Keywords: Biotrophy-Associated Secreted BIC, Biotrophic Interfacial Complex CC, Coiled-Coil CEBiP, Chitin Elicitor-Binding Protein CERK1, Chitin Elicitor Receptor Kinase- Damage-Associated Molecular Patters Effector-Triggered Immunity ETS, Effector-Triggered Susceptibility

REMERCIEMENTS

« Un ciel, la nuit. La voie lactée scintille au loin. La Terre tourne sur elle-même, poursuivant sa course à travers l'espace, parcourant une parcelle infime d'un univers sans fin. Un cosmos décoré de planètes, d'étoiles, de soleils et de galaxies, qui se déploie, jour après jour. De ses origines nous ne savons rien, ni de son devenir, pourtant nous savons qu'il est habité. La Terre a emporté à travers l'espace et le temps le foisonnement changeant des innombrables engendrements du vivant avec les formes les plus belles et les plus merveilleuses qui ont été composée et recomposée depuis l'origine. Chaque créature vivante peut être considérée comme un microcosme, un infime univers, constitué d'une inimaginable multitude de petits organismes qui se reproduisent sans fin et que sont aussi nombreux que les étoiles dans le ciel ». La sculpture du vivant Au mystère de l'origine du cosmos qui nous entoure répond le mystère de l'origine du vivant. Tous les deux enlacent pour un fil invisible de molécules qui sont le coeur de ces univers parallèles. Eh bien… c'était l'irrémédiable pulsion de plonger dans l'univers moléculaire qui m'a propulsé à faire une thèse. C'est pour cela qu'avant tout je remercie la vie. C'est grâce à elle que, pour une succession d'imprévisibles accidents terribles et merveilleux je suis arrivée ici, un coin charmant de la France auprès de gens et paysages en qui j'ai énormément appris. Je les garderai pour toujours dans ma mémoire et mon coeur.

A quelques kilomètres d'ici se trouvent les gens à qui je dois la force que j'ai eu quand l'aventure de la thèse a été difficile à traverser. Padresito, alguna vez escuche que los hijos son la prolongación de la vida y yo lo siento así, es por eso que en cada uno de los pasos que he dado lejos de ti he intentado ser feliz, esta es mi única manera de agradecerte y hacerte parte de un sueño que considero mutuo. A mi madre le agradezco por todos los ángeles que pone cada día en mi destino, son muchos!!! Y sé que todos vienen enviados por ti. Mis hermanos, sobrinos, amigos y demás miembros anexos a la familia, quisiera nombrarlos uno a uno pero no con todo lo que podría decirles no acabaría nunca… en estas cortas líneas solo me queda mencionar que ustedes representan la alegría y la luz de esta loca travesía, los amo profundamente. Camilito, gracias por todo el amor compartido, tu de alguna forma fugaz paso por mi vida se ha inscrito en mi como el paso de una ráfaga de dulzura y ternura inolvidable, tu sabes que nos llevaremos por siempre en la eternidad sin tiempo ni formas.

Thomas je te remercie énormément pour m'avoir donné l'opportunité de faire une thèse sous ton encadrement. Ça fait déjà plus de cinq ans qu'on a commencé à travailler ensemble pour faire d'un rêve une réalité. Tu m'as soutenue toujours, même quand j'ai fait des erreurs avec la candidature à Colciencias! Je ne vais jamais oublier que même à ce moment-là tu m'as fait confiance. Ce sont ces beaux actes qui transforment les gens, alors tout comme Jean Valjean à qui la confiance de quelqu'un a transformé, moi je peux te dire que l'encouragement de ta part LIST OF

a été vital tout au long de cette thèse et que sans cela ce rêve serai une réalité sans saveur. Merci aussi pour toute ta patience et pour tout ce que tu m'as appris et tu m'a poussé à faire. Trois congrès internationaux, un super stage en Angleterre et deux beaux papiers, sont un petit résumé de ton encadrement! En plus je suis très contente d'être ta première thésarde officielle. A Stella d'avoir fait un super travail et faire mon chemin plus facile. Aux membres de mon comité de thèse pour les conseils toujours très constructifs.

Jean Benoit merci beaucoup de m'avoir accueillie dans ton équipe et pour tous tes conseils scientifiques et personnels. Saches que sans le savoir, tes mots d'encouragement sont toujours arrivés dans les moments où j'avais le plus besoin. Merci aussi pour tes gestes qui m'ont fait beaucoup de bien comme par exemple le manioc ou les lumières pour le vélo dans un obscur hiver.

A tout l'ensemble de l'équipe 4 mille et mille mercis pour toute l'aide lors de manips et aussi pour les moments partagés. Véro merci pour toute ton aide avec mes expériences, j'ai trop adoré travailler avec toi. Merci aussi pour les livres et toutes nos conversations « philosophiques » tu vas me manquer. Coco, c'était très agréable recevoir tes câlins et partager notre passion pour le tango, j'espère que tu ne t'arrêteras jamais de danser. Aurélie j'espère que tous les souhaites qu'on a envoyé aux étoiles filantes deviendront réalité. Przemyslaw merci pour ton hospitalité en Pologne et pour les vins partagés. Emilie, quelles vagues ont a surmonté ensemble!!!!!! On est souvent passé de l'euphorie aux larmes… mais bon on est toujours là et tu vois tout va bien… particulière époque de nos vies hein ? Je te remercie pour m'avoir fait découvrir de très belles musiques, je suis contente de te voir rapprocher de plus en plus de ton coté vivant et paisible. Celui que j'ai eu l'opportunité de découvrir et apprécier dans les montagnes Polonaises lorsque tu chantes et joues de la guitare. J'espère que la musique et la science continueront à te procurer autant de bonheur. Elsa, Isabelle, Audrey, Loïc, Huichuan, Bastien, Jing-Jing merci beaucoup pour votre courtoisie, votre aide et vos sourires. Paola, c'était cool d'avoir una compatriota au labo! A ton tour de continuer à faire vibrer la Colombie à BGPI.

Romain Ferdinand qu'est-ce que j'aurais fait à BGPI sans toi !!! Je n'ai pas des mots pour te dire combien je suis contente de t'avoir croisé dans ce coin de la terre. Merci énormément pour tout ton soutien. Je sais que je t'ai embêté un peu trop mais tu sais, j'en avais besoin. Merci aussi pour m'avoir appris le français, m'amener quand il « pleuvait » et enfin d'être toujours là. La facture je te la payerai quand je serai une chercheuse milliardaire. Que la lumière de chaque couché de soleil continue à t'émerveiller. Charlotte, à toi aussi je te remercie profondément pour tes justes mots remplis de beauté et de sincérité. Avoir ton amitié c'est un grand cadeau venu du ciel. En fait ce tour en avion c'était une expérience qui représente très bien tout ce que tu m'as apporté. J'espère qu'on fera d'autres voyages ensemble. Katia, merci pour être à l'écoute et t'investir pour rendre les choses plus simples pour moi et tous les thésards. Merci aussi pour ta joie de vivre, elle est contagieuse!!! Henri, grâce à toi j'ai eu le courage de venir en vélo pendant l'hiver, dans une route sans trop de lumière et avec la menace de sangliers hehe.. quelle aventure !!! mais tu sais ?, pendant que je roulais, je jetais un oeil à la lune et les étoiles et ça devenait un expérience agréable. Bon, quand-même je dois avouer que je suis rassurée de pas devoir le faire à nouveau mais mille merci pour me sauver des heures passées dans les transportes publiques. Merci aussi de nous avoir fait découvrir fillols et le Pyrénées-Orientales c'était top. Sonia, Marlène, Romain Gallet, Agnés, Pierre, Didier, merci beaucoup pour être là dans cette dernière ligne droite. De manière plus générale je remercie vraiment toutes les personnes que j'ai croisées à BGPI et qui ont fait partie de cette belle aventure. A Marie Noel pour m'avoir amenée de retour à ma vraie maison et me rendre mes ailes.

A tous mes amis de Montpellier, Boris mon lien Colombie-France, merci pour m'avoir fait rêver d'étudier dans ton pays. Cela a été sans aucun doute le meilleur choix. On pourrait même dire que t'avoir croisé a changé ma vie! Je t'apprécie très fortement. A la bande de intensos Jesus, Nico, Ponchis, Reini, Raul, Nico, Sonia, Thierry, Miguel, Luis, Sheena, Jean-Philippe, Lili, Ryan et Alvaro, j'ai vécu des très bons moments avec vous et je suis sûre qu'on se retrouvera à nouveau parce que les bons amis restent tout au long d'une vie , vous êtes dans mon coeur. A Elisabeth pour toute son hospitalité. A Dani et à Michel mes « parents » en France, quelle chance j'ai de vous avoir rencontré.

A la nouvelle bande de thésardes avec qui j'ai eu la chance de partager ma dernière année de thèse Coralie, Edwige et Maelle trois filles magnifiques et courageuses, toute mon admiration pour vous! Vous m'avez énormément appris et votre amitié c'est l'une des choses les plus jolies que j'ai eu avant partir de ces murs. Je suis sûre que toutes les trois feront de super thèses ! A mes anciens collègues thésardes Pauline, Deborah, Anne, Souhir merci aussi pour les bons moments et les pauses du vendredi. Votre exemple c'était un moteur de motivation. A Claire Neema pour son inconditionnel soutien aux thésards. Merci à Marie-Agnès, Pierre, Léa et Vincent de m'avoir accueillie avec autant de tendresse dans votre famille.

Et bon… moi qui attendais patiemment la voie de retour à mon pays, comme dans les contes de fée, c'est à BGPI que je suis tombée avec un lobo rêveur qui m'a persuadée d'allonger mon chemin pour découvrir d'autres univers. Je te remercie de m'avoir regardée et de m'inviter à faire partie d'une aventure commune, je suis sûre qu'elle sera magnifique.

INTRODUCTION

Plant immunity

Plant defenses and pathogen infection

Plants are essential organisms of any live food chain and are constantly exposed to abiotic and biotic factors that can positively or negatively affect their development. For instance, during their life cycle plants are in intimate association with microorganisms as diverse as bacteria, fungi, nematodes, oomycetes and viruses and interact with them in different ways. In some cases, this association can lead to beneficial interactions in which both organisms benefit to each other, e.g. the interaction between legumes and rhizobium, a bacteria that, after becoming established inside rood nodules, helps the plant to fix nitrogen [START_REF] Hirsch | What makes the rhizobia-legume symbiosis so special?[END_REF].

However, in other cases microorganism act as pathogens and represent a danger for the plant since their interaction lead to disease. To face this challenge, plants possess preformed defenses to arrest pathogen infection, such as physical barriers and constitutive production of chemical compounds with antimicrobial properties [START_REF] Göhre | Breaking the barriers: microbial effector molecules subvert plant immunity[END_REF]. In addition, plants have evolved a sophisticated innate immune system that confers to individual plant cells the capacity to sense and respond to pathogen attack. Indeed, after pathogen recognition, plants induce complex and multilayered defense responses (Figure 1). At sites with contact to the pathogen, the cell wall is reinforced by the production of various cell wall components such as glycoproteins, lignin, callose or suberin. ROS (Reactive Oxygen Species) are produced and act directly by their antimicrobial activities and the crosslinking of cell wall components as well as indirectly by their role as signaling molecules. The production of pathogenesis-related proteins (PR) and secondary metabolites are induced by complex gene regulatory networks and limit pathogen development by their antimicrobial activities [START_REF] Hammond-Kosack | Resistance Gene-Dependent Plant Defense Responses[END_REF][START_REF] Vanloon | Induced resistance in plants and the role of pathogenesis-related proteins[END_REF][START_REF] Torres | Reactive oxygen species signaling in response to pathogens[END_REF][START_REF] Dong | Battle for survival: plants and their allies and enemies[END_REF][START_REF] Göhre | Breaking the barriers: microbial effector molecules subvert plant immunity[END_REF][START_REF] Lehmann | Reactive oxygen species and plant resistance to fungal pathogens[END_REF].

These plant immune responses lead to the establishment of either nonhost or host resistance.

Nonhost resistance refers to the broad-spectrum plant defense that provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species whereas host resistance is cultivar or accession specific and is activated only in response to some isolates or races of adapted pathogen species.

Pathogens use specialized structures and molecular arms to overcome plant defenses, colonize plant cells and acquire nutrients. For instance, fungi and oomycetes develop specialized cells named appressoria to break the leaf surface and invasive hyphae or haustoria to feed on living plant cells. Nematodes and insects use styletes to penetrate and feed on plant cells. Bacteria possess macromolecular structures like the type III or IV secretion systems to deliver virulence factors inside host cells. Regardless of life style, infection structures and reproduction mode, all plant pathogens use molecular weapons during plant infection corresponding in particular to secondary metabolites or secreted proteins.

Figure 1. Induction of plant defense responses upon pathogen attack.

Adapted from [START_REF] Hammond-Kosack | Resistance Gene-Dependent Plant Defense Responses[END_REF]

Models to explain plant immunity

In all biological systems, immunity relies on the induction of defense responses upon pathogen attack and this is mediated by the recognition of two types of signals, non-self and modified self either inside cells or on the cell surface. In plants, pathogen perception inside cells is exclusively mediated by NLRs (Nucleotide-binding and Leucine-rich repeat proteins) while two different types of immune receptors perceive signals on the cell surface, RLKs (Receptor-Like Kinases) and RLPs (Receptor-Like Proteins) (Figure 2). A detailed description of NLRs is provided in section 3, while basic knowledge on RLKs and RLPs is summarized in Figure 8.

Virulence factors

Defense responses

The Zig-zag model Over the last 10 years, the understanding and interpretation of plant immunity and the action and evolution of the plant immune receptors and their corresponding ligands has been very much influenced by the zig-zag model (Jones & Dangl 2006;[START_REF] Dodds | Plant immunity: towards an integrated view of plant-pathogen interactions[END_REF]. This model (Figure 3) divides the plant immune systems in two layers.

A first layer relies on the recognition of microbial or pathogen associated molecular patterns A second layer of immunity that is based on the recognition of effectors by NLR-type immune receptors and therefore named effector-triggered immunity (ETI) allows the control of these adapted pathogens. The ETI response has been described as an amplification of PTI responses and leads to complete disease resistance. It is frequently associated with a localized programmed cell death called the hypersensitive response (HR). Sometimes, pathogens can adapt to ETI by evolving novel effectors that suppress ETI and establish again ETS.

(
The merit of the Zig-zag model was to provide a first unified view of plant immunity that integrates the phenomena of basal resistance and specific (gene-for-gene type) plant resistance and links it to pathogen effector action. In addition, it provided an evolutionary explanation for the complex interplay between host resistance and pathogen virulence by nicely illustrating the co-evolution between pathogens and plants that both continually modify their weapons to either avoid or induce resistance. In addition, the zig-zag model also provides a good framework for the understanding of the infection process by phytopathogenic bacteria, in particular Pseudomonas sryingae and many Xanthomonas bacteria under artificial conditions. In infection experiments, these bacteria are generally infiltrated into plant leafs where they instantaneously liberate huge quantities of PAMPs and MAMPs and induce rapid PTI responses [START_REF] Katagiri | The Arabidopsis Thaliana-Pseudomonas Syringae Interaction[END_REF][START_REF] Buell | Interactions between Xanthomonas species and Arabidopsis thaliana[END_REF]. Since the type 3 secretion system (T3SS) and T3SS effectors are under transcriptional control and only produced in planta, effector delivery occurs only several hours after infiltration. PTI suppression and ETS establishment is therefore only visible in a second step, typically 3-4 hours after infiltration. Therefore, recognition of effectors and ETI induction also only occurs in a second step [START_REF] Katagiri | The Arabidopsis Thaliana-Pseudomonas Syringae Interaction[END_REF][START_REF] Buell | Interactions between Xanthomonas species and Arabidopsis thaliana[END_REF].

Limitations of zigzag model

Despite its wide popularity and the historical importance of the zig-zag model, research over the last decade showed that major assumptions and conclusions of the zig-zag model miss precision or are erroneous and that there is a need for a novel more unifying model [START_REF] Thomma | Of PAMPs and effectors: the blurred PTI-ETI dichotomy[END_REF]. The fundamental conceptual error of the zig-zag model is the separation of plant immunity in two separated layers. This fits badly with experimental data and has led to many misconceptions and wrong assumptions about the evolution of immune receptors and their ligands. In addition, it generates numerous problems in the classification of virulence factors

PAMPs vs effector as well as the immune response either PTI or ETI triggered by immune receptors.

Problems become e.g. evident when effectors that are defined in the Zig-zag model as highly variable, rapidly evolving and species or lineage specific proteins behave more like, PAMPs meaning they are broadly distributed and present in large ranges of organisms. It is also problematic, when PAMPs that are defined as pathogen molecules involved in general cellular functions behave like effectors since they are highly polymorphic and trigger differential resistance responses [START_REF] Cook | Understanding Plant Immunity as a Surveillance System to Detect Invasion[END_REF]. Indeed, research in multiple pathosystems has shown that not only effectors but also MAMPs/PAMPs evolve to escape recognition and are under selection pressure [START_REF] Michelmore | Impacts of resistance gene genetics, function, and evolution on a durable future[END_REF][START_REF] Vinatzer | Harnessing population genomics to understand how bacterial pathogens emerge, adapt to crop hosts, and disseminate[END_REF]. The rate of evolution of both types of ligands can vary considerably and their phylogenetic distribution can be more or less wide. For example, there is strong variation in the amino acid sequences of the moststudied PAMPs, the flagellin protein, in the bacterial pathogen Xanthomonas campestris pv.

campestris resulting in variation of defense responses on Arabidopsis thaliana possessing the flagellin receptor FLS2 [START_REF] Sun | Within-species flagellin polymorphism in Xanthomonas campestris pv campestris and its impact on elicitation of Arabidopsis FLAGELLIN SENSING2-dependent defenses[END_REF]. Similarly, the significant variation in the residues of the flg22 epitope of Ralstonia solanacearum K60 and Pseudomonas cannabina pv. alisalensis ES4326 resulted in a variation in the resistance response triggered by their corresponding receptor proteins in the host [START_REF] Pfund | Flagellin is not a major defense elicitor in Ralstonia solanacearum cells or extracts applied to Arabidopsis thaliana[END_REF][START_REF] Clarke | Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility[END_REF].

Additionally, the discovery of a second epitope, the flagellin flgII-28, that is sufficient to trigger immune responses in tomato, confirmed the dynamic nature of flagellin-host perception and highlight the importance of PAMP diversification as a virulence strategy of the pathogen [START_REF] Clarke | Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility[END_REF]. In addition, PAMPs can have a patchy distribution. An example for this is the case of the sulfated RaxX peptide recognized by the Xa21 RLK in rice. RaxX is present in a limited number of Xanthomonas species and shows presence absence polymorphism in Xoo isolates (Pruitt et al., 2015).

On the contrary, NLPs (Necrosis-and Ethylene-inducing Peptide1-Like Proteins) are an example of effectors with an extremely wide distribution in prokaryotic and eukaryotic phytopathogens that are recognized in Arabidopsis thaliana by a conserved protein pattern [START_REF] Bohm | A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis[END_REF]Oome & Van den Ackerveken 2014;Oome et al. 2014). Another example of widely distributed effectors, are the LysM domain effector proteins that occur in a broad range of fungal pathogens and that were frequently miss-qualified as PAMPs in the frame of the zigzag model [START_REF] Thomma | Of PAMPs and effectors: the blurred PTI-ETI dichotomy[END_REF].

Another problem of the zig-zag model is that RLPs and RLKS are categorized as PRRs dedicated to MAMP/PAMP detection. They are predicted to be conserved throughout and between plant species and to evolve at slow rates. NLRs, on the contrary, are viewed as receptors of isolate or pathogen-specific effectors and to be present in only limited numbers of plant accessions. This view is again very biased since RLPs may also recognize isolate-specific effectors and act as full, gene-for-gene type R proteins. For example the race-specific Cf-2 receptor from tomato monitors the presence of the effector protein Avr2 from Cladosporim fulvum strains by guarding the target Rcr3 protease [START_REF] Rooney | Cladosporium Avr2 Inhibits Tomato Rcr3 Protease Required for Cf-2 -Dependent Disease Resistance[END_REF]. RLKs, show less diversity but possess marked presence-absence polymorphism within and between species. Indeed most MAMP receptors are redistricted to certain species or phylogenetic classes such as EFR that is limited to Brassicaceae [START_REF] Kunze | The N Terminus of Bacterial Elongation Factor Tu Elicits Innate Immunity in Arabidopsis Plants[END_REF][START_REF] Boller | A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors[END_REF]. As a consequence, contrary to the view given by the zig-zag model that describes PTI as a stable character, there is clear variability in the detection of MAMPs within and between species [START_REF] Robatzek | Molecular identification and characterization of the tomato flagellin receptor LeFLS2, an orthologue of Arabidopsis FLS2 exhibiting characteristically different perception specificities[END_REF][START_REF] Gómez-Gómez | FLS2: an LRR receptor-like kinase involved in the perception of the bacterial elicitor flagellin in Arabidopsis[END_REF]. Finally, the view that ETI responses are stronger and more intense than PTI responses is not always verified since various PAMPs, including flagellin induce an HR.

The invasion model

A recent attempt to describe plant immunity in a more unbiased way, to incorporate a large range of mechanistically diverse interactions and to avoid strict divisions is the invasion model (Figure 3). This model states that plant immunity relies on the recognition of invasion patterns (IP) that can be two different types of ligands, microbe-derived molecules or modified-self ligands by invasion pattern receptors (IPRs) that may be either PRRs or NLRs.

Any molecule that can be recognized by an IPR can act as an IP and induce IP-triggered responses (IPTRs). An important distinction from the zig-zag model is that IPs are only defined with respect to their function in host perception without any restriction or prediction of their physiological or biochemical function. Thus, in the invasion model the probability to activate IPTR depends on several factors such as the ligand's molecular constrains to retain function and the variability of IPs and IPRs across organisms. Thereby, the model incorporates on one hand the diversity of ligands that are important in plant immunity and that englobe MAMPs, damage-associated molecular patters (DAMPs) and effectors and on the other hand since all types of immune receptors fall in the same category, the specificities of recognition are explained in terms of the nature of the response e.g., weak or strong, narrow or broad, specific or common etc., (Figure 3).

Fungal effectors

The importance of fungal plant diseases

Fungi are one of the most important groups of plant pathogens. They cause devastating diseases and are a permanent threat for food production. The use of resistant crops represents the economically and ecologically most sustainable solution for disease control.

However, the durability of crop resistance is frequently broken by the rapid evolution of fungal populations [START_REF] Brown | Durable Resistance of Crops to Disease: A Darwinian Perspective[END_REF]. The current knowledge on the molecular bases of fungal pathogenicity and its evolution is still too limited to obtain durably resistant plants by knowledge-driven breeding or engineering. Indeed, resistance break-down frequently occurs only after few cultural cycles reducing the available genetic resistance resources and limiting the utility of natural resistance. Achieving durable crop resistance is a major goal for a sustainable, knowledge-based agriculture and it cannot be achieved without an improved and detailed understanding of the molecular mechanisms underlying plant-fungal interactions [START_REF] Dangl | Pivoting the plant immune system from dissection to deployment[END_REF]. Since plant immune receptors and effectors molecules are pivotal components of plant defense responses, in this study our main objective is to better understand the molecular bases of the recognition of fungal effector proteins by plant NLRs.

Fungal effectors act either in the plant apoplast or in the cytoplasm

Fungi release an arsenal of highly diverse effectors, here defined as any secreted protein that modulates plant immunity to facilitate infection. Fungal effectors can act in the apoplast or in the cytoplasm (Figure 4) of plant cells and are very variable in sequence and distribution. While most effectors are species or linege-specific, some, in particular apoplastic ones are widespread and have conserved domains. The already mentioned NLP proteins that induce cell death, necrosis and ethylene production when applied to plant leaves are e.g. present throughout a large range of phytopathogenic bacteria, fungi and oomycetes. Effectors containing a sugar-binding lysin motifs (LysMs) occur in many pathogenic and non-pathogenic fungi (Oome et al., 2014;[START_REF] Kombrink | LysM Effectors : Secreted Proteins Supporting Fungal Life[END_REF]. Some of these LysM effectors such as Ecp6 from the fungal tomato pathogen Cladosporium fulvum and Slp1 from the rice blast fungus Magnaporthe oryzae bind chitin with high affinity and sequester by this cell wallderived chitin fragments to interfere with chitin detection by immune receptors and chitin-triggered immunity in the host (Figure 7) [START_REF] Sánchez-Vallet | Fungal effector Ecp6 outcompetes host immune receptor for chitin binding through intrachain LysM dimerization[END_REF][START_REF] Mentlak | Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease[END_REF].

Adapted from Kamoun 2006

Other LysM effectors such as Avr4 from C. fulvum protect fungal hyphae against degradation by hydrolytic enzymes secreted by the host [START_REF] Van Esse | The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor[END_REF] showing that widespread effectors with conserved domains can have versatile functions.

Among the effectors that act in the host cytoplasm, only few show conserved domains that give clues about their function. An example is the secreted chorismate mutase Cmu1 from the maize smut fungus Ustilago maydis that acts as an effector during infection. Chorismate mutase is a key enzyme of the shikimate pathway and catalyses the conversion of chorismate to prephenate the precursor for tyrosine and phenylalanine synthesis. Cmu1 has been shown to be translocated in the host cytoplasm and to reduce levels of the plant defence hormone SA (Salicylic acid) by removing the SA precursor chorismate [START_REF] Djamei | Metabolic priming by a secreted fungal effector[END_REF]. Secreted chorismate mutases are found in many fungal and nematode plant pathogens suggesting that this virulence mechanism to reduce plant SA levels is widespread [START_REF] Bekal | A chorismate mutase from the soybean cyst nematode Heterodera glycines shows polymorphisms that correlate with virulence[END_REF]. Similarly, the effector proteins Psls1 and Vdlsc1 from the two evolutionary distant filamentous pathogens, Phytophthora sojae and Verticillium dahlia, are secreted isochorismatases required for full virulence of the pathogens and acting by reducing the amount of SA in the plant cells.

Another example of a cytoplasmic effector with conserved domains is AVR-Pita from M. oryzae which shows homology to fungal zinc-dependent metalloproteases (Jia et al. 2000;[START_REF] Orbach | A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta[END_REF]. However, the molecular function and role in virulence of AVR-Pita has not been reported.

Effector delivery and translocation

Effector delivery from the pathogen is required to allow effector functions either at the interface with the host or inside plant cells. For this, most fungal effectors harbor an Nterminal secretion signal that directs them in the eukaryotic secretory pathway which involves passage through the Endoplasmic Reticulum (ER) and exocytosis of Golgi-derived secretory vesicles. In this way, effectors are secreted and get into the apoplast [START_REF] Panstruga | Terrific protein traffic: the mystery of effector protein delivery by filamentous plant pathogens[END_REF]. However, effectors acting in the host cytoplasm have to enter into plant cells and to pass the plasma-membrane barrier. To date, this step remains poorly understood. In oomycetes, the conserved motifs RxLR and LxLFLAK are found in a large number of effectors proteins and were shown to be required for host cell translocation [START_REF] Dou | RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery[END_REF][START_REF] Schornack | Ancient class of translocated oomycete effectors targets the host nucleus[END_REF]. It has been proposed that that RxLR motifs enable oomycete effectors to bind phosphatidylinositol-3-phosphate (PI3P) at the host cell surface and subsequently enter host cells through vesicle-mediated endocytosis [START_REF] Kale | External lipid PI3P mediates entry of eukaryotic pathogen effectors into plant and animal host cells[END_REF]. However, this was largely based on results from lipid binding assays and effector uptake assays that have been shown to be very unspecific. Therefore, this model and the corresponding data are largely debated and it is widely considered that the mechanism of RXLR effector uptake remain to be discovered. In fungi, similar conserved sequence motifs have not been identified with the exception of the powdery mildew fungi where 80% of the effector candidates share a conserved Y/F/WxC motif in the N-terminus. A role of this motif in translocation has however not been demonstrated [START_REF] Godfrey | Powdery mildew fungal effector candidates share N-terminal Y/F/WxC-motif[END_REF]. A clear limitation for a better understanding of effector translocation is the lack of reliable effector uptake assays. Their development represents a crucial next step and challenge in fungal and oomycete effector research [START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF].

Evolution of fungal effector proteins

Effectors evolve to optimize the virulence function of the pathogen and to escape from plant recognition. In turn, plants evolve immune receptors that frequently act as resistance proteins to recognize effector and impair pathogen development. This continuous interaction between effectors and plant immune receptors results in certain cases in a boom-and-bust cycle [START_REF] Brown | Plant-parasite coevolution: bridging the gap between genetics and ecology[END_REF]. On the one hand Avr effectors are selected when resistant genes are in low proportion but are eliminated when resistance genes are abundant, on the other hand resistance genes are selected when avr effectors are common and eliminated when avr effectors are rare. As consequence of this boom-and-bust cycle, co-evolution between effectors and resistance genes may be explained by basically two different models [START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF]. The arms race model in which both pathogens and plants will continuously develop new proteins, effectors and immune receptors respectively, that will temporarily fixed in the population (Dawkins & Krebs, 1979) (Figure 5). On the contrary, in the trench warfare model Fungal genome-wide analyses have unrevealed a huge repertoire of effector molecules highly polymorphic in their presence or absence and sequence variation e.g., single-nucleotide polymorphisms (SNPs) or insertions and deletions (indels). Fungal effectors have also been

shown to exhibit signs of positive selection occurring when the ratio between the number of nonsynonymous substitutions and synonymous substitutions (dN/dS) is greater than one [START_REF] Joly | Comparative analysis of secreted protein evolution using expressed sequence tags from four poplar leaf rusts (Melampsora spp.)[END_REF][START_REF] Thines | Gene loss rather than gene gain is associated with a host jump from monocots to dicots in the Smut Fungus Melanopsichium pennsylvanicum[END_REF][START_REF] Hacquard | Mosaic genome structure of the barley powdery mildew pathogen and conservation of transcriptional programs in divergent hosts[END_REF]. Many effectors belong to multigene families that have diversified from a common ancestor [START_REF] Nemri | The genome sequence and effector complement of the flax rust pathogen Melampsora lini[END_REF][START_REF] Pendleton | Duplications and losses in gene families of rust pathogens highlight putative effectors[END_REF][START_REF] Stergiopoulos | In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins[END_REF]. These multigene families are frequently lineage specific but some of them have also been shown to be widespread across the fungal kingdom following multiple expansions [START_REF] Stergiopoulos | In silico characterization and molecular evolutionary analysis of a novel superfamily of fungal effector proteins[END_REF]. Evolution of pathogen effectors by diversifying multigene families is according to the birth-and-death model that proposes that new genes are created by gene duplication, and some duplicated genes are maintained in the genome for a long time, whereas others are deleted or become nonfunctional through deleterious mutations (Nei and Rooney, 2006). An example of effector diversification followed of specialization and host adaptation has been recently shown for the oomycetes pathogens Phytophtrora infestans and Phytophthora mirabilis infecting potato and Mirabilis jalapa respectively. In this work they showed that diversification of an effector belonging to the cystatin-like protease family changed the specificity of the pathogen toward it associated cysteine protease in the host plant leading to a host jump [START_REF] Dong | Effector specialization in a lineage of the Irish potato famine pathogen[END_REF].

Localization of effector proteins in the genome

Many putative fungal effectors have been found to co-localize in distinct genome compartments (Figure 6), such as gene-sparse genomic regions with high genome plasticity and enriched with repetitive elements, accessory chromosomes and AT-blocks [START_REF] Orbach | A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta[END_REF][START_REF] Hogenhout | Emerging concepts in effector biology of plant-associated organisms[END_REF]. The association of effectors with plastic genomic loci constitute an important strategy to increase the genetic variation and allow an accelerated evolution and adaptation to host resistance without affecting the evolution of essential or housekeeping genes evolving a slow rates [START_REF] Guttman | Microbial genome-enabled insights into plant -microorganism interactions[END_REF]. The co-localization of effectors with transposons rich regions also promotes the horizontal gene transfer, gene losses and the chimeras making of this genomic context an important hot spot for evolution of virulence traits (Rafaelle & Kamoun, 2012;[START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF]. The genome of the fungal pathogen

Leptosphaeria maculans represents one of the examples of genome organization and effector compartmentalization. In this genome e.g. the GC-rich blocks are enriched with housekeeping genes whereas the AT-blocks are gene-sparse, harbors mosaics of transposable elements and are enriched in putative effector genes [START_REF] Rouxel | Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations[END_REF][START_REF] Soyer | Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus Leptosphaeria maculans[END_REF]. Effector compartmentalization has also evidenced in the genome of Fusarium oxysporum. In this case it has been found that all known effector genes localize in one of the four F. oxysporum dispensable chromosomes which also contains lineage-specific (LS) genomic regions and transposons. Transferring of LS cromosomes between different F. oxysporum strains conferred pathogenicity to non-pathogenic strains showing that gene horizontal transfer may also be one of the strategies of fungal pathogens to exchange virulence factors and accelerates evolution [START_REF] Ma | Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium[END_REF].

Adapted from Lo Presti et al. 2016 With the advent of genome sequencing over the last 10 years, the genomes of many fungal pathogens became available allowing genome-wide searches for effector candidates that resemble these Avr-effectors. The criteria for such effector candidate searches were generally small size, frequently with a cut-off of <300 aa, presence of a secretion signal and lack of homology to other proteins [START_REF] Wouw | Fungal pathogenicity genes in the age of " omics[END_REF]. With these criteria, hundreds of effector candidates were identified in the genomes of individual fungal phytopathogens. In the powdery mildew fungus Blumeria graminis, 491 effector candidates were identified [START_REF] Pedersen | Structure and evolution of barley powdery mildew effector candidates[END_REF] while in the genome of P. graminis f. sp. Tritici, 1106 effector candidates were predicted [START_REF] Duplessis | Obligate biotrophy features unraveled by the genomic analysis of rust fungi[END_REF]. An important additional criterion to refine the search for effector candidates is in planta expression data since effectors show infection specific expression.

Indeed, in several cases effector expression is specific to particular infection stages or to the invasion of particular plant tissues. This has e.g. been documented for Colletotrichum higgensianum and C. graminearum where different waves of stage specific effectors have been identified [START_REF] O'connell | Lifestyle transitions in plant pathogenic Colletotrichum fungi deciphered by genome and transcriptome analyses[END_REF] as well as in U. maydis where host tissue specific effector repertoires were described [START_REF] Schilling | Virulence of the maize smut Ustilago maydis is shaped by organ-specific effectors[END_REF].

The recent development of dedicated bioinformatic tools allows now more precise and more reliable identification of fungal effectors. Pipelines using Markov clustering of similar sequences and hierarchical clustering with a set of at least 8 defining features for effectors allowed to identify restricted sets of high confidence effector candidates in different rust fungi and Sclerotinia sclerotiorum [START_REF] Saunders | Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi[END_REF][START_REF] Nemri | The genome sequence and effector complement of the flax rust pathogen Melampsora lini[END_REF][START_REF] Guyon | Secretome analysis reveals effector candidates associated with broad host range necrotrophy in the fungal plant pathogen Sclerotinia sclerotiorum[END_REF]. A machine learning approach based on the extraction of sequence-derived properties characteristic for experimentally validated effectors led to the development of the EFFECTORP pipeline that predicts effector candidates in fungal secretome with high accuracy [START_REF] Sperschneider | Methods E FFECTOR P : predicting fungal effector proteins from secretomes using machine learning[END_REF]. Features that discriminate fungal effectors from secreted noneffectors are predominantly protein length, weight and net charge, as well as cysteine, serine and tryptophan content. EFFECTORP is particularly powerful when combined with in planta expression data.

Bioinformatic analysis of fungal genome sequences has also provided valuable information e.g. about important features of effector repertoires. The secretome of 84 plant fungi with different lifestyles was stablished from their genome sequence essentially based on the presence of an N-terminal signal peptide, the absence of transmembrane domains and the assignation of functional domains [START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF]. Further, fungi were grouped according with their feeding strategies and individual secretomes were classified into three different categories; plant cell wall-degrading enzyme (PCWDE), secreted proteins with functional annotation except PCWDE and secreted proteins without functional annotation.

This study led to infer that the effector repertoire was determined by the life fungal style. For example, the proportion of PCWDE was higher in necrotrophs and hemibiotrophs than in biotrophs. This is consistent with the fact that both necrotrophic and hemibiotrophic fungus have to induce plant cell death to colonize plant cells, growth and reproduction. Consequently, hemibiothophs had the largest amount of secreted proteins because they included PCWDEs and putative effectors proteins whereas both biotrophs and necrotrophs had in proportion either putative effectors or PCWDEs respectively

Validation of fungal effectors

The most straightforward strategy to validate candidate effectors is the creation of loss of function mutants, e.g. by gene disruption or RNAi, and further confirmation that such pathogen mutants have reduced virulence. However, in several cases knock-out of an individual effector has little or no impact on virulence probably due to redundancies in effector function. Indeed, it is believed that multiple effectors in the huge effector arsenals of phytopathogenic fungi target redundantly host cellular pathways important for infection. Loss of individual effectors has therefore in most cases only a small impact on virulence [START_REF] Guttman | Microbial genome-enabled insights into plant -microorganism interactions[END_REF]. This is e.g. illustrated in a study in M. oryzae where among mutants for 78 different effector candidates expressed during early infection only one, Δmc69, had an infection phenotype [START_REF] Saitoh | Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens[END_REF].

