red lines, respectively) or differentially regulated between laser-dissected nodule zones (specifically expressed in the nodule apex or differentiation zone: yellow and light blue, respectively). (Pecrix et al., 2018).

Figure 9

 9 Figure 9 Schematic representation the coordinated bacterial infection and nodule organogenesis. Adapted from (Oldroyd et al., 2011).

Figure 10

 10 Figure 10 Plant symbiotic intracellular signalling pathways. Adapted from (Zipfel & Oldroyd, 2017).The Nod-LCOs are recognized by a receptor complex of the LysM receptor kinases NFR1 and NFR5 and the LRR-containing receptor kinase SYMRK (known as DMI2 in Medicago). Pathway activation leads to a burst of reactive oxygen species (ROS) and an influx of calcium across the plasma membrane. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) that associates with SYMRK is involved in the production of mevalonate, which may function as a secondary messenger to activate calcium oscillations in the nucleus. Several ion channels (DMI1, CNGC15 and MCA8) are located at the nuclear membrane, regulating counterflows of potassium and calcium. Nuclear calcium oscillations activate CCAMK, which phosphorylates CYCLOPS to promote the induction of symbiosis gene expression. CYCLOPS may form part of a large complex that contains a number of GRAS-domain-containing transcription factors (NSP1, NSP2 and DELLA).

  Figure 11B: meristem (Zone I); infection zone (zone II) where bacteria are released and infect plant cells; a large nitrogen fixation zone (zone III) where the main nitrogen fixation function of nodules happen; a conically enlargement of senescence zone (zone IV) along with time or induced by stresses.

Figure 11

 11 Figure 11 Overview of nodule zones and bacteroid differentiation stages in indeterminate nodules.

Figure 12

 12 Figure 12 Size, shape, and DNA content of free-living, cultured S. meliloti bacteria and bacteroids isolated from nitrogenfixing M. truncatula nodules. Adapted from (Mergaert et al., 2003). (A) Nomarski (Upper) and fluorescence (Lower) microscopy of DAPI stained bacteria and bacteroids. (B) DNA content of DAPI-stained bacteria and bacteroids measured by flow cytometry.

Figure 13

 13 Figure 13 Endoreduplication of the host plant cells is coupled with bacteroid differentiation in nodules. (A) Development of symbiotic cells in M. truncatula-S. meliloti symbiosis is coupled to genome amplification in both partners and to the production of nodule-specific cysteine-rich (Brenchley et al.) and glycine-rich protein (GRP) symbiotic peptides. Adapted from (Kondorosi et al., 2013). (B) Analysis of nuclear DNA content of nodule cells by flow cytometry at different stages of nodule development. The letters under the bars represent developmental stages as follows: A, nodulation-competent zone of uninoculated root; D, differentiation of the primordium to a nitrogen-fixing nodule; E, nitrogen-fixing nodules at different ages. dpi, days after inoculation. Adapted from[START_REF] Vinardell | Endoreduplication mediated by the anaphase-promoting complex activator CCS52A is required for symbiotic cell differentiation in Medicago truncatula nodules[END_REF].

Figure 14

 14 Figure 14 Nitrogenase regulation, gene and structure.

Figure 15

 15 Figure 15 Idealized growth and viability curves terminated at activating senescence. Adapted from (Thomas, 2013).

Figure 16

 16 Figure 16 Restoration of the atmyb2-1 plants to the wild type (WT), morphologically and in terms of levels of endogenous cytokinin, by intact AtMYB2 or by PAtMYB2-AtCKX1. Adapted from (Guo & Gan, 2011). (A) Phenotypes. (B) Endogenous levels of the ZR-type and IPA-type cytokinin in the basal internodes regions of approximately 8-week-old plants. FW, Fresh weight.

Figure 17

 17 Figure 17 The nitrogen-fixing and senescent nodules.

Figure 18

 18 Figure 18 Microscopic analysis of nodule senescence in M. truncatula. Adapted from (Van de Velde et al., 2006). (A & C) Toluidine blue-stained longitudinal section through a nodule of 35 dpi and 61 dpi, showing a small and more developed senescence zone, Respectively. I, Meristem; II, infection zone; III, fixation zone; IV, senescence zone; y and o, young and old senescence zones, respectively. Bars = 500 μm (B & D) Enlargement of the rectangle in A and C, respectively. Asterisks, arrows, and arrowheads indicate fixing, type I, type II and type III senescent cells, respectively. Bars = 100 μm (E) TEM image of a healthy, nitrogen-fixing cell packed with bacteroids. Bars = 10 μm (F) TEM image of a type I senescent cell with a clearly reduced number of bacteroids and prominent presence of endoplasmic reticulum. Bars = 2 μm (F, H-J)

  post inoculation, TEM: transmission electron microscopy.

Figure 19

 19 Figure 19 Microscopic analysis of the dark-induced nodule senescence in M. truncatula. Adapted from (Perez Guerra et al., 2010) (A) Enlargement of an infected cell at an early stage of developmental nodule senescence. Arrows and arrowhead mark prominent endoplasmic reticulum and a mitochondrion, respectively. Bars = 2 µm (B) Enlargement of an infected cell from a 3-ddt (day of dark treatment) nodule at an early stage of induced nodule senescence. Arrows and arrowheads indicate the enlarged symbiosome space and symbiosome fusions, respectively. Bars = 2 µm (C) Infected cell during developmental nodule senescence, in which all symbiosomes are degraded. From Figure 18H (Van de Velde et al., 2006). Bars = 10 µm (D) Infected cell from a 3-ddt nodule during dark-induced nodule senescence. The bacteroids are degraded, while the PBMs are still visible. (E) Infected cell from a 3-ddt nodule during dark-induced nodule senescence, in which the vacuole is collapsed but the PBMs are intact. Bars = 10 µm (F) Enlargement of an infected cell at the same stage as in E. Arrow indicates an intact PBM. Bars = 2 µm (G) Enlargement of infected cells from a 5-ddt nodule during dark-induced nodule senescence, when bacteria are liberated from the infection threads. Bars = 2 µm (H) Infected cells from a 5-ddt nodule at the last stage of dark-induced nodule senescence and completely filled with saprophytic bacteria. Bars = 10 µm (I-K) Toluidine blue-stained section through a 21-dpi nodule with dark treatment at 0 ddt (I), 3 ddt (J), and 5 ddt (K). The arrow indicates the front of the senescence zone. f, Fixation zone; i, infection zone; m, meristem; s, senescence zone. Bars = 500 μ m

Fig. 1

 1 Fig. 1 Schematic representation of the establishment and of the development of the legume-rhizobium symbiosis, and of the structure of mature indeterminate and determinate nodules. En, endodermis; Co, cortex; dpi, day post-inoculation; Ep, epidermis; Vb, vascular bundle; wpi, week post-inoculation.

Fig. 3

 3 Fig. 3 Developmental and induced senescence in the M. truncatula nodule. (A) Schematic representation of developmental/aging senescence in indeterminate nodules. I, meristematic zone; II, rhizobial infection zone; III, nitrogen fixing zone (in red); IV, senescence zone (in green); V, saprophytic zone (in green). oc, outer cortex; ic, inner cortex; vb, vascular bundle; r, root. (B) Section of a nodule expressing the reporter gene uidA (GUS) under control of the MtCP6 promoter 6 weeks post-inoculation. (C) Schematic representation of nitrate induced-senescence in indeterminate nodules. The arrow shows the extension of the senescent tissues revealed by the pMtCP6:GUS reporter. (D) Section of a nodule expressing the reporter gene uidA (GUS) under control of the MtCP6 promoter 3 days after the nitrate treatment.

  Figure 9.4.1.1 Representation of the developmental senescence in M. truncatula nodule infected cells. In red, schematic of infected cells involved in N-fixation, and in green, representation of typical nodule senescence cells. Dark dotted-lines show potential cell targets of both MtCP6 and MtVPE after release from symbiosomes during nodule senescence. Legends: b, bacteroid; m, mitochondria; n, nuclei.

  Figure 9.4.1.2 Structure of energy-dependent proteases belonging to the AAA+ family.In Clp proteases, proteolytic and ATPase activities are separated on independent polypeptides. ClpP proteolytic subunit is formed of two rings of heptameric ClpP, which associates with hexameric ClpA or ClpX ATPase subunit. In the same way, ClpQ (HslV) proteolytic subunit forms a complex with the ClpY (HslU) ATPase subunit. ClpB doesn't associate with a proteolytic subunit and acts only as a chaperone. Lon and FtsH proteases are hexamers of identical polypeptides which carry both ATPase and proteolytic activity.

Figure 21

 21 Figure 21 Antioxidant enzymes of legume nodules. Adapted from (Matamoros et al., 2003).

FIGURE 1 |

 1 FIGURE 1 | Physiological analysis of the symbiosis between Medicago truncatula and Sinorhizobium meliloti SmgshB mutant strain. (A) Nitrogen fixation capacity of plants with WT strain (grey) and the mutant SmgshB (yellow) at 10-or 20-d post-inoculation (dpi), measured by the acetylene reduction assay per plant (nanomoles of ethylene produced per minute of incubation and per plant). (B) Phenotype of nodules formed with indicated strains at 10-or 20-dpi. Scale bars = 1 mm. (C) Dry weight shoot mass of M. truncatula inoculated with WT (grey) and SmgshB mutant (yellow) strains at 10 or 20 dpi. Error bars are standard errors and * indicates a statistically significant difference (P < 0.05; n = 15).

FIGURE 2 |

 2 FIGURE 2 | Flow cytometry analysis of SmgshB bacteroids. (A) DNA content (PI florescence) of bacteria and bacteroids extracted from nodules obtained with S. meliloti 2011 strain or SmgshB mutant strain after 10 or 20 dpi. DNA content of bacterial and bacteroid populations is indicated in color as: green: 1C, red: 2C, purple: 4C and more. The datasets shown are representative of trends observed in three independent experiments. (B) Percentage of bacteroids with a large SSC (side scatter) value and DNA value of 4C and more. The values shown are the means ± SE of three independent replicates (at least two plants per replicate). Significance was determined using the Mann-Whitney nonparametric test (P < 0.05; n = 3). The * indicates a statistically significant difference (P < 0.05; n = 3).

FIGURE 3 |

 3 FIGURE 3 | Live/dead microscopic analysis of SmgshB nodules. M. truncatula plantlets were inoculated with either WT or SmgshB strains. Bacteroid viability of nodule sections determined live/dead staining by SYTO9 and PI dyes at 10 and 20 dpi. Viable bacteria/bacteroid are stained green by SYTO9 and damaged bacteroids are stained red by PI. The different zones of the nodule are indicated: I, the meristem; II, the infection zone; III, the nitrogen fixation zone; and IV, the senescent zone. Panel (B, D, F, H) represent infected cells in zone III. The images shown are representatives of at least 30 nodules of 15 plants from three independent replicates. Scale bars = 100 µm (A, C, E, G) or = 10 µm (B, D, F, H).