Other common strategies for the validation and functional characterization of effectors are protein-protein interaction analyses, screening for inducers and suppressors of plant cell death or PTI responses and subcellular localization [START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF][START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF]. This have led to the validation of a steadily increasing number of fungal effectors and the elucidation of their role in virulence (reviewed in [START_REF] De Jonge | How filamentous pathogens co-opt plants: the ins and outs of fungal effectors[END_REF][START_REF] Rovenich | Filamentous pathogen effector functions: Of pathogens, hosts and microbiomes[END_REF]Giraldo & Valent 2013;[START_REF] Wit | Fungal effector proteins : past , present and future[END_REF][START_REF] Stergiopoulos | Fungal effector proteins[END_REF]Lo Presti et al. 2015a;[START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF][START_REF] Selin | Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges[END_REF]. In particular large scale screens for effectors in heterologous or homologous experimental systems as cell death inducers or suppressors were quite successful and identified potential cell death inducers or suppressors. However, their localization (apoplasm or host cytoplasm) and their precise molecular activity remain unknown. For example, the transient expression of 70 candidate effector proteins from the necrotrophic apple canker fungus Valsa mali in N. benthamiana identified seven effectors suppressing plant cell death induced by BAX, a pro-apoptotic mouse protein that induces a cell death response similar to the defense-related HR [START_REF] Lacomme | Bax-induced cell death in tobacco is similar to the hypersensitive response[END_REF][START_REF] Li | Candidate effector proteins of the necrotrophic apple canker pathogen Valsa mali can suppress BAX-induced PCD[END_REF]. Knock-out of one of these 7 effectors, VmEP1, resulted in a significant reduction in virulence of the pathogen. This suggests that the suppression of cell death usually associated with ETI, is also important in the interaction of this necrotrophic fungus with its host plant [START_REF] Li | Candidate effector proteins of the necrotrophic apple canker pathogen Valsa mali can suppress BAX-induced PCD[END_REF].

Similar large-scale effector analysis revealed that effectors typically act at specific stages during infection. Colletotrichum higginsianum effectors involved in cell death suppression are e.g. specifically expressed during the early stages of disease whereas effectors involved in cell death induction are expressed in the late stages of disease development [START_REF] Kleemann | Sequential delivery of host-induced virulence effectors by appressoria and intracellular hyphae of the phytopathogen colletotrichum higginsianum[END_REF]. Other effectors like Pep1 from U. maydis that accumulates in the apoplastic space, at sites of fungal cell-to-cell passages are needed during penetration into the host tissue [START_REF] Doehlemann | Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells[END_REF]. Indeed, Δpep1 mutant strains were unable to penetrate epidermal cells and elicited a strong plant defense response revealing an important role for Pep1 in U. maydis virulence.

Function of fungal effectors

Overall, only for a limited number of fungal effectors, we have a detailed understanding of their molecular activity and their role during infection [START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF][START_REF] Selin | Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges[END_REF][START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF]. Further optimization of high-throughput approaches and development of new methods for effector analysis are therefore now a crucial challenge to functionally characterize the large effector repertoires identified in plant pathogen fungi and to get a better understanding of their action [START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF]. Our present knowledge on fungal effectors indicates that they employ highly diverse modes of action and that many of them target common effector host targets such as plant proteases, the ubiquitin-proteasome system or phytohormone signaling and homeostasis.

Apoplastic effectors

Most likely the functions of apoplastic effectors are related to enzyme inhibitors as well as prevention of PTI [START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF][START_REF] Selin | Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges[END_REF][START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF]. For example, C. fulvum secretes Ecp6, an effector containing a LysM chitin-binding domain which binds selectively to the chitin oligosaccharides, preventing their recognition by host PRRs (de Jonge et al., 2010). To limit the release of chitin oligosaccharides, C. fulvum also secretes the Avr4 effector which binds to chitin in the intact fungal cell wall, preventing its hydrolysis by host chitinases (Figure 7) [START_REF] Van Esse | The chitin-binding Cladosporium fulvum effector protein Avr4 is a virulence factor[END_REF]. In other cases, apoplastic effectors act in a more direct manner in the inactivation of plant proteins involved in defense. This is e.g. the case of Avr2 also from C. fulvum that interacts with the plant proteases PIP1 and Rcr3 and inhibits their functions (Figure 7) [START_REF] Van Esse | The Cladosporium fulvum Virulence Protein Avr2 Inhibits Host Proteases Required for Basal Defense[END_REF]. Similarly, Pit2, an effector from U. maydis required for virulence directly inhibits a group of apoplastic maize cysteine proteases that act downstream of SA signaling in the activation of maize defense responses [START_REF] Mueller | Compatibility in the Ustilago maydis-Maize Interaction Requires Inhibition of Host Cysteine Proteases by the Fungal Effector Pit2[END_REF] and Pep1 also from U. maydis inhibits POX12, a peroxidase protein secreted from maize to counteract pathogen attack (Figure 7) [START_REF] Doehlemann | Pep1, a secreted effector protein of Ustilago maydis, is required for successful invasion of plant cells[END_REF].

Cytoplasmic effectors

Cytoplasmic effectors employ different strategies to manipulate host cellular functions. An example is the effector protein Tin2 from U. maydis that targets an important secondary metabolite pathway in plant defense. Tin2 interacts with the cytoplasmic host protein ZmTTK1 that is involved in anthocyanin biosynthesis. Tin2-ZmTTK1 interaction stabilizes ZmTTK1 and increases anthocyanin production provoking a reduction of p-coumaric acid levels required for the lignification of plant cell walls to form physical barriers and avoid pathogen spread (Figure 7) [START_REF] Tanaka | A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize[END_REF]. Cmu1, another effector form U. maydis is a secreted and translocated chorismate mutase that reduces host SA levels by reducing the pool of the biosynthetic precursor chorismate as was mentioned in the previous section (Figure 7). AvrPiz-t an avreffector from M. oryzae is recognized by the rice resistance gene Piz-t (Figure 7) [START_REF] Li | The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t[END_REF]. In absence of the resistance gene, AvrPiz-t interacts with APIP6, a RING E3 ubiquitin ligase involved in plant immunity, resulting in an APIP6 loss of function [START_REF] Park | The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice[END_REF].

Adapted from Lo Presti et al. 2015

Plant immune receptors

Plant surface immune receptors

Plants can detect pathogen invasion by the recognition of self or nonself-derived patterns either on the cell surface or in the cytoplasm. Plant cell surface immune receptors, frequently named Pattern Recognition Receptors (PRR) typically recognize small epitopes from invasion patterns present in the plant apoplast [START_REF] Boller | A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors[END_REF][START_REF] Zipfel | Pattern-recognition receptors in plant innate immunity[END_REF]. The two main classes of PRRs are the Receptor Like Kinases (RLKs) composed of an ectodomain, a transmembrane domain and an intracellular kinase domain and the Receptor Like Proteins (RLPs) that are similar to RLKs in their structural organization but do not possess an intracellular kinase domain [START_REF] Macho | Plant PRRs and the activation of innate immune signaling[END_REF][START_REF] Böhm | Immune receptor complexes at the plant cell surface[END_REF]. The extracellular domains of PRRs are very diverse and can contain e.g. Leucine Rich-Repeats (LRR), lysine motifs (LysMs), lectin motifs or and epidermal growth factor (EGF)-like domains (Figure 8).

LRR-type PRRs usually bind to peptides such as flg22, a fragment of bacterial flagellin whereas LysM and EGF-type PRRs recognize carbohydrate-containing molecules such as chitin, bacterial peptidoglycans, extracellular ATP, or plant-cell-wall-derived oligogalacturonides [START_REF] Brutus | A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides[END_REF][START_REF] Choi | Identification of a plant receptor for extracellular ATP[END_REF][START_REF] Kaku | Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor[END_REF][START_REF] Miya | CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis[END_REF][START_REF] Willmann | Mediate Bacterial Peptidoglycan Sensing and Immunity To Bacterial Infection[END_REF]. PRRs ligand perception occurs via ectodomains and induces the formation of PRR homo-or heterocomplexes, the activation of intracellular kinase domains and the phosphorylation of substrates that contribute to intracellular signal transduction and activation of plant defense responses.

For instance, in the model plant Arabidopsis thaliana, LYK5 (Lysin motif receptor kinase 5) and CERK1 (Chitin Elicitor Receptor Kinase-1) cooperate in the perception of chitin-oligomers [START_REF] Miya | CERK1, a LysM receptor kinase, is essential for chitin elicitor signaling in Arabidopsis[END_REF][START_REF] Wan | A LysM receptor-like kinase plays a critical role in chitin signaling and fungal resistance in Arabidopsis[END_REF][START_REF] Cao | The kinase LYK5 is a major chitin receptor in Arabidopsis and forms a chitin-induced complex with related kinase CERK1[END_REF]). On the contrary to CERK1 that only possess moderate chitin-binding affinity, LYK5 binds chitin oligomers with very high affinity and acts as the primary chitin receptor. Chitin-binding by LYK5 induces formation of a LYK5/CERK1 heterocomplex resulting in phosphorylation of CERK1 and activation of immune signaling.

Interestingly, LYK5 like other LysM RLKs involved in Nod factor receptor (NFR) perception lacks intracellular kinase activity.

In monocotyledonous plants, chitin perception seems to occur by a slightly different mechanism. In rice e.g., CERK1 is recruited by the LysM-RLP CEBiP (Chitin elicitor-binding protein) and LYP4-LYP6 receptors [START_REF] Couto | Regulation of pattern recognition receptor signalling in plants[END_REF]. However, in this case CEBiP appears to be the primary high affinity chitin-binding receptor [START_REF] Kaku | Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor[END_REF][START_REF] Liu | Chitin-Induced Dimerization Activates a Plant Immune Receptor[END_REF]). After chitin binding, CEBiP forms a hetero-oligomeric receptor complex with OsCERK1, the rice ortholog of AtCERK1 which only possess one extracellular LysM domain and does not bind chitin. Subsequently, OsCERK1 activates chitin-mediated signaling and triggers immunity.

These two examples illustrate that pathogen ligand perception mediated by PRRs can be different between plants [START_REF] Shimizu | Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice[END_REF].

Intracellular immune receptors

Plants possess intracellular immune receptors that mediate recognition of both modified 'host-self' and invasion patterns in the cytoplasm [START_REF] Jones | The plant immune system[END_REF]Dodds and Rathjen, 2010c;[START_REF] Cook | Understanding Plant Immunity as a Surveillance System to Detect Invasion[END_REF]. The largest family of plant intracellular immune receptor proteins is the Nucleotide-binding domain and leucine-rich repeat proteins (NLRs). NLRs belong to the Signal Transduction ATPase with Numerous Domains (STAND) super family of proteins that are regulated by nucleotide-binding and intramolecular domain interactions [START_REF] Lukasik | STANDing strong, resistance proteins instigators of plant defence[END_REF][START_REF] Danot | How "arm-twisting" by the inducer triggers activation of the MalT transcription factor, a typical signal transduction ATPase with numerous domains (STAND)[END_REF]. NLRs are present in all eukaryotic organisms and act as molecular switches in the regulation of various processes such as activation of immune responses, regulation of abiotic stresses and apoptosis (Goverse 2012; Jacob et al. 2013;Collier & Moffett 2009;[START_REF] Bernoux | Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model[END_REF]Takken &).

NLRs present a modular architecture with a central nucleotide-binding domain (NB-ARC), a

Leucine-rich repeat domain (LRR) at the C-terminus and a coiled-coil (CC) or a Toll Interleukin-1-like receptor (TIR) domain at the N-terminus (Jacob et al., 2013). Typically, NLRs are arranged in TNL or CNL (TIR (T), CC (C), NB-ARC (N), and LRR (L)) configuration but alternative configurations such as "truncate" forms, TNTNL, TNLT, CNNL, TCNL can be also found (Figure 9) [START_REF] Meyers | Genome-wide analysis of NBS-LRR -encoding genes in Arabidopsis[END_REF][START_REF] Meyers | TIR-X and TIR-NBS proteins: two new families related to disease resistance TIR-NBS-LRR proteins encoded in Arabidopsisand other plant genomes[END_REF]Jacob et al., 2013). In solanaceas an N-terminal domain different to CC and TIR has been frequently found. This domain is called SD (solanacea domain) because is restrict to Solanaceae and is usually found in SDCNL configuration [START_REF] Lukasik-Shreepaathy | Dual regulatory roles of the extended N terminus for activation of the tomato MI-1.2 resistance protein[END_REF].

NLRs can also carry non-canonical domains integrated at low frequencies (Césari, et al. 2014;[START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF][START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF]). These integrated domains correspond to a wide range of molecular and functional categories such as signal transduction proteins, transcription factors or metabolic enzymes. Interestingly, many of them are present in regulators or actors of plant immunity and/or in targets of pathogen effectors. For instance, RIN4 one of the most studied effector targets which interacts with many NLRs including RPS2 and RPM1 has been found to be fused to different NLRs in different plants such as rice, barley and apple [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF].

In addition, many of the integrated domains fused to plant NLRs have been found to interact with effectors in targeted studies or effector interactome screens [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. This suggests that effector recognition is a general feature of integrated domains similar to what has been demonstrated experimentally for Pik-1 and RRS1 (Maqbool et al., 2015a;Le Roux et al., 2015;Sarris et al., 2015). Functional analysis of CC and TIR domains suggest that both domains are involved in downstream signaling and cell death triggering (Qi and Innes, 2013). Transient expression of the TIR 1-248 fragment from the flax resistance proteins L10, L6, L2 and L7 triggered effectorindependent cell death in flax leaves [START_REF] Frost | Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defense responses[END_REF]Bernoux et al., 2011b). Point mutations in conserved amino acids of the L6-TIR 1-248 domain abolished cell death induction but did not affect the interaction between L6 and its cognate avirulence effector protein AvrL567 from Melampsora lini showing that the TIR domain from L6 is not required for effector binding but is necessary and sufficient for immune signaling (Bernoux et al., 2011b).

Furthermore, it was shown that the L6-TIR domain self-associates and forms homodimers that are required for signaling since L6-TIR mutants impaired in self-association lost the ability to trigger cell death (Bernoux, et al. 2011).

Similarly, the CC domain of the barley resistance protein MLA is sufficient to induce cell death and self-associates in vivo, even in the absence of the Avr effector. As with the L6-TIR domain, self-association of the MLA-CC domain is required for immune signaling (Maekawa, et al. 2011). However, isolated CC or TIR domains are not always able to trigger cell death and, in certain cases, others NLR domains seem to activate immune signaling. For example, the overexpression of the of the CC domain of the resistance proteins Rx or RPS5 from potato and A. thaliana respectively did not induce cell death but the overexpression of the Rx NB domain did suggesting that in some cases the NB domain is engaging downstream signaling components and triggers immune signaling [START_REF] Rairdan | The coiled-coil and nucleotide binding domains of the Potato Rx disease resistance protein function in pathogen recognition and signaling[END_REF][START_REF] Ade | Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease[END_REF]. These different results may reflect important differences about the signaling mechanisms used by NLRs to trigger immunity.

Furthermore, many effector targets such as Pto, RIN4 and PBS1 has been shown to interact with the N-terminal domain of resistance proteins [START_REF] Mackey | Arabidopsis RIN4 is a target of the type III virulence effector AvrRpt2 and modulates RPS2-mediated resistance[END_REF][START_REF] Mucyn | The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity[END_REF][START_REF] Ade | Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease[END_REF] indicating that this domain is involved in effector recognition. For instance, N-terminal domain SD of the tomato resistance protein Prf interacts with the kinase effector target protein Pto and mediate recognition to the effector proteins AvrPto and AvrPtoB from

Peudomonas syringae [START_REF] Mucyn | The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity[END_REF][START_REF] Balmuth | Genetic and molecular requirements for function of the Pto/Prf effector recognition complex in tomato and Nicotiana benthamiana[END_REF][START_REF] Saur | The N-Terminal Domain of the Tomato Immune Protein Prf Contains Multiple Homotypic and Pto Kinase Interaction Sites[END_REF]. Similarly, the CC domain of the resistance gene RPS5 has been shown to associate with PBS1 prior to PBS1 cleavage by the effector protein AvrPphB [START_REF] Ade | Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease[END_REF][START_REF] Qi | Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein[END_REF].

To date only the crystal structures of the isolated MLA10 and Rx1 CC domains have been resolved (Maekawa, et al. 2011;[START_REF] Hao | Structural basis for the interaction between the potato virus X resistance protein (Rx) and its cofactor ran GTPase-activating protein 2 (RanGAP2)[END_REF]. Despite the sequence similarity between these domains, structural analyses revealed that both domains adopt different topologies. The MLA10-CC domain is formed by three different α-helices connected by loops and forms homodimers (Figure 10) whereas CC from Rx1 consists of a more compact fold composed of four different α-helices in a helix bundle (Figure 10) that does not dimerize but instead interact with the conserved domain WPP (Trp-pro-pro) of the RanGAP2 protein which is required for Rx1 function.

The crystal structures of the TIR domain from the plant NLRs L6, RPS4 and RRS1 has been determined (Bernoux, et al. 2011;Williams et al. 2014). These domains consist of a flavodoxinlike fold formed by a five-stranded parallel β-sheet surrounded by five α-helices (Figure 10).

Surfaces involved in the formation of L6 and RPS4 TIR domain homodimers and RRS1/RPS4 TIR domain heterodimers has been also identified (Bernoux, et al. 2011;Williams et al. 2014).

NB-ARC the central switch domain

The NB-ARC domain is the most conserved domain in plant and animal NLRs and acts as a molecular switch that translates the recognition of effector proteins into signal initiation through intra and intermolecular interactions and nucleotide binding [START_REF] Hu | Crystal structure of NLRC4 reveals its autoinhibition mechanism[END_REF]Takken & Goverse 2012). Indeed, the NB-ARC domain consists of a nucleotide-binding pocket. In the inactive or "off" state, the nucleotide-binding pocket adopts a "closed" configuration where ADP is preferentially bound and stabilizes the closed structure by mediating intramolecular interactions. The activation or "on" state of the NB-ARC is mediated by the release of the ADP which is replaced by ATP (Bernoux et al., 2011a;Williams et al., 2011b). After ATP-binding the intramolecular interactions of the protein are modified and the pocket adopts an "open" configuration that is required to mediate defense responses. Thus, the central NB-ARC domain functions as a molecular switch that fluctuate between an "off" and "on" state depending on ADP-or ATP-binding respectively. The most conserved part of the NB domain is the Walker-A motif, also called phosphate-binding loop (P-loop) which is a glycine-rich flexible loop that is crucial for ATP-binding. Indeed, the p-loop coordinates together with other amino acids a magnesium cation that binds the β and ϒ phosphates and thereby properly positions ATP. In addition, a highly conserved lysine residue in the P-loop interacts directly with the β and ϒ phosphate groups of the nucleotide and is indispensable for its binding [START_REF] Walker | nucleotide binding fold Figure IA ). Approximately 220 / of amino acid residues along that useATP Homology between a and ( 3 and otherenzymes[END_REF].

Mutations in the NB-ARC domains that weaken ATP binding or stabilize the fixation of ADP result in loss-off function while mutations that weaken ADP-binding or strengthen ATP binding lead to gain of function, autoactive NLR mutants. One of the conserved structural motifs that can be distinguished in the NB-ARC domain is the MHD (Met-His-Asp) motif. Direct mutagenesis in MHD motif of many NLRs result in a spontaneous induction of the defense and cell death responses [START_REF] Bendahmane | Constitutive gain-offunction mutants in a nucleotide binding site-leucine rich repeat protein encoded at the Rx locus of potato[END_REF][START_REF] Howles | Autoactive Alleles of the Flax L6 Rust Resistance Gene Induce Non-Race-Specific Rust Resistance Associated with the Hypersensitive Response[END_REF][START_REF] Van Ooijen | Structure-function analysis of the NB-ARC domain of plant disease resistance proteins[END_REF][START_REF] Williams | An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP[END_REF]. For instance, site-directed mutation of key residues within the MHD motif of the resistance protein M resulted in autoactivation and ATP binding whereas mutations in important residues within the P-loop motif of M resulted in a loss of nucleotide binding and the inactivation of the resistance protein [START_REF] Williams | An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP[END_REF] To date, the three dimensional structure of an NB-ARC domain from a plant NLRs has not been determined but different models have been proposed based in the crystal structures of the NB-ARC domains from the human and Caenorhabditis elegans proteins Apaf-1 and CED-4 respectively, that are both involved in apoptosis [START_REF] Zou | Apaf-1, a human protein homologous to c.elegans CED-4, participates in cytochrome c dependent activation of caspace 3[END_REF]Yan et al. 2005;Shiozaki et al. 2002). These structures show that the NB-ARC domain consists of three structural subunits, notably NB, ARC1 and ARC2 that together form the nucleotide-binding pocket (Figure 10).

LRR the C-terminal domain

The LRR domain is defined by a repeated sequence motif in which hydrophobic residues that are often leucine alternate with hydrophilic residues in a fixed pattern (consensus motif LxxLxLxxNxL). It is the most polymorphic domain of plant NLR proteins and has been demonstrated to play a crucial role in the recognition of effector proteins in several NLR resistance proteins such as RPP1, RPS5 and L [START_REF] Dodds | Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes[END_REF]Qi & Innes 2013;Steinbrenner et al. 2015). For example, seven variants of the effector AvrL567 from the flax rust fungus Melampsora lini are differentially recognized by the extremely similar NLR proteins L5, L6 and L7 from flax. The specificity of this recognition was shown to be determined by polymorphic amino acids in the LRR domains of the different L variants. This matching specificity between AVRL567 alleles and the LRR domain of L alleles may be due to direct, receptor-ligand type interaction since physical interaction between the LRR domain from L and AvrL567 variants was demonstrated by yeast two hybrid assays [START_REF] Dodds | Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes[END_REF]. Similarly, the recognition of different alleles of the ATR1 effector from the downy mildew pathogen Hyaloperonospora arabidopsidis was explained by polymorphic amino-acids and the number of leucine repetitions in the LRR domain of RPP1 alleles from different A. thaliana accessions. The specificity of this recognition was recapitulated by co-immunoprecipitation experiments [START_REF] Rehmany | Differential Recognition of Highly Divergent Downy Mildew Avirulence Gene Alleles by RPP1 Resistance Genes from Two Arabidopsis Lines[END_REF]Krasileva et al. 2010).

In addition to their role in signal perception, the LRR domain plays also an important role in keeping NLR proteins in a "off" state since deletions or mutations in LRR domains frequently result in auto-activation [START_REF] Qi | Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein[END_REF][START_REF] Ade | Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease[END_REF][START_REF] Tameling | RanGAP2 mediates nucleocytoplasmic partitioning of the NB-LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function[END_REF]. This dual role was very convincingly shown by systematic swapping of polymorphic sites between the potato immune receptors Gpa2 and Rx1 that possess high sequence homology but different recognition specificity. A small region in the ARC2 and N-terminal repeats of the LRR domain was found to control the activation state of the proteins whereas the C-terminal LRR repeats were found to determine the specificity of the recognition of the nematode Globodera pallida by Gpa2 and of Potato virus X by Rx1 [START_REF] Slootweg | Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2[END_REF]. Furthermore, in silico modeling combined with structure-informed functional analysis revealed that the opposite charge distribution between the N and C terminal part of the LRR domain determines the dual function of the LRR as an effector sensor and regulator of NLR activity and that intramolecular interactions between the N-terminus of the LRR and the NB-ARC from Gpa2 and Rx1 are mediated by conserved basic residues in the LRR [START_REF] Slootweg | Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2[END_REF]. In RPS5, truncations in the N-terminal part of the LRR showed that the first four repeats are sufficient to inhibit autoactivation but that a full length LRR domain was required to mediate effector recognition.

In addition, swapping of the LRR domain between RPS5 and the closely related NLR RPS2 resulted in autoactivation of RPS5, demonstrating a fine tuned co-evolution between the LRR and NB-ARC domains [START_REF] Qi | Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein[END_REF].

The 3 dimensional structure of LRR domains from plant NLRs has not yet been determined but those from other LRR domain proteins and in particular from animal NLRs are available [START_REF] Hu | Crystal structure of NLRC4 reveals its autoinhibition mechanism[END_REF]. Basically, LRRs display an arc-shape or horse-shoe-like structure where the repeated leucine-rich motifs form a parallel β-sheet at the concave side (Figure 10). The leucine form the hydrophobic core of the β-sheet and the other residues are exposed to the surface. This surface of the concave side is supposed to be engaged in the intramolecular interactions with the NB-ARC domain in the N-terminus of the LRR and to establish protein-protein interactions with ligands that mediate effector recognition in the C-terminus of the LRR. These different Either, they directly bind the effector protein alone, or in complex with a host protein that serves as a co-factor or they recognize host target protein modifications induced by effector proteins [START_REF] Chisholm | Host-microbe interactions: shaping the evolution of the plant immune response[END_REF][START_REF] Jones | The plant immune system[END_REF]van der Hoorn and Kamoun, 2008). In the first case, effector recognition occurs in a receptor-ligand-like mode and in the second case, recognition is indirect and follows the guard or decoy model.

Receptor-ligand model

Direct interaction between effectors and resistance proteins has been demonstrated in few cases. The best documented examples are L6 and Pi-ta from flax and rice respectively (Figure 11) [START_REF] Dodds | Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes[END_REF]Yulin Jia, McAdams, et al. 2000) L6 has been shown to recognize the AvrL567 effector protein in an allele-specific manner and the specificity of this recognition has been recapitulated by the physical interaction of the effector protein with the corresponding resistance protein over a spectrum of alleles and mutants in yeast two hybrids assays [START_REF] Dodds | Direct protein interaction underlies gene-for-gene specificity and coevolution of the flax resistance genes and flax rust avirulence genes[END_REF]. For ATR1 recognition by RPP1, a similar direct interaction model has been proposed since the recognition of ATR1 alleles by matching RPP1 alleles correlates with coimmunoprecipitation of the protein (Krasileva et al. 2010). However, an important role of additional host proteins e.g. co-factors that are required to mediate ATR1 and RPP1

interaction cannot be excluded by these experiments. Involvement of a host-cofactor in effector recognition has been shown in the case of the NLR N from tobacco that recognizes the p50 protein of tobacco mosaic virus. P50 forms a protein complex with N as demonstrated by co-immunoprecipitation assays. However, this association and the resulting immune activation requires the host protein NRIP1 that binds on the one hand p50 and on the other the TIR domain of N [START_REF] Peart | NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein, mediates resistance against tobacco mosaic virus[END_REF][START_REF] Ueda | Direct interaction between the tobacco mosaic virus helicase domain and the ATP-bound resistance protein, N factor during the hypersensitive response in tobacco plants[END_REF][START_REF] Burch-Smith | A novel role for the TIR domain in association with pathogen-derived elicitors[END_REF][START_REF] Caplan | Chloroplastic Protein NRIP1 Mediates Innate??Immune Receptor Recognition of a Viral Effector[END_REF]. Decoys can also be fused to NLRs to allow efficient effector recognition (D). Adapted from [START_REF] Khan | Of guards , decoys , baits and traps : pathogen perception in plants by type III effector sensors[END_REF] The guard/decoy model Alternatively, NLRs can recognize effectors in an indirect manner that does not involve direct association but relies on the sensing of effector-triggered modifications in host proteins.These effector-modified and NLR-guarded host proteins may be either the true effector targets frequently involved in plant immunity or mimics of the real target that solely monitor effector activity . In the first case, the host proteins surveyed by the NLR and modified by the effector are called guardees, in the second case they are called a decoys [START_REF] Dangl | Defence Responses To Infection[END_REF][START_REF] Dangl | Two modes of pathogen recognition by plants[END_REF]van der Hoorn and Kamoun, 2008). Decoy proteins are thought to evolve from the real targets to avoid the conflict of having two opposite selection pressures acting in the same protein (Van der Hoorn & Kamoun 2008). The corresponding modes of recognition are described by the so-called guard model or the decoy model.

One of the best characterized examples of the guard model is the indirect recognition of the effector proteins AvrRpt2 by RPS2 and AvrRpm1/AvrB by RPM1 [START_REF] Chung | Specific threonine phosphorylation of a host target by two unrelated type III effectors activates a host innate immune receptor in plants[END_REF][START_REF] Day | Molecular Basis for the RIN4 Negative Regulation of RPS2 Disease Resistance[END_REF][START_REF] Kim | The Pseudomonas syringae effector AvrRpt2 cleaves its C-terminally acylated target, RIN4, from Arabidopsis membranes to block RPM1 activation[END_REF] (Figure 12). In this case, both immune receptors, RPS2 and RPM1 recognize the presence of their cognate effector proteins by sensing modifications in the host target protein RIN4. RIN4 is involved in plant immunity and contributes to pathogen fitness in absence of RPS2 and RPM1. As a consequence, the guarded effector target RIN4 is exposed to opposing natural selection forces. On the one hand, in the absence of a functional resistance protein, it is favorable to change to evade modification by the effector. On the other hand, when a guarding NLR is present, interaction of the guarded effector target RIN4 with the effector protein is beneficial to enable pathogen perception.

One of the best characterized systems operating according to the decoy model is the tomato NLR Prf that recognizes the effector proteins AvrPto and AvrPtoB from Pseudomonas syringae by sensing the modifications in the protein kinase Pto (Figure 12). Pto does not have a role in immunity and seems to act as a decoy for the true AvrPto and AvrPtoB protein kinase targets.

Indeed, both AvrPto and AvrPtoB target multiple RLKs including CERK1, EFR1 and FLS2 that have an established role in immunity and thereby contribute to virulence of Pseudomonas syringe [START_REF] Mucyn | The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity[END_REF][START_REF] Gutierrez | Prf immune complexes of tomato are oligomeric and contain multiple Ptolike kinases that diversify effector recognition[END_REF][START_REF] Ntoukakis | The changing of the guard: The Pto/Prf receptor complex of tomato and pathogen recognition[END_REF]. Similarly, the NLRs RPS5 and ZAR1 recognize the effector proteins AvrPphB and AvrAC respectively, by sensing perturbations in the decoy kinase proteins PBS1 and PBL2 [START_REF] Shao | Cleavage of Arabidopsis PBS1 by a bacterial type III effector[END_REF][START_REF] Ade | Indirect activation of a plant nucleotide binding site-leucine-rich repeat protein by a bacterial protease[END_REF][START_REF] Wang | The Decoy Substrate of a Pathogen Effector and a Pseudokinase Specify Pathogen-Induced Modified-Self Recognition and Immunity in Plants[END_REF] Integrated decoy model

Recently, a third, intermediate mode of action has been discovered in NLRs that carry unconventional integrated domains (Césari et al. 2014). It was shown that the unusual domains that are found in these NLRs mediate effector recognition by either binding them directly or being modified by them. From this, it was concluded that they act as decoy proteins that are integrated into the immune receptor to mediate pathogen perception [START_REF] Ellis | Integrated decoys and effector traps: how to catch a plant pathogen[END_REF]. An example that supports the integrated decoy model is the recognition of the effector protein

PopP2 by the NLRs pair RRS1/RPS4 (Figure 12). PopP2 is an acetyltransferase that inactivates host WRKY transcription factors involved in plant immunity by acetylating a conserved lysine in their DNA-binding domain (Le Roux et al., 2015;Sarris et al., 2015). RRS1 carries a WRKY domain that is not directly involved in the activation of immunity but is acetylated by PopP2

and is crucial for PopP2 perception. Therefore, the WRKY domain of RRS1 is interpreted as a decoy for the true WRKY targets of PopP2 that has lost its original function as a transcription factor but is still able to assist RSS1 in the perception of the biochemical activity of PopP2 (Figure 12) (Le Roux et al., 2015;Sarris et al., 2015). The RRS1 WRKY domain is also involved in the perception of a second effector, AvrRps4. However, the precise mechanisms of AvrRPS4 recognition and the true targets of this effector remain to be identified.

In rice, the NLR Pik-1 possesses an integrated HMA (heavy metal-associated) domain that is involved in the perception of the effector protein AVR-Pik from M. oryzae. Functional analysis showed that the HMA domain of Pik-1 interacts directly with surface exposed residues of the avirulence effector and that the affinity of this binding determines effector recognition (Maqbool et al. 2015). In addition, recent work in the group of Dr. Terauchi (Iwate, Japan)

showed that rice HMA proteins are virulence targets of AVR-Pik (personal communication, unpublished). Therefore, the HMA domain of Pik-1 seems to act indeed as a decoy for the host targets of AVR-Pik. An evolutionary advantage of this mode of recognition could be that it avoids the segregation between the decoy host target and the NLR receptor.

Frequently, NLRs pairs mediate disease resistance

In many cases single NLRs seem sufficient to mediate disease resistance. However, recently an increasing number of cases were reported where the resistance response to a single Avr gene product is mediated by NLRs pairs (Eitas and Dangl, 2010b). The first example of disease resistance conferred by NLR pairs were RPP2A/RPP2B from A. thaliana that are both required for immunity against certain Hyaloperonospora arabidopsidis isolates [START_REF] Sinapidou | Two TIR:NB:LRR genes are required to specify resistance to Peronospora parasitica isolate Cala2 in Arabidopsis[END_REF].

Similarly, the NLRs pairs, N/NRG1 and RPM1/TAO1 mediate recognition of the effector proteins p50 from Tobacco Mosaic Virus and AvrB from P. syringae respectively [START_REF] Peart | NRG1, a CC-NB-LRR protein, together with N, a TIR-NB-LRR protein, mediates resistance against tobacco mosaic virus[END_REF][START_REF] Eitas | Arabidopsis TAO1 is a TIR-NB-LRR protein that contributes to disease resistance induced by the Pseudomonas syringae effector AvrB[END_REF]. Other NLR pairs such us Lr10/RGA2, Pi5-1/Pi5-2 and Pikm1/Pikm2 mediate resistance to fungal pathogens [START_REF] Loutre | Two different CC-NBS-LRR genes are required for Lr10-mediated leaf rust resistance in tetraploid and hexaploid wheat[END_REF][START_REF] Lee | Rice Pi5-mediated resistance to Magnaporthe oryzae requires the presence of two coiled-coil-nucleotide-binding-leucine-rich repeat genes[END_REF][START_REF] Ashikawa | Two adjacent nucleotide-binding site-leucine-rich repeat class genes are required to confer Pikm-specific rice blast resistance[END_REF]. Furthermore, the pairs RGA5/RGA4 from rice and RRS1/RPS4 from A. thaliana recognize each at least two unrelated Avr-effectors [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF][START_REF] Narusaka | RRS1 and RPS4 provide a dual Resistance-gene system against fungal and bacterial pathogens[END_REF].

These examples indicate that resistance mediated by NLR pairs is widespread and confers immunity to different and in some cases multiple pathogens (Figure 13). The genes coding for NLR pairs are genetically tightly linked and show generally a very typical clustered genomic organization characterized by an inverted tandem arrangement with a short shared 5' intergenic region (Figure 14). The only exception is the RPP2A/RPP2B pair, where both genes are adjacent in the genome but not arranged in an inverted orientation (Figure 14).

This characteristic genomic organization is thought to contribute to a concerted regulation of the transcription of the two genes of one pair and to avoid their separation by segregation and recombination which would cause resistance loss and may result in autoimmune phenotypes (Eitas & Dangl 2010).

Recent studies on RPS4/RRS1 and RGA4/RGA5 gave first insight into the mechanism of effector recognition by paired NLRs. In both cases the NLR pairs function as receptor complexes in which one partner (RRS1 or RGA5) acts as a receptor or sensor of the effector while the other (RGA4 or RPS4) acts as a cell death executor (Bernoux et al., 2014;Williams et al., 2014). In animals, a comparable mechanism of NLR functioning in pairs has been revealed in the case of NAIP/NLRC4 in mice. NAIP act as primary immune receptor and directly bind non-self ligands.

This recognition event induces hetero-complex formation with NLRC4, oligomerization of NLRC4 and activation of defense signaling [START_REF] Kofoed | Innate immune recognition of bacterial ligands by NAIPs determines inflammasome specificity[END_REF][START_REF] Tenthorey | Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes[END_REF].

Activation of NLR resistance proteins

Since NLR proteins participate in the recognition of effector proteins and resistance induction, a fine-tuned regulation of both tasks is needed to avoid autoimmune responses due to unnecessary activation in the absence of a pathogen whereas a rapid response is expected upon pathogen attack [START_REF] Takken | To nibble at plant resistance proteins[END_REF]. In the absence of pathogens, NLRs predominately occur in a closed inactive conformation that is determined on the one hand by binding of an ADP molecule and on the other hand by intra and intermolecular interactions [START_REF] Sukarta | Structure-Informed Insights for Nlr Functioning in Plant Immunity[END_REF]. How NLRs switch upon pathogen perception from this inactive state to the active conformation is still not well understood. In the cases where effectors interact directly with the NLR, the activation has been explained by mostly three different models, a simple "switch" model, the "bait and switch model" and the "equilibrium-based switch" model [START_REF] Sukarta | Structure-Informed Insights for Nlr Functioning in Plant Immunity[END_REF].

The switch model is based on the assumption that the NB-ARC domain acts as a molecular switch to regulate the activity of the NLR receptor (Takken et al., 2006;Faustin et al., 2007).