FIGURE 4 |

 4 FIGURE 4 | Quantitative RT-PCR analysis of nodule gene expression markers in M. truncatula nodules inoculated with WT (grey) and SmgshB strains (yellow). The expression levels of Nodule Cysteine Rich 001 (NCR001), Leghemoglobin (Lb), Thioredoxin s1 (Trx s1), Cysteine Protease 6 (CP6), Vacuolar Processing Enzyme (VPE), Purple Acid Phosphatase (PAP), nifD (nitrogenase molybdenum-iron protein alpha chain) and nifH (nitrogenase iron protein). were analyzed in nodules. Data (technical triplicates of three biological samples) are reported as mean ± standard error. * indicates a statistically significant difference relative to the control (P < 0.05; n = 3).

FIGURE 5 |

 5 FIGURE 5 | General overview of GSH role in S. meliloti bacteroid. GSH plays a central role in the redox networks of glutaredoxin (SmGrx1) and SmLsrB. Defect in GSH accumulation lead to an early nodule senescence whereas deletion in Grx1 impairs bacteroid differentiation. The green color indicates general effects on the root nodule and the orange color indicates direct effects on the bacteroid. Dashed arrow indicates the potential role of g-EC on SmGrx1 in absence of GSH.

Figure S2 :

 S2 Figure S2: Nodule development at 10 and 20 dpi in WT (grey) and GshB mutant (yellow). A. Nodule size (mm 2 ). B. Nodule number per plant. Data (three biological samples) are reported as mean ± standard error. * indicates a statistically significant difference relative to the control (P <0,05; ARA, nodule number and Plant shoot weight were calculated with 15 plants and nodule surface was measured with 75 nodules).

Figure S3 :

 S3 Figure S3: Plant development at 10 and 20 days post infection. Plants were inoculated by S. meliloti 2011 (WT) and S. meliloti 2011 gshB mutant (SmgshB) strains. The image is representative of 15 plants. Scale bar = 1 cm

Figure 22

 22 Figure 22 Expression of gshB in laser-dissected zones of 15-day-old nodules.IZ, interzone; ZIII, zone III; FI, fraction I; FIId, distal fraction II; FIIp, proximal fraction II. Source: Symbimics https://iant.toulouse.inra.fr/symbimics/(Roux et al., 2014).

  Transcriptional regulation is a fundamental biological principle in eukaryotes. It determines divergent cell types and multiple biological functions, nevertheless governed by invariant gene sets. Multiple biological processes of cell life, such as cell proliferation, specification, differentiation, and the final cell death, are initially under the strict control of transcriptional mechanism. Expression of a single gene can be influenced by internal or external signals, as consequences of different spatial localizations in plant tissue, temporal expression under given developmental stages, or responses to environmental stimuli.

Figure 23

 23 Figure 23 Model of the transcriptional machinery. Adapted from (Wasserman & Sandelin, 2004). 1) Transcription factors (TFs) bind to specific sites (transcription-factor binding sites; TFBS) that are either proximal or distal to a transcription start site.

Figure 24

 24 Figure 24 The feedforward loop, single-input module (SIM) and dense-overlapping regulons (DOR) found in transcription networks. Adapted from (Shen-Orr et al., 2002).

Figure 25

 25 Figure 25 Classes of known regulatory elements. Adapted from (Noonan & McCallion, 2010). (a) Promoter sequence shown binding the general transcriptional machinery to mediate basal transcriptional control of a transcribed sequence. (b) Enhancer and (c) repressor sequences are shown to mediate positive and negative effects of transcription through interaction with the promoter sequence. (d) Enhancer blocking and (e) barrier insulator elements block promiscuous activation of gene B by regulatory elements of gene A (enhancer blocking, d) or prevent the spread of chromatin condensation from encroaching on the regulatory sequences controlling a hypothetical gene (barrier, e).

Figure 26

 26 Figure 26 Histochemical analysis of MtCP6 expression throughout nodule development or induced nodule senescence. Adapted from (Perez Guerra et al., 2010; Pierre et al., 2014). (A) GUS staining of MtCP6 expression by means of promoter-GUS (1.7kb) analysis in a 5-wpi (week-post-inoculation) nodule. Bars = 500 µm. Adapted from (Perez Guerra et al., 2010).GUS staining of MtCP6 expression (1.7kb promoter) in nodule sections at 3-wpi (B) and 6-wpi nodules (C), 3d-dark-(D) and 3dnitrogen (NH4NO3, 10mM)-induced (E) nodule senescence on 6-wpi nodules. I, the meristem; II, the infection zone; III, the nitrogen fixation zone; and IV, the senescent zone. Bars = 250 µm. Adapted from(Pierre et al., 2014).

Figure 1 :

 1 Figure 1: Schematic graph of MtCP6 5' promoter deletion constructs, and corresponding GUS expression spatial specificity analysis.A. Schematic depiction of progressive 5'-deletion of MtCP6 promoter constructs(-1,720, -1,467, -1,278, -1,088, -955, -655, -599, -511, -356, -303, -273, -242,-201,-175, -141, -96, -80 and empty vector). Deleted promoter fragments are fused to β-glucuronidase (GUS) gene. Transcription starting site (TSS) is designated with arrow. The position of promoter is indicated in bp relation to TSS. B. nodules were harvested at 4wpi (week-post-inoculation) from A. rhizogenes mediated transgenic M. truncatula roots inoculated with S. meliloti 2011. Overall spatial GUS expression pattern is shown in percentages and classed in specific expression in root nodule zone III-IV (blue, Class A), unspecific expression in other nodule zones (teal blue, Class B), and non-detectable (GUS-, grey, Class C). Total number of nodules analysed is indicated on the right (n=110 to 460). GUS stained nodules are from more than three independent experiments. C. Histochemical localization of GUS activity of MtCP6truncated promoters in nodules at 4wpi, 2d after nitrate treatment (4wpi). The images are from nodule vibrosections (70µm). Nodules sections were stained overnight with X-Gluc(-1,710, -1,450, -1,268, -1,078, -955, -655, -511, -599, -511, -356), or during 4h(-303, -273, -242,-201,-175, -141, -96, -80 and empty vector). Promoter positions are indicated in bp relative to TSS. Overall, GUS staining intensity is denoted as: strong (+++), detectable (+; +/-) or non-detectable (-). Scale bar = 100µm.

Figure 2 :

 2 Figure 2: Gain of function analysis of ProCP6 cis elements. A. Schematic design of gain of function analysis. NS-box (Nodule Senescence) was designed to decipher promoter regulatory region from -242 to -175bp. NS1 (50bp) and NS2 (45bp) are

Figure 3 :

 3 Figure 3: Loss of function of cis-regulatory elements on MtCP6 promoter. A. Design of ΔNS deletion onto CP6 promoter. B. relative distribution of transgenic root nodules with spatiotemporal distribution of zone III/IV, non-specific or undetectable (GUS -).C. Representative nodule sample of GUS histochemical localization of expression fromProCP6 (-1,710bp), ΔNS, and control Pmin35S. D. Fluorometric assay of GUS activity driven by synthetic promoters in nodules at 4wpi from ProCP6 (-1,710bp), ΔNS, and control Pmin35S constructs. Results are obtained from nine independent experimental data points from three biological replicates.

Figure 4 :

 4 Figure 4: In silico analysis of putative cis-regulatory elements present onto NS-box (-242~-175bp).

Figure 5 :

 5 Figure 5: Loss of function of cis-elements on minimal MtCP6 promoter (-242bp).

  Fig. 1

  Fig. 5

  Fig. S1

  Fig. S2

Figure 27

 27 Figure 27 Transcriptional activation of CP genes at the onset of nodule senescence.

  element might shed light on the transcriptional network initiating nodule senescence We have defined in our study the NS-box from 5' flanking sequences of MtCP6 that is related in the early transcription of root nodule senescence. The synthetic promoter composed of an NS tetramer can independently restore the tissue-specific spatiotemporal regulation similar to the promoter of MtCP6. The NS-box is presenting various characteristics with responses to developmental senescence to timescale and induced senescence under changes of abiotic conditions. Due to the fact that developmental and induced senescence might be regulated by distinct cis-elements via multiple pathways, it would be more promising to identify the interacting proteins of the NS-box under different conditions. Thus, this experimentally validated cis-regulatory element might be used to capture the dynamic chromatin interactions orchestrating the nodule senescence-related regulation. Under this perspective, gene-centered approaches could be used to search for interacting proteins, such as the yeast-one-hybrid assay (Fuxman Bass et al., 2016) which we have applied in the next Chapter. Our study should help to decipher the complex biological network that regulates nodule senescence from a detailed description on particular regulators. for NS-box-interacting factors The tetramer of the nodule senescence related cis-regulatory element (4x NS-box), has shown in this study to mediate the expression of MtCP6 during developmental nodule senescence and nodule response to nitrogen status. It is still questioned thereafter which transcription factors (TFs) are interacting with the NS-box. TFs are regulatory proteins that modulate the transcription activity of a given gene by binding to DNA-regulatory sequences and/or via the interaction with other transcriptional regulators, including chromatin remodelling/modifying proteins, to recruit or block the access of RNA polymerases to the DNA template (Udvardi

Figure 28

 28 Figure 28 Schematic representation of the transcriptional machinery under control of activator (A) and repressor (B) TFs, respectively. Adapted from (Alon, 2019).

  box, which is required and sufficient for driving MtCP6-related nodule senescence-associated transcription. By using a tetramer of the NS-box, this chapter describe the yeast one-hybrid screen of a nodule cDNA library. After screening 1.1 X 10 5 yeast clones and confirmation of positive candidates by yeast retransformation, this screen has resulted to date in the identification of 7 ERF transcription factors, among them 5 belonging to the same ERF subgroup IX of ERF transcription factors, which are potential interesting candidates likely involved in the regulation of nodule-associated expression of MtCP6.

Figure 29

 29 Figure 29 Schematic overview of the yeast one-hybrid screening procedure.

Figure 30

 30 Figure 30 Yeast-one-hybrid (Y1H) screening strategy to identify NS-box interacting proteins.

( B )

 B Candidate colonies are selected and classified according to their size as a, b or c types. Candidates a-, b-, c-types were collected and re-plated on SD/-Leu/-His plates with 10mM 3-AT. Subsequent PCR were performed on the yeast clones.

Figure 31

 31 Figure 31 Evaluating the specificity of the interaction of the ERF candidates to the 4xNS reporter in yeast.Purified plasmids (pGAD-ERF091, -ERF092, -ERF073, -ERF0069 and ERN1) or water (-) were used to transform YM4271-4xNFbox (containing a tetramer of the Nod factor-responsive cis-regulatory element, Andrianjkaja) or 4xNS-box (this study) yeast reporter strains. Transformed yeast were diluted (OD600nm=0.1 and 0.01) and spot inoculated on SD/L-, SD/H-/L-and SD/H-/Lplates with 15 mM 3-AT. The images were taken 2.5 days after the LiAc-mediated yeast transformation.

Figure 32

 32 Figure 32 MtERF091 (MtERF1-1) homologues function in leguminous plants. (A) MtERF1-1 (MtERF091) does not interfere root symbiosis effectively with ectopic overexpression driven by the CaMV 35S promoter. Composite A17 plants overexpressing MtERF1-1 (MtERF091) and GFP 14 weeks after transformation and transgenic roots inoculated with S. meliloti. The hash mark indicates non-transgenic root, asterisks indicate transgenic roots. Adapted from (Anderson et al., 2010). (B) Effects of ethylene (ET) and/or JA on the expression of LjERF1 in shoots and root. The relative expression of the indicated LjERF1 in shoots (light gray) and root (dark gray) are shown relative to the nontreated controls (shoots or root). The shoots and roots of hormone-treated seedlings were collected at 3, 6, and 24 h after treatment. Adapted

Figure 33

 33 Figure 33 Analysis of the DNA-binding domain (ERF domain) from the ERF candidates and homologs in Arabidopsis. (A) Conserved motifs found in the ERF candidates in M. truncatula (ERF091, ERF092, ERF069 and ERF073) is covering the amino acid region corresponding to AP2/ERF domain. (B) Phylogenetic tree generated from protein sequences of ERFs in M. truncatula and A. thaliana at the webstite (https://ngphylogeny.fr). (C) Alignment of the 60aa ERF domains of the ERF candidates in M. truncatula (ERF091, ERF092, ERF069 and ERF073) and the homologs in A. thaliana (AtERF1A, AtERF5, AtRAP2.3and AtERF9)[START_REF] Fujimoto | Arabidopsis ethylene-responsive element binding factors act as transcriptional activators or repressors of GCC box-mediated gene expression[END_REF][START_REF] Maruyama | The Arabidopsis transcriptional repressor ERF9 participates in resistance against necrotrophic fungi[END_REF][START_REF] León | RAP2. 3 negatively regulates nitric oxide biosynthesis and related responses through a rheostat-like mechanism in Arabidopsis[END_REF]. The arrangement of the secondary structural elements is annotated as in AtERF1A[START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF].Sequences of the two consensus motifs are at the top. Amino acid residues interacting with nucleotides of GCC-box are underlined[START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF] in the consensus sequence. The structure of ERF domain is shown below with a three-stranded anti-parallel b-sheet and an a-helix. Consensus sequences are shown in red (high), blue (low) and black (neutral). Numbers at right indicate the amino acid position of the ERF domain in each protein. (D) The 3D structure of the ERF domain-DNA complex[START_REF] Allen | A novel mode of DNA recognition by a beta-sheet revealed by the solution structure of the GCC-box binding domain in complex with DNA[END_REF][START_REF] Cerri | Régulation et fonctions de facteurs de transcription ERF ERN au cours de la symbiose entre Medicago truncatula et Sinorhizobium meliloti[END_REF]. The structure of ERF domain is indicated with a three-stranded anti-parallel b-sheet and an a-helix.

  3

  in the consensus sequence. The structure of ERF domain is shown below with a three-stranded anti-parallel b-sheet and an a-helix. Consensus sequences are shown in red (high), blue (low) and black (neutral). Numbers at right indicate the amino acid position of the ERF domain in each protein. (D) The 3D structure of the ERF domain-DNA complex

  Figure 34 A GCCcore-like motif is present in NS-box. (A) Putative TFBS present in NS-box. NS-box is identified from the MtCP6 promoter region between -242 and -175bp from TSS. Previously identified cis-elements area indicated as: ARR1-binding-site (NGATT) in dashed line, NSP1 consensus motif (consensus motif AATTT) underlined (Hirsh et al., 2009); or annotated with name: GCC-like motif(Andriankaja et al., 2007), CAAT-box(Shirsat et al., 1989), WRKY (W-box)(Eulgem et al., 1999; Zhang et al., 2004; Xie et al., 2005); core binding site of Dof proteins (AAAG)(Yanagisawa & Schmidt, 1999; Yanagisawa, 2000).

B

  and GCC-related sequence of NS-box are shown in red (high consensus), blue (low consensus) and black (neutral). Nicotiana spp. pathogenesis-related (PR) genes M. truncatula gene ENOD11 (NFbox) M. truncatula gene MtCP6 (NS-box) A. thaliana gene COR15a A. thaliana genes COR78/RD29ATCP20 is involved in plant responses to nitrate availability and jasmonic acid (JA) signalling pathwaysIt was interesting that one positive clone is identical to TCP20 which might be involved in nodule response to nitrate status and other environmental stresses. However, caution should be taken here by the fact that only one TCP clone was found out of this screening.Nonetheless, it is necessary to keep in mind that the cDNA library we used in the Y1H screening were not specifically designed to study nodule senescence. It is therefore possible that the interactive TFs of NS-box are not yet highly abundant in the cDNA library from nodules of 4-and 19-days. The output of Y1H screenings might be affected by different reasons: 1)

Finally

  , no transcription factors putatively binding to the WRKY/CAAT box identified in Chapter II was isolated from Y1H. Further experiments might be needed to understand the roles of these DNA regulatory sequences in gene regulation. In Chapter II, we have shown a dramatic decline of GUS activity driven by the tetramer of NS-box in synthetic promoter and the minimal functional MtCP6 promoter down to -242bp. A potential transcriptional activator is thus presumably present in the minimal MtCP6 promoter besides NS-box.

Figure 35

 35 Figure 35 Summary of transcriptional elements presumably involved in the expression of MtCP6 during nodule senescence. TF proteins are indicated in blue oval shape. The potential transcriptional regulations are indicated in grey arrows with dashed lines. Biological processes are indicated with black arrows. The corresponding cellular signals (hormones or small molecules) are indicated on the top of proteins. NIN-Like Proteins (NLPs); ABA: abscisic acid (ABA); jasmonic acid (JA); NO3-(nitrate); NO2-(Nitrite); NO (nitric oxide).

Figure 36

 36 Figure 36 Flow chart of perspectives to characterize TF candidates.

•

  Transient expression in N. benthamiana. (TF nuclear localization; co-infiltration with 4NS::GUS) Protein-DNA binding Phenotyping • DNA pull down, EMSA, CHIP, etc. • Mutation in GCC-related-sequence of NS-box • Mutation in DNA-binding domain of TFs • Phenotyping of TF candidates • Promoter activity (4NS::Pmin35S) in TF mutants the consequence of the reciprocal nutrient exchange between legume plants and rhizobia. The success or failure of a symbiotic relationship is under the control of both partners. In this thesis, the involvement of the plant host genetic regulation and the rhizobial performance was investigated in the nodule senescence process.

  assay, thereof Hence, transcriptional regulation of nodule senescence, e.g. NS-box mediated expressions, is in the downstream of both developmental and induced nodule senescence pathways. We assume that multiple transcriptional pathways may orchestrate the activation of senescence proteases and eventually trigger the nodule senescence terminating the final death of the symbiotic organ. In this context, the identification of other TFs involved in different steps of the developmental or induced nodule senescence is an interesting scientific topic. The analysis of the transcriptional regulation of MtCP6 has allowed to show that the MtCP6 promoter includes multiple regulatory elements which modulate the transcription of the gene. In this context, identification of the transcription factors which regulates positively or negatively MtCP6 is crucial to initiate the definition of the transcriptional network regulating the nodule senescence process. A better definition of the gene targets of the already identified TFs is also necessary to implement our knowledge of the senescence transcription network. Nevertheless, the transcriptional efficiency might be also conferred via other transcriptional factors such as DNA methylation and chromatin conformation. Analysis of TF interacting factors via tap-tag experiments may allow to identify these factors. Other approaches have been developed to capture interacting proteins with a priori knowledge of the target loci. Using a bait nucleotide probe, its associated proteins could be isolated and identified via a protocol called Proteomics of Isolated CHromatin segments (PICh)

  E. coli strains DH5a and DH10b can be used for Gateway cloning. In addition to these two strains, E. coli XL1_blue could be used in restriction cloning (not for Gateway). Strains should be recovered from glycerol stock at -80°C 3-4 days in advance. An overnight pre-culture (5ml in LB) was prepared 37°C, 225 rpm. 1ml (O.D.600nm at ~0.2) was used to inoculate in 500ml 2xTY medium (Tryptone16 g/L, Yeast Extract10 g/L, NaCl 5.0 g/L, pH=6.8) or LB broth. When O.D.600nm reached 0.5-0.6 (for ~6h shaking at 30°C), culture was harvested by centrifugation (4000g, 15min, 4°C). The E. coli cells is necessary to be kept at low temperature after harvesting (on ice or in cold room 4°C). The pellets were rinsed two times with cold sterile water by the same centrifugation. Then the pellet was resuspended in 25ml cold sterile glycerol 10% and centrifuged again. After discarding the supernatant, the pellet was finally suspended in 2ml cold sterile glycerol 10% and the cell concentration was verified with 1µl cells in 1ml glycerol 10% resulted in O.D.600nm at ~0.2-0.3. The competent cells were aliquoted in 50µl (one for one reaction) and frozen in liquid nitrogen before stored at -80°C.Transformation efficiency was evaluated before used in subsequent transformation.For transformation, 1-5µl Gateway-recombination-mix or ~50ng plasmids could be added into ice-thawed competent cells (50µl). The mix was transferred into the pre-cold electroporation cuvettes (0.1 or 0.2cm) and tapped down to the bottom. The cells were pulsed (set at 'Ec1' or 'Ec2' for 0.1cm or 0.2 cm cuvettes, respectively) using the MicroPulser TM Electroporation Apparatus (Bio-Rad) with a constant ~5millisecond. Immediately, 950ml S.O.C. medium was added into the cuvettes and the cells were gently resuspended. [S.O.C. medium: 1) S.O.B. medium: Tryptone 20g/L, Yeast extract 5g/L, NaCl 0.5g/L, KCl 2.5mM; pH=7.0, autoclave; sterile magnesium solution: MgSO4 to 10mM and MgCl2 to 10mM; 2) add before use glucose (1M; 0.22µm filter-sterile) to a final concentration of 20mM]. The transformants were then transferred in a 10ml polypropylene tube and shaken for 1h at 37°C and 100µl were spread on LB plates with appropriate antibiotics. Colony PCR was performed using specific primers to select the positive clones. Plasmids were purified with Wizard Plus SV Miniprep kit (ref A1460, Promega). The cloning results were finally confirmed by sequencing. Similar methods were used to prepare competent cells for Agrobacterium Sp. and subsequent transformations with the medium (LB for A. tumefaciens, TY/Ca for A. rhizogenes) at 28°C.

  of 16h:8h, light:dark, and thermoperiod of 20/20°C, respectively. After one-week growth at 20°C (which allows efficient T-DNA insertion), plants were transferred at thermoperiod of 23/20°C. Three weeks later, transgenic roots were selected by hypocotyl localization (adventitious roots) (Figure S 1) and green fluorescence using a stereo-microscope (Leica MZ FL III). Non-transgenic roots were discarded, and chimeric plants were re-planted in nitrogen-free pots. At this time, it could be better to allow around one-week of recovery time for the disturbed plants. Then plants were inoculated with S. meliloti inoculum (O.D. 600nm=0.1).Nodules were harvested for at 4 or 6 week-post-inoculation. Parts of the plants were used for nitrate treatment.