Recognition of an effector by the inactive NLR which is in the ADP-bound/OFF state has consequences on the entire protein and results in a conformational change that triggers exchange of ADP for ATP and transition to the active/ON state and activate defense signaling (Figure 15). The conformational change is supposed to affect in particular the surfaces of the N-terminus of the LRR and the ARC2 that mediate inhibitory interactions and the alteration in the nucleotide binding status, from ADP to ATP is believed to lead to an open configuration of the NLR that exposes the N-terminal domain and facilities additional intermolecular interactions ultimately resulting in signal initiation.

Effector recognition may occur by direct binding like in the case of L6 and mainly involve the LRR domain or require co-factors that may be guardees, decoys or other host proteins (also defined as molecular baits of effectors) for effector recognition and activation. This latter situation has been further developed in the bait and switch model. This model state that cofactors or baits prime NB-LRR proteins to be functional and retain, in addition, the molecular switch in an inactive conformation until the effector-induced modification of the bait triggers release of autoinhibition. This bait modification can have multiple forms, such as posttranslational modification, cleavage, degradation or simply complex formation with the bait protein. However, at least some baits, such as NRIP1, interact with their cognate NB-LRR proteins only in the presence of the effector (Figure 15). In these cases, priming of the NLR has to occur independently of the bait and it is the binding of the effector-bait complex to the NLR that triggers the switch. This binding may involve as in the case of N multiple interactions between the effector-bait complex and different domains of the NLR.

"Equilibrium-based switch" model

Recent investigation of the mode of activation of L6 led to the development of the alternative equilibrium-based switch model. It's baseline is that even in the resting state, in the absence of effector, a small but significant part of the NLRs are in the ATP-loaded "ON" state and that effectors bind to these NLRs and not to the ADP-bound NLRs that are "OFF" as in the simple switch model [START_REF] Williams | An autoactive mutant of the M flax rust resistance protein has a preference for binding ATP, whereas wild-type M protein binds ADP[END_REF][START_REF] Bernoux | Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model[END_REF]). In the resting state, most NLRs are OFF and this equilibrium is maintained by rapid hydrolysis of ATP and slow release of ADP.

Upon binding of the effector to the ON state, ATP hydrolysis is slowed down preventing the receptor to switch towards an inactive configuration and stabilizing the ATP-loaded form. By this, the equilibrium is shifted efficiently to the side of the ON state in the presence of effector and defense responses are triggered (Figure 15). This model is supported by two main findings. On the one, hand the effector AvrL567 binds preferentially to the ATP-loaded ON, state and not to the ADP-bound OFF/ state of the receptor protein L. On the other hand it was shown that auto-active mutants of the L6 and L7

proteins preferentially bind ATP molecules [START_REF] Bernoux | Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model[END_REF].

The role of the intramolecular interactions in the NLR activation

The activation of NLR resistance proteins not only relies in the direct or indirect interaction with their cognate effectors but also depends on the strength of the intramolecular, inhibitory interactions between the different NLR domains. Indeed, modifications of NLR sequences can result in expanded effector recognition or even effector-independent activation suggesting that the modification of the intramolecular interactions alters the control of the active and inactive states of NLRs. More recently it was demonstrated that the specific modification of residues involved in negative regulatory intramolecular interactions, e.g. in the NB-ARC or the TIR, can lead to NLR variants that are efficiently activated by Avr effector alleles that not or only weakly activate the non-modified NLRs [START_REF] Giannakopoulou | Tomato I2 immune receptor can be engineered to confer partial resistance to the oomycete Phytophthora infestans in addition to the fungus Fusarium oxysporum[END_REF][START_REF] Segretin | Single amino acid mutations in the potato immune receptor R3a expand response to Phytophthora effectors[END_REF][START_REF] Harris | Stepwise artificial evolution of a plant disease resistance gene[END_REF]. Such 'trigger-happy' NLR variants can thus have extended recognition spectra and illustrate that receptor activation results from an interplay between the sensitivity in sensing the effector and the ease with which inhibitory intramolecular interactions are released. This is nicely illustrated by the role of the L-TIR domain in effector recognition specificity.

Usually, the specificity of the recognition of different AvrL567 variants by different alleles of L is explained by the polymorphic residues concentrated in the LRR domain. However, the alleles L6 and L7 differently recognize AvrL567 variants even though they possess identical LRR domains and only differ in 10 polymorphic residues confined to the TIR domain [START_REF] Bernoux | Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model[END_REF]). AvrL567 variants expressed in flax plants carrying L7 or L6 trigger a weak or strong cell death response respectively and AvrL567-A showed a weak interaction with the full length of L7 protein compared to L6. Swaps between L6 and L7 showed that two polymorphic regions in TIR and in NB domain are involved in the regulation of cell death and demonstrated that negative functional interaction between the TIR and NB domains favors the inactive ADPbound state impairing the receptor to switch to an active ATP-bound state [START_REF] Bernoux | Comparative analysis of the flax immune receptors L6 and L7 suggests an equilibrium-based switch activation model[END_REF].

NLRs and their signaling components

NLRs are modular proteins mediating effector recognition and downstream signaling to induce defense responses. Transient expression of a truncated form of L10 encoding the N-terminal TIR homology region of the L10 protein led to cell death induction and suggested for the first time a role of the N-terminal part of the plant NLRs in signaling induction [START_REF] Frost | Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defense responses[END_REF].

Further analysis showed similar results e.g. transient expression of different truncations of RPP1A in tobacco revealed that its TIR-NB-ARC portion was sufficient to induce an elicitorindependent cell death [START_REF] Weaver | The Arabidopsis thaliana TIR-NB-LRR R-protein , RPP1A ; protein localization and constitutive activation of defence by truncated alleles in tobacco and Arabidopsis[END_REF]. Similarly, transient expression of the TIR 1-45 and TIR 1-80 fragments of RPS4 induced cell death in tobacco leaves. Furthermore, the cell death mediated by TIR 1-80 required EDS1, SGT1 and HSP90 showing that this fragment of the protein seems have the same genetic requirements than the full length protein [START_REF] Swiderski | The TIR Domain of TIR-NB-LRR Resistance Proteins Is a Signaling Domain Involved in Cell Death Induction[END_REF].

Over-expression of the CC of MLA10 also resulted in cell death induction in barley (Maekawa et al. 2011). However, not always the transient expression of CC or TIR domains leads to cell death induction. For example, the transient expression of the TIR 1-80 fragment of RPP2A or RPP2B did not induce cell death [START_REF] Swiderski | The TIR Domain of TIR-NB-LRR Resistance Proteins Is a Signaling Domain Involved in Cell Death Induction[END_REF]. Likewise, in the resistance proteins Rx, the over-expression of the NB domain and not the CC domain was sufficient to induce cell death. This lack of uniformity suggests that the NLR domains and downstream components that mediate signaling vary from one NLR to another. In addition, specific intra-and intermolecular interactions may be required to activate NLR signaling platforms (Eitas & Dangl 2010;[START_REF] Sukarta | Structure-Informed Insights for Nlr Functioning in Plant Immunity[END_REF].

Role of complexes in NLR signaling

Homotypic or heterotypic interactions between the N-terminal domains of NLRs seem crucial for their function. This has in particular been demonstrated for MLA10 and L6 where the formation of homotypic complexes of their TIR or CC domains is crucial for downstream signaling. MLA10 and L6 mutants with single amino acid substitutions in the CC or TIR interaction interfaces are impaired in homodimer formation and fail to induce cell death (Bernoux et al. 2011;Maekawa et al. 2011).

The role of heterotypic TIR domain interactions has been investigated in the case of the RRS1/RPS4 pair. RRS1 and RPS4 TIR domains form homo-and heterodimers and structure -function analysis revealed the interaction surfaces (Williams et al., 2014). The RPS4-TIR domain induces like the L6 TIR domain effector-independent cell death and for this homodimer formation is crucial since mutants that fail to dimerize are impaired in cell death induction. The RRS1 TIR domain, on the contrary, does not cause cell death but represses cell death caused by the RPS4 TIR domain. For this repression, formation of RRS1-RPS4 TIR domain hetero-complexes are required since RRS1 TIR mutants that fail in hetero-complex formation do not repress the cell death induced by the RPS4 TIR domain (Williams et al., 2014).

The tobacco NLR protein N oligomerizes in the presence of the effector protein p50. This oligomerization not only involves the TIR domain but also requires an intact P-loop suggesting that the NB-ARC domain may be involved in mediating NLR homo-complexes formation [START_REF] Mestre | Elicitor-mediated oligomerization of the tobacco N disease resistance protein[END_REF]. In animals NLR activation leads to the creation of supramolecular structures that act as signaling platforms that enable the recruitment of additional adaptors and signaling components [START_REF] Hu | Structural and biochemical basis for induced self-propagation of NLRC4[END_REF]. Similar plant NLR superstructures mediating cell signaling have not yet been identified.

Role of accessory proteins in NLR signaling

Recruitment of executing signaling proteins by NLRs is thought to be a key step for the activation of the resistance responses. In plants, several signaling proteins have been shown to associate with NLRs and contribute to the activation of NLR-triggered defense. However, direct interaction between NLR receptors and signaling partners has been poorly demonstrated and little is known about the mechanism NLRs use to activate downstream signaling components.

A recent study showed that the CC domains of several NLRs with homologies to the tomato NLR I2 induce cell death and interact with the chloroplastic protein Tylakoid Formation 1 (THF1) [START_REF] Hamel | The chloroplastic protein THF1 interacts with the coiled-coil domain of the disease resistance protein N' and regulates light-dependent cell death[END_REF]. In addition, they have a negative effect on the accumulation of THF1. Further functional analysis with the NLR protein N indicated that THF1 functions as a negative regulator of the cell death and that the activation of N by P50 results in the destabilization of THF1. THF1 is crucial in chloroplast homeostasis and the cell death induced by N was light-dependent indicating a link between light, chloroplasts and the cell death induced by I2-like NLRs [START_REF] Hamel | The chloroplastic protein THF1 interacts with the coiled-coil domain of the disease resistance protein N' and regulates light-dependent cell death[END_REF]. Other signaling components in NLR-triggered immunity are NDR1, EDS1 and PAD4 [START_REF] Bibliography Aarts | Quel sont les rôles des autres domaines de RGA5 dans la reconnaissance de AVR-Pia ? Afin de mieux comprendre la reconnaissance d'AVR-Pia par RGA5, nous avons caractérisé individuellement les interactions de AVR-Pia avec les différents domaines de RGA5 (CC, NB-ARC, LRR et RATX1) par des techniques de double hybride et co-immunoprécipitation de complexes protéiques[END_REF][START_REF] Parker | Unravelling R gene-mediated disease resistance pathways in Arabidopsis[END_REF]. NDR1 is a hub to mediate CNL defense responses whereas EDS1 and PAD4 are required for TNL-mediating signaling. However, the mechanistic link between NLRs and these signaling components and their molecular activity are still unknown.

Transcriptional regulators have also been shown to play a role in NLRs-mediate signaling. For example, in tobacco, association between N and the transcription factor SLP6 (squamosal promoter binding protein (SBP)-domain) was shown to be required for N-mediate signaling and defense responses [START_REF] Padmanabhan | Novel Positive Regulatory Role for the SPL6 Transcription Factor in the N TIR-NB-LRR Receptor-Mediated Plant Innate Immunity[END_REF]. Similarly, after effector perception and activation, the NLR protein MLA10 translocates into the nucleus where it interacts with WRKY transcription factors that act as repressors of resistance and with the transcription factor MYB6 that acts as an activator [START_REF] Shen | Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses[END_REF][START_REF] Chang | Barley MLA immune receptors directly interfere with antagonistically acting transcription factors to initiate disease resistance signaling[END_REF]. In rice, the CNL protein Pb1 was shown to interact with the transcription factor WRKY45 in the nucleus. Disruption of the Pb1-WRKY45 interaction mediated by the CC domain of Pb1 compromised Pb1-mediated resistance (Inoue et al., 2013a). Association of NLR domain with transcriptional repressors and activators may be a widespread strategy to rapidly modify gene expression for the activation of resistance responses.

Some NLRs, rely for their function on so-called helper NLRs (Eitas and Dangl, 2010b).

Therefore, helper NLRs are thought to complement the function of NLRs that do not possess functional signaling domains and that probably act only as sensor proteins. Helper NLRs have been found to be not only involved in NLR signaling but also to be associated with RLK and RLP signaling. Thus, this group of proteins probably acts as a molecular bridge connecting these two different layers of plant immunity. Among the best characterized examples of helper NLRs are ADR1 that co-operates with several TNLs in A. thaliana and NRG1 which acts downstream of the TNL N (Bonardi et al., 2011;[START_REF] Collier | Cell Death Mediated by the N-Terminal Domains of a Unique and Highly Conserved Class of NB-LRR Protein[END_REF]. Unlike classical NLRs receptors, NGR1

and ADR1 family members are conserved among different plant species and do not require an entire P-loop to induce cell death (Bonardi et al., 2011).

Compartmentalization of NLR proteins

NLRs can act in different cellular compartments. The immune receptors RPS2, RPS5 and RPM1

possess N-terminal motifs that determine their localization at the plasma membrane (Figure 16). This membrane localization correlated to their activity as guards of proteins located in the plasma membrane where they are targeted by their corresponding effector proteins [START_REF] Qi | Structure-function analysis of the coiled-coil and leucine-rich repeat domains of the RPS5 disease resistance protein[END_REF][START_REF] Gao | Plant intracellular innate immune receptor Resistance to Pseudomonas syringae pv. maculicola 1 (RPM1) is activated at, and functions on, the plasma membrane[END_REF]. The flax rust resistance proteins L6 and M carry N-terminal signal sequences that address them to different endomembranes (Figure 16). L6 to the Golgi and M to the tonoplast [START_REF] Takemoto | N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance[END_REF]. Swapping of the N-terminal sequences changes the localization of the proteins but does not interfere with their function. However, complete deletion of the N-terminal signal sequence of L6 affects its stability and function [START_REF] Takemoto | N-terminal motifs in some plant disease resistance proteins function in membrane attachment and contribute to disease resistance[END_REF].

Other plant NLRs move between the cytoplasm and other cellular structures (Figure 16). For instance, prior to pathogen attack, the potato NLR protein R3a is in the cytoplasm and after perception on the effector AVR3a it relocates to the endosomes [START_REF] Engelhardt | Relocalization of Late Blight Resistance Protein R3a to Endosomal Compartments Is Associated with Effector Recognition and Required for the Immune Response[END_REF]. The inhibition of endocytotic trafficking impairs R3a function suggesting that its re-localization is required for R3a-mediated defense activation.

Several plant NLRs re-localize to the nucleus upon pathogen attack and effector recognition (Figure 16). This is the case of the resistance protein MLA10 which in the absence of pathogen locates in the cytoplasm but after recognition of the effector Avr10, it is addressed to the nucleus where it interacts with different transcription factors [START_REF] Shen | Nuclear activity of MLA immune receptors links isolate-specific and basal disease-resistance responses[END_REF][START_REF] Chang | Barley MLA immune receptors directly interfere with antagonistically acting transcription factors to initiate disease resistance signaling[END_REF]. Other NLRs such as Rx1, N, RPS4 and RRS1 have also been found to move from the cytoplasm to the nucleus upon effector recognition (Figure 16). Nucleo-cytoplasmic partioning of several NLRs has been found to be an important mechanism to differentially activate downstream signaling. For example, the NLR protein RPS4 confers along with RRS1 the recognition of the effector proteins AvrRps4 and PopP2. RPS4 accumulates in the endomembranes and the nucleus [START_REF] Wirthmueller | Nuclear Accumulation of the Arabidopsis Immune Receptor RPS4 Is Necessary for Triggering EDS1-Dependent Defense[END_REF]. The forced accumulation of AvrRps4 in the nucleus leads to RPS4-mediated resistance but impairs RPS4-mediated HR.

However, when AvrRps4 is retained in the cytosol RPS4 is able to mediate both resistance and HR (Heidrich et al., 2011[START_REF] Heidrich | Molecular and spatial constraints on NB-LRR receptor signaling[END_REF]. The NLR Rx of potato recognizes the effector protein CP (coat protein) from Potato Virus X. The sequestration of Rx in the nucleus resulted in an impaired function of the resistance protein and the forced accumulation of Rx in the cytosol enhanced its function [START_REF] Slootweg | Nucleocytoplasmic distribution is required for activation of resistance by the potato NB-LRR receptor Rx1 and is balanced by its functional domains[END_REF]). However, when CP is forced to accumulate in the nucleus,

Rice-M.oryzae pathosystem: a model of host-fungal pathogen interactions

Rice: an important crop in food security

Rice (Oryza sativa) is a diploid, annual grass of the genus Poaceae (grass family) that selfpollinates resulting in a limited degree of outcrossing (< 0.5%). Oryza sativa possess two different subspecies indica and japonica both domesticated about 9.000-10.000 ago in India (Ganges plain) and China (middle and lower Yangtze valley) [START_REF] Civá | Three geographically separate domestications of Asian rice[END_REF]. Today, rice is cultivated in wet tropical, semi-tropical, and warm temperate areas around the world for the production of its cereal grain. There is high genetic diversity in rice germplasm and thousands of cultivars exist that have different grain color, size, and shape, as well as environmental tolerances and seasonality. In most of the cases rice is grown in fields that are flooded by either irrigation, rain-fed or floodplain systems. Moreover some varieties can be cultivated without flooding but they account for only 4% of rice cultivated worldwide [START_REF] Garris | Genetic structure and diversity in Oryza sativa L[END_REF].

Rice together with wheat is one of the most important cereal crops for human consumption in the world. Its production in 2012 was about 697.225 million tons in 158 million hectares that means 4.40 tons/hectare (http://ricestat.irri.org:8080/wrsv3/entrypoint.htm). To reach this tremendous production, the use of improved high yielding varieties, adequate irrigation, use of fertilizers and other complementary inputs are necessary. Despite the significant progress in enhancing rice productivity since the green revolution, population growth has outpaced increase in rice production [START_REF] Hossain | Technological progress for sustaining food-population balance: Achievement and challenges[END_REF]. Estimations state that crop yields must be increased by 150% before 2030 to satisfy the global food demand, nevertheless rice yield production has already stuck [START_REF] Rao | Recent progress on molecular breeding of rice in China[END_REF]. Rice production is frequently threat for pathogen diseases. Among them blast disease caused by the fungus M. oryzae represents one of the most serious and widespread constraints.

Rice blast disease: a major threat for rice production

Rice blast, caused by Magnaporthe oryzae, is the most destructive disease of rice worldwide and represents a major economic concern and a serious threat for food security (Pennisi 2010;Dean et al. 2012). Globally, annual yield losses provoked by this disease could feed 60 million of people (Pennisi, 2010).

M. oryzae attacks all aerial parts of the plants and causes characteristic necrotic lesions on leaves, stems and panicles (Figure 17) [START_REF] Faivre-Rampant | Characterization of the model system rice-Magnaporthe for the study of nonhost resistance in cereals[END_REF]. Management of rice blast disease is based on a combination of blast resistant cultivars, fungicides and agricultural practice such as limitation of nitrogen fertilization (Wang & Valent, 2009). The selection pressure by continuous use of fungicides results in the emergence of fungicide-resistant populations, limiting its efficiency. In addition, fungicides used increase production costs and raise increasing concern due to their impact on the environment and human health. The use of disease resistant rice varieties represents the economically and ecologically most favorable solution for blast disease control. However, frequently, the resistance of new cultivars breaks down within a couple of years after release due to the rapid evolution of M. oryzae populations [START_REF] Tharreau | World population structure and migration of the rice blast fungus, Magnaporthe oryzae[END_REF] So far, the bases of durable disease resistance remain largely elusive and the elucidation of the mechanisms underlying fungal pathogenicity and plant resistance is believed to be important element to achieve of this goal. Because of the genetic and molecular tools available for rice and M. oryzae (molecular markers, genetic maps, whole genome sequence, and transformation facilities) as well as the agronomic relevance of rice for world population, the rice-M. oryzae pathosystem is one of the most suitable models to study plant-fungus interactions.

The fungus: Magnaporthe oryzae

Magnaporthe oryzae causes blast disease on a broad range of cereal and grass plants including food security crops such as rice, wheat, barley and millet [START_REF] Couch | Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice[END_REF]. So, while the M.

oryzae species complex can cause disease on almost hundred different plant species, individual isolates have very narrow host ranges and can infect generally only one single plant species. Rice isolates e.g. only infect rice whereas wheat isolates are only pathogenic on wheat. Isolates that share the same host plant are genetically more related than isolates with other host specificities [START_REF] Couch | Origins of host-specific populations of the blast pathogen Magnaporthe oryzae in crop domestication with subsequent expansion of pandemic clones on rice and weeds of rice[END_REF][START_REF] Chiapello | Deciphering Genome Content and Evolutionary Relationships of Isolates from the Fungus Magnaporthe oryzae Attacking Different Host Plants[END_REF].

Recently, the comparison of genome sequences and morphological structures of 6 isolates of Magnaporthe species from three different host plants, showed that the host specificity and adaptation of different pathotypes of Magnaporthe were not influenced by inherent morphological structures, but rather that host specificity and adaptation are evolutionary traits acquired by Magnaporthe species under strong selection pressures predetermined their host plants [START_REF] Zhong | Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species[END_REF]. Another comparative genomic study of host-specific M. oryzae isolates showed that gain and loss of genes is a major mechanism in host specialization [START_REF] Yoshida | Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements[END_REF]. Transposable elements seem to facilitate this process of gene evolution potentially by enhancing the rearrangement of chromosomes and other forms of genetic variation [START_REF] Yoshida | Host specialization of the blast fungus Magnaporthe oryzae is associated with dynamic gain and loss of genes linked to transposable elements[END_REF].

The rice blast fungus M.oryzae is currently found in the literature under two different names.

Pyricularia oryzae, the anamorph (asexual) form of the fungus is the prevalent form in the field and is associated with the infection. However, the nomenclature of M. oryzae, the teleomorph (sexual) form, has been widely adopted by the rice blast scientific community during the last years. Hence, in this study we will refer to the recognizable name M. oryzae.

M. oryzae is an ascomycete haploid, heterotallic and hemibiotrophic fungus. After germination of conidia on leaf surfaces, germinating hyphae differentiate into a specialized cell called appressoria that produces tremendously high turgor pressure to mechanically break the leaf surface (cuticule and cell walls) to invade biotrophically one underlying epidermal cell. Thin primary invasive hyphae characterized by hyphal tip growth change rapidly their growth pattern and become more bulbous and multiply in a budding-like manner until the first invaded cell is completely filled with fungal hyphae. Subsequently, the fungus will invade adjacent cells in a similar biotrophic manner probably by exploiting pit fields for cell to cell passage. During this initial biotrophic phase, M. oryzae actively suppress the host immune system and cell death allowing the invasive hyphae to colonize living plant tissue from where nutrients are taken up. This is followed by a necrotrophic phase that is initiated by the induction of host cell death by the fungus, accompanied by the growth of thin extracellular hyphae in the dying plant tissue and terminates in asexual spore production (Figure 18).

Figure 18. Magnaporthe oryzae infection cycle.

The M. oryzae infection cycle begins when a conidia from the fungus lands on rice leaves. Conidia attach firmly and germinate after few hours. Germ tube stops polar growth and forms a highly melanized dome-shape infection structure named appressorium. During the next hours, turgor pressure increases in the appressorium resulting in cell penetration via the penetration pore from which a narrow primary invasive hyphae emerges. After penetration, biotrophic growth starts and the primary hypha differentiate into a series of bulbous hypha those continuously divide in a budding type manner to completely fill the first invaded cell.

Before expand the infection to other cells. After 4-5 days the fungus initiates necrotrophic growth that results in host tissue killing, development of disease symptoms and finally, clonal reproduction and sporulation. Adapted from Ribot et al. 2008

Effector proteins in M. oryzae; key determinants of virulence

Effector proteins are key elements in M. oryzae virulence and are particularly important during the biotrophic phase of fungus infection (Valent & Khang 2010;Giraldo & Valent 2013;[START_REF] Koeck | The role of effectors of biotrophic and hemibiotrophic fungi in infection[END_REF]. M. oryzae has a genome of approximately 38 Mb coding about 11.000 genes [START_REF] Dean | The genome sequence of the rice blast fungus Magnaporthe grisea[END_REF]. Depending on the annotation pipeline, 750 to 1570 putative secreted proteins were identified in the genome of the rice blast fungus [START_REF] Dean | The genome sequence of the rice blast fungus Magnaporthe grisea[END_REF][START_REF] Yoshida | Association genetics reveals three novel avirulence genes from the rice blast fungal pathogen Magnaporthe oryzae[END_REF][START_REF] Chiapello | Deciphering Genome Content and Evolutionary Relationships of Isolates from the Fungus Magnaporthe oryzae Attacking Different Host Plants[END_REF][START_REF] Sperschneider | Methods E FFECTOR P : predicting fungal effector proteins from secretomes using machine learning[END_REF]. More than 750 of them have no functional annotation and approximately 40% are smaller than 250 amino acids resulting in huge sets of potential M. oryzae candidate effectors even if more refined analysis are performed. For instance, a recent genome-wide effector search relying on multiple parameters and a machine learning approach identified 485 effector candidates [START_REF] Sperschneider | Methods E FFECTOR P : predicting fungal effector proteins from secretomes using machine learning[END_REF]. An important additional criteria to further refine the M. oryzae candidate effector lists is in planta expression. For example, by comparing the expression of M. oryzae genes in earlyinfected rice leaf sheaths with their expression in in vitro grown mycelium, 58 candidate effectors specifically expressed during infection and named BAS (biotrophy-associated secreted) proteins were identified. However, the functional analyses of hundreds of candidate effectors remains a tremendous challenge and so far, not more than 35 M. oryzae effectors and effector candidates, including 10 Avrs, have been further characterized.

The crucial role of small secreted proteins in the M. oryzae effector repertoire is underscored by the finding that all cloned M. oryzae Avr genes (Table 1) with the exception of ACE1, a cytosolic polyketide synthase/peptide synthetase and AVR-Pita a secreted zinc protease, code for small secreted proteins. Analysis of their localization during infection revealed that all these AVR effectors are translocated into host cells indicating that they act as cytoplasmic effectors (Giraldo and Valent, 2013). For six of them as well as for AVR-Pita a corresponding intracellular NLR rice immune receptor has been identified (Table 1).

Characterization of putative effectors in M. oryzae

Four BAS (Biotrophy-Associated Secreted) proteins, BAS1-4, that are specifically and highly expressed during early biotrophic infection were confirmed to be secreted in compatible but not incompatible interactions [START_REF] Mosquera | Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease[END_REF]. Furthermore, BAS1 and BAS2 were shown to be translocated into host cells and to accumulate in BICs (biotrophic interfacial complexes) a structure characteristic for the biotrophic phase (more details on the BIC further in this chapter). BAS3 and BAS4 are extracellular effector and while BAS3 specifically colocalizes with cell wall crossing points, BAS4 uniformly outlines invasive hyphae [START_REF] Mosquera | Interaction transcriptome analysis identifies Magnaporthe oryzae BAS1-4 as Biotrophy-associated secreted proteins in rice blast disease[END_REF]. However, disruption of these four BAS genes had no measurable impact on the virulence of M. oryzae.

851 putative effectors were identified in transcriptome profiles of rice blast infected leaves by using an integrative genome expression profiling approach that combines robust-long serial analysis of gene expression (RL-SAGE) with massively parallel signature sequencing (MPSS) and sequencing by synthesis (SBS) [START_REF] Chen | Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice[END_REF]. 42 of these predicted effector proteins were selected and transiently expressed in rice protoplast to evaluate their ability to induce plant cell death. Five of the 42 effectors called MoCDIP1-5 (M. oryzae cell death-inducing proteins) induced cell death in rice protoplast when they contained the signal peptide for secretion to the extracellular space [START_REF] Chen | Identification and characterization of in planta-expressed secreted effector proteins from Magnaporthe oryzae that induce cell death in rice[END_REF]. In addition, four of them also induced cell death in tobacco plants, suggesting that these effectors from M. oryzae may be key elements for facilitating colonization of the fungus during infection.

Disruption of 78 M. oryzae genes coding for putative secreted proteins was carried out to identify effector proteins mediating fungal pathogenicity during the early stages of infection [START_REF] Saitoh | Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens[END_REF]. With the exception of one putative effector protein named MC69, the knock-out of these putative effectors did not affect growth, conidiation or pathogenicity of M.

oryzae probably because they have redundant functions or no functions in these conditions.

The mc69 mutants, on the contrary, showed significant reduction of blast disease symptoms on rice suggesting that it has a crucial role in M. oryzae pathogenicity. Further, M. oryzae infection analyses showed that MC69 is dispensable for appressorium formation but is required for invasive hyphae development in rice leaf sheath. This suggests that MC69 is important in the interaction of the blast fungus with living rice cells during early infection. Live-cell imaging indicates that MC69 is not translocated into the rice cytoplasm. Targeted mutagenesis of two cysteine residues (Cys36 and Cys46) in MC69 did not affect secretion but blocked its function, suggesting that disulfide bonds may be required for proper folding, stability or the molecular function of the MC69 in pathogenicity. Knock-out of an orthologue of MC69 in Colletotrichum orbiculare responsible of cucumber anthracnose disease, also reduced fungal pathogenicity showing that MC69 is a secreted protein required for infection in at least two different plant pathogens [START_REF] Saitoh | Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens[END_REF].

A set of 247 candidate M. oryzae effectors was generated by a combination of expression studies, in silico identification of secreted proteins, a size limit of 250 amino acids and an elevated cysteine content [START_REF] Sharpee | Form and function of fungal and oomycete effectors[END_REF]. From these 247 putative effectors, 73 were

Mode of action of M. oryzae effector proteins

The molecular function of one apoplastic effector and two cytoplasmic avirulence effector proteins from M. oryzae has been recently characterized in more detail.

The effector protein Slp1 (Secreted LysM Protein1) is a secreted LysM protein crucial for M. oryzae pathogenicity. Live-cell imaging of infected rice tissue showed that Slp1 accumulates at the plant-fungal interface during the early stages of infection where it specifically binds chitin oligomers with high affinity. Competition assays showed that Slp1 actually out-compete the rice immune receptor CEBiP for chitin binding and suppresses by this chitin-induced plant immune responses [START_REF] Mentlak | Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease[END_REF]. Deletion of Slp1 considerably reduced the ability of M. oryzae to proliferate in rice tissue and cause disease suggesting that the suppression of chitin-triggered immunity by this apoplastic effector plays crucial roles in M. oryzae virulence.

The avirulence effector AvrPiz-t is a cytoplasmic effector recognized by the intracellular NLR immune receptor Piz-t [START_REF] Li | The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t[END_REF]. In the absence of Piz-t, AvrPiz-t contributes to M.

oryzae virulence since transgenic rice plants expressing AvrPiz-t showed enhanced susceptibility to the blast fungus [START_REF] Park | The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice[END_REF]. This was correlated with suppressed immune responses in response to flg22 and chitin. Furthermore, AvrPiz-t interacts with the rice RING E3 ubiquitin ligase APIP6 and induces APIP6 degradation. APIP6 ubiquitinates AvrPizt and promotes its degradation indicating that both proteins stimulate the degradation of each other when co-expressed together [START_REF] Park | The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice[END_REF]. APIP6-silencing in rice plants reduced PTI response and basal resistance to M. oryzae suggesting that APIP6 positively regulates rice innate immunity. However, APIP6 is not required for Piz-t-mediated resistance [START_REF] Park | The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice[END_REF].

In a separate study, AvrPiz-t was shown to target APIP10, another rice E3 ligase different from APIP6. AvrPiz-t suppressed the ubiquitin ligase activity of APIP10 which, in turn, ubiquitinates AvrPiz-t in vitro [START_REF] Park | The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice[END_REF]). In the absence of Piz-t, silencing of APIP10 compromised basal defense against M. oryzae. However, in a Piz-t background, silencing of APIP10 triggered Piz-t accumulation, cell death and enhanced resistance to M. oryzae. This seems due to promotion of Piz-t degradation via the 26S proteasome by APIP10. Therefore, it seems that APIP10 acts as a negative regulator of Piz-t and functionally connects AvrPiz-t and its cognate NLR Piz-t. Collectively, these 2 studies indicate that AVR-Piz-t targets the host ubiquitin proteasome system to suppress PTI [START_REF] Park | The Magnaporthe oryzae effector AvrPiz-t targets the RING E3 ubiquitin ligase APIP6 to suppress pathogen-associated molecular pattern-triggered immunity in rice[END_REF][START_REF] Park | The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice[END_REF].

The effector AVR-Pii from M. oryzae is recognized by the paired NLRs Pii-1 and Pii-2 coded by the resistance locus Pii. AVR-Pii has been shown to form complexes with two rice Exo70 proteins, OsExo70-F2 and OsExo70-F3 [START_REF] Fujisaki | Rice Exo70 interacts with a fungal effector , AVR-Pii , and is required for AVR-Pii-triggered immunity[END_REF]. Exo70 proteins are components of the exocyst complex mediating tethering and fusion of vesicles and plasma membrane at sites of polarized exocytosis. However, in the absence of Pii, the over-expression of AVR-Pii or the knockdown of both Exo70 genes did not affect virulence of M. oryzae [START_REF] Fujisaki | Rice Exo70 interacts with a fungal effector , AVR-Pii , and is required for AVR-Pii-triggered immunity[END_REF].

Therefore, an important role of AVR-Pii in M. oryzae virulence could not be demonstrated.

However, knock-down of, OsExo70-F3 but not OsExo70-2, abolished defense responses and resistance triggered by AVR-Pii and dependent on Pii. This suggests that the interaction of AVR-Pii with OsExo70-F3 plays an important role in Pii-mediated immunity to blast and OsExo70-F3 is expected to act as a decoy or a helper in Pii-mediated AVR-Pii recognition.

Delivery of M. oryzae effector proteins

Live-cell imaging of chimeric effector proteins expressed by transgenic M. oryzae isolates and labelled with fluorescent proteins such as green fluorescent protein (GFP), monomeric red fluorescent protein (mRFP) or monomeric Cherry (mCherry) has been useful to monitor effector localization during infection (Giraldo et al., 2013). This assays showed that apoplastic effectors, such as Bas4, Bas113 and Slp1 are retained within the extra-invasive hyphal membrane (EIHM) compartment located between the fungus and the plant plasma membrane and outline the entire IH in a uniform manner. On the contrary, translocated cytoplasmic effectors such as Pwl2, AVR-Pita, Bas1 and Bas107 accumulate in a punctate structure in the biotrophic interface named the biotrophic interfacial complex (BIC). The BIC is a plant membrane-derived interfacial structure initially localized on the tip of primary invasive hyphae and later positioned subapically, beside the first bulbous IH cell (Giraldo et al., 2013).

Translocated effectors are supposed to be delivered from the BIC inside host cells by unknown mechanisms. Some of these intracellular effectors subsequently move up to three or four cells away from the invading hyphae. The movement from cell to cell observed for some cytoplasmic effectors possibly occurs through plasmodesmata and likely contributes to preparing host cells for pathogen colonization and infection (Figure 19) (Khang et al., 2010;Giraldo et al., 2013).

Fungal effectors are usually secreted into the plant-fungus interface via the classical ER-Golgi secretory pathway [START_REF] Mentlak | Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease[END_REF]. This involves guidance of nascent secreted proteins to the rough, ribosome-rich ER by their N-terminal hydrophobic signal peptide and subsequent co-translational translocation into the lumen of the ER. From there, secreted proteins move by vesicle-mediated transport through different compartments of the Golgi-apparatus to finally reach the plasma membrane where transport vesicles fuse with the plasma membrane to deliver the secreted proteins to the extracellular space. Fusion of transport vesicles with the plasma membrane is a highly controlled process and involves two key steps: tethering of the vesicle to the plasma membrane by the exocyst complex and membrane fusion mediated by SNARE (soluble N-ethylemaleimide sensitive factor attachment protein receptors) protein. The exocyst complex is an octameric protein complex (in yeast Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) composed of subunits initially associated to the vesicle (e.g. Sec15), the plasma membrane (e.g. Sec3 and EXO70) or cytoplasmic. It assembles when the vesicle reaches the plasma membrane. Subsequent membrane fusion is mediated by v-and t-SNAREs located respectively on the vesicle and in plasma membrane [START_REF] Mentlak | Effector-Mediated Suppression of Chitin-Triggered Immunity by Magnaporthe oryzae Is Necessary for Rice Blast Disease[END_REF].

While apoplastic effectors secreted from invasive hyphae into the extracellular space are believed to follow this conventional secretory pathway, cytoplasmic effectors were recently described to follow a novel pathway of secretion that is independent of the Golgi. This pathway, that also relies on exocyst components and the t-SNARE Sso1 is mainly defined by insensitivity to Brefeldin A an inhibitor of vesicle flow from the ER to Golgi vesicles. Indeed, Brefeldin A inhibits secretion of apoplastic effectors but not the secretion of cytoplasmic effectors that therefore seem to follow another secretion pathway that does not involve the

Rice Immune receptors

Rice plants possess an arsenal of resistance genes that counteract M. oryzae attack. More than 100 blast resistance genes have been genetically identified and 25 genes from 11 loci have been cloned (Table 1). All rice blast immune receptors cloned so far code for NLRs with the exception of Pi-d2 that encodes a receptor-like kinase [START_REF] Chen | A B-lectin receptor kinase gene conferring rice blast resistance[END_REF], suggesting that blast resistance mainly relies on the recognition of cytoplasmic effectors by NLR immune receptors.