Figure S 1

 1 Figure S 1 Example of transgenic roots of M. truncatula generated by A. rhizogenes Arqua1.

  N2 using RNAzol ® RT Reagent (Sigma-Aldrich) following the manufacturer's instructions. The grounded nodule powder (100mg for each sample) was added with 1ml RNAzol ® RT and 400µl DEPC H2O, then vortexed 15s and incubated at room temperature for 15min. The reaction mix was centrifuged (12 000g, 15min, 4°C) and the supernatant was transferred into a new tube. then added 500µL of isopropanol, mixed by inverting and incubate at room temperature for 10min. The RNA was then precipitated by centrifuge for 10min and rinsed two times with 75% ethanol (500µl and 400µl, respectively) by centrifuge. After elimination of the supernatant, RNA was finally eluted in 50µl Nuclease-free H2O and stored at -80°C. RNA quantity and quality were checked with Nanodrop 2000 (Thermo Scientific). After RQ1 RNase-Free DNase (ref M6101, Promega) treatment (37°C, 30min) in a final volume of 10µl (stop with 1µl RQ1 DNase Stop Solution, 65°C, 10min), gel electromigration was performed to confirm the RNA integrity and the absence of genomic contamination.cDNA synthesis by reverse transcription (RT)The DNase treated total RNA (1µg in 11µl) was used to synthesize cDNA with GoScript TM Reverse Transcriptase (ref A6001, Promega): 1) pre-heating (70°C, 5min) with oligo (dT)15 primer (1µl); 2) reverse transcription (25°C, 5min; 42°C, 1h; 70°C, 15min) in a total volume of 20µl (1µl Nuclease-Free H2O, 4µl GoScript™ 5X Reaction Buffer, 0.5µl MgCl2 25mM, 1µl PCR Nucleotide Mix, 0.5µl Recombinant RNasin® Ribonuclease Inhibitor and 1µl

  was grown at 28-30℃ on YPD [Difco peptone(BD 211677) 20g/L, yeast extract (BD 212750) 10g/L, pH=5.8, glucose 2%]. The sterilization of the media requires a special autoclave condition (temperature at 110℃, 20min) to avoid glucose degradation. Alternatively, a filter (0.2μm)-sterilized glucose mother stock solution (40%) is prepared and added to a final concentration of 2% to the autoclaved media (without glucose). For the One hybrid screen, the yeast was grown in the selective "synthetically defined" (SD) media, composed of yeast nitrogen base (Sigma, 6.7g/L), yeast synthetic Drop-out (Clontech, 0.62g/L) without Histidine (-His), without Histidine and Leucine (-His/-Leu) or without Leucine(Zhao et al.) supplemented with 2% glucose and an adjusted pH of 5.8. 2% Bacto-Agar (BD 214010) was added to media for the preparation of solid YPD or SD plates.Transformation of yeast was done according to a PEG/Lithium acetate transformation protocol (according to the Clontech protocol handbook and minor modifications by F. de Carvalho-Niebel, LIPM). Firstly, in advance of 3-5 days before transformation, yeast YM4271 strains were recovered from the glycerol stock at -70℃ and streaked on YPD plates and grown overnight at 28℃. A liquid pre-culture in YPD (50ml) is initiated in YPD in the next day for about 5-6 hours until the culture reaches a O.D.600nm = 0.5-0.6 (Note: DO were measured with a 10X dilution of the yeast culture). The culture is pelleted by using a swing centrifuge rotor at 3500rpm (5min) and resuspended in water before being centrifuged again and resuspended in 0.1 M Lithium Acetate (1ml freshly made). These "competent yeast cells" are then transferred to a 2 mL Eppendorf before centrifugation at maximum speed for 15 seconds. The pelleted yeast cells are then resuspended in 400μl of 0,1 M Lithium Acetate (enough for ~8 transformations) and aliquoted in 2 mL Eppendorf tubes (50 µl/tube). For each tube (50μl yeast competent cells aliquot) add 360μl of the Transformation Mix [containing 20μl of DNA (1μg cDNA library or prey plasmids), and a 340µl solution comprising 240µl PEG 50%, 36μl LiAc 1M, 14μl sterile dH2O, 50μl carrier DNA (ref 1467140; Roche, Basel, Switzerland) 2mg/ml (boiled for 5min in advance)]. After vortexing vigorously for 30s, the transformation mix were incubated in two steps at 30°C for 30 min (invert mix every 10min) and then at 42°C for 45min.After centrifugation at 7000 rpm for 15s, the pelleted yeast cells were recovered in 200µl sterile dH2O and incubated at room temperature for 10-30 min. (Note: yeast competent cells should be tenderly treated and homogenized by flicking the tube). Finally, 100μl of transformed yeast was plated on selective plates by using glass beads. For the generation of the bait 4xNS-box strain, they were plated on SD/-His while for selective prey screen they were plated on SD/-Leu/-His plates with or without different concentrations of 3-AT (ref A-8056, Sigma-Aldrich; stock solution (1M) was filter sterilized and stored at 4℃). For evaluating the yeast transformation efficiency, 1μl of the transformed yeast culture was diluted with 99μl sterile of dH2O and plated on SD/-Leu plates. The plates were thereafter incubated at 28℃ and yeast colony growth was monitored 3 to 5 days post-transformation. 4x NS-box bait sequence into the yeast YM4271 genomeThe tetramer of tandem NS-box sequences (4x NS-box) was synthesised (Genewiz corporation, Takeley, United Kingdom) and inserted into the EcoRI/MluI restriction sites of the pHISi vector (Clontech) to generate the 4xNS-box-HIS3 construct. This construct was sent to Toulouse and L. Frances (LIPM) has linearized it by XhoI digestion and used it to transform and select YM4271 strains carrying the construct integrated in the yeast genome. Yeast positive colonies growing in selective SD/-His plates and carrying the 4xNS-box sequence (after validation by PCR and DNA sequencing) were selected to test their aptitude to grow in selective screen conditions.

  to Medicago Mt4.0 and MtrunA17r5.0-ANR genomic databases. Plasmids of the candidates were thereafter prepared from a 3ml overnight culture of the candidate yeast clones grown in YPD, using the Miniprep kit (Promega) and eluted in 50μl dH2O. The extracted plasmid DNA preparations were used for E. coli DH5ɑ transformation for subsequently extraction of plasmid DNA of higher quality and yield for subsequent DNA sequencing and retransformation of yeast. 1 µg of plasmid preparations of ERF091, ERF092, ERF073 and ERF069 clones belonging to the ERF-IV, VII and VIII groups (Table

  , total protein (10µg) extraction was incubating shaking at 37°C, 400rpm for a kinetics of 4h in presence of 1mM substrate [4methylumbelliferyl-β-D-glucuronide (4-MUG) (Biosynth AG)]. Upon hydrolysis of βglucuronidase enzyme (GUS), the fluorescent compound 4-methylumbelliferone (4-MU) is produced, thus the detected florescent intensity (FI) are used in this assay. The excitation/emission maxima for 4-MU was about 355/460nm. GUS activities were measured using a microtiter fluorimeter (FLUOstar OMEGA, BMG Labtech, Germany) in 96-well microplates (COSTAR). Standard curves were prepared with a range of increasing concentrations of 4-MU (Sigma-Aldrich). The GUS activity is calculated as follows: Live/Dead Staining Molecular Probes' LIVE/DEAD ® BacLight TM Bacterial Viability Kits (ref. L7007, Invitrogen) was used to test bacterial viability with a two-color fluorescence assay. All bacteria (with/without intact membranes) will be labelled by the green-fluorescent nucleic acid stain SYTO ® 9. In contrast, bacteria with damaged membranes are penetrated by the redfluorescent nucleic acid stain propidium iodide (PI), causing a reduction in the SYTO 9 stain fluorescence when both dyes are present. Thus, bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red.

Figure S 2

 2 Figure S 2 Heterogenous nodule size appears at 20-and 28dpi following declining nitrogen fixation capacity. (A) Nodule size (Feret's diameter) inoculated with SmgshB compared with WT strain at 10-, 20-and 28-dpi, respectively. (B) Nitrogen fixation capacity of plants with WT strain (white) and the mutant SmgshB (black) at 10-or 20-and 28-day post-inoculation (dpi), measured by the acetylene reduction assay per plant. (C) Dry weight shoot mass of plant aerial parts and (D) nodule number per plant inoculated with WT (black) and SmgshB mutant (black) strains. Error bars are standard errors and asterisk indicates a statistically significant difference (P < 0.05; n = 15).

Figure S 3

 3 Figure S 3 Kinetics of histochemical localization of β-glucuronidase (GUS) activity in nodules carrying promoter-GUS constructions for the MtCP6.(A) Nodules were stained with X-Gluc (blue; for GUS reporter) and Magenta-Gal (red; for bacteria localization). Promoter-GUS constructions of MtCP6 (-1720bp, -242bp and empty vector) were selected showing kinetics of temporal transcriptional activities as full promoter, minimal functional promoter and negative control. Staining with X-Gluc was carried out for 4h. One biological replicate with at least 20 independent nodules of 10 plants were sampled at 1-, 2-, 4and 6-wpi. Promoter positions are indicated relative to TSS. Scale bar = 500µm.(B) Nodule numbers stained blue by X-Gluc (blue) or only stained red by X-Gal (orange) corresponding to different constructs at 1-, 2-, 4-and 6-wpi. One biological replicate with at least 20 independent nodules of 10 plants were sampled at 1-, 2-, 4-and 6-wpi. Promoter positions are indicated relative to TSS.

Figure S 4

 4 Figure S 4 Putative cis-regulatory elements present in the minimal functional MtCP6 promoter (-242bp).

Figure S 5

 5 Figure S 5 Re-growth and yeast colony PCR of Y1H candidates. (C) Regrowth for 2 days of yeast candidates on selective medium SD H-/L-added with 10mM 3-AT. 88 yeast clones were recovered in 10µl H2O and 2µl was spotted for each clone.(D) Yeast colony PCR. Green colored candidates were gel-purified and sequenced. Blue colored candidates contain only pGAD vector backbone sequences with PCR products at ~250bp.

Figure S 6

 6 Figure S 6 Alignment of ERF candidate clones (translated protein sequences) to the identical putative ERFs (protein sequence from Mt4.0). Consensus sequences are shown in red (high), blue (low) and black (neutral).

Figure S 7

 7 Figure S 7 Expression profile of ERF091, -092, -069 and -073 on MtGEA: M. truncatula Gene Expression Atlas / Noble Foundation (https://mtgea.noble.org/v3).

Table 1 Examples of organisms that can carry out nitrogen fixation. Adapted from (Taiz et al., 2015).

 1

	Symbiotic nitrogen fixation
	Host plant	N-fixing symbionts
	Legumes, Parasponia	Rhizobia
	Actinorhizal	Frankia
	Gunnera	Nostoc
	Azolla	Anabaena
	Sugarcane	Acetobacter
	Free-living nitrogen fixation
	Type	N-fixing genera
	Cyanobacteria	Anabaena, Calothrix, Nostoc
	Aerobic:	Azospirillum, Azotobacter, Beijerinckia, Derxia
	Facultative:	Bacillus, Klebsiella
	Anaerobic:
	Nonphotosynthetic	Clostridium, Methanococcus (archaebacterium)
	Photosynthetic	Chromatium, Rhodospirillum

Table 2 Associations between rhizobia and host plants. Adapted from (Poole et al., 2018).

 2

	Rhizobia	Plant host species
	Azorhizobium caulinodans	Sesbania rostrata
	Azorhizobium doebereinerae	Sesbania virgata
	Bradyrhizobium japonicum	Glycine max
	Bradyrhizobium spp.	Cajanus cajun, Glycine max, Aeschynomene americana, Aeschynomene indica,
		Aeschynomene rudis, Arachis duranensis and Arachis hypogaea
	Burkholderia spp.	Mimosa pudica
	Cupriavidus sp. AMP6	Mimosa asperata
	Cupriavidus taiwanensis	Mimosa pudica
	Mesorhizobium haukuii	Sesbania sesban
	Mesorhizobium loti	Lotus japonicus and Lotus corniculatus
	Rhizobium etli	Phaseolus vulgaris
	Rhizobium gallicum	Phaseolus vulgaris
	Rhizobium leguminosarum bv. phaseoli	Phaseolus vulgaris
	Rhizobium leguminosarum bv. trifolii	Trifollium spp.
	Rhizobium leguminosarum bv. viciae	Pisum sativum, Lens culinaris, Vicia cracca, Vicia hirsuta and Vicia faba
	Rhizobium sp. strain IRBG74	Sesbania cannabina, Sesbania bispinosa, Sesbania cannabina, Sesbania
		exasperata, Sesbania formosa, Sesbania grandiflora, Sesbania
		madagascariensis, Sesbania macrantha and Sesbania pachycarpa
	Rhizobium tropici	Phaseolus vulgaris
	Sinorhizobium fredii NGR234	112 genera
	Sinorhizobium fredii USDA257	79 genera
	Sinorhizobium meliloti and	Medicago sativa and Medicago truncatula
	Sinorhizobium medicae

Table 3

 3

	) (Van

Table 3 Cell types of developmental nodule senescence. Adapted from (Van de Velde et al., 2006).

 3

Table 4 Comparison of developmental and dark stress induced nodule senescence. Adapted from (Perez Guerra et al., 2010). Developmental senescence (DS) Dark stress induced senescence (IS)

	Factors	progressive with time	fast progressing upon dark stress
	Vesicles	numerous vesicles	not pronounced vesicle mobilization
	Plant cells	prominent endoplasmic reticulum,	absent symptoms
		mitochondria and Golgi apparatus
	Symbiosome	symbiosome degradation,	bacteroid content condensed,
		mixed with the cytoplasm	peribacteroid space increased
	PBMs	degraded	remained intact, fused
	Saprophytic bacteria		colonize in senescent cells

  , defoliation(Vance

						N 2
	Plant infected cell
	H + H +	Nitrogenase ④ PBS H + Bacteroid H +	Hmp NH 3 H + H +	+ NH 4	GS	Amides Ureides
		H +	Proteases	H +	Nitrate
	NCR					treatment
			O 2 .-			NO	NO 2 -	NO 3 -
	(h)GSH
	Malate Redox state ①		Lb	ASC/GSH Mitochondria
			②
					③
		Sucrose	O 2
	Dark stress			Drought stress
	Fig. 2 Major processes triggering nodule senescence in response to environmental
	constraints. (1), Modification of the redox balance; (2), alteration of the bacteroid car-
	bon nutrition; (3), alteration of O 2 homeostasis; (4), alteration of N 2 fixation (nitroge-
	nase). Red lines indicate down-regulation. Black dotted lines indicate scavenging of
	NO or O 2 by Lb or Hmp proteins. The pink color of the plant cell cytoplasm illustrates
	the Lb content. ASC: Ascorbate; Hmp: Flavohemoglobin; GS: Glutamine; (h)GSH: (homo)
	Glutathione; synthetase; Lb: Leghemoglobin; PBS: Peribacteroid space; Proteases:
	Cysteine proteases; NCR: Nodule cysteine-rich peptides.

Table 1

 1 Genes involved in symbiotic nodule senescence.

	Gene (mutant)	Species	Gene characteristics	Reference
	LjIPT3	Lotus japonicus	Reduced nitrogen fixation observed in RNAi	Chen et al., 2014
			plants. Coding for an Isopenthyl-Transferase, a
			rate limiting step in cytokinin biosynthesis
	MtCP6 and MtVPE	Medicago truncatula	RNAi plants are delayed in the apparition of	Pierre et al., 2014
			senescence in nodule
	MtNAC969	Medicago truncatula	Accumulation of senescence marker transcripts in	de Zélicourt et al., 2012
	Hmp (hmp þþ )	Sinorhizobium meliloti	RNAi plants Hmp overexpression delays nodule senescence.	Cam et al., 2012
			Hmp encodes a flavohemoglobin involved in
			NO detoxication
	nnrS1	Sinorhizobium meliloti	nnrS1/2 mutants induce early nodule senescence.	Blanquet et al., 2015
	nnrS2		nnrS1/2 encode for cooper-containing
			membrane proteins involved in NO
			detoxication
	norB	Sinorhizobium meliloti	norB mutants induce early nodule senescence.	Meilhoc et al., 2013
			norB encodes for a NO reductase in the
			denitrification pathway
	LjGlb1-1	Lotus japonicus	Glb1-1 overexpression delays nodule senescence.	Fukudome et al., 2019
			Glb1-1 encodes a Phytoglobin involved in NO
			detoxification
	vapBC-5	Sinorhizobium meliloti	Delayed senescent phenotype. Coding an active	Lipuma et al., 2014
			bacteria toxin

IPT: IsoPentenyl Transferase, CP: Cystein Protease, VPE: Vacuolar Processing Enzyme, NAC:NAM ATAF CUC transcription factor, Hmp: flavohemoglobin, nnrS: nitrite and nitric oxide reductase S, norB: NO reductase B, Glb1: Class 1 Phytoglobulin, vapBC: toxin-antitoxin module.

Table 5 Relative expressions of cysteine protease encoding genes and putative DNA-binding protein encoding genes during root nodule senescence. Adapted from (Van de Velde et al., 2006).

 5 Three clusters were identified with senescence-associated differential expression in cDNA-AFLP data of nodule senescence in M. truncatula.

	GENBANK ACCESSION	CONSENSUS BLAST HIT	CLUSTER	R	N1	cDNA-AFLP EXPRESSION DATA N2 ASN1 ASN2	PSN1	PSN2
	NUMBER DQ455144	putative MYB-related transcription activator	1	0.89	-1.00	-0.75	1.49	0.71	-0.56	-0.78
	DQ455180	DNA-directed RNA polymerase subunit A	1	-0.95	-0.82	-0.23	1.95	0.48	0.14	-0.57
	DQ455329	probable DNA-binding protein RAV2	1	-0.90	-0.99	-0.69	1.81	0.51	-0.16	0.43
	DQ455430	putative AP2 domain-containing protein	1	-1.09	-0.73	-0.85	-0.04	1.17	0.88	0.91
	DQ455155	DEAD-box protein/RNA helicase	1	-1.09	-0.55	-0.65	-0.32	1.93	0.22	0.46
	DQ455119	putative DNA-binding protein	2	-1.48	0.25	-0.67	-0.23	1.75	-0.07	0.45
	DQ455187	no apical meristem (NAM) family protein	2	0.42	-0.89	-0.78	-0.82	1.44	-0.54	1.18
	DQ455231	putative DNA-directed RNA polymerase	2	-0.96	-0.84	-0.44	-0.20	1.92	-0.16	0.67
	DQ455352	AW574143 putative bHLH transcription factor	2	-1.51	-0.38	-0.69	-0.05	1.52	0.27	0.85
	DQ455403	BG454808 bZIP transcription factor	2	-1.03	-0.71	-0.39	-0.64	1.07	0.05	1.65
	DQ455442	Homeobox-leucine zipper protein HAT22	2	-0.43	-0.68	-0.54	-0.81	2.01	-0.14	0.59
	DQ455436	probable DNA-binding protein	2	1.98	-0.89	-0.39	-0.93	0.42	-0.31	0.13
	DQ455381	MYB-related transcription factor MYB4	2	-0.21	-0.98	-0.49	-0.99	0.48	0.34	1.85
	DQ455183	bZIP transcription factor ATB2	2	-0.30	-0.68	-0.72	-0.82	0.36	0.12	2.03
	DQ455114	cysteine protease	2	-1.57	0.22	-0.13	-1.05	0.59	0.67	1.26
	DQ455113	TC100437 cysteine protease MtCP6	2	-0.75	-0.56	-0.74	-0.72	0.85	0.10	1.82
	DQ455173	TC100443 cysteine protease	2	-0.54	-0.50	-0.39	-0.71	-0.01	-0.05	2.19
	DQ455264	TC100440 cysteine protease	2	-0.57	-0.52	-0.49	-0.54	0.06	-0.13	2.20
	DQ455343	BE999342 cysteine protease	2	-0.77	-0.61	-0.69	-0.61	0.39	0.32	1.98
	DQ455362	TC100436 cysteine protease	2	-0.72	-0.39	-0.40	-0.57	-0.05	-0.06	2.20
	DQ455093	TC98625 cysteine protease	3	-0.54	-0.45	-0.46	-0.59	-0.12	-0.07	2.22
	DQ455112	TC93993 cysteine protease 15A precursor	3	0.07	-0.50	-0.52	-0.73	0.02	-0.50	2.16
	DQ455138	TC102742 cysteine protease	3	-0.06	-0.37	-0.30	-0.51	-0.59	-0.39	2.24
	DQ455162	TC106338 vacuolar processing enzyme	3	-0.42	-0.57	-0.35	-0.54	-0.01	-0.33	2.23
	DQ455107	putative RNA-binding protein	3	-0.59	-0.23	-0.68	0.38	-0.58	-0.42	2.12
	DQ455169	transcription factor TGA1 homolog	3	-0.13	-0.09	-0.83	-0.70	-0.18	-0.24	2.17
	DQ455195	putative NAC domain protein NAM	3	-0.57	-0.39	-0.52	-0.50	-0.14	-0.12	2.23
	DQ455302	DNA-binding protein	3	-1.12	-0.75	-0.04	-0.33	-0.31	0.66	1.89
	DQ455321	DNA-directed RNA polymerase	3	0.65	-0.77	-1.01	-0.85	0.71	-0.34	1.62
	DQ455354	ERF domain protein 12	3	0.16	-0.73	-0.17	-0.70	-0.92	0.37	1.98
	DQ455358	GAI-like protein 1	3	-1.59	0.43	-0.41	-0.03	-0.37	0.26	1.71

AFLP samples for transcriptome analysis nodule senescence in M. truncatula. Adapted from (Van de Velde et al., 2006).