Rice PRRs

Rice RLPs and RLKs contribute to basal resistance against the blast fungus, however with the exception of immune receptor Pi-d2 they appear not to confer complete resistance. Pi-d2 has a unique structure composed of a predicted extracellular bulb-type mannose-specific binding lectin (B-lectin) and an intra-cellular serine-threonine kinase domain. Pi-d2 has been shown to confer resistance to the Chinese rice blast strain ZB15 in transgenic rice plants [START_REF] Chen | A B-lectin receptor kinase gene conferring rice blast resistance[END_REF][START_REF] Chen | Genetic Transformation of Rice with Pi-d2 Gene Enhances Resistance to Rice Blast Fungus Magnaporthe oryzae[END_REF]. However, the ligands and the precise function of Pi-d2 remain elusive.

Among the best understood examples of rice PRR function is the perception of chitin, one of the main structural components of fungal cell walls, by the rice immune receptor complex CEBiP/CERK1 (Chitin oligosaccharide elicitor-binding protein/Chitin Elicitor Receptor Kinase).

CEBiP is an RLP protein containing a transmembrane domain and three LysM motifs that bind chitin (more details about PRR receptors above in this chapter). Rice CEBiP mutants display an increased susceptibility to attack by the rice blast fungus as well as a reduced chitin-triggered immune response. Upon chitin-binding, CEBiP forms heterodimers with CERK1, a RLK protein with LysM motifs in the extracellular domain and an intracellular Ser/Thr kinase domain.

Chitin-bound CEBiP/CERK1 heterodimers represent a signaling-active receptor complex that activates chitin-triggered immunity in rice [START_REF] Shimizu | Two LysM receptor molecules, CEBiP and OsCERK1, cooperatively regulate chitin elicitor signaling in rice[END_REF].

Additional rice PRRs such as LYP4 and LYP6 binds both chitin and peptidoglycan. Knockdown of CEBiP, OsCERK1, LYP4 or LYP6, all containing at least one LysM domain, resulted in a reduced chitin-triggered immune responses and compromised the resistance against M. oryzae suggesting that LysM domain has an crucial role in chitin oligosaccharides perception in rice plants [START_REF] Couto | Regulation of pattern recognition receptor signalling in plants[END_REF].

Other rice RLPs or RLKs have also been shown to be involved in blast resistance. For instance, the knockout of several rice wall-associated kinases (OsWAK), a sub-family of receptor-like kinases, compromised resistance responses to the rice blast fungus [START_REF] Hu | Cloning and characterization of the Oryza sativa wall-associated kinase gene OsWAK11 and its transcriptional response to abiotic stresses[END_REF][START_REF] Delteil | Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus[END_REF][START_REF] Harkenrider | Overexpression of Rice Wall-Associated Kinase 25 (OsWAK25) alters resistance to bacterial and fungal pathogens[END_REF]. Their mode of action and their ligands are however not known.

Rice NLRs

Genome-wide investigation of NLRs in two sequenced rice varieties, Nipponbare (japonica)

and 93-11 (indica) identified 623 and 725 NLRs respectively [START_REF] Luo | Dynamic Nucleotide-Binding Site and Leucine-Rich Repeat-Encoding Genes in the Grass Family[END_REF]. Among them, 347 (55.7%) in Nipponbare and 345 (47.6%) in 93-11 are pseudogenes caused either by large deletions, nonsense point mutation or small frame-shift insertion/deletions (indels). 75% of the NLRs are organized in clusters in the rice genome with two or more NLRs separated by no more than eight non-NLRs. In addition, NLRs are also found as single-copy genes. Thus, from 279 NLR loci identified in Nipponbare, 160 were single-copy loci and 119 were multiple-copy loci [START_REF] Luo | Dynamic Nucleotide-Binding Site and Leucine-Rich Repeat-Encoding Genes in the Grass Family[END_REF]. Many NLRs conferring rice blast resistance are localized in NLR clusters.

An important cluster for rice blast resistance is e.g. located on chromosome 6 an harbours Pi2, Pi9, Pi50 and Piz-t [START_REF] Jiang | Molecular mapping of the Pi2/9 allelic gene Pi2-2 conferring broad-spectrum resistance to Magnaporthe oryzae in the rice cultivar Jefferson[END_REF].

Rice NLRs can be classified in two main types based on their nucleotide diversity. Type I NLRs are rapidly evolving genes with extensive chimeric structures and high diversity among rice accessions [START_REF] Luo | Dynamic Nucleotide-Binding Site and Leucine-Rich Repeat-Encoding Genes in the Grass Family[END_REF]. Frequently, orthologous relationships cannot be established between Type I NLRs from different varieties since there is copy number variation between different varieties and intense recombination between more or less closely related Type I NLRs. Type II NLRs are slowly evolving genes without sequence exchanges between different

NLRs and highly conserved in different accessions [START_REF] Luo | Dynamic Nucleotide-Binding Site and Leucine-Rich Repeat-Encoding Genes in the Grass Family[END_REF]. Therefore, for Type II NLRs, orthologous can be identified in different rice varieties. Type II NLRs are the predominant class of NLRs in the genome of Nipponbare and almost all the NLR R genes cloned so far belong to this category. Although type I NLRs only account for a small proportion of all NLRs, e.g. 5.8% in Nipponbare, these NLRs significantly contribute to the diversity of NLRs in the rice genome. Furthermore, conserved rice NLRs are mostly single-copy and/or localize as a singleton whereas NLRs contributing to rice genetic variation are usually part of complex clusters [START_REF] Yang | Genome-wide investigation on the genetic variations of rice disease resistance genes[END_REF]. Presence and absence (P/A) polymorphisms are also frequent in rice NLR loci. For instance, 84 of the 323 NLR loci in 93-11 are absent from the Nipponbare genome. Similarly, about 40 NLR loci of Nipponbare are absent from 93-11. In total, at least 124 NLR loci exhibit P/A polymorphism between the two rice genomes suggesting that gain and loss of NLRs is an important mechanism in the evolution of rice immunity [START_REF] Luo | Dynamic Nucleotide-Binding Site and Leucine-Rich Repeat-Encoding Genes in the Grass Family[END_REF].

Bioinformatic searches of highly polymorphic NLRs associated with rapid molecular evolution enabled recently the massive identification of novel, functional blast resistance NLRs (Yang et al. 2013). 60 NLRs from three rapidly evolving NLR families were randomly chosen in maize, sorghum and brachypodium and 20 from 5 different rice varieties. After cloning and transformation into 2 different blast susceptible rice varieties, transgenic rice plants were inoculated with 12 different isolates of M. oryzae to test for sensitivity or resistance to the fungus (Yang et al., 2013b). By this, in total, 28 functional blast resistance NLRs were obtained, 15 from non-rice grasses and 13 from rice. This suggests that rapidly evolving NLR gene family are important for the resistance to fast evolving fungal pathogens such as M. oryzae and present an outstanding resource for resistance breeding (Yang et al. 2013). [START_REF] Kanzaki | Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions[END_REF]. The interaction is allele-specific and individual NLR alleles recognize only particular subsets of the AVR-Pik alleles. AVR-PikD is e.g. recognized by Pikp, Pik and Pikm while AVR-PikA is only recognized by Pikm [START_REF] Kanzaki | Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions[END_REF]. Detailed molecular and structural analysis demonstrated that AVR-Pik binds directly to the unconventional HMA domain of Pik-1 and that this physical interaction is required for recognition. The binding surface in AVR-Pik has been structurally characterized and it was shown that polymorphisms in this surface as well as in the Pik-1 HMA domain explain differences in AVR-Pik-HMA binding and immune activation (Maqbool et al. 2015). This work also showed that the HMA domain has been integrated into Pik-1 to mediate effector recognition.

R genes only

Pi36 NB-LRR -- -- Pi37 NB-LRR -- -- Pi50 NB-LRR -- -- Pi64 NB-LRR -- -- Pit NB-LRR -- -- Pi5 b NB-LRR -- -- Pid3 NB-LRR -- -- Pid3-A4 NB-LRR -- -- Pi54 NB-LRR -- -- Pi25 NB-LRR -- -- Pi1 NB-LRR -- -- Pb1 NB-LRR -- -- Pish NB-LRR -- -- Pi2 NB-LRR -- -- pi21 Proline-containing protein -- -- Pi-d2 B lectin
The other well-studied example of NLR proteins functioning in pairs to recognize the blast fungus are RGA5 and RGA4. The corresponding genes show the characteristic tandem inverted clustered organization of paired NLRs. RGA5/RGA4 mediate recognition to two different avirulence effectors AVR-Pia and AVR1-CO39 [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Functional analyses showed that RGA5 works as a sensor NLR protein to recognize effector proteins and as a repressor of RGA4, that acts as a cell death executor (Césari et al. 2014). Furthermore, RGA5 contains like Pik-1 an HMA/RATX1 domain which is also involved in effector binding [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF], confirming that integrated domains such as HMA/RATX1 play important roles in blast effector recognition.

The interaction of the blast resistance NLR Pi-ta and the M. oryzae effector AVR-Pita was the first report of direct interaction of fungal Avrs and NLRs (Jia et al 2000). This model was supported by the physical interaction between AVR-Pita with the Pi-ta-LRR which is disrupted by a single amino acid substitution in the Pi-ta-LRR domain. However, a second NLR named Ptr seems to be required for full Pi-ta function [START_REF] Jia | Identification of a new locus, Ptr(t), required for rice blast resistance gene Pi-ta-mediated resistance[END_REF]. Furthermore, an splice variant of Pi-ta encoding a TRX (thioredoxin) integrated domain in its C-terminal has been shown to present the highest level of expression while the originally described Pi-ta splice variant is of minor importance [START_REF] Costanzo | Alternatively spliced transcripts of Pi-ta blast resistance gene in Oryza sativa[END_REF]. Whether the TRX domain found in the Pi-ta variant is involved in AVR-Pita binding or whether Pi-ta cooperates with Ptr to mediate effector recognition remains to be determined.

The investigation of RGA4/RGA5 and Pik-1/Pik-2 indicates that the recognition of effector proteins by NLR pairs containing integrated domains and following the, "integrated decoy model" (Césari, et al. 2014), is an important mode to sense M. oryzae attack. The investigation of the Piz-t/AvrPiz-t and Pi-ta/AVR-Pita systems suggests also a role for indirect recognition following the guard or decoy model as well as direct ligand-receptor type interactions. Further investigation of these latter systems and additional NLR/blast effector couples such as Pib/AVR-Pib, Pi9/AvrPi9 or Pii/AVR-Pii are required to get a better understanding of the role and mode of action of NLRs in rice blast resistance.

Downstream responses in blast resistance

Relatively few rice proteins required for NLR-triggered immune signaling have been characterized so far. One example is OsRac1 that belongs to a family of small GTPases that function as molecular switches in different signaling cascades [START_REF] Kawano | Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity[END_REF]. OsRac1

participates to PTI signaling and is also required for Pit, a blast NLR, to mediated resistance to M. oryzae. OsRac1 therefore seems to be a key regulator in rice immunity (Figure 20) [START_REF] Kawano | Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity[END_REF].

Most other proteins identified to signal downstream of NLRs are either protein kinases or transcription factors. Among them, MAPKs are particularly well established as important immune signaling components. For example, OsMAPK6 kinase activity is induced by a sphingolipid elicitor derived from M. oryzae [START_REF] Lieberherr | A Sphingolipid Elicitor-Inducible Mitogen-Activated Protein Kinase Is Regulated by the Small GTPase OsRac1 and Heterotrimeric G-Protein in Rice[END_REF]. Furthermore, OsMAPK6

interacts with OsRac1 to mediate resistance responses and is also required in signaling transduction triggered by Pit and the OsRac1, RAR1, HSP90 and SGT1 protein complex (Figure 20) [START_REF] Kawano | Activation of a Rac GTPase by the NLR family disease resistance protein Pit plays a critical role in rice innate immunity[END_REF].

Many transcription factors of the WRKY family have been shown to be involved in rice blast resistance responses. Under M. oryzae attack the expression of 15 OsWRKY is increased.

Overexpression of OsWRKYs such as OsWRKY30 and OsWRKY45 enhances resistance to the blast fungus. OsWRKY45 is regulated by the ubiquitin proteasome system (UPS). Interaction of OsWRK45 with Pb1, a blast resistance NLR, avoids OsWRKY45 degradation and is required for Pb1 to mediate resistance to M. oryzae (Figure 20) (Inoue et al., 2013b).

Different rice hormones such as salicylic acid (SA), jasmonic acid (JA), gibberellic acids (GAs), abscisic acid (ABA) cytokinine (CK) and brassinolide (BL) are important components in rice immunity. Rice plants treated with BL or JA show e.g. increased resistance to M. oryzae and induction of defense gene expression while ABA treatment enhances susceptibility (Yang et al. 2013). Recently, CKs were reported to be required for oxidative stress tolerance to the rice blast fungus [START_REF] Chanclud | Cytokinin Production by the Rice Blast Fungus Is a Pivotal Requirement for Full Virulence[END_REF]. However, the understanding of hormone function in rice immunity remains limited and requires further studies.

Adapted RGA4 and RGA5 functionally and physically interact to recognize AVR-Pia. CC domain homo and hetero-complex formation has been reported (Césari et al., 2014c). In addition, it was observed that RGA4 mediates cell death activation, while RGA5 acts as RGA4 repressor and AVR-Pia sensor.

In this study, to better characterize RGA5 and RGA4 interactions, we evaluated the physical interaction between these two NLRs in presence of AVR-Pia WT . Next, we tested the hetero- infestans have been used to identify new resistance genes in potato germplasm, to assist resistance selection using them as "molecular markers" and finally to deploy resistance genes in potato commercial crops by monitoring the spacio-temporal distribution of effector proteins in pathogen populations [START_REF] Vleeshouwers | Effector genomics accelerates discovery and functional profiling of potato disease resistance and phytophthora infestans avirulence genes[END_REF][START_REF] Win | In Planta Expression Screens of Phytophthora infestans RXLR Effectors Reveal Diverse Phenotypes , Including Activation of the Solanum bulbocastanum Disease Resistance Protein Rpi-blb2[END_REF][START_REF] Boyd | Plant-pathogen interactions: Disease resistance in modern agriculture[END_REF] In recent years, structural biology has proven to be a particularly powerful approaches for the understanding of effectors [START_REF] Wirthmueller | On the front line : structural insights into plant -pathogen interactions[END_REF]. For example, the three-dimensional structure of an NLP from P. aphanidermatum was a key element to uncover the mechanism of NLP phytotoxicity [START_REF] Ottmann | A common toxin fold mediates microbial attack and plant defense[END_REF][START_REF] Küfner | Cytolytic toxins as triggers of plant immune response[END_REF]. The NLP structure revealed a fold formed by a central β-sandwich surrounded by α-helices and loops that exhibits structural similarities to soluble proteins produced by marine organisms named actinoporins [START_REF] Ottmann | A common toxin fold mediates microbial attack and plant defense[END_REF]. Actinoporins are cytosolic toxins that form trans-membrane pores via their Nterminal regions [START_REF] Črnigoj Kristan | Molecular mechanism of pore formation by actinoporins[END_REF]. In NLPs the N-terminal region is also required to induce necrosis and plant defense activation [START_REF] Fellbrich | NPP1, a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis[END_REF] suggesting that NLPs and actinoporins share a cytolytic, membrane-disintegrating mode of action.

complex
Comparison of the structures of the four oomycete RXLR effector proteins AVR3a4, AVR3a11, PexRD2 and ATR1 that share less than 20% sequence identity; revealed a common three-αhelix fold, termed the 'WY domain' that appear to provide a flexible, stable scaffold that supports surface diversification of RXLR effectors [START_REF] Win | Sequence divergent RXLR effectors share a structural fold conserved across plant pathogenic oomycete species[END_REF]. Similarly, analyses of the structure of two alleles of the flux rust effector AvrL567 revealed a β-sandwich fold that has slight structural similarity to the host-selective toxin ToxA from the fungal pathogen Pyrenophora tritici-repentis. In addition, two patches of positive charge on the AvrL567 surface were identified as potential DNA-binding sites [START_REF] Wang | Crystal structures of flax rust avirulence proteins AvrL567-A and -D reveal details of the structural basis for flax disease resistance specificity[END_REF][START_REF] Wirthmueller | On the front line : structural insights into plant -pathogen interactions[END_REF].

The crystal structures of ATR1, AVR-Pik, AvrL567-A and AvrL567-D were also highly instrumental to map polymorphic residues involved in the interaction with their cognate NB-LRRs and, therefore, in mediating recognition [START_REF] Wirthmueller | On the front line : structural insights into plant -pathogen interactions[END_REF].

Altogether In this chapter, we describe the structure as well as important biochemical surface properties of the M. oryzae AVR-Pia and AVR1-CO39. We also present the discovery of a family of sequence-unrelated effector proteins with a structurally conserved fold we that seems particularly abundant and important in M. oryzae.

Article I

Supporting information

Supplemental Table S1 The rmsd (upper right) and the Z-score (lower left) are from the pairwise superposition. The low RMSDs and the high Z-scores indicate that the ToxB(b) structure is the closest to the other three structures.

MGG_00954

GTACCCAAGCCCTGGAAAGT CCAGGACAATGCTCAAATCA

MGG_02207

GTGCCAATTGGTGGCTTACT TCTTTCGCTTAGCGTCGTAAC

MGG_02546

AAGCTACGCCGTTTGTGAAC CGGGTAATTTCTCGCTGTCA

MGG_04735

GCAAAGTCGTCACCGAGATT CGTCCATTGTCCCTTCAATTA

MGG_05896

GACGTTTGCCTGGGTTAAAG GGCAAGTGTAGCCGTTGATAA

MGG_08414

CGTCGTGGGTGATTTGAAC GTACCAATTCCACGCCAGTC

MGG_08482

ACCATGCAAGGTCACGATTT TGATATGTGTGGCCGGTAGA

MGG_08944

CCTGACCAATGGCAACAAG AAATTCGCACTTGGAGTTCA

MGG_08992

AAGAGACGGGAACGTTGATG ATCCACGTCGTACCGCTTAT

MGG_09675

CCGTCAAAGAGGAAGGATTC TTTGGCAGAATGCAATTCAC

MGG_11967

ATGGGCTCAAAGCTCTTGTC TGGATTTCTTGGTCCCAGTT

MGG_13133

AGTCGAAAGGGTTGGGAGAT ATCGGATTGCACTCCTTGTC

MGG_15207

CCCACCTCAATCCTGAGAGA TATCTCGCTCCCACGGTTAC

MGG_15625

CAGCAAAGAAGGTGGGAAGA GCCGATACACATTTGCACTG

MGG_15972

TCGAGAGCGAGACCCTAACT CCCAGTTTGGACCAACTTTC

MGG_16175

CGATTGCACACTTGGTTGTAA TGCATCCTCTTCCGATCTTT

MGG_16619

GAAGCTAGGCGATCGTTCTG GCAATCCTGTTCCACATGAA

MGG_16939

ATTGCTGGCCAAGGTTGAG CGGCAGTTTCTGAACATGAG

MGG_17266

AAACACCAGCTCCCATCAAC GCTGGAACTAGGCGACTCTG

MGG_18019

TGCCTTCCAGCCTACAGAGT CTTTCGGAACGCTCTTCTTG

MGG_18062

CAACAGCAGCTTCGATATCAT CCAACTTTGGCAAGCAATTC

M_TH16_00000541

GGAAATCAGCGGTTTCAAGT CAAACTTCCAGCTCGTGACA

M_TH16_00027411

AGAGTCGAAGCAGGCGAAAT TTCGCGGAGACTGTACATGA

M_TH16_00034081

AATCAGACGCCTGAGCAAGT GCGATCGCAGGTAATGTAGC

M_TH16_00040131

GCTTGGCTGGGTTAAGATTG TTTGCAGTTGGTGTCCTCTG

M_TH16_00079081

GGTTGGTGCAAGGTCAAAGT CCGTCGAGATTCCAATGAGT

M_TH16_00079311

AAAGCTGCATGGCAAGACT AACCTCCGATTGTTCCACTG

M_TH16_00104561

CGTCGTCGATTCAGCAAATA GCTGCTTCCTTATCCCACAA

M_TH16_00119711

CAGCGGTGTAGTGGCAATAA TCCTTCCCAGCTTTCACAGT

M_TH16_00120731

ACCGTGGGAATGGGTTACTT TAAACTCCCGGCTGGATAAG

M_TH16_00124981

GCTTGGGTTGCACCTACAGT TTCAAACTCGCAGGTGTCTG

M_TH16_00127871

GTTGCCTGTGCAGCCTATCT GATTCGGCTGGAATCATCTC

M_TH16_00136331

AAGGAAGAAGTCGAGGGTGAG AACGATATCCCAACCGTCAG

MGG_14793

ACAGCCGTCTGCGACTTTAT GTCCGCTCAGGCTAAGTTTG

MGG_07184

AGCGTGTCTGCAAAGCTGTA AGACCTCCCAACGGTTCTCT

Actin, MGG_03982

TCTTCGAGACCTTCAACGCC ACCGGAGTCGAGCACGATAC

EF1α , MGG_03641

GCC CGG TAT GGT CGT TAC CT AGC TGC TGG TGG TGC ATC TC

ORF3, MGG_08381

GGTGAGGGTGTTGGAGGTAGTG TGGAGCTGCCCAACATGTC

MGG_01147

CGACGACCTACCTGCTGACT CAATGCTCCTCTTCCTGGAG

BAS3, MGG_11610

CCCGTGTTTGAGGAATTGTG CTTGAGGTTGTCGGTGCTCT 01

MGG_00821

2 D Q R 0 M A G O R A v r P i z -t / 1 -1 0 8 2 l w 6 -A v r P i z -t

SUMMARY POINTS (CHAPTER I)

 Structural insights into effector proteins start to reveal common patterns hidden in highly diverse amino acid sequences.

 MAX-effectors are a large family of effectors characterized by a common βsandwich structure and unrelated sequences.

 The common β-sandwich structure of MAX-effectors is specifically found in effectors from Ascomycete fungi and has massively expanded and diversified only in M. oryzae and M. grisea.

 MAX-effectors seem to evolve under diversifying selection.

 MAX-effector candidates could act as virulence effectors since the majority of them are express specifically during biotrophic infection.

 MAX-effectors present very different surface properties and activities despite their similar topology.

 The conserved architecture found in MAX-effectors allows effector proteins to change and adapt to new functions as well as to face general constrains such as stability in fungus-host interfaces.

INTRODUCTION

Since the initial cloning of genes conferring disease resistance in plants and coding for NLR immune receptors, tremendous efforts have been made to better understand the mechanistic basis of their function. One long-term goal in this research is the engineering of NLRs with defined novel specificities that confer durable and broad spectrum disease resistance in economically important crop plants. Unfortunately, until now attempts for resistance engineering have generally failed owing to the complexity of effector recognition as well as NLR signaling. To date, only a few NLRs that recognize a broader range of alleles of the same pathogen effector have been successfully engineered through in vitro evolution and rational design. However, the development of NLRs with completely new specificities has not yet been achieved.

In many cases NLRs monitor host proteins, to sense pathogen attack. Hence, an alternative approach in which the host target protein of the pathogen effector is engineered rather than the NLR itself has recently been successfully implemented [START_REF] Kim | Using decoys to expand the recognition specificity of a plant disease resistance protein[END_REF]. PBS1 from Arabidopsis thaliana is a "decoy" kinase protein targeted by the protease effector AvrPphB.

The proteolytic cleavage of PBS1 by AvrPphB results in the activation of the NLR protein RPS5.

A three amino-acid insertion at the cleavage site of PBS1 protein also results in the activation of RPS5 in absence of effector proteins showing that RPS5 can sense different PBS1 modifications. Based on these findings, the AvrPphB cleavage site in PBS1 was substituted with new sequences that corresponded to the cleavage sites of two different proteases: AvrRpt2

from Pseudomonas syringae or NIa from tobacco etch virus (TEV). In both cases, the cleavage of PBS1 resulted in RPS5-mediated resistance to the corresponding pathogen, showing that decoy proteins can be engineered to function in a very specific manner as a target of effector proteins from very different classes of pathogens [START_REF] Kim | Using decoys to expand the recognition specificity of a plant disease resistance protein[END_REF].

Work on the rice NLR pair RGA4/RGA5 lead to the development of the integrated decoy model and the demonstration that the integration of potential decoy domains is frequent (Cesari et al., 2014). Key hypothesis of the integrated decoy model were confirmed by functional studies with the NRLs RRS1 and Pik-1 and large scale searches of NLRs with integrated domains (NLR-IDs) revealed that NLRs occur in roughly 5% of the NLRs of land plants [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF][START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF]. Based on these findings, it was proposed that the modular architecture of NLR-IDs presents an alternative and extremely promising opportunity for the engineering of NLRs with novel specificities. Indeed, by changing the integrated decoy and introducing a novel effector trap, an NLR-ID with a novel recognition specificity may be generated. (Cesari et al., 2014;[START_REF] Ellis | Integrated decoys and effector traps: how to catch a plant pathogen[END_REF]. However, to really reach this goal, a much better understanding of the mode of function of NLR-IDs is required.

Previous work had shown that the RATX1/HMA domain in RGA5 establishs physical interactions with the effectors AVR1-CO39 and AVR-Pia in planta and in yeast two hybrid assays [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]). Furthermore, it was suggested that the physical interaction between the effectors and RGA5 RATX1 is required for recognition. Indeed, AVR-PiaH3, an AVR-Pia allele that is not recognized in resistant rice plants, does not interact with the RATX1 domain. These results prompted us to go forward in the molecular characterization of the interaction of AVR-Pia and RGA5 RATX1 .

In this chapter we present new insights into the role of integrated decoys in the recognition of effector proteins. In particular, we delimit the interface with which AVR-Pia binds the RGA5

RATX1 domain and we propose a model that explains the role of AVR-Pia-RGA5 RATX1 binding in RGA5/RGA4-mediated effector recognition.

ARTICLE 2 INTRODUCTION

Plant disease resistance largely relies on inducible immune responses that are triggered upon receptor-mediated recognition of pathogen molecules and that often involve a localized programmed cell death called the hypersensitive response (HR). Particularly important are cytoplasmic nucleotide-binding oligomerization domain-like receptors (NLR) that present a multidomain architecture composed of a C-terminal leucine-rich repeat (LRR) domain and a central nucleotide-binding (NB-ARC) domain (Takken and Goverse, 2012;Jacob et al., 2013;Qi and Innes, 2013). In most cases, they carry in addition an N-terminal coiled-coil (CC) or TOLL/interleukin-1 receptor (TIR) domain that have both been reported to mediate NLR homo complex formation and to be crucial for the activation of downstream signalling (Bernoux et al., 2011;Maekawa et al., 2011). Plant NLR proteins specifically recognize pathogen-derived effectors that act inside plant cells [START_REF] Cui | Effector-Triggered Immunity: From Pathogen Perception to Robust Defense[END_REF].

145

Traditionally, both effector recognition and activation of resistance signalling are thought to be mediated by single plant NLRs but recent studies revealed an increasing number of cases where different NLRs cooperate in pathogen recognition and resistance (Eitas and Dangl, 2010;Césari et al., 2014a). Frequently, the genes coding for these paired NLRs occur in a paired, inverted tandem arrangement in the genome. In the few cases that have been investigated in more detail, the NLR pairs seem to act as hetero-complexes where only one of the paired NLRs acts directly in effector recognition while the other is crucial for the activation of downstream signalling (Williams et al., 2014;[START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF]. In other cases, helper NLRs that act downstream of several NLRs with different recognition specificities were shown to be required for resistance and pathogen detection [START_REF] Gabriëls | An NB-LRR protein required for HR signalling mediated by both extraand intracellular resistance proteins[END_REF]Bonardi et al., 2011;[START_REF] Wu | Helper NLR proteins Protein extraction and Western blotting Yeast proteins were extracted with a post-alkaline extraction method (Kushnirov, 2000), resuspended in Laemmli buffer, boiled 10 min at 90°C and separated in 12% Tris-tricine SDS-PAGE gels (GE Healthcare). After transfer to nitrocellulose membrane (Millipore), protein detection was performed using a-AVR-Pia antibodies (1000x dilution) raised against purified recombinant AVR-Pia protein in rabbits (Eurogentec). Proteins from fungal culture filtrates were dialyzed against water (cut-off 1kDa, 1 part of medium by 5 parts of water), lyophilized, resuspended in 10 mM Tris-HCL pH 7.5, separated on 16% Tristricine NuPAGE gels (GE Healthcare) and, after blotting, analyzed using a-AVR-Pia antibodies[END_REF].

In certain cases, plant NLRs recognize effectors in an indirect manner. They detect either a modification of the effector's host target protein called 'guardee', or modifications of a host protein that mimics the effector target and is called a 'decoy' (van der Hoorn and Kamoun, 2008).

In other cases, effectors are recognized in a direct manner by binding, either alone, or in complex with a cofactor that may be a guardee or a decoy, to the NLRs (Takken and Goverse, 2012;Collier and Moffett, 2009). In these cases, the LRR domain plays a crucial role in recognition specificity and was frequently shown to mediate direct effector binding [START_REF] Ellis | Flax Rust Resistance Gene Specificity is Based on Direct Resistance-Avirulence Protein Interactions[END_REF]Krasileva et al., 2010;Jia et al., 2000). Alternatively, direct effector recognition can also be mediated by noncanonical domains integrated into NLRs at low frequencies [START_REF] Kanzaki | Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions[END_REF]Sarris et al., 2015;Maqbool et al., 2015;Le Roux et al., 2015;[START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Recent work lead to the hypothesis that these highly diverse integrated domains are mimics of effector targets and can therefore be considered as integrated decoy domains (Le Roux et al., 2015;Sarris et al., 2015;Césari et al., 2014a). However, the molecular mechanisms of effector recognition by integrated domains and eventual advantages of this mode of recognition remain largely unknown.

Rice blast, caused by the fungus Magnaporthe oryzae is a highly destructive crop disease and a serious threat for food security (Pennisi, 2010;Dean et al., 2012;[START_REF] Skamnioti | Against the grain: safeguarding rice from rice blast disease[END_REF]. NLRmediated pathogen recognition is the major mechanism in rice blast resistance. Among 25 different blast resistance genes cloned over the last 20 years, 24 code for NLRs [START_REF] Liu | Novel Insights into Rice Innate Immunity Against Bacterial and Fungal Pathogens[END_REF].

146

A particular feature is that blast-resistance is frequently conferred by paired-NLRs with clustered tandem organisation in the genome. Among them, the NLR pair RGA4/RGA5 conferring Pi-CO39

and Pia resistances has been developed as a model for the molecular understanding of paired NLRs [START_REF] Okuyama | A multifaceted genomics approach allows the isolation of the rice Pia-blast resistance gene consisting of two adjacent NBS-LRR protein genes[END_REF][START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF][START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF]. In this pair, RGA4 acts as a constitutively active disease resistance and cell death inducer that is repressed by RGA5 in the absence of pathogen [START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF]. In addition to its repressor function, RGA5 acts as a receptor for the M. oryzae effectors AVR1-CO39 and AVR-Pia. Direct binding of RGA5 to these effectors results in de-repression of RGA4 and activation of resistance signalling. Effector binding involves the unconventional C-terminal domain of RGA5 that is related to ATX1 (RATX1 domain), a heavy metal-associated (HMA) domain protein from Saccharomyces cerevisiae, that acts as a cytoplasmic copper chaperone [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Interestingly, the RATX1 domain of RGA5 is dispensable for RGA4 repression and seems exclusively dedicated to effector binding [START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF]. Since the rice RATX1/HMA protein Pi21 is a blast susceptibility factor required for full disease development [START_REF] Fukuoka | Loss of function of a proline-containing protein confers durable disease resistance in rice[END_REF], it has been hypothesised that AVR1-CO39 and AVR-Pia target RATX1/HMA proteins for disease development and that the RATX1 domain is an integrated decoy domain [START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF][START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Interestingly, an HMA domain 53% identical to the RGA5 RATX1 domain is also present in another rice NLR, Pik-1 that acts together with the NLR Pik-2 in the specific recognition of the M.oryzae effector AVR-Pik. Like in RGA5, this domain acts by binding directly the effector and is crucial for its recognition [START_REF] Kanzaki | Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions[END_REF].

However, on the contrary to the C-terminal RATX1 domain of RGA5, the HMA domain of Pik-1 is located between the CC and NB domains indicating independent integration of the same domains in the two unrelated NLRs [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Recently, the determination of the crystal structure of the AVR-PikD/Pikp-1 HMA domain complex allowed the precise identification of the AVR-PikD surface mediating binding to the Pikp-1 HMA domain (Maqbool et al., 2015).

Although AVR-Pik, AVR-Pia and AVR1-CO39 do not share sequence similarities, they share a highly similar three dimensional structure characterized by a six β-sandwich fold also present in two other effectors: AvrPiz-t from M. oryzae and ToxB from the wheat pathogenic fungus Pyrenophora tritici repentis [START_REF] Yang | Rapidly evolving R genes in diverse grass species confer resistance to rice blast disease[END_REF][START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF]Maqbool et al., 2015;[START_REF] Nyarko | Solution NMR Structures of Pyrenophora tritici-repentis ToxB and Its Inactive Homolog Reveal Potential Determinants of Toxin Activity[END_REF]. The corresponding, structurally related Magnaporthe Avr and ToxB effectors were termed MAX effectors. MAX effectors are present in other, sometimes only distantly related phytopathogenic fungi and the MAX effector family underwent strong expansion in M. oryzae

where it accounts for roughly 10% of the effectors [START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF]. In the present study, we investigated the molecular and structural bases of AVR-Pia recognition by RGA5 with a focus on the role of the RATX1 domain in effector-binding and recognition. We show that AVR-Pia interacts with RGA5 RATX1 domain through a precise surface that shares some similarity, but also important differences, with the HMA-binding surface of AVR-Pik. We demonstrate that binding to the RATX1 domain is required for effector recognition but that strong reduction in binding strength is tolerated. We also provide evidence that the RATX1 domain is not required for association of AVR-Pia with RGA5 and that it interacts with additional sites in the NLR which could explain the high tolerance of recognition to reduced AVR-Pia-RATX1 binding strength. Based on our results, we propose a model illustrating advantages of effector recognition by integrated decoy domains as well as additional simultaneously occurring interactions with NLR receptors.

RESULTS

The F24S and T46N substitutions in the non-recognized AVR-Pia-H3 allele affect surface properties but not structure

We previously described the naturally occurring AVR-Pia allele AVR-Pia-H3 that carries two nonsynonymous polymorphisms leading to the F24S and T46N substitutions [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. M.

oryzae isolates carrying the AVR-Pia-H3 allele are virulent on rice varieties carrying the Pia resistance locus and AVR-Pia-H3 does not interact in Y2H assays with the C-terminal part of the rice NLR immune receptor RGA5 containing the RATX1 domain (RGA5C-ter). The NMR structure of AVR-Pia showed that both the F24 and T46 residues are surface-exposed and suggested that the corresponding substitutions only affect AVR-Pia surface properties without major structural 148 rearrangements (de [START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF]. To test this hypothesis, the structures of AVR-Pia-H3

and the single mutants AVR-Pia F24S or AVR-Pia T46N were analyzed by NMR spectroscopy. We performed sequential assignments using 15 N-labeled AVR-Pia samples and the 13 Cα and 13 Cβ assignments were performed using 13 C-1 H 2D experiments with a 13 C-natural abundance simple in D 2 O (Supplemental Table 1).

When compared to AVR-Pia wild type, 1 H-15 N chemical shifts differed more in AVR-Pia-H3 than in AVR-Pia F24S to AVR-Pia T46N single mutants (Figure 1A). The NMR structure of AVR-Pia-H3

proved to be very similar to the structure of AVR-Pia (PDB code 5JHJ) (Figure 1B, supplemental Table 2). The backbone RMSD for superposition of the AVR-Pia and AVR-Pia-H3 structures is 1.53 Å and drops to 0.93 Å when the ß1-ß2 loop is excluded and the superposition starts at residue R23. Like the AVR-Pia wild type protein, AVR-Pia-H3 shows the MAX-effector topology characterized by 6 antiparallel β-strands (Figure 1C). The 1 H-15 N chemical shift data for AVR-Pia F24S and AVR-Pia T46N indicate that both single mutants probably also keep the MAX-effector fold (Figure 1A). The analysis of the relaxation data indicates that both AVR-Pia wt and AVR-Pia-H3 have rigid structures with average S2 values of 0.8 and similar S2 profiles indicating similar protein dynamics (Supplemental Figure 1). The 3D structure of AVR-Pia-H3 therefore supports that the F24S and T46N substitutions do not result in conformational changes but rather alter AVR-Pia surface properties required for strong interaction with the RATX1 domain of RGA5 (RGA5 RATX1 ) and disease resistance activation.

AVR-Pia binds RGA5 RATX1 with intermediate affinity

To characterize the AVR-Pia/RGA5 RATX1 interaction, in vitro binding assays with recombinant RGA5 RATX1 and AVR-Pia or AVR-Pia-H3 were performed using isothermal calorimetry (ITC). For AVR-Pia, specific and direct binding to RGA5 RATX1 with a one site model and a Kd of 7,8 µM was detected (Figure 2A). For AVR-Pia-H3, no binding was detected under identical conditions, suggesting that its affinity to RGA5 RATX1 is at least 10 fold smaller than that of AVR-Pia.

β-strands 2 and 3 and residues R23, F24, E56 and E58 constitute a candidate RGA5 RATX1interaction surface in AVR-Pia

To test the hypothesis that the residues F24 and T46 are part of the AVR-Pia surface mediating direct contacts to RGA5 RATX1 and to identify other residues in direct contact to RGA5 RATX1 or located in the close vicinity of the binding interface, NMR-titration experiments were performed. This technique consists in recording the 1 H-15 N-HSQC NMR spectra of 15 N-labelled AVR-Pia in the presence of increasing amounts of unlabeled RGA5 RATX1 . When protein-protein binding occurs, it modifies the chemical environment of the amino acids located on the binding surface and eventually mediating the binding. This results in a change of the chemical shift in NMR experiments. Depending on the transition rate of the complex formation, expressed by the exchange rate constant kex, and the chemical shift difference ∆ω between the unbound and bound states (∆ω difference between the resonance frequencies of the exchanging sites), different exchange regimes occur. NMR titration shows that the AVR-Pia-RGA5 RATX1 complex is in slow exchange with kex << ∆ω since separate resonances appear for individual species (bound and unbound states) (Figure S2A). Residues with important chemical shift changes between free AVR-Pia (R=0) and AVR-Pia bound to RGA5 RATX1 (molar ratio R=2) were almost exclusively surface-exposed and located in a region formed essentially by β-strands 2 and 3 and including, in addition, residues R23 and F24 from β-strand 1 as well as E56 and E58 from β-strand 4

(Figures 2B andC). No peaks were observed for residues Y27, V37, Y41, I44 and T51 in the complex. This candidate interaction surface largely overlaps with an extended, solvent-exposed patch of hydrophobic residues formed by F24, V26 and Y28 in β1, V37, L38 and Y41 in β2, and Y85 in β6. The residues on the other side of the AVR-Pia structure were not shifted in the NMR titration and seem therefore not involved in the interaction with RGA5 RATX1 (Figure 2D).

Two exceptions are E83, which probably senses a perturbation of the residue Y41 that is close in space, and the I69 residue, which may be involved in local conformational rearrangement of the short β5 strand. RGA5 RATX1 -titration experiments were also performed with 15 N-labelled AVR-Pia-H3 that shows no binding in Y2H [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF] and ITC analysis (Figure 2A). Spectral perturbations were strongly reduced and only few and limited changes of chemical shifts occurred when titrating AVR-Pia-H3 with RGA5 RATX1 (Figure 2B and E and Figure S2B). Signals for the R23, S24, V42, R43 and E83 residues were still observed at the end of the titration while they were mostly lost at a molar ratio of 0.5 in the case of AVR-Pia (Figure S2). Similarly, signals for E58, V59 and T47 were much less perturbed. Nevertheless, the peaks for Y41, N46 and T51

were also perturbed indicating a weak residual interaction between RGA5 RATX1 and AVR-Pia-H3 ( Figure 2B and E and Supplemental Figure 2).

In summary, NMR titration identified a candidate interaction surface formed by β-strands 2 and 3 and including, in addition, residues R23, F24, E56 and E58 (Figure 2B). This surface overlaps extensively with an extended hydrophobic patch on the AVR-Pia surface that contains F24 and has T46 on its border and that may be crucial for RGA5 RATX1 -binding.

Y2H experiments with structure-informed AVR-Pia mutants confirm an important role of the candidate interaction surface in RGA5C-ter-binding

To test whether the AVR-Pia candidate interaction surface identified in vitro mediates binding to RGA5C-ter in vivo, we performed yeast two-hybrid (Y2H) assays using AVR-Pia variants bearing point mutations in critical residues identified by NMR titration. Individual surface-exposed hydrophobic (M40, Y41, Y85) or charged (R23, D29, R36, E56, E58) amino acids, located in or at the border of the candidate interaction surface, were replaced by alanine. In addition, naturally occurring AVR-Pia polymorphisms located within the candidate interaction surface were tested:

F24S and T46N from AVR-Pia-H3 and R43G from AVR-Pia-H2 identified in M. oryzae isolates pathogenic on Setaria species (Supplemental Figure 3A) [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. As controls, mutants where surface-exposed charged residues located outside the candidate interaction surface are replaced by alanine (D63A, K67A, K74A, D78A) were generated (Figure 3A).

As previously reported, yeasts co-expressing BD-AVR-Pia and AD-RGA5C-ter or AD-RGA5C-ter and BD-RGA5C-ter grew on selective medium indicating physical binding between AVR-Pia and RGA5Cter, and homo-interaction of the RGA5C-ter domain (Figure 3A) [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]Cesari et al.,2014). Yeasts co-expressing AD-RGA5 C-ter and AVR-Pia F24S , AVR-Pia R43G , AVR-Pia R36A fused to the BD domain did not grow on selective medium indicating that these mutations abolish binding to RGA5C-ter. Isolates expressing BD-fusions of AVR-Pia variant carrying the mutation R23A, D29A, T46N, E58A or D63A showed reduced growth compared to wild type BD-AVR-Pia indicating that these mutations also affect AVR-Pia-RGA5C-ter interaction. AVR-Pia Y41A , AVR-Pia E56A , AVR-Pia K67A , AVR-Pia K74A , AVR-Pia D78A , AVR-Pia M40A and AVR-Pia Y85A isolates showed stronger growth. All BD-AVR-Pia variants were expressed at similar levels as the wild-type BD-AVR-Pia (Figure 3B). Taken together, these Y2H data show that the replacement of all charged amino acids in the interaction surface, with the exception of E56, either abolish or reduce binding of AVR-Pia to RGA5C-ter while exchanging hydrophobic residues within the interaction surface seems to abolish the interaction in the case of F24S, or to increase the binding in the case of M40A and Y85A.

To rule out that reduced binding of AVR-Pia mutants to RGA5C-ter is due to major changes in protein structure, the AVR-Pia mutants R23A, D29A, R36A, R43G and E58A were expressed in E.

coli, purified to homogeneity and analyzed by 1 H-1D-NMR experiments (Supplemental Figure 3B).

All mutant proteins showed similar spectra as AVR-Pia wild-type, indicating that they are well structured and only locally disturbed. Recombinant AVR-Pia D63A could not be expressed. Taken together, these results suggest that most residues of the AVR-Pia interaction surface identified by NMR titration play an important role in RGA5C-ter-binding.

Co-IP experiments identify key residues in the AVR-Pia interaction surface that are crucial for RGA5 RATX1 -binding in planta

To investigate the role of the AVR-Pia interaction surface in in planta binding to RGA5C-ter, coimmunoprecipitation (co-IP) experiments were performed. HA-tagged RGA5C-ter and YFP-tagged AVR-Pia mutants with reduced binding in Y2H were co-expressed in N. benthamiana by Agrobacterium tumefaciens-mediated transient transformation. In addition, AVR-Pia M40A that, according to Y2H experiments, has increased affinity for RGA5C-ter was also analyzed. As a negative control, a YFP fusion of the cytoplasmic M. oryzae effector PWL2 was used (Khang et al., 2010). Immunoblotting using anti-GFP and anti-HA antibodies showed proper expression of all proteins (Figure 4). However, AVR-Pia mutants with reduced binding to RGA5C-ter in Y2H reproducibly accumulated at lower levels than AVR-Pia wt while AVR-Pia M40A was expressed at similar levels (Figure 4). All YFP fusion proteins were efficiently precipitated with anti-GFP antibodies but only AVR-Pia M40A co-precipitated RGA5C-ter as strong as AVR-Pia wt . The other mutants showed various degrees of impairment ranging from slightly (AVR-Pia R23A AVR-Pia E58A , AVR-Pia D63A ) to strongly reduced (AVR-Pia D29A AVR-Pia R36A , AVR-Pia R43G ) or even completely abolished RGA5C-ter co-precipitation (AVR-Pia F24S ) (Figure 4A). The specificity of the interactions was confirmed with PWL2 that does not interact with RGA5C-ter.

152

It has previously been shown that the interaction of AVR-Pia with RGA5C-ter relies on interaction with the RATX1 domain (RGA5 RATX1 ) [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. To verify that interaction specificities of the AVR-Pia mutants with RGA5C-ter correlates with interaction strength with RGA5 RATX1 , co-IP experiments were performed using HA-tagged RGA5 RATX1 . AVR-Pia wt and AVR-Pia M40A strongly coprecipitated HA-RGA5 RATX1 , while the other mutants showed reduced (R23A and D63A), strongly reduced (D29A, R36A and E58A) or no co-precipitation of RGA5 RATX1 (F24S and R43G) (Figure 4B).

Taken together, these data indicate that AVR-Pia wt and AVR-Pia M40A RGA5C-ter and RGA5 RATX1 , while mutants affected in direct binding to RGA5C-ter in Y2H showed reduced association with RGA5C-ter and RGA5 RATX1 in planta. Complete absence of association with RGA5 RATX1 for AVR-Pia F24S and AVR-Pia R43G , both in planta and in Y2H, indicates a crucial role of these residues in the binding interface and suggests that they are pivotal for AVR-Pia recognition.

Direct binding to the RATX1 domain is required for AVR-Pia recognition

To determine the role of the RATX1-binding surface of AVR-Pia in specific recognition by the RGA4/RGA5 pair, AVR-Pia mutants were co-expressed in N. benthamiana with RGA4/RGA5 and cell death activation was monitored. Since tagged versions of AVR-Pia proved inactive in this assay, un-tagged AVR-Pia mutants were used. AVR-Pia mutants with wildtype binding to RGA5 RATX1 induced cell death indicating they are recognized by RGA5/RGA4 (Supplemental Figure 4A andB).

Weakly or non-binding mutants lost cell death inducing activity but were also less abundant than AVR-Pia wt or recognized AVR-Pia mutants (Supplemental Figure 4A, B andC). Therefore, no clear conclusions can be drawn for these mutants since lack of recognition may be due to low protein abundance. Similar differences in the protein level of AVR-Pia mutants were previously observed with YFP-tagged variants expressed in N. benthamiana (Figure 4) but not upon expression in E.

coli or yeast (Figure 3B). Therefore, differences in the accumulation of AVR-Pia variants seem not related to an intrinsic destabilization of these proteins but rather result from reduced stability in N. benthamiana.

AVR-Pia interacts with RGA5 outside of the RATX1 domain

The high resilience of RGA4/RGA5-mediated AVR-Pia recognition to reduction of AVR-Pia-RGA5 RATX1 interaction strength suggested that AVR-Pia might interact with additional sites in RGA5. To test this hypothesis, in planta association of the AVR-Pia mutants with the RGA5 fulllength protein was assayed by co-IP. All AVR-Pia mutants, including AVR-Pia F24S and AVR-Pia R43G co-precipitated RGA5 as efficiently as AVR-Pia wt (Figure 6A). This indicates that lack of binding to RGA5 RATX1 does not abolish association with RGA5. To test whether binding of AVR-Pia to RGA5 is truly independent of the RATX1 domain, association of AVR-Pia with an RGA5 construct lacking the RATX1 domain (RGA5 ΔRATX1 ) was tested by co-IP. All AVR-Pia variants co-precipitated RGA5 ΔRATX1 (Figure 6B) and AVR-Pia mutants with reduced or no binding to RGA5 RATX1 interacted as strongly with RGA5 ΔRATX1 as AVR-Pia wt demonstrating that the RATX1 domain is not necessary for formation of RGA5/AVR-Pia complexes. These results suggest that AVR-Pia interacts with additional sites in RGA5 outside of the RATX1 domain and that the region of AVR-Pia that mediates interaction with RGA5 ΔRATX1 lies outside of the RATX1-binding surface.

It was previously shown that RGA5 ΔRATX1 inhibits RGA4-triggered cell death and that therefore the RATX1 domain is not required for RGA5-mediated repression of RGA4 [START_REF] Césari | The NB-LRR proteins RGA4 and RGA5 interact functionally and physically to confer disease resistance[END_REF]. Since AVR-Pia still associates with RGA5 ΔRATX1 in planta, we tested whether AVR-Pia would be recognized by RGA5 ΔRATX1 /RGA4 and trigger cell death independently of the RATX1 domain.

Neither co-expression of RGA4, RGA5 ΔRATX1 and AVR-Pia, nor expression of these three proteins together with the isolated RATX1 domain triggered cell death (Figure S6). This indicates that interaction of AVR-Pia with regions outside of the RATX1 domain is not sufficient to release RGA5-mediated RGA4 repression and further confirms that binding of AVR-Pia to the RGA5 RATX1 is required for de-repression of RGA4. In addition, these results suggest that AVR-Pia has to interact with the RATX1 domain in the context of the full length RGA5 protein since an isolated RATX1 domain does not complement RGA5 ΔRATX1 for AVR-Pia recognition

DISCUSSION

Identification of a RGA5RATX1-binding surface in AVR-Pia

In this study, we provide evidence that AVR-Pia interacts with the RATX1 domain of RGA5 through a precise binding surface consisting of β-strands 2 and 3, residues R23 and F24 from β-strand 1, and residues E56 and E58 from β-strand 4 (Figure 2C). This interaction surface, identified by NMR titration experiments with recombinant AVR-Pia and the RATX1 domain, was confirmed by mutant analysis. Indeed, replacement of residues R23, F24, D29, R43, T46 or E58 strongly reduced or abolished binding to RGA5 RATX1 in Y2H and Co-IP experiments while replacement of residues M40 and Y85 increased interaction in Y2H (Figures 3 and4). Substitutions outside of the candidate interaction surface had no impact on binding with the exception of residues R36 and D63. Residue R36 is located in the loop joining β1 and β2 and might also be involved in RATX1-binding.

Alternatively, it may play an important role in defining the positions of β-strands 1, 2 and 6

through the salt bridge it forms with residue E83 in β6. The D63A polymorphism seems to destabilize the overall structure since AVR-Pia D63A could not be expressed in E. coli or M. oryzae.

Actually, D63 seems important to structure the loop ß4-ß5 as its side-chain carboxyl group forms a hydrogen bond with the side-chain amid group of N65. This may be required to position properly cysteine C66 that forms a disulfide bridge with C25 linking the two β sheets β1, β2, β6 and β3, β4, β5 and thereby impact global folding.

AVR-Pia and AVR-PikD have distinct RATX1/HMA-binding surfaces that are situated at similar positions The 3 dimensional structure of a AVR-PikD-Pikp-1 HMA complex was determined by crystallography and showed that, like the formation of the AVR-Pia-RGA5 RATX1 complex, binding of AVR-PikD to Pikp-1 HMA involves β-strands 2 and 3 (Supplemental Figure 7A) (Maqbool et al., 2015). However, in AVR-PikD, the residues of β-strand 2 that are crucial for Pikp-1 HMA -binding, R64 and D66, are charged and establish, respectively, hydrogen bonds and salt bridge interactions. In contrast, in AVR-Pia, surface-exposed residues of β-strand 2 are hydrophobic and probably establish hydrophobic interactions (Figure 2C). In addition, unlike AVR-Pia, AVR-PikD possesses an Nterminal extension of 32 amino acids that is crucial for physical binding to Pikp-1 HMA and recognition by Pikp-1/Pikp-2 (Supplemental Figure7B). In particular, residue H46 from this extension establishes important interactions with matching residues in Pikp-1 HMA . These interactions are necessary for binding and, together with the neighboring residues P47 and G48, for matching specificities with alleles of Pikp-1 [START_REF] Kanzaki | Arms race co-evolution of Magnaporthe oryzae AVR-Pik and rice Pik genes driven by their physical interactions[END_REF]Maqbool et al., 2015). These residues are missing in AVR-Pia but similarly important interactions are established with the amino acid F24 from the very hydrophobic β-strand 1. Therefore, recognition of the sequence-unrelated, but structurally similar, effectors AVR-Pia and AVR-PikD seems to involve similar structural elements but relies on distinct and highly specific mechanisms.

Whether the effector interaction surfaces of the RATX1/HMA domains of RGA5 and Pikp-1 are similar or completely different remains an open question. Crystal structures show that Pikp-1 HMA , has a typical HMA α/β-sandwich fold composed of two α-helices and a 4-stranded anti-parallel β-sheet, that mediates interaction with AVR-PikD (Supplemental Figure 7C) (Maqbool et al., 2015). We used molecular modeling to evaluate whether AVR-Pia-binding may involve similar regions in RGA5 RATX1 but no consensus docking model could be generated for the AVR-Pia-RGA5 RATX1 complex (Supplemental Figure 7C). Interestingly, none of the docking models predicted an interaction surface in RGA5 RATX1 similar to the effector-binding surface of Pikp-1 HMA . This suggests that the RGA5 RATX1 -AVR-Pia complex differs significantly from the Pikp-1 HMA -AVR-Pik-D complex.

Taken together, recognition of the structurally similar MAX effectors AVR-Pia and AVR-Pik by independently acquired NLR-integrated HMA domains seems to rely on distinct molecular mechanisms. Future work is required to test this hypothesis through functional studies of the Pikp-1 HMA interaction surface identified by structural analysis and the identification of the surface that mediates effector binding in RGA5 RATX1 .

Binding of AVR-Pia to the integrated RATX1 domain is required for recognition but of moderate affinity

The mutants, AVR-Pia F24S and AVR-Pia R43A , showed drastically reduced RGA5 RATX1 -binding and triggered, respectively, no or reduced resistance indicating that the AVR-Pia-RGA5 RATX1 interaction is required for RGA4/RGA5-mediated recognition. The presence of these polymorphisms in naturally occurring AVR-Pia alleles [START_REF] Ribot | The Magnaporthe oryzae effector AVR1-CO39 is translocated into rice cells independently of a fungal-derived machinery[END_REF] suggests that AVR-Pia is undergoing selection for mutations in the RATX1-interaction surface and escape from RGA4/RGA5-mediated recognition.

These results therefore provide further support for a crucial role of non-conventional, integrated decoy domains in effector recognition and NLR specificity. However, we also found high resilience of AVR-Pia recognition to a reduction in RGA5 RATX1 -binding strength since weakly-binding AVR-Pia mutants were still able to trigger resistance. Similar observation was made in the case of AVR-PikD, the only other case where the affinity of an effector to the integrated decoy domain of its NLR receptor has been determined (Maqbool et al., 2015). Indeed, AVR-PikD A67D and AVR-PikD P47A-G48D mutants showed drastically reduced binding to Pikp-1HMA but were nevertheless perfectly well recognized by Pik-1/Pik-2.

A possible explanation for this tolerance to a reduction in the affinity between effectors and integrated decoys could be that effectors interact with multiple independent sites in NLR receptors. Indeed, our study suggests that, besides the RATX1 domain, AVR-Pia interacts with other, not yet defined, regions in RGA5. In the simplest case, this interaction relies on direct physical binding, but since it was solely detected by co-IP experiments, it cannot be excluded to be indirect and to involve additional co-factors. This interaction seems mediated by other AVR-Pia surfaces than those involved in RGA5 RATX1 -binding since mutants with reduced binding to RGA5 RATX1 are not affected in interaction with RGA5 ΔRATX1 . Since the RATX1 domain is covalently linked to the rest of the RGA5 receptor, AVR-Pia-binding to these other sites has the potential to increase the overall effector binding affinity to RGA5 despite the low affinity binding to RGA5 RATX1 (Kd=7µM). In this context, further mutation-induced reduction of AVR-Pia-affinity toward the RATX1 domain may not have a dramatic effect unless it completely abolishes AVR-Pia/RGA5 RATX1 interaction. This situation highlights an advantage of the integration of the decoy domain into the NLR receptor over a situation where the decoy is a separate molecule and has to bind to the effector before subsequent binding to the NLR receptor. In the latter case, low affinity of the effector-decoy interaction leads to drastically reduced receptor occupancy and renders the corresponding resistance very vulnerable to effector mutations affecting decoy binding.

Interaction of effectors with multiple independent sites is a hallmark of NLR receptor activation

Effector recognition by RGA4/RGA5 differs from other well-studied NLR models. Indeed, RGA5

has no inherent signaling activity and functions, on the one hand, by repressing RGA4 signaling activity and, on the other, by releasing repression upon AVR-Pia binding (Césari et al., 2014a).

Interestingly, the RATX1 domain is only required for de-repression and not for repression (Supplemental Figure 6) (Césari et al., 2014).

Providing the RATX1 domain separately in the presence of RGA5 ΔRATX1 and AVR-Pia does not relieve repression despite the fact that AVR-Pia interacts with the separate partners, RGA5 ΔRATX1 and RGA5 RATX1 . To explain this result, we propose the hypothesis that simultaneous binding of AVR-Pia to different sites in RGA5, including the RATX1 domain, is required to trap RGA5 in a conformation unable to repress RGA4 (Figure 7). That effectors have to establish simultaneously several independent interactions with NLRs or NLRS and co-factors to be recognized and trigger resistance has been frequently observed with effectors from various origins (Collier and Moffett, 2009). In the Pseudomonas syringae effector AvrRPS4 two different surface areas on opposite and distant sites of the molecule are required for recognition by the RRS1/RPS4 pair [START_REF] Sohn | Distinct regions of the Pseudomonas syringae coiled-coil effector AvrRps4 are required for activation of immunity[END_REF]. One of these sites is crucial for binding to the integrated WRKY decoy domain of RRS1 while the other seems to interact with other not yet identified regions in RRS1 (Sarris et al., 2015). Similarly, recognition of the Hyaloperonospora arabidopsidis effector ATR1-EMOY2 by the NLRs RPP1-NdA or RPP1-WsB from A. thaliana relies on two different surface areas from two different domains and on opposite sides of the molecule suggesting simultaneous interaction with independent binding sides in RPP1-NdA and RPP1WsB [START_REF] Chou | Hyaloperonospora arabidopsidis ATR1 effector is a repeat protein with distributed recognition surfaces[END_REF]Steinbrenner et al., 2015). Also in NLRs that recognize effector-co-factor complexes, simultaneous binding of these complexes to different parts of the NLR, generally involving the N-terminus and the LRR have been frequently described (Collier and Moffett, 2009). Therefore, we propose the hypothesis that effectors or effector-co-factor complexes forcing or trapping NLRs in an activated state by simultaneously binding to multiple binding sites and inducing or stabilizing by this major conformational changes is a widespread mechanism in NLR activation and in particular NLRs with integrated domains. Future, structural and functional analysis will be necessary to test this model and elucidate in more detail how activation occurs at the molecular level.

METHODS

Growth conditions of plants and fungi and infection assays

N. benthamiana plants were grown in a growth chamber at 22°C with a 16-h light period. Rice plants (Oryza sativa) were grown as described [START_REF] Faivre-Rampant | Characterization of the model system rice-Magnaporthe for the study of nonhost resistance in cereals[END_REF]. Transgenic M. oryzae GUY 11 strains were grown at 25°C during 5 days on rice flour agar for spore production (Berruyer et al., 2003) and in Tanaka complete culture medium (Villalba et al., 2008) agitated at 60 rpm and 25°C during 5 days for liquid culture.

For the analysis of interaction phenotypes, a suspension of M. oryzae conidiospores in water with 0.1% of gelatin and adjusted to 5 x 104 spores ml -1 was sprayed on the leaves of 3-week-old rice plants (Berruyer et al., 2003). Symptoms were analyzed 7 days after inoculation on the youngest leave that was fully expanded at the time of inoculation. For quantitative analysis leasons were classified and counted; resistant lesions, visible as small brown spots (type 1); weakly susceptible/partially resistant lesions characterized by a pronounced brown border and a small grey centre (type 2); fully susceptible lesions characterized by a large grey centre (type 3).

Constructs

Plasmids were generated by Gateway cloning (ThermoFisher, Waltham, USA), restriction/ligation, site-directed mutagenesis using the QuickChange Lightning kit (Agilent, Santa Clara, USA) or gaprepair cloning in yeast (Bruno et al., 2004). Gateway entry clones were generated using the pDONR207 plasmid (ThermoFisher, Waltham, USA). Gateway destination vectors were modified pBIN19 plasmids for expression of tagged proteins in N. benthamiana [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF] or modified pGAD-T7 or pGBK-T7 plasmids (Clontech, Mountain View, USA) for yeast two hybrid experiments (Bernoux et al., 2011). For protein expression, the pET-15b vector (Merck-Millipore, Darmstadt Germany) was used. For M. oryzae transformation constructs were based on the pDL02 plasmid (Bruno et al., 2004). For details on PCR and mutagenesis primers and generation of plasmid refer to Supplemental Tables 3 and4.

Nuclear magnetic resonance spectroscopy

Spectra were acquired on a 700 MHz Avance Bruker spectrometer equipped with triple-resonance

( 1 H, 15 N, 13 C) z-gradient cryo-probe at 305 K. Experiments were recorded using the TOPSPIN pulse sequence library (v. 2.1) (Supplemental Table 1). All spectra are referenced to the internal reference DSS for the 1 H dimension and indirectly referenced for the 15 N and 13 C dimensions [START_REF] Wishart | 1H, 13C and 15N chemical shift referencing in biomolecular NMR[END_REF]. Spectra were processed using Topspin (v. 3.2) and analyzed using strip-plots with Cindy in house software and CCPN (Vranken et al., 2005) [analysis v 2.3]. The 15 N and 13 C assignments were derived from the 2D and 3D spectra at 700 MHz listed in Table S1.

NMR titration

For the assignments, protein samples (1mM) in 20 mM potassium-sodium phosphate, pH 5.4 and 150 mM NaCl, were used. For the titrations of 15 N-labeled AVR-Pia proteins, different samples with constant concentrations of AVR-Pia WT or H3 (50µM) and various concentrations of unlabeled RATX1 (ratios 2:1, 1:1, 0.5:1, 0.25:1, 0:1 for the reference) were prepared. HSQC spectra were recorded at 305K on a Bruker Avance 700 MHz spectrometer. Chemical shift differences were measured from the HSQC spectra of AVR-Pia or AVR-H3 alone and the AVR-RATX1 complex at R = 2. They are reported as Hamming distance weighted by the magnetogyric ratios (Schumann et al., 2007).

Coimmunoprecipitation and Yeast two hybrid interaction assays

Protein-protein interaction analyses by co-immunoprecipitation were performed with protein extracts from N.benthamina leaf discs harvested 2 days after Agrobacterium infiltration [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. For the interaction of AVR-Pia variants with RGA5C-ter and RGA5 For immunodetection of proteins, rat anti-HA-horseradish peroxidase (clone 3F10 ; Roche) or mouse anti-GFP (Roche) and goat anti-mouse-horseradishperoxidase (Sigma-Aldrich) were used in combination with the Immobilon western kit (Millipore).

Binding domain (BD) fusions of AVR-Pia variants in pGBKT7-53 and activation domain (AD) fusions of RGA5C-ter in pGADT7 were transformed in Gold and Y187 yeast strain respectively.

Interactions assays were performed according to the Matchmaker Gold yeast two-hybrid system protocol (Clontech).

Transient Protein Expression and HR assays in N. benthamiana

For agroinfiltration in N. benthamiana pBIN19 binary vectors containing either AVR-Pia, PWL2 or RGA5 variants were transformed into Agrobacterium tumefaciens strains GV3101 by electroporation. Individual clones were selected and grown in Luria-Bertani liquid medium containing 50 mg ml-1 rifampicin, 15 mg ml-1 gentamycin, and 50 mg ml-1 kanamycin at 28°C for 24h before agroinfiltration. Co-inoculation mixtures adjusted to an OD600 of 1.0 were infiltrated in 4 weeks old N. benthamiana plants. The infiltrated plants were incubated for 48 or 96 h in growth chambers under controlled conditions for coimmunoprecipitations or cell death assays respectively. Three days post infiltration, N. benthamiana leaves were scanned using a Typhoon AVR-Pia binds to the RATX1 domain of RGA5 with a defined interaction surface and interacts, in addition, through independent surfaces with other sites in RGA5. These additional interactions are not sufficient to relieve the repression RGA5 exerts on RGA4. Indeed, AVR-Pia mutants that associate with RGA5, but do not bind RGA5 RATX1 as well as RGA5 mutants that lack the RATX1 domain, do not permit activation of resistance. We propose that simultaneous interactions of AVR-Pia with different parts of RGA5, including the RATX1 domain, stabilize conformational changes that activate the RGA4/RGA5 complex. shows the peaks present at the beginning of the titration for Y27, Y41, T51, N46 (in black or green for R=0 or R=0.5) that are missing at the end of the titration (in pink, R=2).

Supplemental

Supplemental Figure 3. AVR-Pia mutants affected in RGA5 RATX1 -binding are well-structured.

(A) AVR-Pia NMR structure [START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF] showing the amino acids that were replaced in the AVR-Pia variants carrying mutations in the candidate interaction surface (left) and, for control Structures were calculated using CYANA, refined using CNS, and analysed using PROCHECK.

(a) Residues in regular secondary structures were derived from the chemical shifts using TALOS+ software. (b) Main chain atoms (N, C, C) over the residues 20-85. Supplemental Table 3 this study [START_REF] Ribot | The Magnaporthe oryzae effector AVR1-CO39 is translocated into rice cells independently of a fungal-derived machinery[END_REF] this study this study this study this study this study this study this study

Structure Calculation

The programs CYANA [START_REF] Güntert | Automated NMR structure calculation with CYANA[END_REF][START_REF] Güntert | Automated NMR structure calculation with CYANA[END_REF] and CNS [START_REF] Brunger | Version 1.2 of the Crystallography and NMR system[END_REF] were used for automatic NOE assignments and structure calculations. The NH, Ha, 15 N, 13 Ca and 13 Cb chemical shifts were converted into F/Y dihedral angle constraints using TALOS+ (v. 1.2) [START_REF] Shen | TALOS+: a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts[END_REF].

Final structure calculations were performed with CYANA (v. 2.1) using 1366 distance restraints and 100 F/Y dihedral angle constraints. The 20 conformers with lowest target function starting from 200 initial structures, were refined by CNS (v. 1.2) using the refinement in water of RECOORD [START_REF] Nederveen | RECOORD: A recalculated coordinate database of 500+ proteins from the PDB using restraints from the BioMagResBank[END_REF]. These are the structures discussed herein and deposited (PDBs, 5JHJ).

The final 20 structures contained no NOE violations greater than 0.4 Å and no dihedral angle constraint violations greater than 2°. Structures were validated using PROCHECK [START_REF] Laskowski | Main-chain bond lengths and bond angles in protein structures[END_REF] (Table S2). The following AVR-Pia residues were restrained to lie closer than 12 Å from the RATX1 residues using Rosetta site constraints: R23, F24, V37, L39, M40, Y41, V42, R43, T46, T47, A48, T49, T51, T56 and E58. These residues correspond to the AVR-Pia central surface displaying the highest chemical shift differences from the NMR titration with RATX1. For each of the 3 RATX1 models, 3000 complexes were built with the same protocol. The 2% best scored complexes (180/9000) were clustered according to the orientation of AVR-Pia relatively to RATX1. All complex interfaces of the resulting clusters were evaluated using Pisa from the CCP4 suite. Some conclusions arising from these simulations could be stressed:

AVR-Pia

1) In all but one clusters, the central residue on AVR-Pia interface is M40, which is also central in the NMR mapped surface. The last cluster has a slightly displaced interface whose central residue is Y41.

2) The complex clusters display various interface orientations on RATX1 and none of them can be clearly distinguished neither from the number of poses, the Rosetta scores nor from the Pisa ΔGs.

3) None of the best clusters has an interface oriented as in the AVR-Pik / HMA complex. Other docking simulations done using the Zdock server (http://zdock.umassmed.edu) instead of Rosetta also failed to reproduce an interface oriented as seen in the AVR-Pik / HMA complex.

CHAPTER III

Analysis of the interaction of AVR-Pia with RGA5 domains other than RATX1

CONTEXT

In Chapter II, we describe the characterization of the AVR-Pia surface that directly binds to the RATX1 domain of the RGA5 immune receptor. We demonstrated that the AVR-Pia-RATX1

interaction is required for AVR-Pia recognition but that reduced binding affinity is welltolerated. Next we showed that RATX1 is dispensable for the binding of AVR-Pia to RGA5.

Indeed, the RATX1 deletion mutant RGA5 ΔRATX1 strongly associates with AVR-Pia in in planta experiments. Based on these results, we proposed a model to explain a robust effector recognition mediated by integrated decoy domains in combination with additional effector-NLR interactions. Furthermore, [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF] showed that in addition to AVR-Pia, RGA4/RGA5 mediate recognition of AVR1-C039 and that the RATX1 domain is also involved in the binding of this effector protein. Furthermore, a weak interaction between RGA5 RATX1 and RGA5 NB-ARC but not with RGA5 LRR or RGA5 CC was reported in yeast two hybrid experiments by Césari et al. 2014.

To evaluate the role of other RGA5 domains in effector recognition, we performed yeast two-hybrid (Y2H) and co-immunoprecipitation (co-IP) assays using different RGA5 domains: RGA5 ΔRATX1 , RGA5 CC , RGA5 NB , RGA5 CC-NB , RGA5 CC-NB-ARC , RGA5 NB-ARC , RGA5 LRR-C-ter and RGA5 C-ter (Figure 21) and we analyzed their interaction with the effector protein AVR-Pia and AVR1-CO39.

To investigate the role of RATX1 interactions with others RGA5 domain (Figure 21) in effector recognition and activation of the resistance, we analyzed the association of RATX1 domain with different RGA5 domains before and after AVR-Pia recognition by co-IP assays.

AVR-Pia associates with NB-ARC, LRR and RATX1 domains of RGA5

To analyze the interaction of AVR-Pia with other RGA5 domains than the RATX1 domain, BD and AD fusions of RGA5 ΔRATX1 , RGA5 CC , RGA5 NB , RGA5 CC-NB , RGA5 CC-NB-ARC , RGA5 NB-ARC and RGA5 LRR-C-ter were generated (Figure 21). AD and BD fusions of RGA5 C-ter were used as positive controls and, as previously reported, yeasts co-expressing either BD-AVR-Pia and AD-RGA5 C-ter or AD-AVR-Pia and BD-RGA5 C-ter grew on selective medium indicating physical binding between AVR-Pia and RGA5 C-ter (Figure 22). Yeasts co-expressing AD-or BD-AVR-Pia in combination with different AD-or BD-RGA5 domains, other than RGA5 C-ter , did not grow on selective medium (Figure 22) suggesting that AVR-Pia does not bind to those RGA5 domains in yeast. The BD-and AD-AVR-Pia fusions were correctly expressed and most of the BD-and AD-RGA5 domains were expressed at similar levels except for BD-and AD-RGA5 ΔRATX1 , BD-and AD LRR-Cter , BD-RGA5 NB and AD-RGA5 CC that were not expressed (Figure 23). Therefore, for these constructs, the lack of yeast growth could be explained by a failed protein expression.

To test whether AVR-Pia associates with RGA5 domains in planta, we used HA-tagged RGA5 domains and YFP-tagged AVR-Pia. A YFP fusion of PWL2, a M. oryzae effector that does not interact with RGA5 was used as a negative control. All proteins were properly expressed and both YFP fusion proteins were efficiently precipitated with anti-GFP agarose beads (Figure 24). All RGA5 domains but RGA5 CC were co-precipitated by YFP-AVR-Pia and not by PWL2, suggesting that AVR-Pia associates with RGA5 NB-ARC in planta. Since the isolated LRR domain of RGA5 could not be expressed in N. benthamiana (data not shown) and the RGA5 LRR-C-ter construct contains the C-ter domain that binds AVR-Pia we could not conclude whether AVR-Pia also interacts with the RGA5 LRR domain. To address this question, we tested the association with AVR-Pia F24S that does not interact with RGA5 Cter . For this, we tested the co-IP of HA-tagged RGA5 fragments by YFP-AVR-Pia F24S in comparison with the positive and negative controls YFP-AVR-Pia WT and YFP-PWL2. The three YFP fusion proteins were well precipitated. YFP-AVR-Pia F24S co-precipitated RGA5 LRR-Cter as did YFP-AVR-Pia WT but failed to co-precipitate RGA5 C-ter , suggesting that AVR-Pia associates with the LRR domain of RGA5. As observed for YFP-AVR-Pia WT , RGA5 CC was not co-precipitated by YFP-AVR-Pia F24S . Strikingly, YFP-AVR-Pia F24S efficiently co-precipitate RGA5 CC-NB and RGA5 CC-NB-ARC but not RGA5 NB (Figure 25) .

Altogether, these results indicate that AVR-Pia associates with the RATX1, NB-ARC and LRR domains of RGA5 in in planta assays. For the NB-ARC and LRR domains, these interactions were not detected in yeast two hybrid assays. Therefore, it remains to be elucidated by other approaches whether additional proteins are required for these interactions or whether they rely on direct physical binding. Yeast co-expressing BD-AVR1-CO39 with AD-RGA5 CC-NB and with AD-RGA5 CC-NB-ARC efficiently grew on elective medium and to similar levels as the positive control (Figure 26). In contrast, yeast expressing BD-AVR1-CO39 in combination with AD-RGA5 NB showed a very weak growth suggesting that AVR1-CO39 interacts with the NB domain of RGA5 and that the ARC domain is important to stabilize this interaction or establishes additional interactions with the effector (Figure 26). Under higher selection stringency, on -LWH + 3AT medium, only yeasts co-expressing BD-AVR1-CO39 and AD-RGA5 CC-NB-ARC grew but this growth was weaker than that of yeasts co-expressing BD-AVR1-CO39 and AD-RGA5 C-ter (Figure 26), suggesting that at least in yeast the interaction of AVR1-CO39 with RGA5 C-ter is stronger than with the NB-ARC domain.