  , Nitrogen-starved roots; N1, nitrogen-fixing, round, indeterminate nodule without senescence zone; N2, nitrogen-fixing, more elongated, indeterminate nodule without senescence zone; ASN1 and PSN1, apical and proximal part of nodule with small, young green senescence zone, respectively; ASN2 and PSN2, apical and proximal part of nodule with large green senescence zone, respectively. Lines indicate transversal sectioning of senescent nodule samples. Bar = 1 mm

	R

The involvement of proteases in the nodule senescence is more described in the following review (Yang, Li et al., 2020).

Yang L, Syska C,

Garcia I, Frendo P, Boncompagni E 2020

. Involvement of proteases during nodule senescence in leguminous plants. In: Bruijn Fd ed. The Model Legume Medicago truncatula, 683-693.

Table 9 .4.1.2

 9 Characterization of cysteine proteinases (CP, Cyp, VPE) related to nodule senescence in legume plants

	Genes	Plant	Nodule type	Expression	Expression location	Possible Function	References
	MtCP1-5	Medicago	Indeterminate	Nodule-specific	Periphery of the	PCD and induced	Van de Velde et al.
		truncatula			conical senescence	senescence; protein	2006; Perez Guerra
					zone IV	synthesis and	et al. 2010
						processing, protein
						degradation
	MtCP6	Medicago	Indeterminate	Nodule-specific	Zone III -IV	PCD and induced	Van de Velde et al.
		truncatula				senescence; protein	2006; Perez Guerra
						synthesis and	et al. 2010; Pierre
						processing, protein	et al. 2014
						degradation
	MtVPE	Medicago	Indeterminate	Nodule-nonspecific	Meristematic and	PCD and induced	Van de Velde et al.
		truncatula			infection zones;	senescence; protein	2006; Pierre et al.
					weak in zone III -IV	synthesis and	2014
						processing, protein
						degradation
	PsCyp1	Pisum sativum	Indeterminate	Nodule-nonspecific	Zone III, weak in zone	PCD, early senescence	Kardailsky and Brewin
					IV, root	stage	1996; Serova et al.
					parenchymal cells		2017
	PsCyp15a	Pisum sativum;	Indeterminate	Nodule-nonspecific	Meristem and cortex,	PCD and induced	Jones and Mullet
	MsCyp15a	Medicago sativa;			vascular bundles,	senescence;	1995; Kardailsky
	MtCyp15a	Medicago			infecting	adaptation to	and Brewin 1996;
		truncatula			ramification, zone	changes in cell	Vincent et al. 2000;
					II-IV	turgor, protein	Vincent and Brewin
						turnover; nodule	2000; Sheokand
						organogenesis and	et al. 2005; Serova
						maturation	et al. 2017
	NODf32	Astragalus sinicus	Indeterminate	Nodule-specific	Zone IV to II-III	PCD, protein turnover,	Naito et al. 2000; Li
						remodeling of	et al. 2008
						tissues, putative
						vacuolar-targeting
	TrCPs	Trifolium repens	Indeterminate	Nodule-specific	Zone IV	PCD, putative	Asp et al. 2004
						vacuolar-targeting
						signal
	GmCP	Glycine max	Determinate	Nodule-nonspecific	Periphery of the	PCD, protein	Alesandrini et al. 2003
					central zone, central	degradation
					infected cells
	GmCYSP1	Glycine max	Determinate	Nodule-specific	Not determined	PCD, nodule	Oh et al. 2004
						development

Table 9 .4.1.3

 9 RNA-seq gene expression of some bacterial proteases coupled to laser microdissection of M. truncatula nodule from the apical meristem to the nitrogen fixation zone

	Protease family	Gene	FI	FIID	FIIP	IZ	FIII
	AAA+
	Lon	SMc01905	802.4	834	2053.9	1331.5	1466.2
	ClpP1	SMc01903	287.1	275.2	397.5	348.8	230
	ClpP2	SMc02720	223.7	234.5	391.8	497.1	498.3
	ClpP3	SMc03841	75.4	22.7	17.9	160.4	1621.9
	ClpX	SMc01904	1853.8	2601.5	3479.9	2363.6	1016.3
	ClpA	SMc02109	656.1	459.8	1399.4	1019.3	953
	ClpB	SMc02433	591.2	439.3	2289.8	1585.2	3835
	ClpS1	SMc02110	270.2	195.2	418.6	458.3	207.2
	ClpS2	SMc02694	68.1	131.6	279.7	180.1	194.4
	HsIU	SMc02577	82.9	77.5	944.5	380.5	100.2
	HsIV	SMc02575	37.4	89.3	707.3	331.7	78.8
	FtsH	SMc04459	1512.4	1309.4	3023.6	2099.1	1048.6
	HflK	SMc01441	226.1	231	557.8	447.6	286.6
	HflC	SMc01440	382.8	480.5	931.6	812.8	558.3
	Carboxy terminal protease
	CtpA	SMc03783	1118.8	901.3	718.9	579.4	2155.7
	0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

  Li Yang 1 † , Sarra El Msehli 2 † , Sofiane Benyamina 1 , Annie Lambert 1 , Julie Hopkins 1 , Julie Cazareth 3 , Olivier Pierre 1 , Didier Hérouart 1 , Samira Achi-Smiti 2 , Eric Boncompagni 1 and Pierre Frendo 1 *

	CHAPTER I
	Glutathione Deficiency in
	Sinorhizobium meliloti Does Not
	Impair Bacteroid Differentiation But
	Induces Early Senescence in the
	Interaction With Medicago truncatula

Pierre, O., Hérouart, D., Achi-Smiti, S., Boncompagni, E., and Frendo, P. (2020)

. Glutathione Deficiency in Sinorhizobium meliloti Does Not Impair Bacteroid Differentiation But Induces Early Senescence in the Interaction With Medicago truncatula. Front. Plant Sci. 11. doi:10.3389/fpls.2020.00137 1 Université Côte d'Azur, INRA, CNRS, ISA, Sophia-Antipolis, France, 2 Laboratoire de Physiologie Végétale, Faculté des Sciences de Tunis, Campus Universitaire El Manar II, Tunis, Tunisia, 3 Institut de Pharmacologie Moléculaire et Cellulaire, CNRS UMR 7275, Université Côte d'Azur, Valbonne, France

Table S2 :

 S2 expression analysis of pap isoforms up regulated in nodules compared to roots. Pap isoforms were found in symbimics (https://iant.toulouse.inra.fr/symbimics/) website using "purple acid phosphatase" as a key word. Amongst the 52 isoforms, 5 were significantly more expressed in nodules than in roots. The pap Mt0031_00308 is significantly more expressed in zone III than in all the other zones. Numbers correspond to measures from nodule laser dissection (deseq-normalized-RNA-seq reads).

	symbimics accession	Nodule	Root	ZI	ZIId	ZIIp	IZ	ZIII
	Mt0009_11080	788	3	3	23	1	10	35
	Mt0010_10703	2355	912	25	30	45	43	112
	Mt0025_10286	217	19	1	0	0	0	0
	Mt0028_00268	1532	389	98	189	114	73	59
	Mt0031_00308	1020	48	1,3	2,2	0	0	143

Table 6 Phenotypes of GSH deficient rhizobia strains Strains Free-living phenotype Symbiotic phenotype compared to WT strain Ref.

 6

	Nodulation	Nitrogen fixation & plant biomass	Senescence

Li Yang 1 , Lisa Frances 2 , Fernanda De Carvalho-Niebel 2 , Pierre Frendo 1 , Éric Boncompagni 1

	1 Institut Sophia Agrobiotech, UMR INRAE 1355, Université Côte d'Azur, CNRS 7254, 400
	Route de Chappes, BP167, 06903 Sophia Antipolis, France.

2 LIPM, Université de Toulouse, INRA, CNRS, 31326 Castanet-Tolosan, France Short title: Transcriptional Regulation of MtCP6 One-sentence summary: Identification of cis-acting regulatory elements of MtCP6 during nodule senescence

  MtCP6 expression (MtGEA), in which MtCP6 induction was detected as early as 10dpi. However, only a small proportion of nodules carrying MtCP6 promoters were stained blue in young nodules, whilst more than 94% of nodules were detected as GUS positive. Hence, we confirmed the temporal transcriptional activity MtCP6 is induced in later stage of nodule development. It appears that spatial and temporal expressions are correlated in determinate nodules. In addition, we have also observed occasional expression in nodule meristems and roots. This might be due to rare expression of MtCP6 in roots or its response to A. rhizogenes, or it could result from the promoter leakage.

	To further clarify
	the temporal pattern of MtCP6 gene expression, we carried out kinetic study at 1, 2, 4 and
	6wpi to show temporal expression on transgenic nodules carrying MtCP6 promoters (-1720bp
	and -242bp), as well as empty vector. (Annex IV, Figure S 3). The induction of tested MtCP6
	promoter expressions has started in young nodules since 1wpi. This result is comparable to
	microarray data of

Table 8 List of AD-In frame His+ candidates 1 .