Results with AD-RGA5 LRR-Cter , AD-RGA5 CC or AD-RGA5 ΔRATX1 are not informative because there is insufficient protein production for these RGA5 constructs (Figure 23). The lack of growth observed for yeasts co-expressing BD-AVR1-CO39 and AD-RGA5 LRR-Cter , AD-RGA5 CC or AD-RGA5 ΔRATX1 therefore does not indicate lack of interaction. Strikingly, when we co-expressed AVR1-CO39 fused to AD with RGA5 domains fused to BD we only detected interaction between AD-AVR1-CO39 and BD-RGA5 C-ter (Figure 26) . A possible reason is that the AD domain interferes with the physical binding of AVR1-CO39 to the NB-ARC domain of RGA5 that was efficiently expressed in yeast (Figure 23).

To investigate the interaction between AVR1-CO39 and RGA5 domains in planta we performed co-IP of different HA-tagged RGA5 domains with YFP-tagged AVR1-CO39 or PWL2 with the same co-IP conditions used for AVR-Pia. All proteins were properly expressed and anti-GFP agarose beads efficiently immunoprecipitated YFP-AVR1-CO39 and YFP-PWL2

(Figure 27). Under these conditions, YFP-AVR1-CO39 exhibit strong association with HA-RGA5 CC-NB-ARC and HA-RGA5 NB-ARC . Association with HA-RGA5 CC-NB or HA-RGA5 NB is also observed but was very weak. With HA-RGA5 LRR-C-ter, HA-RGA5 CC or HA-RGA5 C-ter no association was observed (Figure 27). For the constructs containing the RATX1 domain, HA-RGA5 LRR-C-ter and HA-RGA5 C-ter this is surprising since RGA5 C-ter -AVR1-CO39 interaction was observed in Y2H and was reported previously (Césari et al., 2014c). However, in these in planta association studies between AVR1-CO39 and RGA5 C-ter less stringent condition for co-IP experiments had been used [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. Taken together these results suggest that AVR1-CO39 binds directly to RGA5 NB-ARC in addition to RGA5 C-ter. RGA5 CC , indicating that RATX1 interacts with RGA5 NB-ARC and self-associates which has also previously been found in yeast two hybrid analysis (Césari, et al. 2014) (Figure 28).

To determine whether the interactions between RATX1 and RGA5 domains are modified by AVR-Pia recognition, we tested the co-IP of HA-RATX1 and YFP-tagged RGA5 domains (RGA5 C-term , RGA5 CC, RGA5 NB-ARC or RGA5 ΔRATX1 ) in the presence of AVR-Pia. In the presence of AVR-Pia, the association between HA-RGA5 RATX1 with YFP-RGA5 NB-ARC or YFP-RGA5 C-term is weaker than in its absence (Figure 29), indicating that AVR-Pia modifies these interactions.

However, the co-IP of HA-RGA5 RATX1 by YFP-RGA5 ΔRATX1 was not affected in the presence of AVR-Pia potentially because association is stabilized by the LRR domain. Therefore, the association between RATX1 and LRR should be analyzed in the future.

To get a better view of the strength of the RATX1 self-interaction and the interaction of RATX1 with the NB-ARC domain we also used more stringent conditions for coimmunoprecipitation of HA-RATX1 with YFP-RGA5 NB-ARC , YFP-RGA5 ΔRATX1, YFP-RGA5 CC or YFP-RGA5 RATX1 (used here instead YFP-RGA5 C-ter ,) in presence of AVR-Pia. Under these conditions, a strong association was only observed between HA-RATX1 and YFP-RATX1 in the absence of AVR-Pia (Figure 30). In addition, a weak association of HA-RATX1 with YFP-RGA5 NB-ARC and YFP-RGA5 ΔRATX1 was detected and in all cases the interactions were lost in the presence of AVR-Pia.

Taken together these results suggest that the RATX1 domain interacts with itself and with the NB-ARC domain of RGA5. Both, RATX1-RATX1 and RATX1-NB-ARC interactions seem to be affected by AVR-Pia.

DISCUSSION

The immune receptor RGA5 interacts with different effector proteins through multiple domains

In this study, we provide evidence that RGA5 interacts with AVR-Pia and AVR1-CO39 through its NB-ARC and RATX1 domains. In addition we showed that AVR-Pia also interact with the LRR domain of RGA5. Since association of AVR-Pia with RGA5 NB-ARC and RGA5 LRR was only detected in in planta assays and not in yeast, we cannot exclude that additional proteins are required to mediate this interaction and that the interaction is indirect. Interaction between AVR1-CO39 and RGA5 NB-ARC was detected in both in planta and in yeast two hybrid assays suggesting that it relies on direct physical binding. Altogether, these results strongly support that both AVR-Pia and AVR1-CO39 bind not only to the RATX1 domain of RGA5 but also to its NB-ARC domain. In chapter II we propose a cooperative model for AVR-Pia-RGA5 interaction in which effector recognition depends on simultaneous binding to the integrated domain and other NLR domains. Here, we provide additional support for this model and show that at least one second domain, RGA5 NB-ARC, binds or indirectly associates with the two structurerelated effectors AVR-Pia and AVR1-CO39. This opens now the way to a more precise characterization of this interaction including the identification of the interaction surfaces and the individual residues that mediate this interaction. Subsequently, these residues can be mutated to verify if the effector-NB-ARC interactions are required for effector recognition and to determine their contribution to the recognition specificity. This would give also some domain [START_REF] Leipe | STAND, a class of P-loop NTPases including animal and plant regulators of programmed cell death: Multiple, complex domain architectures, unusual phyletic patterns, and evolution by horizontal gene transfer[END_REF][START_REF] Van Ooijen | Structure-function analysis of the NB-ARC domain of plant disease resistance proteins[END_REF]. Interestingly, this domain confers ligand recognition specificity to NAIP proteins and e.g. NAIP2 directly interacts through its nucleotide-binding domain with PrgJ a type III secretion system (T3SS) component of the bacteria Salmonella [START_REF] Tenthorey | Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes[END_REF]. Therefore it is tempting to speculate that like NAIP NACHT , the RGA5 NB-ARC domain has evolved the ability to interact with pathogen-derived ligands which would be an interesting case of convergent evolution.

RGA5 RATX1 self-association and RGA5 RATX1 -RGA5 NB-ARC interaction is perturbed by AVR-Pia

The interaction of RGA5 RATX1 with RGA5 NB-ARC and RGA5 RATX1 in the presence of AVR-Pia was characterized in planta by co-immunoprecipitation assays and showed that the interaction or RGA5 RATX1 with RGA5 NB-ARC as well as its self-association is weakened by AVR-Pia.

Interestingly, AVR-Pia associates with both domains suggesting that AVR-Pia interacts with the RATX1 surfaces mediating self-association. In addition, they suggest that AVR-Pia and RATX1 may interact with similar RGA5 NB-ARC surfaces or that the AVR-Pia-binding surface in the RATX1 domain is also involved in RGA5 NB-ARC -binding. Since the RATX1 domain of RGA5 is not required for the repression of RGA4 it can be speculated that it is not the removal of the RGA5 RATX1 -RGA5 NB-ARC association by AVR-Pia that triggers activation of resistance. Rather, the simultaneous interaction of AVR-Pia with the RATX1 and the NB-ARC domain could force RGA5 in a conformation that no longer allows RGA4 repression and thereby triggers resistance. The role RGA5 RATX1 self-association in effector recognition requires further analyses since the stoichiometry of the RGA4-RGA5 complex is unknown and it is unclear whether it has biological relevance. It would be particularly interesting to better characterize the composition of the RGA4/RGA5 complex and to investigate whether RGA5 RATX1 mutants impaired in the formation of homo-complexes are affected in AVR-Pia or AVR1-CO39 recognition.

MATERIAL AND METHODS

The material and methods of this chapter are detailed in the annex.

SUMMARY POINTS (CHAPTER II and III)

 AVR-Pia binds the RGA5 RATX1 domain through multiple residues located in a specific surface exposed region of the effector protein.

 M. oryzae effector proteins with homologous structures can bind similar domains through different protein surfaces.

 The RATX1-interaction surface of AVR-Pia undergoes mutations to escape recognition by the RGA4/RGA5 complex.

 The RATX1 domain is crucial to mediate recognition of AVR-Pia by the RGA5/RGA4 complexes.

 AVR-Pia binds the RGA5 RATX1 domain with intermediate affinity and reduced binding is well tolerated in recognition.

 The RATX1 domain is dispensable for the association of AVR-Pia with RGA4/RGA5 complexes.

 AVR-Pia associates with RGA5 domains other than RATX1.

 Simultaneous binding of AVR-Pia to different RGA5 domains could explain the high resilience of RGA4/RGA5-mediated resistance to mutations in the effector.

INTRODUCTION

One of the most amazing features of NLRs is their ability to confer resistance to a large number of pathogens from different kingdoms such as insects, oomycetes, bacteria, viruses and nematodes. To respond to these highly diversified groups of pathogens, NLRs possess flexible structures and varied modes of action that allow the recognition of a maximum number of effector proteins, one of the most powerful molecular arms of pathogens to cause infection.

Cooperativity between NLRs appears to be one way to maximize pathogen detection by using a limited set of resistance proteins (Williams et al., 2014). The NLR proteins RRS1 and RPS4 e.g. act together to mediate resistance to three different pathogens; Pseudomonas syringae, Ralstonia solanacearum and Colletotrichum higginsianum. Functional analyses have shown that RRS1 and RPS4 proteins interact in part via their TIR domains to form heterocomplexes that are essential for defense activation. Further, it has been shown that effector proteins PopP2 from R. solanacearum and AvrRPS4 from P. syringae exclusively interact with RRS1 and not with RPS4 showing that RRS1 acts as the effector sensor (Williams et al., 2014). P-loop motif of many NLRs is required for nucleotide binding and cell death triggering. Mutations in the P-loop motif in RPS4 and not in RRS1 impaired recognition of PopP2 and AvrRPS4 and cell death induction. Furthermore overexpression of RPS4 induced cell death showing that this NLR act as a cell death executor (Williams et al., 2014).

Together these results present an example of cooperation between NLRs, in which one protein act as an "effector plataform" that facilities the perception to several pathogens by using a second protein that mediates the defense signaling.

The NLRs RGA4 and RGA5 from rice are another couple of resistance proteins working together to mediate pathogen recognition and the molecular bases of their mode of function started to be elucidate by Césari, et al., in 2014. It has been shown that RGA5

mediate the recognition to the sequence unrelated effector proteins AVR-Pia and AVR1-CO39 and the acts an effector sensor whereas RGA4 is a cell death executor as was also observed in RRS1 and RPS4 respectively. In addition, it was demonstrated that RGA5 acts as a RGA4 repressor and therefore in the presence of the AVR-Pia, RGA5 binds the effector protein relieving RGA4 which in turn trigger cell death.

In this chapter, we will provide details about the physical interaction between RGA5 and RGA4 domains and the nature of complexes before and after effector binding.

CONTEXT

Previously it was shown that RGA4 and RGA5 functionally interact to mediate effector recognition (Césari, et al. 2014). Furthermore, it was demonstrated that RGA5 and RGA4 form homo and hetero-complexes and that they physically interact through their CC domains. However it has not yet been elucidate whether domains in RGA4 and RGA5 others than CC also interact. In the absence of effector proteins, RGA5 interacts with RGA4 and repress its activity. Effector recognition by RGA5 releases RGA4 repression but it is unknown whether the active form of RGA4 still interacts with RGA5 or whether the interaction between RGA5 and the effector protein disrupts the RGA5-RGA4 hetero-complex.

To better understand the link between physical RGA4-RGA5 interaction and the activation of resistance after effector perception we evaluated the interaction of different RGA4 and RGA5 domains (Figure 31) as well as the interaction of RGA4 and RGA5 in the presence of AVR-Pia.

RESULTS

RGA5 and RGA4 interact through multiple domains

Interaction between the CC domains of RGA5 and RGA4 was previously reported by Césari et al. (2014). To investigate whether other domains also interact, we generated mutants for RGA4 and RGA5 where the CC domains is deleted and fused them to HA and GFP or YFP tags:

RGA4 ΔCC -HA, RGA4 ΔCC -GFP, HA-RGA5 ΔCC and YFP-RGA5 ΔCC . Then, we tested their interaction by co-IP assays in comparison with the co-IP of the full length proteins RGA4-HA, RGA4-GFP, HA-RGA5 and YFP-RGA5. The Co-IP of RGA4 ΔCC -HA and HA-RGA5 ΔCC by YFP was used as a negative control.

All proteins were properly expressed and proteins tagged with GFP or YFP were efficiently immunoprecipitated by anti-GFP agarose beads (Figure 32). Both RGA4 ΔCC -HA and HA-RGA5 ΔCC were co-immunoprecipitated by YFP-RGA5 ΔCC and RGA4 ΔCC -GFP respectively (Figure 2). The interaction of HA-RGA5 Δcc with RGA4 ΔCC -GFP was weaker than that of HA-RGA5 with RGA4 ΔCC -GFP probably due to a reduced quantity of RGA4 ΔCC -GFP in protein extracts (input Figure 32). Taken together, these results indicate that the CC domains of RGA4 and RGA5 are dispensable for association and that additional domains in these NLRs seem to interact to mediate hetero-complex formation.

The RGA5 C-ter does not interact with the NB-ARC and LRR-C-ter domains of RGA4

To investigate what domains of RGA5 are involved in the interaction with RGA4, we analyzed the interaction of HA-RGA5 NB-ARC , HA-RGA5 LRR-C-ter and HA-RGA5 C-ter with RGA4 NB-ARC -GFP and RGA4 LRR-C-ter -GFP by co-IP assays in N. benthamiana. The interactions between YFP and RGA5 variants were used as negative controls.

All proteins were properly expressed and the proteins fused to YFP or GFP were efficiently immune-precipitated (Figure 33). HA-RGA5 NB-ARC and HA-RGA5 LRR-C-ter were well coprecipitated by RGA4 NB-ARC -GFP and RGA4 LRR-C-ter -GFP respectively, suggesting that the NB-ARC and LRR domains of RGA4 and RGA5 are involved in hetero-complex formation, in addition to the previously reported interaction between their CC (Césari, et al in 2014).

On the contrary, HA-RGA5 C-ter was not co-precipitated by either RGA4 NB-ARC -GFP or RGA4 LRR-C- ter -GFP. Taken together these results suggest that all RGA5 domains but the C-ter participates in the interaction with RGA4. The interaction between RGA5 C-ter and RGA4 CC will have to be tested in the future.

DISCUSSION

RGA5 and RGA4 present a robust interaction mediated by at least three different domains

In this work we investigated the molecular details of the interaction of RGA5 with RGA4 and we showed that RGA5 NB-ARC and RGA5 LRR domains interact with their corresponding domains RGA4 NB-ARC and RGA4 LRR domains. In addition we showed that RGA5 RATX1 does not interact with neither RGA4 NB-ARC nor with RGA4 LRR . These data led us to the conclusion that RGA4 and RGA5 establish tight and multiple interactions mediated by three different domains: the CC domain, as shown by Césari et al 2014 and the NB-ARC and LRR as showed here. Only the RGA5 RATX1 domain appears not to participate directly in RGA4/RGA5 complex formation.

However, since we have not yet characterized the interaction between RGA5 C-ter and RGA4 CC we cannot reject the possibility that RGA5 RATX1 somehow interacts with this domain.

RGA5 RATX1 is dispensable for repression of RGA4 by RGA5 and AVR-Pia association but is required to mediate AVR-Pia recognition. On the other hand, RGA5 RATX1 interacts with neither RGA4 NB-ARC nor RGA4 LRR . Taken together these suggests that binding of AVR-Pia to the RATX1 domain and association to the NB-ARC and eventually the LRR domain induce the release of RGA4 repression, rather than the interaction of RGA5 RATX1 with RGA4 CC or other RGA4 or RGA5 domains. Further confirmation and investigation of the relevance of AVR-Pia association with RGA5 NB-ARC and RGA5 LRR domains will therefore be of great importance to better understand the role of inter and intramolecular interactions of RGA4/RGA5 complex in effector recognition and activation of the resistance.

An RGA4/RGA5 complex seems to mediate effector depend cell death induction

Here we provide further evidence that the robust physical interaction between RGA4 and RGA5 is not abolished in the presence of AVR-Pia. Previously, Césari et al 2014 showed that -RGA5 represses RGA4-triggered cell death and that AVR-Pia relieves this repression. We show that YFP-RGA5 co-precipitates RGA4-HA in the presence of AVR-Pia, suggesting that the RGA4/RGA5 complex is not disrupted upon AVR-Pia recognition but that rather conformational changes and modifications of intermolecular interactions between RGA4 and RGA5 occur. In this way, the surfaces in RGA4 required to induce cell death may become available to interact with other uncharacterized proteins or to form a signaling competent complex with RGA5.

In RRS1/RPS4 e.g., effector recognition by RRS1 leads to conformational changes in the RRS1/RPS4 complex that result in homotypic interactions of the RPS4 TIR domain required for cell death but not in disruption of the complex. In animals NAIP/NLRC4 NLR pairs form a wheel-shaped oligomer called the inflammasome after signal recognition and during defense signaling that contains one NAIP-ligand dimers and up to 11 NRC4 molecules [START_REF] Tenthorey | Molecular Basis for Specific Recognition of Bacterial Ligands by NAIP/NLRC4 Inflammasomes[END_REF][START_REF] Bentham | Animal NLRs provide structural insights into plant NLR function[END_REF]. Formation of the oligomer is initiated by formation of a NAIP-ligand complex. Then, the NAIP-ligand dimer binds to NLRC4, which triggers association of additional NLRC4 molecules and formation of the inflammasome that transduces the immune signal. However in this case, NAIP and NLRC4 interact only after ligand perception suggesting that the mechanism to activate signaling markedly different between plant and animal NLR pairs. Further analyses are therefore required to better understand the nature of NLR heteropair complexes in plants during effector recognition and defense signalling.

Material and methods

The material and methods of this chapter are detailed in the annex.

GENERAL DISCUSSION AND PERSPECTIVES

During their co-evolution, plants and pathogens employ a large repertoire of molecular arms to interact with each other. Pathogens deploy effector proteins that act inside or outside host cells to manipulate plant cellular processes, to promote pathogen growth and cause disease while plants rely on the continuous surveillance of the cell by using immune receptors to avoid disease. A major class of plant immune receptors are the NLR proteins that recognize cytoplasmic pathogen effectors and this recognition results in the activation of defense responses protecting plants from the disease.

In this work, we studied the molecular factors in both effector and NLR proteins that determine recognition specificity and the molecular bases of the activation of the NLR proteins after effector perception. In addition, we investigated the diversity of fungal effector proteins.

Fugal effectors that are highly variable in sequence may be related by structural homologies and common evolutionary origin

The huge effector complements of fungi are extremely diverse and mainly lineage or species specific suggesting that the effectors of different fungal linages evolve rapidly and independently from one another. This can, e.g., be nicely observed in the powdery mildew fungi where genotypes that infect barley and closely-related genotypes that attack wheat share about 500 candidate effectors while they have only few in common with more distantly related powdery mildew species that attack dicotyledonous plants [START_REF] Pedersen | Structure and evolution of barley powdery mildew effector candidates[END_REF][START_REF] Spanu | Genome Expansion and Gene Loss in[END_REF]. As a consequence, the massive identification of fungal candidate effector proteins in the last decade has unraveled thousands of unique proteins of unknown functions and origin.

In this work, we determined by NMR the 3-dimensional structure of the sequence-unrelated M. oryzae effector proteins AVR-Pia and AVR1-CO39. This, in combination with bioinformatic searches and in planta expression analyses, led us to the identification of a family of effector proteins that we named MAX-effectors (Magnaporthe Avrs and ToxB like). MAX-effectors are the first family of effectors with conserved structure and unrelated sequence identified in fungi. Their discovery is of great importance for effector biology since it suggests that many of the huge number of apparently unrelated effectors in fungi may actually be structurally and phylogenetically related and gathered in structurally defined families.

Highly similar 3-dimensional structures have been also found in effector proteins with limited sequence homology in oomycetes [START_REF] Boutemy | Structures of Phytophthora RXLR effector proteins: A conserved but adaptable fold underpins functional diversity[END_REF][START_REF] Win | Sequence divergent RXLR effectors share a structural fold conserved across plant pathogenic oomycete species[END_REF]. The crystal structures of the effector proteins AVR3a11 and PexRD2 from Phytophthora capsici and Phytophthora infestans revealed that despite extremely weak sequence identity, below 20%, these two effectors displayed a conserved α-helical fold termed the "WY-domain" [START_REF] Boutemy | Structures of Phytophthora RXLR effector proteins: A conserved but adaptable fold underpins functional diversity[END_REF]. Bioinformatic analyses suggest that the core α-helical fold actually occurs in 44% and 26% of annotated Phytophthora and H. arabidopsis RXLR effectors respectively either as a single domain or in tandem repeats [START_REF] Boutemy | Structures of Phytophthora RXLR effector proteins: A conserved but adaptable fold underpins functional diversity[END_REF].

The identification of structurally related effector families such as MAX-effectors and WY-RXLR-effector that are highly diversified provides a molecular framework to better understand the way effectors evolved and tolerate sequence hyper-variability. For example, we observed that the β-sandwich fold of the MAX-effectors allows insertion or deletion of amino acids in the loops or the exchange of surface exposed amino acids. A flexible structure like the MAX-effector β-sandwich fold therefore facilitates the generation of multiple effectors with different shapes and surface properties, molecular activities and host targets that nevertheless maintain a stable overall structure.

The identification of fungal candidate effectors relies mainly on bioinformatic sequence analyses and is not very precise because it is usually based on relatively broad criteria. Here we showed that a combination of structure-informed pattern-based searches led to the identification of huge numbers of MAX candidate effectors mainly in M. oryzae and M.

grisea. In addition we showed that a large majority of MAX candidate effectors in M. oryzae are expressed specifically during early infection indicating that this group of effectors may be important during the biotrophic colonization of the plant host. This analysis demonstrates that structure determination and structure-informed bioinformatics analysis are extremely powerful approaches in the analysis of fungal effectors.

Interestingly, three of the best characterized MAX-effector proteins AVR-Pia, AVR1-CO39

and AVR-Pik in M. oryzae interact with a HMA/RATX1 domain integrated in an NLR protein;

Pik-1 in the case of AVR-Pik and RGA5 in the case of AVR-Pia and AVR1-CO39. This suggests that in addition to sharing a conserved structure, some MAX-effectors may also interact with highly similar domains in host proteins, despite the big differences in their surface properties. The structure of the AVR-Pik-HMA complex has been determined (Maqbool et al. 2015) and it is now a priority to generate also the structure of AVR-Pia and AVR1-CO39 in complex with the RGA5 RATX1 domain and compare whether the interaction between these avirulence proteins and HMA/RATX1 involves homologous β-strands or loops or whether the residues that determine the specificity and strength of the interaction possess similar properties.

61 HMA/RATX1-like proteins are present in the rice genome according to Panther database (www.pantherdb.org/panther/family.do?clsAccession=PTHR22814) [START_REF] Cesari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. In a mid-term, it would be very interesting to analyze e.g. by yeast two hybrid assays whether other MAX-effectors interact with some of these HMA/RATX1-like proteins in rice. These studies would provide an overview about the conservation of MAX-effector functions and targets. However, from present data it is already clear that MAX effectors target also other host proteins than HMA/RATX1 proteins, since AvrPiz-t interacts with E3 ubiquitin ligases. To get a broader picture of MAX effector functions, it will in the future be important to determine the host targets of a greater number of MAX effectors by approaches without a priori such as pull down or yeast two hybrid screens.

The two MAX effector proteins AVR-Pia and AVR-Pik have similar RATX1-binding surfaces

In this thesis, we deciphered in detail the interaction between AVR-Pia and the RATX1 domain of RGA5. We identified the RATX1-binding surface in AVR-Pia and found that this surface resembles to the surface of AVR-Pik that binds the HMA domain of Pik-1. The HMA and the RATX1-domains have similar sequences (55% identity) and according to homologybased structure modeling have very similar structures. However, they seem to have significant differences in the HMA/RATX1-binding surfaces of AVR-Pia and AVR-Pik. Surface properties such as charge distribution and hydrophobicity are different between both MAX effectors and, in particular, an N-terminal extension of AVR-Pik absent from AVR-Pia is crucial for interaction with Pik-1 HMA . One interesting question now is how AVR1-CO39, a sequence-unrelated but structurally similar effector to AVR-Pia and AVR-Pik, interacts with the RATX1 domain? It is e.g. possible that the binding surface in AVR1-CO39 involves like in AVR-Pia and AVR-Pik the β-strands 2 and 3 and loop 2. However, these surfaces have very different properties in AVR-Pia and AVR1-CO39 which makes it difficult to understand how they could bind the same target sequence. In addition, AVR1-CO39 has a C-terminal extension that could be involved in RATX1-binding in a similar way that the N-terminal extension of AVR-Pik that binds the HMA domain. Hence, the question with which surface AVR1-CO39 binds the RATX1 is clearly open.

Another important question is: which are the surfaces of the RATX1 that interact with AVR1-CO39 or AVR-Pia. One possibility is that both AVR1-CO39 and AVR-Pia interact with the same binding surface in the RATX1/HMA structure. However, if this is the case it will be particularly interesting to learn how this is accomplished since both effectors differ strongly in surface properties and shapes. Alternatively, they could interact with different surfaces in the RATX1 domain that may in addition be different from the homologues surface in Pik-1 HMA bound by AVR-Pik. Docking models for the AVR-Pia/RATX1 complex support this hypothesis since no model of this complex resembles the structure of the Avr-Pik/Pik HMA complex (chapter II) .

Finally, it would be interesting to identify the virulence targets of AVR1-CO39, AVR-Pia and AVR-Pik, and determine whether they are in all cases small HMA proteins and to investigate by which molecular mechanism these three effectors act on their corresponding virulence targets. Work in the group of R. Terauchi (IBRC, Iwate, Japan) indicates that AVR-Pik targets a clade of small HMA proteins similar to the Pik HMA and RGA5 RATX1 domains and establishes with them the same type of interaction they set with Pik HMA . These interactions stabilize the small HMA proteins which contribute to increased susceptibility and reduction of ROS production (personal communication). Whether AVR-Pia and AVR1-CO39 target the same clade of sHMAs and whether they target them by the same type of mechanism remains to be determined.

Additional NLR domains cooperate with integrated domains in effector binding and recognition

In this work, we found that AVR-Pia mutants that lost the capacity to bind the RGA5 RATX1 domain are able to fully interact with RGA5 ΔRATX1. , This indicates that other domains of RGA5 than RGA5 RATX1 interact with AVR-Pia and this probably through AVR-Pia surfaces that are different from the RATX1-binding surface. Indeed further analyses suggested that these additional interactions involve the RGA5 NB-ARC and RGA5 LRR domains and that similar interactions occur with AVR1-CO39 . The recognition of different effector surfaces by independent binding sites in NLRs, including interactions with IDs, is expected to increase specificity and robustness in effector recognition since reduction in the affinity to one binding side has minor impact when a second binding site is present. Such coordinated binding to several binding sites, including the integrated decoy could constitutes an efficient strategy to avoid the loss of recognition by rapid mutations in the effector and be one of the key advantages of NLR-IDs over a situation where NLRs and integrated decoys are separated.

To validate this model it will be important to demonstrate in the future that the interaction between the effector and the NLR domains other than the integrated domain are required for effector recognition. For this, we will further characterize the interaction of AVR-Pia and AVR1-CO39 with the NB-ARC and LRR domains. Then we are planning to identify AVR-Pia mutants that do no longer associate with either or both of these domains and to analyze the impact of the loss of this association in effector binding and avirulence activity.

Integrated domains are crucial for effector recognition and promise to be useful guides for the identification of plant effector targets.

In the present study we found that binding of AVR-Pia to the RATX1 domain is required for effector recognition. This is an important support for the integrated decoy model that states that NLR integrated domains (NLR-ID) serve as effector detector modules. Similarly compelling evidence for this model has for the moment only been provided by the investigation of AVR-Pik/Pik-1, PopP2/RRS1 or AvrRPS4/RRS1. In AVR-Pik/Pik-1 and AvrRPS4/RRS1 effector-binding to the decoy domain have been identified as in AVR-Pia/RGA5 as key events for NLR activation. In PopP2/RRS1, the NLR is rather activated by an effector-mediated acetylation of the integrated decoy domain. This highlights that integrated domains, very likely as classical guardees, decoys or co-factors, can function in effector recognition by different manners: they can be involved in complex formation or acquire post-translational modifications that are sensed by the associated NLR.

The systematic analysis of plant NLRs has shown that the integration of uncommon domains is frequent; it occurs at a rate of approximately 5%, and is widespread since NLR-IDs have been found in all land plants from mosses to angiosperms (Césari, et al. 2014;[START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF][START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]). In addition, the diversity of integrated domains is very high. For instance, [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF] identified about 265 different integrated domains in 750 NLR proteins carrying integrated domains [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. The most highly represented domains are protein kinases, DNA-binding and protein-protein interaction domains [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. Interestingly, many of the integrated domains fused to plant NLRs have been found to interact with effectors in targeted studies or effector interactome screens [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. This suggests that effector recognition is a general feature of integrated domains similar to what has been demonstrated experimentally for RGA5, Pik-1 and RRS1.

Furthermore, many of the integrated domains have not yet been associated with plant diseases or immunity and represent an outstanding guide for the identification of novel effector targets and immune components [START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF][START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF][START_REF] Ellis | Integrated decoys and effector traps: how to catch a plant pathogen[END_REF][START_REF] Malik | Commentary Inspirational decoys : a new hunt for effector targets[END_REF] Perspectives for plant immune receptor design exploiting the modular structure of NLR-IDs

The uncovered role of integrated domains in pathogen perception as well as the modular structure of NLR-ID has raised the possibility that the recognition specificity of this group of NLR receptors could be modified by simply exchanging the integrated domain. For instance it could be conceivable to plug cellular hubs of immune signaling that are targeted by multiple effectors into NLR-IDs to generate versatile large spectrum immune receptors. However, our study suggests that the design of NLR-IDs with completely novel specificities will not be as simple as that. Indeed, we show that the RATX1 domain as well as AVR-Pia interact with additional RGA5 domains. Indeed, we think that these interactions are required for RGA5 function, and, in particular, for proper RGA4 de-repression in the presence of effect Therefore, we believe that these additional interactions will have to be taken into consideration when integrating novel decoy domains. The work on AVR-Pik/Pik-1 suggests that NLR-IDs with extended recognition specificities that recognize additional alleles of the same effector may be relatively easy to generate by changing single residues in the integrated domains.

Another first step can be the creation of RGA5 variants where residues that are crucial for AVR-Pik recognition have been introduced into the RATX1 domain to create an RGA5 variant that recognizes AVR-Pik. However, according to our model such variants would require probably additional modifications outside the RATX1 domain to interact correctly with the effector and be completely functional. However, a much more detailed understanding of effector-NLR-ID interactions and intramolecular interaction in NLR-IDs will be required before novel specificities can be designed by integration of completely new decoy domains

NLR pairs interact through multiple domains

Another open and challenging question is how RGA4 and RGA5 and more generally other paired NLRs interact to mediate effector recognition. In the TNL pair RRS1/RPS4, the important role of TIR domain homo and heterotypic interactions in activation and repression has been documented (Williams et al., 2014). In RGA4/RGA5, previously shown CCinteractions could play similar roles (Césari, et al. 2014). In the present study, we provide evidence that additional domains of RGA4 and RGA5 contribute to their hetero-association.

Indeed, the NB-ARC and LRR domains of RGA4 seem to interact with the NB-ARC and LRR domains in RGA5 respectively. To evaluate the importance of these interactions for the function of the hetero-complex, it will be important to further characterize these interactions and to create specific mutations within RGA4 and/or RGA5 CC, NB-ARC or LRR domains that prevent their hetero-association and analyze whether these mutations also affect effector recognition and defense signalling.

The RGA4/RGA5 hetero-complex seems not to be disrupted by the presence of the effector It was previously demonstrated that RGA4 and RGA5 interact in the absence of recognized AVRs (Césari, et al. 2014). In this study, we provide preliminary results suggesting that the presence of the effector does not disrupt the RGA4/RGA5 hetero-complex. However, additional experiments are required to confirm this hypothesis and it remains to be determined whether the functional complex is indeed a tripartite complex constituted by AVR-Pia/RGA4/RGA5.

One perspective is to perform additional co-IP experiments where AVR-Pia is immunoprecipitated and the co-precipitation of RGA4 and RGA5 is evaluated. In addition, it should be important to confirm whether RGA4 co-precipitates AVR-Pia in presence of RGA5 in in planta experiments. Alternatively, BiFC analyses and Fret-Flim experiments can monitor the in planta interaction of RGA4 and RGA5 in the absence and the presence of AVR-Pia.

Defense signalling mediated by plant NLR hetero-complex is poorly understood. In the case of RGA4/RGA5 it was shown that RGA4 is able to induce cell death either in absence of RGA5

or after the recognition of AVR-Pia through RGA5 (Césari, et al. 2014). However, how RGA4 mediates defense signaling is still an open question and the downstream signaling partners are not known. In animals it has been shown that a functional hetero-complex of NLRs functioning in pairs actually involves a multiprotein complex called inflammasome. The last, has a wheel-like structure that creates one oligomerization surface required to defense signaling [START_REF] Hu | Structural and biochemical basis for induced self-propagation of NLRC4[END_REF]. For example, in mouse and humans, the NLRs called NAIPs (Neuronal Apoptosis Inhibitor Protein) interact with NLRC4 (NLR family CARD domaincontaining protein 4) to mediate resistance to bacterial pathogens. In mice, NAIP2 directly recognizes the rod protein PrgJ, a principal component of the type III secretion systems in bacteria. This recognition induces NAIP2 interaction with NLRC4 that result in the oligomerization of NLRC4 in complex with NAIP and PrgJ. The structure of the PrgJ-NAIP2-NLRC4 complex has been determined by cryogenic electron-microscopy and have revealed that this multiprotein complex is constitute by one molecule of PrgJ, one molecule of NAIP2 and 10-12 molecules of the NLRC4 [START_REF] Hu | Structural and biochemical basis for induced self-propagation of NLRC4[END_REF]. To investigate whether plant heterocomplex can form similar structures, it would be interesting to elucidate the stoichiometry of the active RGA4/RGA5 complex.

CONCLUSION

In this work, we identified the first family of structure-related fungal effector proteins and established new lights for a better understanding of effector evolution and diversification in fungi. Furthermore, we characterized the AVR-Pia surface that is recognized by the integrated RATX1 decoy domain of RGA5. In this way, we expanded the knowledge about how effector proteins, in particular MAX-effectors, bind integrated domains. Next we showed that RATX1 domain is required for effector recognition and that the NB-ARC and LRR domains of RGA5 also associate with effector proteins. Based on these results we proposed a cooperative model in which the recognition of the effector proteins and the activation of defense signalling depend on the association/binding of multiples NLR domains with the effector protein. We believe that this mode of recognition constitute a good strategy to maximize the recognition of rapidly evolving effector proteins and to avoid the segregation between the NLR and the integrated decoy. Finally we reveled that RGA5 and RGA4 interact through their CC, NB and LRR domains and that some of these interaction seem not to be disrupted by the effector since we observed hetero-complex formation in the presence of AVR-Pia. However, additional experiments are required to confirm these preliminary results.

Collectively, these results provide important insights about the recognition of effector proteins mediated by NLR integrated decoys. Nevertheless, we need to put much more effort in the study of the molecular bases of this recognition before the engineered of NLRs with integrated decoys could be considered.

Model of effector recognition and resistance activation by the RGA4/RGA5receptor complex

Based on the work presented in chapter II, we proposed a model to describe the recognition of AVR-Pia by RGA5 and RGA4 on the molecular level. The basis of the model is that the AVR-Pia-RGA5 RATX1 interaction is absolutely required for effector recognition. In addition, it takes into consideration that the strength of AVR-Pia-RGA5 RATX1 binding can be rather low and that AVR-Pia associates with additional RGA5 domains, outside the RATX1 domain. We hypothesized that those interactions are important to occur simultaneously with binding of AVR-Pia to the RATX1 domain to increase overall binding strength and affinity of the effector to RGA5 .

In chapter III we describe further dissection of the AVR-Pia/RGA5 interaction and present evidence that the additional RGA5 domains mediating AVR-Pia-binding are the NB-ARC and the LRR domains. In addition, we show that also AVR1-CO39 interacts with other domains of RGA5 than the RATX1 domain and that again the NB-ARC domain is involved. When analyzing for intramolecular interactions of the RATX1 domain, we found that RATX1 forms homo-dimers and associates in addition with RGA5 NB-ARC . Interestingly, both interactions are perturbed by AVR-Pia.

Since RGA5 mainly acts through RGA4 the molecular bases of the interaction between RGA4 and RGA5 were also investigated in chapter IV. This provided evidence that in addition to previously known interactions between the CC domains, the NB-ARC and the LRR domains are engaged in heterotypic interactions. Interestingly, the RATX1 domain seems not to interact with RGA4, neither with RGA4 NB-ARC nor with RGA4 LRR-C-ter suggesting that the Cterminus of RGA5 C-ter is not directly involved in RGA4 and RGA5 complex formation.

Altogether these results led to a refined model for effector recognition by RGA5 and RGA4.

Model for effector recognition by the RGA4/RGA5 receptor complex

Questions de recherche, approches et résultats principaux

Le projet de thèse s'est penché sur les questions suivantes. tridimensionnelles une hélice α conservée appelée "WY-domain" [START_REF] Boutemy | Structures of Phytophthora RXLR effector proteins: A conserved but adaptable fold underpins functional diversity[END_REF]. Des analyses bioinformatiques complémentaires ont suggéré que ce WY-domain était présent dans les 44% des effecteurs décrits chez P. infestans [START_REF] Boutemy | Structures of Phytophthora RXLR effector proteins: A conserved but adaptable fold underpins functional diversity[END_REF]. L'analyse systématique des NLR végétaux a montré que l'intégration des domaines « non conventionnels » se produit à un taux d'environ 5%. De plus des NLR-ID ont été trouvés dans toutes les plantes terrestres, des mousses aux angiospermes (Cesari, et al 2014;[START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF][START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. En outre, il a été observé que la diversité des domaines intégrés est très élevée. En effet, environ 265 domaines intégrés différents ont été identifiés dans des protéines NLR [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. Les domaines les plus représentés sont des protéines kinases, des domaines de liaison à l'ADN ou des domaines d'interaction protéine-protéine [START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. De manière intéressante, plusieurs études sur l'interaction protéineprotéine montrent qu'un grand nombre de domaines intégrés pourraient agir comme cibles des effecteurs. Ceci suggère que la reconnaissance des effecteurs est une caractéristique générale des domaines intégrés. Ceci a justement été démontré expérimentalement pour RGA5, Pik-1 et RRS1. Par ailleurs, un grand nombre de domaines intégrés n'ont pas encore été associés à des cibles impliquées dans l'immunité de l'hôte, et constituent donc une liste exceptionnelle de candidats pour l'identification de nouveaux composants immunitaires (Cesari et al., 2014 ;[START_REF] Kroj | Integration of decoy domains derived from protein targets of pathogen effectors into plant immune receptors is widespread[END_REF][START_REF] Sarris | Comparative analysis of plant immune receptor architectures uncovers host proteins likely targeted by pathogens[END_REF]. L'hétéro-complexe RGA5/RGA4 ne semble pas être perturbé par la présence de l'effecteur

Le modèle coopératif

AVR-Pia

Il a été précédemment démontré que RGA4 et RGA5 forment un hétéro-complexe en l'absence de AVR-Pia (Cesari, et al., 2014). Dans cette étude, nous avons obtenu des résultats préliminaires suggérant que la présence de l'effecteur ne modifiait pas la formation de l'hétéro-complexe. Cependant, des expériences supplémentaires sont nécessaires pour tester cette hypothèse. Des expériences possibles consisteraient à des analyses BiFC dans lesquelles l'interaction de RGA4 et RGA5 serait évaluée en l'absence ou en présence de la protéine d'avirulence. En outre, il reste à vérifier si le complexe fonctionnel est bien un complexe tripartite constitué par AVR-Pia/RGA4/RGA5. Dans ce but, on pourrait tester si AVR-Pia co-précipite RGA4 et RGA5 dans des expériences in planta.

La signalisation de défense induite par des hétéro-complexes NLR est mal comprise

Dans le cas du couple RGA4/RGA5 il a été montré que RGA4 est capable d'induire la mort cellulaire, soit en l'absence de RGA5, soit en présence d'AVR-Pia (Césari, et al. 2014).

Cependant, la manière dont RGA4 induit la signalisation de défense reste une question ouverte. Chez les animaux, il a été démontré que les protéines NLR fonctionnant par paires peuvent former un complexe tripartite avec le ligand du pathogène. Ce triple complexe est ensuite capable d'induire le recrutement de protéines supplémentaires, ce qui donne lieu à la formation d'un complexe multi-protéique appelé inflammasome [START_REF] Hu | Structural and biochemical basis for induced self-propagation of NLRC4[END_REF]. Cet inflammasome est alors composé par une molécule ligand, une protéine NLR qui agit comme récepteur, et 10 à 12 molécules NLR qui agit comme adaptateur. Ce complexe multi protéique prend la forme d'une roue et crée une surface d'oligomérisation nécessaire à la l'activation de l'immunité innée [START_REF] Hu | Structural and biochemical basis for induced self-propagation of NLRC4[END_REF]. Afin de déterminer si les NLR hétérocomplexes chez la plante peuvent former des structures similaires, il serait donc intéressant d'élucider la stoechiométrie du complexe RGA4/RGA5 actif.

ANNEX 1

Materials & methods chapter III and IV Plant material and constructs N. benthamiana plants were grown in a growth chamber at 22°C with a 16-h light period.

Plasmids were generated by Gateway cloning (ThermoFisher, Waltham, USA), restriction/ligation, Gateway entry clones were generated using the pDONR207 plasmid (ThermoFisher, Waltham, USA) (Table 1). Gateway destination vectors were modified pBIN19 plasmids for expression of tagged proteins in N. benthamiana [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]Césari et al. 2014) or modified pGAD-T7 or pGBK-T7 plasmids (Clontech, Mountain View, USA) for yeast two hybrid experiments (Bernoux et al., 2011) (Table 1).

Coimmunoprecipitation and Yeast two hybrid interaction assays

Protein-protein interaction analyses by co-immunoprecipitation were performed with protein extracts from N.benthamina leaf discs harvested 2 days after Agrobacterium infiltration [START_REF] Césari | The rice resistance protein pair RGA4/RGA5 recognizes the Magnaporthe oryzae effectors AVR-Pia and AVR1-CO39 by direct binding[END_REF]. For the interaction of AVR-Pia and AVR1-C039 with RGA5 variants as well as the interactions between RGA5 and RGA4 domains, 5 leaf disks per that unconventional domains in NLRs act as decoys in the recognition of effector proteins. By detailed structure-informed analysis of AVR-Pia, I could confirm the pivotal role of the AVR-Pia-RATX1 interaction for effector recognition lending important support to the integrated decoy model. In addition, I could precisely characterize the interaction surface with which AVR-Pia binds to the RGA5 RATX1 domain. Finally, I detected interactions of AVR-Pia with other parts of RGA5, in particular the NB-ARC and the LRR domains. Based on these results, I developed a model that explains how such binding to several independent sites in NLRs leads to high overall affinity and robust effector recognition that is resilient to effector mutations. Taken together, this study provides important novel insight into NLR function and effector recognition and contributes by this to a better understanding of plant immunity which is crucial for generating durable disease resistance in crops.

Figure 2 .

 2 Figure 2. Schematic view of plant immunity. Plant immunity relays in two different classes of immune receptors located in the plasma membrane and cytoplasm where they detect virulence factors and modified-self. Adapted from Dodds & Rathjen 2010.

  MAMPs or PAMPs) such as bacterial flagellin or chitin from fungi. MAMPs and PAMPs are defined as conserved microbial molecules with low variability and wide distribution in a phylogenetically large range of organisms. They allow thus the recognition of wide ranges of potentially deleterious microorganisms by activating pattern-triggered immunity (PTI). This provides basal protection against non-adapted pathogens and potentially damaging microorganisms and attenuates the virulence of adapted pathogens. The recognition of MAMPs or PAMPs in plants is mediated by pattern recognition receptors (PRRs) that are transmembrane receptor proteins of the RLK and RLP type. However, adapted pathogens are able to overcome PTI by the deployment of effectors that suppress or circumvent this first layer of immunity. They are therefore able to infect their host plant and to establish effectortriggered susceptibility (ETS).

Figure 3 .

 3 Figure 3. Plant immunity explained by Zig-zag model and Invasion model. In the Zig-zag model plant immunity is divided in two separates branches depending on the biochemical function of the ligand recognized by immune receptors in the host (A). In the invasion model plant immunity result after the stimulation of immune receptors by ligands with any biochemical function (B). The invasion model proposes a more wide view of plant immunity and incorporates diverse interactions that are strictly separate in the Zig-zag model. Adapted from Jones & Dangl 2006; Cook et al. 2014.

Figure 4 .

 4 Figure 4. Plant pathogenic fungi secrete apoplastic effectors into the plant extracellular space and cytoplasmic effectors inside the plant cell

  effectors and immune receptors are maintained in populations and their frequencies oscillate over the time(Stahl et al. 1999) (Figure5). One of the main signs of an arms race way of evolution is the high variability observed in the interacting proteins. To date, the majority of fungal pathosystems studied in agricultural context support the arms race model what may in part be explained by the constant human intervention on host varieties[START_REF] Brown | Plant-parasite coevolution: bridging the gap between genetics and ecology[END_REF][START_REF] Lo Presti | Fungal Effectors and Plant Susceptibility[END_REF].

Figure 5 .

 5 Figure 5. Co-evolutionary principles driving effector and plant target evolution. Populationwide allele frequencies of a pathogen-derived effector molecule and a host-derived interactor are represented by red and green lines respectively. Allele fixation and recurrent development of new alleles are indicated by a light-colored line in the arms model (A) and contrast with the fluctuation of allele frequencies in the trench warfare model (B). Adapted from Lo Presti et al. 2015.

Figure 6 .

 6 Figure 6. Effector genes reside in distinct genome compartments

Figure 7 .

 7 Figure 7. Effector proteins have different function and different targets in the extra and intracellular space of plant cells.

Figure 8 .

 8 Figure 8. Plant surface immune receptors belong to two main different classes: receptor like kinases (RLK) and receptor like proteins (RLP). RLKs are composed of an ectodomain, a transmembrane domain and an intracellular kinase domain. RLP also have an extracellular and transmembrane domain but lack intracellular kinase domain. After invasion pattern perception both RLKs and RLPs hetero-complexes with downstream acting RLKs. This leads to the

Figure 9 . 3 . 3

 933 Figure 9. Intracellular immune receptors present flexible structures with different arrangements of conserved domains.

functions of the 2

 2 different parts of the LRR are reflected, as already mentioned, by the dual structure of the LRR with an N-terminal part dominated by positively charged amino acids and a C-terminal part enriched in aromatic residues probably involved in hydrophobic interactions (Takken & Goverse 2012). The sequences between the LRR repeat motifs form the convex side of the arc and can form different secondary structures. Compared to other LRR proteins, LRRs in plant NLRs are diverse and irregular presenting strong variation in the number and the length of repeats.

Figure 10 . 3 . 4

 1034 Figure 10. Structure of NLR domains. Cartoon representing the crystal structure of the CC domain of RX and MLA10 (A) and the TIR domain of L6 (B). MLA10 and L6 forms homodimers that are represented in green and blue. The residues that have been shown to be important for stability and/or dimer formation in each protein are highlight with sticks. Threedimensional models are represented for the structure of the NB-ARC (C) and LRR domains (D) of Gpa2 generated by homology modeling based on the Apaf1 crystal structure. The subdomains NB, ARC1 and ARC2 are represented by different shades of blue and the main residues contributing to the nucleotide-binding pocket are indicated in red (C). The NB-ARC domain is represented in a closed conformation bound to a yellow ADP molecule. The N and C terminal parts of LRR domain from Gpa2 involved in NB-ARC interaction and effector recognition respectively are represented by different colors (D). Adapted from[START_REF] Slootweg | Structural determinants at the interface of the ARC2 and leucine-rich repeat domains control the activation of the plant immune receptors Rx1 and Gpa2[END_REF][START_REF] Hao | Structural basis for the interaction between the potato virus X resistance protein (Rx) and its cofactor ran GTPase-activating protein 2 (RanGAP2)[END_REF] Maekawa, et al. 2011

Figure 11 .

 11 Figure 11. Modes of effector protein recognition by NLRs. In the absence of NLRs acting as resistance proteins, effectors target host proteins to cause disease (A). Resistance plants possess NLR immune receptors that recognize effector proteins by direct association (B) or by sensing modifications such as degradation or phosphorylation of target or decoy proteins (C).Decoys can also be fused to NLRs to allow efficient effector recognition (D). Adapted from[START_REF] Khan | Of guards , decoys , baits and traps : pathogen perception in plants by type III effector sensors[END_REF]

Figure 12 .Figure 13 .

 1213 Figure 12. Examples of the different recognition model. (A) L6 directly interacts with AvrL567 to activate resistance. (B) RPS2 senses the cleavage of the guard protein RIN4 by AvrRpt2 and induces ETI. (C) Prf monitories the phosphorylation of the decoy protein Pto induce by AvrPto and AvrPtoB to induce resistance. (D) The interacted WRKY domain in RRS1 mediates recognition to AvrPps4 and PopP2 to trigger ETI. Adapted from Khan et al 2016

Figure 14 .

 14 Figure 14. Examples of NLRs genetically linked. (A) Arabidopsis TNL pair in tail-to-head orientation. (B) Arabidopsis TNL pair in head-to-head orientation. Adapted from Griebel et al. 2014.

Figure 15 .

 15 Figure 15. Models of resistance protein activation. The activation of resistance proteins after effector perception has been illustrated by three different models. (A) Switch model, the effector directly binds to an inactive protein inducting a conformational change orquestrated by the release of an ADP molecule and binding of ATP resulting in the activation of the protein. (B) Bait and switch model, the effector binds an inactive protein through a co-factor and as in (A) active the resistance protein. (C) The equilibrium-base switch model state that resistance proteins exist in equilibrium between an active and inactive configuration. Therefore, in this model the effector binds directly to the active protein, stabilizes its configuration and shifts the equilibrium towards activation. Adapted from Sukarta et al. 2016.

  Rx is impaired to activate resistance responses showing that recognition of effector protein and Rx-mediated signaling occurs in the cytoplasm[START_REF] Tameling | RanGAP2 mediates nucleocytoplasmic partitioning of the NB-LRR immune receptor Rx in the Solanaceae, thereby dictating Rx function[END_REF]. The role of Rx accumulation in the nucleus is still an open question.

Figure 16 .

 16 Figure 16. Localization of resistance proteins in plant cell compartments. NLR proteins can localize in the plasma membrane and endo-membranes (A) or accumulate in the cytoplasm and/or the nucleus (B) Adapted from Qi & Innes 2013

Figure 17 .

 17 Figure 17. Symptoms of rice blast disease caused by M. oryzae. Symptoms on aerial parts of rice plants infected by M. oryzae (A). Complete destruction by blast of a susceptible rice variety in an experimental field (B). Photo from Didier Therrau

  successfully cloned and transiently expressed in tobacco plants to identify suppressors of BAX1 or Nep1-induced cell death. 11 of them named SPDs (suppressors of plant cell death) blocked cell death induced by the Nep1 and with exception also cell death triggered by BAX. Five of the eleven SPDs have been previously characterized, and are either essential for disease development, secreted during biotrophic infection, or cell death suppressors. Sequence analysis of the 11 SPD genes from 43 re-sequenced M. oryzae genomes showed that SPD2, SPD4, and SPD7 are particularly polymorphic when compared with other M. oryzae effector proteins suggesting that these effectors are under diversifying selection pressure to escape recognition from the host and have important roles in blast infection.

  Figure 19. Secretion and accumulation of M. oryzae effector proteins. A) Apoplastic effectors (blue), accumulate in the EIHM compartment surrounding the IH, resulting in uniform outlining of the IH. These apoplastic effectors follow the conventional, BFA-sensitive, Golgi-dependent secretion pathway. In contrast, cytoplasmic effectors (magenta), accumulate in the BIC beside the first-differentiated bulbous IH cell. Cytoplasmic effectors follow a nonconventional, BFAinsensitive secretion pathway B) Schematic representation of the effector accumulation during the differentiation of a filamentous primary invasive hypha into a pseudohyphal-like bulbous invasive hypha in the first-invaded rice cells. Cytoplasmic effectors show preferential accumulation in the BIC (black arrows), which is first located in front of the growing primary hyphal tips, and then remains behind beside the first-differentiated bulbous IH cell. Typical accumulation patterns for cytoplasmic (magenta) and apoplastic (blue) effectors are shown within the EIHM (tan) compartment enclosing the IH. The EIHM appears to lose integrity when the fungus has moved into neighbor cells (dotted line). Adapted from Giraldo et al. 2013

Figure 20 .

 20 Figure 20. Signaling pathways in rice blast resistance. The rice GTPase OsRac1 forms a complex with the rice NB-LRR protein Pit. OsRac1 is required for Pit-mediated disease resistance to the rice blast fungus. The active form of Pit induces the activation of OsRac1 at the plasma membrane.OsRac1 functions in rice innate immunity through the RAR1-SGT1-HSP90 cytosolic complex. OsMAPK6 transduces the signaling mediated by Pit, OsRac1 and RAR1-SGT1-HSP90 complex. Pb1 interacts with a transcription factor WRKY45 through its CC domain and this interaction protects the degradation of WRKY45 by the ubiquitin-proteasome system. Pb1-mediated blast resistance is dependent on SA. Adapted from kawano et al. 2013

  formation of RGA4 ΔCC and RGA5 ΔCC and the association of individual domains (NB, CC, NB-LRR) of RGA4 and RGA5 by co-IP experiments (Chapter IV).INTRODUCTIONEffectors are among the most powerful arms of plant pathogens to cause disease. Their study provides deep insight into the mechanisms that pathogens employ to provoke disease and into the co-evolution with their hosts. In addition, effectors are key elements for disease resistance breeding since their recognition by immune receptors is exploited for the development of resistant varieties. Recently, effector-informed breeding has been developed as a powerful tool to accelerate the identification, functional characterization and deployment of resistance genes in commercial crops. Effector-assisted breeding has been particularly successfully applied in late blight control(Whisson et al. 2011). Effector proteins from the oomycete P.

  , these examples illustrate how structural studies can lead to the elucidation of effector functions and mechanisms of effector action as well as the identification of interaction interfaces in effectors and hidden similarities between effector by sequences. The M. oryzae effectors AVR-Pia and AVR1-CO39 are both recognized by the rice NLR pair RGA5/RGA4 through direct binding with the C-terminal domain of RGA5 containing the RATX1 domain. Interestingly, despite recognition by the same NLRs, AVR-Pia and AVR1-CO39 have very different amino acid sequences, suggesting molecular factors other than amino acid sequence are important determinants in effector recognition in this case.

  The line width is proportional to the NOE intensity. The dots (•) indicate slow exchange NH observed in 2D-NOESY in D2O. Grey arrows indicate the ß-strands determined from the structure analysis (vide infra). S4 Fig. Solution structures of (A) AVR-Pia and (B) AVR1-CO39. Superposition of the backbone atoms of the 20 lowest energy conformers used to calculate the final structures. Only mature chains are shown, from residues Ala20 and Trp23 for AVR-Pia and AVR1-CO39, respectively. S5 Fig. Structure-guided alignment and diversity of MAX-effector homologs identified by Blast. A) Homologs of AVR1-CO39, AvrPiz-t and AVR-Pia identified by Blast in M. oryzae and M. grisea genomes and ToxB homologs identified by Psi-Blast in the GeneBank database were aligned to the structural alignment of mature ToxB, AVR1-CO39, AvrPiz-t and AVR-Pia. (B) A diversity tree was constructed by the neighbor-joining method using the alignment in (A). It highlights the high diversity of MAX-effector homologs. Branch supports are based on 1000 bootstraps and horizontal branch length reflects sequence divergence. Accession numbers of non-Magnaporthe sequences were completed by a 2 letter identifier for the species: BO for Bipolaris oryzae, CF is for Colletotrichum fioriniae, CH for C. higgensianum, CG for C. gloeosporioides, CO for C. rbiculare, LM for Lepthosphaeria maculans, PT for Pyrenophora triticirepent is and PB for Pyrenophora bromi. 4 5 9 3 6 . 1 C H / 2 -8 7 E N H 8 4 6 2 1 . 1 C O / 2 -8 7 E Q B 5 7 5 1 6 . 1 C G / 2 -9 3 X P 0 0 7 6 0 0 1 4 4 . 1 C F / 2 -8 6 M B R 2 9 E u G e n e 0 0 1 1 3 5 3 1 / 1 -9 6 M C D 1 5 6 E u G e n e 0 0 1 3 8 5 0 1 / 1 -9 9 M B R 2 9 E u G e n e

  -effector homologs identified in the UniRef90 database by a low stringencyHMM search. (A) Histogram showing the numbers of MAX-effectors identified by an HMM pattern search in a non-redundant UniRef90 database. (B) MAX-effectors identified by HMMpattern search were aligned to the structural alignment of mature ToxB, AVR1-CO39, AvrPizt and AVR-Pia. (C) A diversity tree was constructed by the neighbor-joining method using the alignment in (B). This highlights the high diversity of MAX-effector homologs. Branch supports are based on 1000 bootstraps and horizontal branch length reflects sequence divergence. Accession numbers contain the following information on the species: MAGGR, MAGO7, MAGOP and MAGOR are from M . oryzae , COLGC and COLGN from C . gloeosporioides , COLHI from Colletotrichum higginsianum , 9PEZI from C . fioriniae and COLOR from Colletotrichum orbiculare , 9PLEO from P . tritici-repentis or P . bromi , ARTOA from Arthrobotrys oligospora , COCMI from Bipolaris oryzae , LEPMJ from Leptosphaeria maculans , MYCGM from Zymoseptoria tritici , 9PSED from Pseudomonas sp . StFLB209 and SOLLC from Solanum lycopersicum. -effector homologs identified by a low stringency HMM search in the fungal genomes database. (A) MAX-effectors identified by an HMM pattern search in a redundant database comprising the small secreted proteins of 25 ascomycete fungi, 8 additional M. oryzae and one M. gisea isolate were aligned to the structural alignment of mature ToxB, AVR1-CO39, AvrPiz-t and AVR-Pia and gaps were removed. (B) A diversity tree was constructed by the neighbor-joining method using the alignment in (A). Branch supports are based on 1000 bootstraps and horizontal branch length reflects sequence divergence. Accession numbers of non-Magnaporthe sequences were completed by a 2 letter identifier for the species: BO for Bipolaris oryzae, CF for Colletotrichum fioriniae, CH for C. higgensianum, CG for C. gloeosporioides, CO for C. orbiculare, GF for Fusarium fujcuroi, LM for Lepthosphaeria maculans, PT for Pyrenophora tritici-repentis, PB for Pyrenophora bromi and ZT for Zymoseptoria tritici. of M . oryzae MAX-effector candidates and marker genes during rice infection and in in vitro grown mycelium. mRNA levels of M. oryzae genes coding for MAX-effectors (A, B and C) and marker genes (D) was determined by q-RT-PCR in rice leaf samples harvested 16, 24, 48 or 72 h after inoculation and mycelium grown liquid medium for 72 hours. (A) Infection specific MAX-effectors identified in the HMM search, (B) infection specific MAX-effectors identified in the Psi Blast search but nor in the HMM search, (C) constitutively expressed MAX-effectors identified in the HMM search and (D)marker genes for appressorium and very early infection (ORF3 of the ACE1 cluster, MGG_08381), biotrophic infection (BAS3, MGG_11610), late infection (MGG_01147), constitutive expression (EF1α, MGG_03641). Relative expression levels were calculated by using expression of a constitutively expressed Actin (MGG_03982) as a reference. Mean values and standard deviation were calculated from three independent biological samples.The analyzed genes, were in most cases not or extremely weakly expressed in the mycelium. For genes with significant expression in the mycelium (ratio gene versus actine > 0,01) a T-test was performed to determine if in planta expression was significantly different from expression in the mycelium. In these cases (MGG_11967, MGG_14793, MGG_15207, , M_TH16_00124981), a star or two stars (* or **) mark conditions where the expression was different from expression in the mycelium at respectively p<0,05 or p<0,005. S10 Fig. Prediction of the secondary structure of M. oryzae MAX effectors. The secondary structures of the MAX-effectors from the 70-15, TH16 and BR29 genomes was predicted with SSPRO5 [93].The predictions are shown at the bottom of the figure and are aligned onto the corresponding primary sequence alignment shown at the top of the figure. Sequence identifiers for the secondary structure predictions are suffixed with « .2d.SSPRO5 ». Blue »H », red « E » and yellow « C » correspond respectively to helix, extended sheet and coil predictions. The sequences of the 4 MAX effectors with experimentally determined structures are displayed at the top of the multiple sequence alignment and, for clarity, the alignment positions corresponding to shared gaps in the known structures were removed.

FLA9000Figure 1 .

 1 FIGURE LEGENDS

Figure 2 .

 2 Figure 2. AVR-Pia binds RGA5 RATX1 with intermediate affinity and a well-defined interaction surface

Figure 3 .

 3 Figure 3. Mutations in the binding surface of AVR-Pia affect binding to RGA5 C-ter in yeast two hybrid assays.

Figure 4 .

 4 Figure 4. AVR-Pia mutants with reduced RGA5 C-ter binding in yeast are also impaired in binding to RGA5 C-ter and RGA5 RATX1 in planta.

Figure 5 .

 5 Figure 5. Effector recognition by RGA5 requires binding to the RATX1 domain

Figure 6 .

 6 Figure 6. AVR-Pia interacts with RGA5 outside the RATX1 domain

Figure 7 .

 7 Figure 7. Model of AVR-Pia recognition by the RGA4/RGA5 receptor complex

Figure Legends Supplemental Figure 1 .Supplemental Figure 2 .

 12 Comparison of NMR relaxation of AVR-Pia and AVR-Pia-H3.Generalized order parameter (S2) obtained from 15N relaxation data at 500 MHz for AVR-Pia (circles) and AVR-Pia-H3 (triangles) determined from Lipari-Szabo formalism with the program DYNAMOF(Barthe et al., 2006). The different colors indicate the use of the "simple" Lipari-Szabo formalism by black triangles for AVR-Pia-H3, and by white circles for AVR-Pia, with possible additional exchange contributions shown by the symbols in grey color. HSQC spectra of AVR-Pia and AVR-Pia-H3 recorded upon titration with RGA5 RATX1 .HSQC spectra are shown for 50 μM AVR-Pia (A) and AVR-Pia-H3 (B) without RGA5 RATX1 (black) and various concentration of RGA5 RATX1 at 12.5 μM (R=0.25 in grey), 25 μM (R=0.5 in green), 50 μM (R=1 in orange) and 100μM (R=2 in pink). Residues belonging to the N-terminal tag are indicated by negative numbering and (*). For AVRPia (A) some of the largest chemical shift differences between the free (R=0) and bound (R=2) form are shown in the zoom panels at the upper left corner. With AVR-Pia-H3 (B), most of the HSQC peaks remain unperturbed and the enlarged plot

BFigure 1 .Figure 3 .Figure 4 .Figure 5 .Figure 4 .

 13454 Figure 1. The AVR-Pia-H3 NMR structure is similar to the structure of wild-type AVR-Pi

  //zhanglab.ccmb.med.umich.edu/ITASSER), providing the chain B of the 5A6W PDB structure as an explicit homologous template. The obtained Itasser models were docked onto the AVR-Pia NMR structure (PDB identifier 2MYW) using the protein-protein docking program RosettaDock from the Rosetta modeling suite[2015.12.57698]. Each docking simulation was initialized by randomizing the relative positions and orientations of both partners and was performed with default parameters.

Figure 21 .

 21 Figure 21. Borders of RGA5 domains analyzed for their interaction with AVR-Pia and AVR1-CO39

Figure 22 .

 22 Figure 22. AVR-Pia binds specifically to the RGA5 C-ter domain in yeast. The interaction between AD-and BD-AVR-Pia fusion proteins and different BD and AD-RGA5 fusion domains respectively was tested by a yeast two-hybrid experiment. Three dilutions (1/10, 1/100, 1/1000) of yeast cultures adjusted to an OD of 0.2 were spotted on synthetic double drop out (DDO) medium (-LW) to control proper growth and on synthetic TDO (-LWH) either without or supplemented with 3-amino-1,2,4-triazole (3AT) to test the strength of the interaction. Photos were taken after 4 days of growth.

Figure 23 .

 23 Figure 23. Western blotting of transgenic yeast protein extracts. Equal production of AD and BD-AVR-Pia and AVR1-CO39 fusion proteins was determined by immunoblotting with anti-HA and anti-BD antibodies respectively. Similar quantities of AD and BD-RGA5 fusion domains were specifically detected with anti-HA and anti-BD antibodies, except for the constructs indicated with one asterisk.

Figure 24 .

 24 Figure 24. Different RGA5 domains associate with AVR-Pia in planta. HA-RGA5 domains were transiently expressed with YFP-AVR-Pia or YFP-PWL2 in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated AVR-Pia and PWL2. Co-precipitated HA-RGA5 domains proteins were detected using α-HA antibody. RGA5 domains not co-precipitated by AVR-Pia are indicated by one asterisk

Figure 26 .

 26 Figure 26. AVR1-CO39 binds to RGA5 NB-ARC and RGA5 C-ter domains in yeast. The interaction between AD-and BD-AVR-Pia fusion proteins and different BD and AD-RGA5 fusion domains respectively was tested by a yeast two-hybrid experiment. Three dilutions (1/10, 1/100, 1/1000) of yeast cultures adjusted to an OD of 0.2 were spotted on synthetic double drop out (DDO) medium (-LW) to control proper growth and on synthetic TDO (-LWH) either without or supplemented with 3-amino-1,2,4-triazole (3AT) to test the strength of the interaction. Photos were taken after 4 days of growth.

Figure 27 .

 27 Figure 27. AVR1-CO39 associates with RGA5 NB-ARC in planta and in yeast. HA-RGA5 domains were transiently expressed with YFP-AVR1-CO39 and YFP-PWL2 in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and

Figure 28 .

 28 Figure 28. RATX1 associates with RGA5 NB-ARC and RGA5 C-ter but not with RGA5 CC in planta. HA-RATX was transiently expressed with YFP-RGA5 domains and YFP in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated RGA5 domains. Co-precipitated HA-RAXT1 was detected using α-HA antibody. RATX1 not coprecipitated by RGA5 domains is indicated by one asterisk.

Figure 29 .

 29 Figure 29. RATX1 association with RGA5 NB-ARC and RGA5 C-ter is affected by AVR-Pia. HA-RATX was transiently expressed with YFP-RGA5 domains and YFP in presence of AVR-Pia without tag in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated RGA5 domains. Co-precipitated HA-RAXT1 was detected using α-HA antibody. The associations of RATX1 with RGA5 domains affected by AVR-Pia are indicated by one asterisk.

Figure 30 .

 30 Figure 30. RATX1 strongly self-associates in absence of AVR-Pia. HA-RATX was transiently expressed with YFP-RGA5 domains and YFP in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated RGA5 domains. Coprecipitated HA-RAXT1 was detected using α-HA antibody. *In this experiment the expression of YFP-CC failed.

  clues about the question to what extent synchronous binding to the integrated decoy and the NB-ARC domain contributes to the extended recognition specificity of RGA5 that recognizes two different effectors. The NACHT domain from animal immune receptors such us NAIP (Neuronal Apoptosis Inhibitor Protein) or CIITA (MHC class 2 transcription factor) is closely related to the NB-ARC

Figure 31 .

 31 Figure 31. RGA4 and RGA5 domains used to analyze their interaction in planta.

Figure 32 .

 32 Figure 32. The CC domain of RGA4 and RGA5 is dispensable for their interaction. Full length and delta-CC of both RGA5 and RGA4 proteins tagged with HA, YFP or GFP were transiently expressed in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection

Figure 33 .

 33 Figure 33. RGA4 and RGA5 interact through their NB-ARC and LRR domains. RGA4 NB-ARC -GFP and RGA4 LRR-Cter were transiently expressed with HA-RGA5 variants in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immuneprecipitated RGA4 NB-ARC -GFP, RGA4 LRR-Cter and YFP. Co-precipitated RGA5 variants were detected using α-HA antibody.

Figure 34 .

 34 Figure 34. RGA4 and RGA5 interact in presence of HA-tagged AVR-Pia protein. RGA4-GFP was transiently expressed with HA-RGA5 and HA-AVR-Pia in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated RGA4-GFP and YFP. Co-precipitation of HA-AVR-Pia and HA-RGA5 was verified by using α-HA antibody.

Figure 35 .

 35 Figure 35. RGA4 and RGA5 interact in presence of the un-tagged AVR-Pia protein. YFP-RGA5 was transiently expressed with RGA4-HA and AVR-Pia in N. benthamiana. Protein extracts were analyzed by immunoblotting with anti-HA (α-HA) and anti-GFP antibodies (α-GFP) (Input). Immunoprecipitation (IP) was conducted with anti-GFP beads (IP GFP) and analyzed by immunoblotting with α-GFP for the detection of immunoprecipitated YFP-RGA5 and YFP. Co-precipitation of RGA4-HA was verified by using α-HA antibody.

1.