 8

	Gene locus	Occurrence	Genome annotation	Name	Group
	Medtr4g100380.1		Ethylene-Responsive Factor	MtERF091 (MtERF1-1)	ERF IX
	Medtr4g100420.1		Ethylene-Responsive Factor	MtERF092	ERF IX
	Medtr2g435590.1		Ethylene-Responsive Factor	MtERF069 (MtERFB2.3)	ERF VII
	Medtr4g078710.1		Ethylene-Responsive Factor	MtERF073	ERF VIII
	Medtr7g028160.1		TCP family transcription factor	MtTCP20	TCP class I
	Medtr1g068600.1		histone H2B
	Medtr7g099960.1		histone H2B

of rhizobia to M. truncatula plants for nodulation

  at 37°C with appropriate antibiotics. Agrobacterium rhizogenes ARqua1was grown at 28°C in TY medium (pH = 7.2, 5g/L bacto-tryptone, 3g/L yeast extract, 6mM CaCl2) added with 100μg/ml streptomycin. Agrobacterium tumefaciens was grown in LB Broth(Miller) at 28°C with appropriate antibiotics. For making solid medium, agar (1.5%, 15g/L) was added in the liquid medium before autoclaving.For nodulation studies, strains are recovered from -80°C on LBMC agar plates with appropriate antibiotics and incubate at 30°C for 3d. Pre-culture (5ml) was prepared in LBMC with appropriate antibiotics, and shaken overnight at 30°C. Inoculate 100ml LBMC with appropriate antibiotics, shake overnight at 30°C. Harvest the overnight culture by centrifuge (8000rpm, 5min) and rinse two times with sterile water. The inoculum is obtained by resuspending the pellet in sterile water, with O.D. 600nm adjust to 0.05-0.1. Inoculation at the bottom of each plant with 10ml inoculum with 50ml syringe in pots. In case of in vivo culture on plates of Fahraëus medium, bacteria suspension was prepared with optical density at O.D.

	Inoculation

600nm =0.05. Seedlings were inoculated with 100μl S. meliloti inoculum for each plant two days after planting.

Table S 2 Examples for BP and three-entry multisite LR reaction mix.

 S

	Multi-GW	DNA/vector	bp	fmol	ng	ng/ul	ul	TE ul
		PCR Y1	1480	25	24.4	20	1.2	2
	BP	PCR C187	242	25	4	20	0.2	3
	(Qsp 5ul)	PCR C0 (attB4::attB1)	54	25	0.9	2	0.4	2.8
		pDONR P4-P1r	4777	25	78.8	100	0.8
		pENTR TM GUS	3841	10	25.4	50	0.5
		pENTR (P2R-P3) T35s	2856	10	18.8	50	0.4
	LR plus	PKM43-rolD::egfp	11588	20	153	100	1.5
	(Qsp 8ul)	P4-P1r Y1	4125	10	27.2	50	0.5	4.1
		P4-P1r C187	2833	10	18.7	50	0.4	4.2
		P4-P1r C0 (empty vector)	2645	10	17.5	50	0.3	4.2
		"ng=(x fmols)(bp)(660fg/fmols)(1ng/1000000fg)"

Table S 3 List of primers used in yeast-one-hybird screen strategy

 S

	Name	Sequence	Source	Comments
	4NSHindIII-MluI R	GATCACGCGTATCCCAAGCTTATTCAACGG	Li Y.	multiple binding in tetramers
	pHIS fw (LS) (Lisa F)	TTCCCAGTCACGACGTTG	Lisa F.
	pHIS rev (LS) (Lisa R)	ATATTCTTCGAAGAAATCAC	Lisa F.
	SacIEcoRI4NS F	CAGGAGCTCAGCCGGAATTCTTGATTGTG	Li Y.	multiple binding in tetramers
	pGAD10-R1	CAC AGT TGA AGT GAA CTT GC	Lisa F.	pGAD Sfi vecteur AD-levure
	pGADT7-For1	AGT ACC CAT ACG ACG TAC CAG	Lisa F.	pGAD Sfi vecteur AD-levure

Advances in Botanical Research, Volume 94 ISSN 0065-2296 https://doi.org/10.1016/bs.abr.2019.09.013 © 2020 Elsevier Ltd. All rights reserved.

ThéophileKazmierczak et al.

The Model Legume Medicago truncatula, First Edition. Edited by Frans J. de Bruijn. © 2020 John Wiley & Sons, Inc. Published 2020 by John Wiley & Sons, Inc.

Frontiers in Plant Science | www.frontiersin.org March 2020 | Volume 11 | Article 137

Acknowledgements

Acknowledgments

The three laboratories involved (IPS2, ISA and LIPM) are funded by a common "Agence Nationale de la Recherche" (ANR) project (STAYPINK ANR-15-CE20-0005). The IPS2 institute benefits from the support of the LabEx "Saclay Plant Sciences" (SPS) and of the Lidex "Plant Phenotyping Pipeline" (3P). T.K. was supported by Université Paris-Saclay and ANR fellowships. The ISA was supported by the Institut National de la Recherche Agronomique, the Center National de la Recherche Scientifique, the University of NiceeSophia-Antipolis, and the French Government (National Research Agency, ANR) through the LABEX SIGNALIFE program (ANR-11-LABX-0028-01). L.Y. was supported by a doctoral fellowship from China Scholarship Council (CSC). The LIPM was supported by the French Laboratory of Excellence project TULIP (ANR-10-LABX-41). E.M. was supported by the National Institute for Applied Science (INSA-Toulouse).

Acknowledgments

L.Y. is supported by a doctoral fellowship from the China scholarship council (CSC). C.S. is the recipient of a doctoral grant from the "Ministère de l'Education Nationale, de l'Enseignement supérieur et de la Recherche." This work was supported by the French Government (National Research Agency, ANR) through the "STAYPINK" project (reference # ANR-15-CE20-0005) and "Investments for the Future" LABEX SIGNALIFE (program reference # ANR-11-LABX-0028-01).

ACKNOWLEDGMENTS

We are largely indebted to the symbiosis team members for numerous nodule harvests. We thank Renaud Brouquisse for critical reading of the manuscript.

FUNDING

LY is supported by a doctoral fellowship from the China scholarship council (CSC). SE was financed by IMAGEEN program from the European Communities and the Tunisian government (bourse d'alternance). This work was supported by the "Institut National de la Recherche Agronomique", the "Centre National de la Recherche Scientifique", the University of Nice-Sophia Antipolis and the French Government (National Research Agency, ANR) through the "STAYPINK" project (reference # ANR-15-CE20-0005) and the "Investments for the Future" LABEX SIGNALIFE: program reference # ANR-11-LABX-0028-01.

DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

CHAPTER I

Analysis of bacterial GSH deficiency on nodule functioning

SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpls.2020.00137/ full#supplementary-material

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

CHAPTER II

The Nodule Senescence (NS) related cis-regulatory elements MtCP1 to MtCP6 were shown to be involved in both developmental and dark-stressinduced nodule senescence (Perez Guerra et al., 2010). Compared with the 3wpi nodules without signs of senescence, MtCP1 to MtCP6 share an elevated transcript accumulation in nodules with an increased senescence zone (Perez Guerra et al., 2010). In situ hybridizations revealed the tissue-specific expressions of MtCP1 to MtCP6 genes in infected cells located at the periphery of the conical senescence zone starting in the centre of the fixation zone (Perez Guerra et al., 2010).

I.2.1 Role of cysteine protease 6 (MtCP6) in nodule senescence

MtCP6 is induced during both developmental and induced nodule senescence Among the CPs, MtCP6 were further characterized and evidenced to be amongst the best early molecular marker for nodule senescence. MtCP6 is predicted as an SPG31-like protease of the papain like protease family (http://www.ebi.ac.uk/merops/) [START_REF] Rawlings | The MEROPS database of proteolytic enzymes, their substrates and inhibitors in 2017 and a comparison with peptidases in the PANTHER database[END_REF]. Enzymatic characterization has determined its cathepsin-L activity using various protease substrates and inhibitors (Pierre et al., 2014). The papain family is homologous to a well-known Arabidopsis leaf senescence gene marker, AtSag12 (Lohman et al., 1994;Van de Velde et al., 2006). The involvement of MtCP6 in nodule senescence has been evidenced via functional characterization. Down-regulation of MtCP6 steady state mRNA by RNAi is correlated to a delayed senescence, resulted in increased nitrogen fixation capacity and nodule size (especially an extension of zone III) (Pierre et al., 2014). Conversely, sequence (NS2, 45bp) added to a minimal CaMV 35S promoter (Pmin35S) and fused to GUS reporter gene in pLP100 binary vector (Fig. 2A). The expression pattern of the resulting chimeric genes was analysed in transgenic root nodules at 4wpi with or without nitrogen application. Representative images of transgenic nodules expressing chimeric gene constructs are presented in Fig. 2B. GUS activity was observed in the basal part of the nodule (Class A)

when GUS was under the control of NS tetramer (Fig. 2B,2C). GUS staining was not detected when the gene was under the control of NS1 and NS2 tetramers. Nitrate treatment induced a stronger GUS staining in nodule with NS tetramer which was localized in zone III and in the beginning of zone IV (Fig. 2C). As during developmental senescence, GUS staining was not detected when the gene was under the control of NS1 and NS2 tetramers. The GUS activity of 4xNS construct was detected in 12% of the analysed samples with an appropriate localisation of the expression in 79% of the positive samples (Fig. 2B). Under nitrate treatment, 20% of nodules were stained and all the GUS positive nodules presented an appropriate GUS staining localization (Fig. 2B). Detectable GUS activity related to 4xNS1 construct at 4wpi appeared only in one biological experiment with only 8% of nodules (77% in Class A) and the nitrate treatment did not increase the GUS activity in nodules. No GUS activity was observed for 4xNS2 construct. The control (Pmin35S) with an empty pLP100 vector does not showed any stained nodules in the same treatment conditions.

Quantification of GUS activity using fluorometric assay was performed to compare the transcriptional activity of the different constructs (Fig. 2D). 4xNS construct allowed a significant higher GUS activity (124±13 pmol MU/min/mg protein) than 4xNS1 construct (41±2 pmol MU/min/mg protein), 4xNS2 construct (17±2 pmol MU/min/mg protein). After nitrate treatment, GUS activity of 4xNS construct increased 6-fold to 786±110 pmol MU/min/mg protein, while the increase was less than 1.8-fold for 4xNS1 construct and 4xNS2 construct with 73±2 and 31±2 pmol MU/min/mg protein, respectively. The GUS activities for the Pmin35S construct were 18±2 and 20±2 pmol MU/min/mg protein at 4wpi PI or 4wpi PI with nitrate treatment, respectively (Fig. 2D).

Taken together, these data show that the tetramer of the NS-box fused to minimal 35S promoter is able to provide the nodule senescence tissue-transcription specificity to GUS supporting the hypothesis that the NS-box contains cis-regulatory elements involved in the gene regulation during nodule senescence process.

Loss of the NS-box impairs the promoter

SUPPLEMENTARY DATA:

Table S1: List of primers used in this study.

CHAPTER III

Searching for NS-box-interacting transcription factors

II. Material and Methods

Yeast-One-Hybrid screening

The Yeast-One-Hybrid screening of the tetramer of NS-box (4x NS-box) for interactive TF candidates was done as previously described (Andriankaja et al., 2007). The tetramer of 3-Amino-1,2,4-triazole (3-AT) conditions, was selected to be used in the further one-hybrid screen. The One-hybrid screen was done using a nodule AD fusion cDNA library, generated before by the Dualsystems company (http://www.dualsystems.com/) from RNA samples extracted from 4 and 19 days-old nodules (and kindly provided by A. Niebel) [START_REF] Baudin | A phylogenetically conserved group of NF-Y transcription factors interact to control nodulation in legumes[END_REF]. This screen used a concentration of 10mM 3-AT in the yeast media. Yeast transformation methods and media composition are all detailed in the Method section (ANNEX III) of this PhD thesis.

His + yeast colonies were recovered in 10µl of water and 2-3 µl were re-spotted on SD/-Leu/-His plates with 15mM 3-AT to confirm growth in selective conditions. Positive candidates were analysed by PCR on 2µl of yeast suspension using primers (ANNEX III Table S3). After sequencing using the same primers, clones exhibiting ORFs in frame with the AD activation domain were further analysed by BLASTN and BLASTX algorithms [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] using Medicago Mt4.0 (http://blast.jcvi.org/Medicago-Blast/index.cgi) and MtrunA17r5.0-ANR (https://medicago.toulouse.inra.fr/MtrunA17r5.0-ANR/) genomic databases. Among the 29 in frame clones (Table 8), eight encode for putative TFs mostly of the ERF TF family. We therefore decided to try to validate these candidates by retransformation of the yeast 4xNS-box strain.

Plasmid DNAs from selected His + candidate clones were prepared and used to transform Escherichia coli DH5a. Plasmids of the NS-interacting ERF candidates and ERN1

ERF domain binds to GCC-related cis-regulatory elements

The DNA binding domain of ERF1-like transcription factors to the typical GCC-box motif [START_REF] Ohme-Takagi | Structure and expression of a tobacco beta-1,3-glucanase gene[END_REF][START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF]. The GCC-box was firstly identified from the 228-bp-long cis-regulatory elements known as the Ethylene-Responsive Element (ERE) [START_REF] Broglie | Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco[END_REF] from ethylene inducible pathogenesis-related (PR) genes in Nicotiana spp., as the 11-bp sequence (TAAGAGCCGCC) (Ohme-Takagi & Shinshi, 1990;

Ohme-Takagi & Shinshi, 1995). AtERF1 has been shown to recognize GCC-box [START_REF] Franco-Zorrilla | DNA-binding specificities of plant transcription factors and their potential to define target genes[END_REF], but with only marginal binding to other GCC-like variants [START_REF] Godoy | Improven protein-binding microarrays for the identification of DNA-binding specificities of transcription factors[END_REF]. A GCC-like sequence (TGCAGGCCT) has been identified from the upstream region of MtENOD11 to mediate gene activation upon Nod factor treatment (Andriankaja et al., 2007) and this sequence is recognized by group V ERF TFs. C-repeat/DRE that contains similar sequences has been found in promoters of COR (cold-regulated) genes in A. thaliana, such as COR15a [START_REF] Baker | The 5′-region of Arabidopsis thaliana cor15a has cisacting elements that confer cold-, drought-and ABA-regulated gene expression[END_REF], COR78 (Yamaguchi-Shinozaki & Shinozaki, 1994) and Crepeat/DRE Binding Factor 1 (CBF1) [START_REF] Stockinger | Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[END_REF]. Inside NS-box, a 11-bp GCrich sequence (TTTGGCCGTT) could be found in addition to other cis-regulatory elements discussed in Chapter I (CAAT-box, W-box and Dof, etc.) (Figure 34A). This sequence region is here termed as GCCcore-like, which is similar to previously identified GCC-like or Crepeat/DREB sequences (Figure 34B) [START_REF] Ohme-Takagi | Structure and expression of a tobacco beta-1,3-glucanase gene[END_REF][START_REF] Ohme-Takagi | Ethylene-inducible DNA binding proteins that interact with an ethylene-responsive element[END_REF][START_REF] Stockinger | Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit[END_REF]Andriankaja et al., 2007).

(SmgshB). Under this perspective, it might be better to carry out a preliminary experiment to observe nodule phenotypes (nodule number, size, colour, etc.) in comparison to the wild-type nodules (WT). Given by effective nodule phenotypes caused by genetic perturbation, some additional conventional studies on nodule symbiosis might be carried out: 1) Acetylene

Reduction Assay (ARA) to quantify the nitrogen fixation capacity; 2) RT-qPCR to investigate expression of nodule function marker genes or related transcriptional regulating genes (e.g.

CPs)

; 3) histological analysis such as Toluidine blue-or live/dead-staining; 4) spatial localization of transcripts and proteins; 5) plant phenotyping provided with homozygous mutants; etc. Finally, it might be interesting to evaluate the 4NS-box-Pmin35S synthetic promoter activities with provided TF mutants. Possibly, it could be interesting to investigate promoter activity (using the luciferase reporter) in M. truncatula protoplasts for various purposes (e.g. nitrate response, transactivation with TF candidates, hormone responses).

Men say they know many things;

But lo! they have taken wings, -

The arts and sciences, And a thousand appliances;

The wind that blows Is all that anybody knows.

Henry David Thoreau

suggesting that bHLH2 is the transcriptional repressor of CP77 (Deng et al., 2019). However, the authors suggested bHLH2 as a repressor deduced from an enhancing transcriptional activity of CP77 in bhlh2 mutants via RT-qPCR and GUS staining. It might be interesting to exam if there is a down-regulation of CP77 expression by overexpressing bHLH2. Other than that, expression of CP77 (Medtr4g065077.1) is upregulated in zone IZ and zone III according to the RNA-seq data of laser-dissected nodules (Roux et al., 2014), and prominently expressed in seeds according to the M. truncatula Gene Expression Atlas (Benedito et al., 2008). The expression pattern of CP77 is not reminiscent of that in the MtCPs (MtCP1 to MtCP6) identified from nodule senescence transcriptome (Van de Velde et al., 2006;Perez Guerra et al., 2010). In addition, numerous TFs were characterized to mediate nodule senescence under various environmental changes. NAC969 was evidenced to be upregulated upon abiotic stresses with nitrate, but it was negatively related to nodule developmental senescence (de Zelicourt et al., 2012). The divergent transcriptional activities of NAC969 imply a distinct regulation pathway of developmental nodule senescence and nodule response to abiotic stresses. NIN-Like Protein 1 (NLP1) appears to compete with the nodulation gene NIN to bind the nitrate responsive cis-regulatory element NRE, and thus modulates nodulation or nitrate-triggered nodulation inhibition (Lin et al., 2018). Notably, NLPs in Arabidopsis are well known to bind this nitrate responsive cis-regulatory elements NRE which is conserved in NIR promoters (Konishi & Yanagisawa, 2013). ERFB2.1/B2.11, which are in the same group (ERF VII) of our candidate ERF069, were related to O2 sensing with a potential role in maintaining anaerobic condition in nodules, whereas silencing these two genes did not affect MtCP6 expression [START_REF] Rovere | ERF-VII family as key players in hypoxic signaling and adaptation in Medicago truncatula[END_REF].

These data and our results suggest that multiple transcription factors and thus cisregulatory elements are involved in the senescence process which includes a degradation process of the plant and the bacterial cells and a recovery process of nutrients by the plant host. Moreover, there is also a saprophytic zone where bacteria are still present in the nodule.

Therefore, it is likely that modulators of defence response in plant immunity, cell death and recovery of nutrients are likely to be found in nodule senescence regulation. Overall, it might be thus explained that some TFs such as NAC969 exhibited divergent regulation patterns in case of developmental and induced nodule senescence. On the contrary, the NS-box cisregulatory element, which we identified from the proximal 5' flanking sequence of MtCP6, is possessing transcriptional information positively responsive to both developmental and induced nodule senescence. These results indicate that nodule senescence is likely to culminate with proteolytic activities initiated by the co-elevation of cysteine protease genes.

ANNEX I

Materials & Methods

I.2 Nicotiana benthamiana plant growth conditions

Seed of N. benthamiana should be vernalized 4°C for one week and geminated in growth chamber for one week. The pots were filled with growth media as sand:peat at ratio 1:1, and complete fertilizer was used in N. benthamiana plant growth. The growth chamber is with a photoperiod of 16/8h and thermoperiod of 25/20°C, respectively for day and night. The plantlets were transferred into new pots and grown for 3 weeks before infiltration.

Table S 1 Strains and plasmids used during the course of this thesis

Strains/plasmids Characteristics References

Strains

A. rhizogenes ARqua1

Sm R , derivative of R1000 strain, moderate virulence (Quandt, 1993) A. rhizogenes A4RS Rif R , high virulence, originally used to transform coffee roots [START_REF] Alpizar | Efficient production of Agrobacterium rhizogenes-transformed roots and composite plants for studying gene expression in coffee roots[END_REF]) pKM43-rolD::GFP Sp R , destination vector for three entry multisite Gateway cloning [START_REF] Karimi | GATEWAY™ vectors for Agrobacterium-mediated plant transformation[END_REF] pLP100 Km R , -47bp Camv35S (pmin35S)::GUS Fernanda de Carvalho-Niebel pHISi Amp R , HIS3, reporter vector for Yeast-One-Hybrid (Clontech) Fernanda de Carvalho-Niebel pGAD-HA Amp R , Leu, prey vector for Yeast-One-Hybrid (Clontech) Fernanda de Carvalho-Niebel

I.3 Bacterial materials

Table S 1 is the list for microbe strains and plasmids used in the course of this thesis.

Sinorhizobium meliloti 2011 was growing in LBMC (LB Broth, added with 2.5mM CaCl2 and 2.5 mM MgSO4) with 100μg/ml streptomycin at 30°C. Bacteria strains wild type Sinorhizobium meliloti 2011 and S. meliloti 2011 gshB was growing with LBMC medium with appropriate sealed vials (10ml). In each vial, 10% of the atmosphere (1ml) was substituted with fresh prepared acetylene (1ml). After 1h of incubation at 28°C, gas samples were collected with 1ml syringes and needles were blocked with rubber before use. The gas samples (200µl) were analysed with a gas chromatograph system. Twelve plants were tested per replicate and at least three different biological replicates were analysed.

Nodule imaging

All the images of nodules were treated with the software Fiji (Schindelin et al., 2012).

The brightness/contrast was adjusted in the post-processing of images. Confocal images of each channel were treated independently and then merged. As for nodule size analysis, nodule images were transformed in 8-bit ('image' -> 'Type') and converted to black and white ('make binary'). Th parameters of selected particles (nodules) can be analysed under the menu 'Analyze'.

Flow cytometry

Nodules (equal amount) were crushed in 1.5ml Eppendorf in phosphate buffer (pH=7.5) with 4% mannitol (m/v, 320~400 mOSM/L). The mixture was centrifuged 3min at 1500 rpm at 4°C to separate bacteria/bacteroids from plant tissue by differential density gradients. 500μl

of supernatant was transferred into a new Eppendorf tube and the debris was discarded.

Bacteroids were then harvested from the pellets after centrifugation (10min, 8000rpm, 4°C).

Resuspended bacteroids were heat-killed (70°C, 15min) to allow the penetration of PI and The first of the line is tied to a tree and the last is being eaten by the ants.

Gabriel García Márquez