  Est-ce que la structure tridimensionnelle des effecteurs est un facteur clé de leur fonction, leur diversité et leur évolution ? Pour répondre à cette question nous avons tout d'abord déterminé la structure tridimensionnelle des effecteurs AVR1-CO39 et AVR-Pia par résonnance magnétique nucléaire (RMN). Puisque ces deux effecteurs ont des structures similaires, qu'ils partagent, en outre, avec deux autres effecteurs protéiques : respectivement AvrPiz-t et ToxB chez M. oryzae et Pyrenophora tritici-repentis, nous avons identifié une famille d'effecteurs de séquences différentes mais qui possèdent une structure conservée. Cette famille a été appelée MAX-effectors (Magnaporthe Avrs and ToxB like). Leur analyse bioinformatique a montré que les effecteurs MAX sont distribués dans de nombreux taxons phylogénétiques, domaines NB-ARC et LRR de RGA5, en plus de celle précédemment décrite avec RATX1 (Chapitre III). 5. Comment se forme le complexe de récepteurs RGA4/RGA5 ? RGA4 et RGA5 interagissent physiquement et fonctionnellement pour reconnaître AVR-Pia. La formation d'homo-et d'hétéro-complexes de leur domaines CC a été rapportée (Césari et al. 2014). De plus, il a été montré que RGA4 active la mort cellulaire programmée, alors que RGA5 agit comme un inhibiteur de RGA4 ainsi comme un récepteur de AVR-Pia. Dans cette étude, pour mieux caractériser les interactions entre RGA4 et RGA5, nous avons évalué l'interaction physique entre ces deux récepteurs de type NLR en présence de l'effecteur sauvage AVR-Pia WT . Ensuite, nous avons testé la formation de l'hétéro-complexe RGA4 ΔCC et RGA5 ΔCC , et son association avec les domaines individuels de RGA4 et RGA5 (NB, CC, NB-LRR) par des techniques co-immunoprécipitation. Ces expériences préliminaires ont mis en évidence une interaction entre les trois diffèrent domaines de RGA4 et RGA5 et il a été observé que la association du complexe RGA4/RGA5 ne est pas perturbé par l'effecteur AVR-Pia (Chapitre IV).DISCUSSION GENERALE ET PERSPECTIVESDans ces travaux de thèse, j'ai tout abord étudié la diversité des effecteurs fongiques chez les Ascomycètes. J'ai ensuite analysé les déterminants moléculaires de la spécificité de reconnaissance d'effecteurs fongiques par des récepteurs intracellulaires du système immunitaire des plantes. Enfin, j'ai approfondi l'étude de l'activation des récepteurs immunitaires intracellulaires des plantes qui agissent en paires pour induire la résistance.Des effecteurs fongiques de séquences très variables peuvent présenter des homologies structurales et posséder une origine évolutive commune.Les champignons possèdent un répertoire d'effecteurs très large et diversifié. De plus, ces effecteurs sont spécifiques d'une espèce ou d'une lignée, ce qui suggère que les effecteurs appartenant à différents lignages fongiques évoluent rapidement et indépendamment les uns des autres. Par exemple, dans le cas de l'oïdium, les génotypes infectant l'orge sont étroitement liés à ceux infectant le blé et partagent avec ces derniers environ 500 effecteurs candidats, alors qu'ils ont très peu d'effecteurs en commun avec les génotypes d'oïdium qui infectent les plantes dicotylédones[START_REF] Pedersen | Structure and evolution of barley powdery mildew effector candidates[END_REF][START_REF] Spanu | Genome Expansion and Gene Loss in[END_REF]. En conséquence, l'identification récente et massive de nouveaux effecteurs fongiques a mis en évidence des milliers de protéines dont les fonctions et les origines sont largement méconnues. Dans ce travail, nous avons déterminé par RMN la structure tridimensionnelle des effecteurs AVR-Pia et AVR1-CO39 chez Magnaporthe oryzae, le champignon pathogène responsable de la pyriculariose du riz. Cette analyse, combinée à des recherches bioinformatiques et des analyses d'expression chez la plante, nous a conduit à l'identification d'une famille de protéines effectrices que nous avons nommée « effecteurs MAX » (Magnaporthe Avrs and ToxB like). Chez les champignons, les effecteurs MAX constituent la première famille identifiée d'effecteurs comportant une structure conservée malgré des séquences en acides aminés très différentes. Cette découverte est d'une grande importance pour l'étude de la biologie des effecteurs car elle suggère qu'un grand nombre d'effecteurs, apparemment sans aucun lien entre eux, peuvent en réalité être liés au niveau structural, être rassemblés en familles et partager une origine phylogénétique commune. En effet, chez les oomycètes, une famille d'effecteurs possédant des structures similaires a également été trouvée (Boutemy et al 2011; Win et al 2012). C'est notamment le cas des effecteurs AVR3a11 et PexRD2 de Phytophthora capsici et Phytophthora infestans, respectivement. En dépit d'une identité de séquence inférieure à 20%, ces deux effecteurs possèdent dans leurs structures

L

  'identification de familles d'effecteurs structurellement liés, telles que celles des effecteurs MAX et WY-RXLR, fournit un cadre moléculaire permettant de mieux comprendre l'évolution des effecteurs vers une meilleure tolérance de l'hyper-variabilité des séquences en acides aminés. Nous avons par exemple observé que les structures en feuillets β-sandwich des effecteurs MAX permettaient l'insertion ou la délétion d'acides aminés dans les boucles, ou la substitution d'acides aminés en surface. Une structure souple des effecteurs MAX, comme celle en feuillet β-sandwich, facilite donc la génération d'effecteurs avec des surfaces de formes et de propriétés différentes, tout en préservant leur activité moléculaire, leurs cibles et une structure globale stable. Jusqu'à présent, l'identification d'effecteurs fongiques reposait principalement sur l'analyse bioinformatique de séquences et pouvait manquer de précision car généralement ces analyses sont basées sur des critères relativement larges. Ici, nous avons montré que la recherche d'effecteurs candidats basée sur leurs structures peut conduire à une identification très précise d'un très grand nombre d'effecteurs. En outre, nous avons montré chez M. oryzae que la grande majorité des effecteurs candidats MAX sont exprimés spécifiquement au cours de l'infection précoce, suggérant que ce groupe d'effecteurs a un rôle important dans la colonisation de l'hôte pendant les premières étapes de l'infection. L'analyse fonctionnelle des effecteurs candidats MAX, par des approches visant notamment à identifier leurs partenaires protéiques dans les cellules végétales, ou à déterminer leur localisation, permettront de mieux comprendre le ou les modes d'action de cette famille d'effecteurs. De manière intéressante, AVR-Pia, AVR1-CO39 et AVR-Pik, les effecteurs MAX les mieux caractérisés, interagissent avec un domaine appelé RATX1/HMA, intégré dans les récepteurs immunitaires Pik-1 pour AVR-Pik, et dans RGA5 dans le cas d'AVR-Pia et AVR1-CO39. Ceci suggère qu'en plus de posséder une structure conservée, certains effecteurs MAX peuvent également interagir avec des domaines très similaires qui se trouvent dans les protéines de l'hôte, malgré les grandes différences dans les propriétés de surface de ces effecteurs. La structure du complexe AVR-Pik-HMA a été déterminée récemment par Maqbool et al. (2015). Il serait donc également intéressant de générer la structure du complexe formé par AVR-Pia ou AVR1-CO39 avec le domaine RATX1 afin d'évaluer si ces différentes interactions AVR-RATX1/HMA font intervenir des sites d'interaction similaires, ou si la spécificité et la force de l'interaction sont déterminées par des interfaces ou des résidus différents. Les effecteurs MAX AVR-Pia et AVR-Pik ont des surfaces de liaison au domaine RATX1/HMA très similaires Dans cette thèse, nous avons disséqué en détails la surface d'AVR-Pia qui se lie au domaine RATX1, intégré dans le récepteur immunitaire RGA5. Nous avons également souligné que cette surface est similaire à la surface de liaison d'AVR-Pik avec le domaine HMA, intégré dans le récepteur immunitaire Pik-1. Curieusement, bien que 34 des 74 résidus qui constituent les domaines HMA et RATX1 diffèrent, ces domaines sont très similaires. De plus, les propriétés de la surface, telles que la distribution des charges ou l'hydrophobie, sont différentes entre les deux effecteurs MAX. En particulier, une extension N-terminale d'AVR-Pik, cruciale pour l'interaction avec Pik-1 HMA , est absente chez AVR-Pia. Il serait intéressant de découvrir comment AVR1-CO39, un effecteur dont la séquence en acides aminés est très différente de celles d'AVR-Pia et d'AVR-Pik, mais qui partage une structure similaire, peut se lier au domaine RATX1. Il est possible qu'à la différence d'AVR-Pia et d'AVR-Pik, la surface de liaison d'AVR1-CO39 implique les feuilles β 2 et 3 ainsi que la boucle 2. Néanmoins, il est également possible que l'extension C-terminale d'AVR1-CO39, qui ressemble à l'extension Nterminale d'AVR-Pik, soit impliquée dans l'interaction avec RATX1. D'autre part, nous avons relativement peu d'indications concernant la surface de RATX1 qui interagit avec AVR1-CO39 ou AVR-Pia. Une possibilité est que ces deux effecteurs interagissent avec la même surface de liaison de RATX1. Toutefois, si tel est le cas, il sera particulièrement intéressant d'en expliquer les raisons, puisque les deux effecteurs présentent des propriétés de surface très différentes. Alternativement, ils pourraient interagir avec des surfaces différentes du domaine RATX1, surfaces qui pourraient également différer de la surface de son homologue HMA (qui se lie à AVR-Pik). La modélisation structurale du complexe AVR-Pia/RATX1 soutient cette dernière hypothèse car aucun modèle de ce complexe ne ressemble à la structure du complexe AVR-Pik/Pik HMA . Dans le futur, il sera intéressant d'identifier les cibles de la virulence d'AVR1-CO39, AVR-Pia et AVR-Pik, et déterminer si elles sont systématiquement de petites protéines HMA/RATX1. Il sera aussi nécessaire d'étudier, au niveau moléculaire, comment ces trois effecteurs agissent sur leurs cibles de virulence. Le travail réalisé par le groupe de recherche de R. Terauchi (IBRC, Iwate, Japon) indique qu'AVR-Pik cible un clade de petites protéines HMA (sHMA) semblables aux domaines intégrés Pik-HMA ou RGA5-RATX1. Sur la base de ces résultats, ils ont pu déterminer qu'AVR-Pik établit le même type d'interaction avec ses cibles et avec le domaine HMA de Pik. Ces interactions ont également montré que les petites protéines HMA contribuent à l'augmentation de la sensibilité du riz à M. oryzae et à la réduction de la production d'espèces réactives de l'oxygène (R. Terauchi, communication personnelle). Il reste toutefois à déterminer si AVR-Pia et AVR1-CO39 ciblent le même clade de sHMAs, et si elles interagissent par le même type de mécanisme. Les domaines intégrés des NLRs sont impliqués dans la reconnaissance des effecteurs et peuvent être utilisés pour l'identification de cibles d'effecteurs chez la plante Dans cette étude, nous avons constaté que la liaison d'AVR-Pia au domaine RATX1 est nécessaire pour la reconnaissance de l'effecteur. Ceci conforte le modèle de leurre intégré, qui prédit que les domaines intégrés aux NLR (NLR-ID) servent de « leurres » pour piéger et détecter les effecteurs. En effet, jusqu'à ce jour, la seule preuve convaincante appuyant ce modèle avait été fournie par les études de recherche sur les interactions AVR-Pik/Pik-1, PopP2/RRS1 ou AvrRPS4/RRS1. Dans le cas de AVR-Pik/Pik-1 et AvrRPS4/RRS1, il a été démontré que la liaison de l'effecteur au domaine leurre est nécessaire, comme dans le cas de AVR-Pia/RGA5, pour l'activation du NLR. Dans le cas de PopP2/RRS1, le NLR est plutôt activé par une acétylation du domaine leurre intégré par l'effecteur. Cela met en évidence que les domaines intégrés, tout comme les protéines de garde classiques, les protéines leurres ou les cofacteurs, peuvent permettre la reconnaissance d'effecteurs par différentes manières, par exemple en étant impliqués dans la formation du complexe ou en acquérant des modifications post-traductionnelles détectées par le NLR associé.

  pour la reconnaissance des effecteurs par NLR-ID Dans le cadre de ce travail, nous avons découvert que les mutants d'AVR-Pia qui perdent la capacité à se lier au domaine RGA5 RATX1 sont pourtant capables d'interagir avec les protéines RGA5 ΔRATX1 . Ceci indique que différentes surfaces d'AVR-Pia sont impliquées dans l'interaction avec RGA5 via d'autres domaines que RATX1. Plus précisément, nous avons montré qu'AVR-Pia et AVR1-CO39 interagissent avec les domaines RGA5 NB-ARC et RGA5 LRR .L'interaction de l'effecteur reconnu avec de multiples sites de liaison indépendants au sein de la protéine NLR peut constituer un mécanisme moléculaire de résistance efficace pour éviter une perte de reconnaissance qui pourrait être engendrée par des mutations rapides de l'effecteur. Ainsi, au travers d'interactions avec le domaine de leurre intégré et avec d'autres domaines de la protéine NLR, l'effecteur peut être reconnu de manière très robuste et spécifique. Ces multiples interactions pourraient constituer l'un des principaux avantages des protéines NLR-ID.Pour valider ce modèle, il serait important de démontrer que l'interaction entre l'effecteur et les domaines de la protéine NLR, autres que le leurre intégré, est nécessaire pour la reconnaissance de l'effecteur. Pour cela, il sera nécessaire d'identifier des mutants d'AVR-Pia qui perdent l'association avec RGA5 ΔRATX1 mais conservent l'interaction avec RGA5 RATX1 , puis d'évaluer si la perte de cette interaction a un impact sur la reconnaissance de l'effecteur.Les NLR qui agissent en duo pour la reconnaissance des effecteurs peuvent interagir au travers de différents domainesL'une des questions abordées lors de cette thèse était de comprendre comment RGA4/RGA5 et plus généralement d'autres NLR qui forment des hétéro-complexes interagissent pour conférer la reconnaissance de l'effecteur. Dans l'hétéro-complexe RRS1/RPS4, un rôle important des interactions homo et hétérotypiques entre les domaines TIRs dans l'activation et la répression du complexe, a été documenté(Williams et al. 2014). Dans le cas de RGA4/RGA5, des interactions entre leurs domaines CC ont déjà été décrites(Cesari, et al. 2014) ; il est possible qu'elles jouent un rôle similaire à celles des domaines TIR du complexe RRS1/RPS4. Dans la présente étude, nous mettons en évidence que des domaines supplémentaires de RGA4 et RGA5 contribuent à leur hétéro-association. En effet, les domaines NB-ARC et LRR de RGA4 interagissent avec les domaines correspondants dans RGA5. Pour évaluer l'importance de ces interactions dans la fonction de l'hétéro-complexe, il serait intéressant d'identifier des mutations spécifiques dans RGA4 ou RGA5 au sein des domaines CC, NB-ARC ou LRR qui empêchent leur hétéro-association et d'analyser si ces mutations affectent également la reconnaissance de l'effecteur tout comme la signalisation de la défense.

  sample were homogenized in extraction buffer (50mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 1.0% IGEPAL CA-630, 0.05% sodium deoxycholate, and 0.1% SDS, supplemented with a complete protease inhibitor cocktail (Roche) and polyvinylpolypyrrolidone 0.5%). And co-IP was performed with 8 uL of agarose GFP_trap_A suspension (Chromotek) and four washes with the modified protein extraction buffer. For the interactions between RGA5 RATX1 domain and RGA5 variants we used the same extraction buffer described above but we also used a modified extraction buffer with 0.5% IGEPAL CA-630.Bound proteins were eluted by boiling for 10 min at 70°C in 50 uL of Nupage sample buffer, separated by polyacrylamide gel electrophoresis using NuPAGE 4-12% gels (Invitrogen, Carlsbad, CA, USA), transferred to nitrocellulose membrane (Millipore), and analyzed by immunoblotting. For immunodetection of proteins, rat anti-HA-horseradish peroxidase (clone 3F10; Roche) or mouse anti-GFP (Roche) and goat anti-mouse-horseradishperoxidase (Sigma-Aldrich) were used in combination with the Immobilon western kit (Millipore).Binding domain (BD) fusions of AVR-Pia, AVR1-CO39 and PWL2 as well RGA5 variants in surface d'interaction avec laquelle AVR-Pia lie le domaine RATX1. De plus, j'ai détecté des interactions entre AVR-Pia et d'autres parties de RGA5, indépendantes du domaine RATX1, notamment les domaines NB-ARC et LRR. Ceci a permis de développer un modèle qui explique comment la liaison d'un effecteur à un récepteur NLR comportant un leurre intégré par différentes interactions indépendantes conduit à une reconnaissance très sensible et spécifique qui est peu affectée par des mutations ponctuelles de l'effecteur. En résumé, cette étude a produit des connaissances nouvelles sur la fonction des récepteurs des plantes de type NLRs et sur leur capacité à reconnaitre des effecteurs. Ceci contribue à une meilleure compréhension du système immunitaire des plantes, ce qui est un élément important pour l'obtention de cultures durablement résistantes aux maladies.

Table 1 . Cloned blast rice resistance genes and M. oryzae avirulence genes Resistance genes Avirulence genes

 1

		R gene	Encoded protein	Avr gene	Encoded protein
		Pib	NB-LRR	AvrPib	75 aa secreted protein
		Pi-ta a	NB-LRR	AvrPi-ta	224 aa secreted protein
		Pi9	NB-LRR	AvrPi9	91 aa secreted protein
	R-Avrs	Piz-t	NB-LRR	AvrPiz-t	108 aa secreted protein
	pairs	Pik	NB-LRR	Avr-Pik/km/kp 113 aa secreted protein
		Pikm	NB-LRR	Avr-Pik/km/kp 113 aa secreted protein
		Pikp ab	NB-LRR	Avr-Pik/km/kp 113 aa secreted protein
		Pia abc	NB-LRR	AVR-Pia	85 aa secreted protein
		Pi-CO39 abc NB-LRR	AVR1-CO39	89 aa secreted protein

  RKL

		ACE1	Polyketide synthase
	AVRs only	AvrPii	70 aa secreted protein
		PWL2	145 aa secreted protein
		Adapted from Liu & Wang 2016
	a= These genes have been reported to have integrated domains

b= These genes requires two NLRs members to function c= These genes recognized two unrelated effector proteins 4.6 Recognition of effector proteins by NLRs Rice blast resistance can be mediated by either single NLRs such as Piz-t, Pi9 and Pib or by functional NLRs pairs such as Pik-1/ Pik-2, RGA4/RGA5 and Pi5-1/ Pi5-2 (Liu, 2016). Effector recognition has been particularly well studied in the case of Pik-1/Pik-2 and RGA4/RGA5. Pik and the corresponding AVR effector gene, AVR-Pik are highly polymorphic. Pik has six different alleles (Pik, Pikm, Pikp, Piks, Pikh and Pi1) while AVR-Pik has five different alleles (AVR-Pik A, B, C, D and E)

  . NMR experiments acqui red for structur e cal cul ations and chemical shi ft assignments DALI Statistics for structural alignment of AVR-Pia, AVR1-CO39, AVRPiz-t and ToxB.

	a) AVR-Pi a
				Size			Sweep width (ppm)
	Experiments	nuclei		F3	F2	F1	F3	F2		F1	Mix(ms)	NS D1(s) B o (MHz)
	15 N-HSQC	1 H, 15 N		1024	128	-	-	-		-	-	16	1
	HNCO(*)	1 H, 15 N, 13 C 1024	64	64	12.98	36	17.67	-	8	0.001
	HNCA(*)	1 H, 15 N, 13 C 1024	64	80	12.98	36	31.81	-	16	0.1
	HNCOCACB(*)	1 H, 15 N, 13 C 1024	64	128 12.98	36	75.02	-	32	0.2
	HNCACO(*)	1 H, 15 N, 13 C 1024	64	80	12.98	36	17.67	-	64	0.001
	HNCACB(*)	1 H, 15 N, 13 C 1024	64	128 12.98	36	75.02	-	32	0.2
	CON(**)	13 C, 15 N		1024	200	-	30	36		-	-	4	1
	CACO(**)	13 C, 13 C		1024	256	-	30	50.32		-	-	8	1
	15 N-NOESY-HSQC RM SD (Å)	1 H, 15 N, 1 H 1500 AVR-Pia	76 AVR1-CO39 360 14	35 AVRPiz-t	14	120 ToxB	8	1
	15 N-TOCSY-	1 H, 15 N, 1 H 1500	70	320	14	35		14	56	8	1
	HSQC Z-score
	NOESY (D 2 O)	1 H, 1 H		2048	512	-	15.00	15.00		-	100	128	1
				4096	512		12.01	12.01		-	150	128	1
	TOCSY (D 2 O) COSY-dqf (D 2 O) 1 H, 1 H 1 H, 1 H AVR-Pia		4096 4096	512 800	-2.3	12.01 12.01	12.01 12.01 2.8		--	39.6 -2.2	64 64	1 1
	1 H, 15 N NOE	1 H, 15 N		1024	128	-	14	35		-	Sat. 3s	64	6
	R 1	1 H, 15 N		1024	128	-	14	35		-		16	2.5
	R 2	1 H, 15 N		1024	128	-	14	35		-		16	2.5
	b) AVR1-CO39 AVR1-CO39		2.9					3.0			2.2
				Size			Sweep width (ppm)
	Experiments	nuclei		F3	F2	F1	F3	F2		F1	Mix(ms)	NS D1(s) B o (MHz)
	15 N-HSQC	1 H, 15 N		1500	160	-	12.02	28		-	-	16	1
	1 3 C-HSQC	1 H, 13 C		2048	256	-	15.00	80		-	-	512	1.5
	HNCO(*) AVRPiz-t	1 H, 15 N, 13 C 1024 3.3	64	64 3.1	12.02	28	17.67	-2.2	8	0.001
	HNCA(*)	1 H, 15 N, 13 C 1024	64	80	18.02	28	31.81	-	16	0.1
	HNCOCACB(*)	1 H, 15 N, 13 C 1024	64	128 18.02	28	75.02	-	32	0.2
	HNCACO(*)	1 H, 15 N, 13 C 1024	64	80	18.02	28	17.67	-	64	0.001
	HNCACB(*)	1 H, 15 N, 13 C 1024	64	128 18.02	28	75.02	-	32	0.2
	CON(**) CACO(**) ToxB	13 C, 15 N 13 C, 13 C	4.4	1024 1024	200 256	--4.2	30 30	28 50.32 5.4		--	--	4 2	1 1
	15 N-NOESY-	1 H, 15 N, 1 H 1500	76	360	12	28		12	120	8	1
	HSQC
	15 N-TOCSY-	1 H, 15 N, 1 H 1024	72	320	14	35		14	56	8	1
	HSQC
	NOESY (D 2 O)	1 H, 1 H		2048	512	-	15.00	15.00		-	100	64	1
				2048	512		15.00	15.00		-	200	32	1
	TOCSY	1 H, 1 H		2048	512	-	15.00	15.00		-	40	32	1
	COSY-dqf	1 H, 1 H		2048	800		15.00	15.00		-	-	32	1
	1 H, 15 N NOE	1 H, 15 N		1024	128	-	14	35		-	Sat. 3s	64	6
	R 1	1 H, 15 N		1024	128	-	14	35		-		16	2.5
	R 2	1 H, 15 N		1024	128	-	14	35		-		16	2.5

Experiments were recorded using the TOPSPIN Library (v. 2.1) at 305 K. (*) Best pulse sequences (**)IPAP scheme for virtual decoupling S2 Table

.

  S3 TableMAX-effector candidates identified by psi-Blast in the genomes of the M. oyzae isolates 7015 and TH16 and the M. grisea isolate BR29.

	S 4 Table Primers used for qRT-PCR
	GENE	Forward		Reverse
	geneID	localization	AVR-Pia*	AVR1-CO39* ToxB*
	M_BR29_EuGene_00004921	scaffold00001	9,0E-12	-	6,0E-12
	M_BR29_EuGene_00041131	scaffold00007	-	9,0E-06	-
	M_BR29_EuGene_00043011	scaffold00008	-	9,0E-06	-
	M_BR29_EuGene_00060181	scaffold00013	-	4,0E-08	-
	M_BR29_EuGene_00081821	scaffold00023	-	3,0E-07	-
	M_BR29_EuGene_00082031	scaffold00023	-	1,0E-05	-
	M_BR29_EuGene_00085071	scaffold00025	-	-	3,0E-15
	M_BR29_EuGene_00087671	scaffold00027	4,0E-13	4,0E-12	8,0E-11
	M_BR29_EuGene_00088411	scaffold00027	-	-	5,0E-12
	M_BR29_EuGene_00091361	scaffold00030	3,0E-12	-	-
	M_BR29_EuGene_00091681	scaffold00031	-	2,0E-05	-
	M_BR29_EuGene_00095641	scaffold00035	-	3,0E-13	5,0E-13
	M_BR29_EuGene_00106461	scaffold00049	7,0E-23	-	-
	M_BR29_EuGene_00107481	scaffold00051	-	2,0E-06	-
	M_BR29_EuGene_00112111	scaffold00059	-	6,0E-09	-
	M_BR29_EuGene_00113041	scaffold00061	-	7,0E-08	-
	M_BR29_EuGene_00118801	scaffold00076	-	2,0E-05	-
	M_BR29_EuGene_00119491	scaffold00079	-	3,0E-22	-
	M_BR29_EuGene_00119511	scaffold00079	-	9,0E-17	-
	M_BR29_EuGene_00121691	scaffold00087	-	1,0E-13	-
	M_BR29_EuGene_00125811	scaffold00145	-	7,0E-08	-
	M_BR29_EuGene_00126081	scaffold00163	-	-	2,0E-20
	MGG_00821	Chromosome_8.5	-	6,0E-14	-
	MGG_04384	Chromosome_8.2	-	2,0E-04	-
	MGG_08482	Chromosome_8.4	-	1,0E-06	-
	MGG_08944	Chromosome_8.2	-	-	5,0E-09
	MGG_10120	Chromosome_8.4	-	5,0E-04	-
	MGG_14793	Chromosome_8.2	4,0E-19	3,0E-06	9,0E-15
	MGG_14834	Chromosome_8.4	-	2,0E-05	-
	MGG_15207	Chromosome_8.3	-	5,0E-08	-
	MGG_15459	Chromosome_8.1	-	8,0E-08	-
	MGG_16058	Chromosome_8.1	-	1,0E-05	-
	MGG_16113	Chromosome_8.1	-	3,0E-09	-
	MGG_16175	Chromosome_8.1	-	5,0E-08	-
	MGG_16619	Chromosome_8.3	-	3,0E-06	-
	MGG_17132	Chromosome_8.4	2,0E-06	7,0E-04	9,0E-10
	MGG_17255	Chromosome_8.4	-	1,0E-06	-
	MGG_18019	Chromosome_8.7	-	7,0E-05	8,0E-11
	MGG_18060	Chromosome_8.7	5,0E-14	5,0E-14	4,0E-06
	M_TH16_EuGene_00000541	scaffold00001	-	1,0E-14	-
	M_TH16_EuGene_00027191	scaffold00004	4,0E-10	-	-
	M_TH16_EuGene_00027411	scaffold00004	4,0E-12	-	-
	M_TH16_EuGene_00034081	scaffold00004	-	3,0E-08	-
	M_TH16_EuGene_00040131	scaffold00005	2,0E-25	-	-
	M_TH16_EuGene_00045871	scaffold00007	-	3,0E-12	-
	M_TH16_EuGene_00079081	scaffold00016	6,0E-10	-	-
	M_TH16_EuGene_00079311	scaffold00016	-	2,0E-08	-
	M_TH16_EuGene_00099371	scaffold00026	-	3,0E-08	-
	M_TH16_EuGene_00101881	scaffold00028	-	2,0E-05	2,0E-08
	M_TH16_EuGene_00106621	scaffold00033	-	-	5,0E-14
	M_TH16_EuGene_00120731	scaffold00052	3,0E-06	-	-
	M_TH16_EuGene_00124981	scaffold00063	-	7,0E-11	-
	M_TH16_EuGene_00127871	scaffold00072	-	2,0E-21	-
	M_TH16_EuGene_00134971	scaffold00110	-	9,0E-14	1,0E-14
	M_TH16_EuGene_00135161	scaffold00112	-	-	2,0E-19

Orthologs of 7015 proteins in TH16 are not listed. * Values correpond to e-values in psi-blast search with the corresponding protein

benthamiana Supplemental Table 1 .

 Supplemental1 NMR experiments acquired for structure calculations and chemical shift assignments of AVR-Pia-H3

			Size		Sweep width (ppm)
	Experiments	nuclei	F3	F2	F1	F3	F2	F1	Mix(ms)	NS D1(s) Bo(MHz)
	15 N-HSQC 15 N-NOESY-	1500 1 H, 15 N, 1 H 1500 1 H, 15 N	256 64	-280	-14	-40	-14	-150	8 8	1 1
	HSQC
	NOESY (H2O)	1 H, 1 H	1500	512	-	15.95	15.95	-	150	96	1
	TOCSY (H2O)	1 H, 1 H	1500	512	-	15.95	15.95	-	41.2	64	1
	13 C-HSQC(D2O) 13 C-TOCSY(D2O)	1 H, 13 C 1 H, 13 C	2048 2048	400 400	-	14 12.03	160 100	--	-40.25	240 320	1 1
	1 H, 15 N NOE R1 R2	1 H, 15 N 1 H, 15 N 1 H, 15 N	1024 1024 1024	128 128 128	---	14 14 14	35 35 35	---	Sat. 3s	64 16 16	6 2.5 2.5
	Supplemental Table 2. Statistics for 20 NMR structures of AVR-Pia-H3
				AVR-Pia-H3
	NOE restraints			1366
	Short range (|i-j|≤1)		891
	Medium range (1<|i-j|<5)		133
	Long range (|i-j|≥5)		342
	H-bond restraints		20
	Dihedral restraints (a)		100
	Average Number of NOE
	Violations per structure
	> 0.1 Å			23.75
	> 0.2 Å			1.0
	> 0.3 Å			0.05
	> 0.4 Å			0
	Dihedral violations
	> 2°			0
	Ramachandran plot statistics
	most favourable regions (%)		84.3
	additionally allowed regions (%) 15.7
	generously allowed regions (%)	0.0
	disallowed regions (%)		0.0
	Pairwise RMSD (Å) (b)
	Backbone			0.77 ± 0.19
	Heavy atoms			1.43 ± 0.16

PROJET DE THESE ET DISCUSSION GENERALE INTRODUCTION Dans

  l'agriculture moderne, le développement de variétés exprimant une résistance à la fois durable et efficace contre une large gamme d'agents pathogènes est essentiel à la productivité et la durabilité des systèmes de production. L'une des principales stratégies variétales pour la lutte contre les maladies consiste au déploiement de gènes de résistance (gènes R). Ces gènes sont spécifiquement activés par des protéines appelées « effecteurs », codées par des gènes dits « de virulence » chez les agents pathogènes. Cependant, l'efficacité des gènes R est souvent de courte durée du fait de la grande diversité génétique dont disposent les populations d'agents pathogènes, et de leur capacité à évoluer de manière extrêmement rapide. Ainsi, le déploiement seul de gènes R en monocultures mène bien souvent à la sélection des variants (d'agents pathogènes) dont l'effecteur concerné a été muté ou désactivé, induisant alors une réaction compatible avec la plante pour le déclenchement de la maladie. Il est donc essentiel de développer des techniques de sélection génétique et des stratégies de déploiement innovantes, afin d'approvisionner les systèmes agricoles avec des gènes de résistance efficaces et durables. Une approche prometteuse consiste à générer de manière synthétique des gènes de résistance spécifiques à de nouveaux effecteurs, avec un large spectre d'action (c'est-à-dire capables de cibler plusieurs agents pathogènes), et ce à un rythme au moins aussi rapide que celui auquel évoluent les agents pathogènes. Pour cela, il est nécessaire d'explorer la riche diversité des gènes de résistance des plantes, et d'approfondir la caractérisation fonctionnelle de leur mode d'action afin de comprendre comment les produits de ces gènes perçoivent l'attaque d'un agent pathogène et déclenchent les réactions immunitaires de la plante. Le principal objectif de cette thèse est de contribuer à une meilleure compréhension des déterminants moléculaires de l'immunité des plantes. Plus précisément, nous avons cherché à élucider les bases moléculaires de la reconnaissance des effecteurs de champignons phytopathogènes par les récepteurs de type NLR des plantes, et le lien entre cette reconnaissance et la réaction immunitaire. Pour cela, nous avons caractérisé la structure des effecteurs protéiques AVR1-CO39 et AVR-Pia du champignon Magnaporthe oryzae, et étudié la diversité et l'évolution des effecteurs fongiques définis par leur structure chez les champignons ascomycètes. Nous avons également étudié les déterminants moléculaires permettant la reconnaissance de AVR-Pia par les récepteurs immunitaires RGA4/RGA5. Enfin, nous avons analysé la composition et la formation du complexe de récepteurs RGA4/RGA5. Puisque le pathosystème Riz -M. oryzae est un système modèle pour les interactions plantes -champignons, les résultats générés dans le cadre de cette thèse viennent plus généralement alimenter nos connaissances sur les maladies végétales causées par les champignons.

	OBJECTIF GENERAL ET QUESTIONS DE RECHERCHE
	Objectif général

Accesion Numbers

Sequence data from this article correspond to those previously published by (Cesari, et al., 2013) and can be found in the the GenBank/EMBL databases under the following accession numbers: AVR-Pia (AB498873), AVR-Pia-H3 (KC777366), PWL2 (U26313), RGA4 (AB604622), Sasanishiki RGA5-A (AB604627), Sasanishiki RGA5-B (KC777365). The PDB accession number for the AVR-Pia_H3 structure is 5JHJ.

Acknowledgments

This work was supported by the French Infrastructure for Integrated Structural Biology (FRISBI) ANR-10-INSB-05-0 and the ANR project Immunereceptor (ANR-15-CE20-0007). D. Ortiz-Vallejo was supported by a PhD grant from the ministry of research of Colombia (Colciencias). This work benefited from interactions promoted by COST Action FA 1208 https://www.cost-sustain.org.

Christian Roumestand is acknowledged for fruitful discussions.

THESIS OUTLINE

General objective

The main objective of this thesis is to contribute to a better understanding of the molecular determinants of plant immunity. More specifically, we were interested in the elucidation of the molecular bases of the recognition of fungal effector proteins by plant NLR immune receptors and the link between effector recognition and immune activation. For this, we structurally characterized the M. oryzae effector proteins AVR1-CO39 and AVR-Pia and studied the diversity and evolution of structure-related effector proteins in ascomycete fungi. Furthermore, we investigate the molecular determinants mediating the recognition of AVR-Pia by the immune receptors RGA4/RGA5. Finally we analyzed the composition and formation of the RGA4/RGA5 receptor complex.

Since the M. oryzae-rice pathosystem is a model system for fungal-plant interactions, the knowledge generated in this work is highly relevant for a better comprehension of plant diseases caused by fungi.

Research questions and approaches

The following specific questions were addressed to reach the general objective of the PhD project 1. Is the three-dimensional structure of effector proteins a key determinant of their function, diversity and evolution?

To answer this question we first determined the three-dimensional structure of the M. oryzae effector proteins AVR1-CO39 and AVR-Pia using Nuclear magnetic resonance (NMR). Since both effectors had similar structures that they shared, in addition, with two other fungal effector proteins, AvrPiz-t and ToxB, from M. oryzae and Pyrenophora tritici-repentis respectively, we could define the novel MAX-effector family (M. oryzae Avr and ToxB effectors) by structure-informed sequence patterns. Bioinformatic searches exploiting this pattern showed that MAX effectors are phylogenetically widely distributed, expanded and diversified specifically in the order of the Magnaporthales (Chapter I).

How does AVR-Pia bind the RGA5 RATX1 domain?

Previously, our research group demonstrated that the M.oryzae effector AVR-Pia directly interacts with the C-terminal RATX1 domain of RGA5. In addition, it was shown that AVR-Pia-H3, a natural AVR-Pia allele, lost the interaction with the RATX1 domain and does not trigger blast resistance in rice plants carrying RGA4/RGA5.

In this study, we deepened the characterization of the AVR-Pia-RGA5 RATX1 interaction. We compared the NMR structures of AVR-Pia and AVR-Pia-H3 and identified a candidate interaction surface by NMR-titration. We characterized in vitro AVR-Pia-RGA5 RATX1 complex formation by isothermal calorimetry (ITC) and validated the candidate interaction surface as well as two key residues by yeast two hybrid and co-immunoprecipitation experiments (Chapter II).

What is the role of AVR-Pia -RGA5 RATX1 binding in AVR-Pia recognition?

To determine the importance of AVR-Pia-RGA5 RATX1 binding for recognition and avirulence activity of AVR-Pia, structure-informed AVR-Pia mutants were analyzed in transgenic M. oryzae isolates and transient N. benthamiana cell death induction assays. These experiments showed that AVR-Pia-RGA5 RATX1 binding is required for resistance but that even weakly binding AVR-Pia mutants are still efficiently recognized (Chapter II).

How are other RGA5 domains involved in AVR-Pia recognition?

To better understand RGA5-mediated recognition of AVR-Pia, interactions of AVR-Pia with individual RGA5 domains (CC, NB-ARC, LRR, RATX1) were characterized by yeast two hybrid and co-immunoprecipitation assays. This revealed interactions of AVR-Pia with the RGA5 NB-ARC and LRR domains in addition to RATX1 (Chapter III). Chapter II

CHAPTER I

Structural analyses of

AVR-Pia recognition by the decoy domain of rice the NLR immune receptor RGA5

Since transient heterologous experiments failed to determine the importance of the binding of AVR-Pia to RGA5 RATX1 for recognition and disease resistance, the biological activity of AVR-Pia mutants was assayed in the homologous rice/M. oryzae system. Transgenic M. oryzae isolates were generated that carried the different mutant alleles under the control of the constitutive RP27 promoter (RP27Pro) [START_REF] Bourett | Reef coral fluorescent proteins for visualizing fungal pathogens[END_REF]. As a control, transgenic Guy11 isolates carrying a RP27Pro:mRFP construct were generated and proved to be fully virulent (Figures 5 and Supplemental Figure 5B). For three different PCR-validated transgenic isolates per construct, the accumulation of AVR-Pia variants was verified in culture filtrates by immunoblotting with anti AVR-Pia antibodies (Supplemental Figure 5A). All AVR-Pia mutants were detected in at least one transgenic isolate except AVR-Pia D63A that may be instable in M. oryzae. For AVR-Pia D29A and AVR-Pia E58A , only two and one isolate expressed the mutant protein (Figure S5A).

The transgenic isolates were analyzed on the rice cultivars Kitaake carrying the Pia locus and Maratelli lacking Pia. All isolates were highly virulent on Maratelli indicating that they were not affected in virulence (Supplemental Figure 5B). On Kitaake plants, the isolates expressing AVR-Pia wt , AVR-Pia R23A , AVR-Pia D29A , AVR-Pia R36A or AVR-Pia E58A were completely avirulent and produced either no symptoms or small HR lesions characteristic of resistance (Figure 5 and Supplemental Figure 5B). This indicates that these AVR-Pia variants are fully active and recognized by RGA4/RGA5. Consistent with the absence of protein expression, AVR-Pia D63A

isolates did not induce resistance and were fully virulent on Kitaake plants. Isolates producing AVR-Pia R43G were partially virulent and formed disease lesions characterized by a grey center that were however smaller and less frequent than those provoked by the control mRFP isolates.

Isolates expressing AVR-Pia F24S were highly virulent on Kitaake and produced large numbers of disease lesions (Figures 5 and Supplemental Figure5B).

Taken together, these results indicate that interaction of AVR-Pia with the RGA5 RATX1 domain is required for recognition but that a reduction of this interaction as in AVR-Pia R23A , AVR-Pia D29A , or AVR-Pia E58A does not impair recognition. Only the R43G and F24S polymorphisms that abolished RGA5 RATX1 interaction both in planta and in yeast affected AVR-Pia recognition, with AVR-Pia F24S being completely inactive.

Author contributions

Conceptualization

Supplemental Methods

Production and purification of recombinant proteins for ITC and NMR

The expression and purification of recombinant AVR-Pia mutant proteins were carried out as previously described for the AVR-Pia wild type (de [START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF].

NMR samples

The NMR samples for assignments and structure calculations were prepared with 1mM purified protein at 10% D2O and 0.5 mM DSS as a reference. The purification buffer was exchanged with phosphate buffer (20 mM potassiumsodium phosphate, pH 5.4 and 150 mM NaCl), by filtrating with Centricon®. For the D2O experiments, a 15Nlabeled sample was lyophilized and dissolved in D2O.

N backbone amide NMR Relaxation data

Relaxation data for AVR-Pia-H3 were acquired at 305K on a Bruker Avance 500 MHz spectrometer using R1, R2 and 15 N 1 H heteronuclear NOE pulse sequences (TOPSPIN library, v 2.1) with a protein sample at 1 mM and the experimental settings already given in (de [START_REF] Guillen | Structure analysis uncovers a highly diverse but structurally conserved effector family in phytopathogenic fungi[END_REF].

Relaxation parameters, R1, R2 and NOEs were determined from the analysis module of CCPN (Vranken et al., 2005). Lipari-Szabo analysis was performed using the DYNAMOF software (Barthe et al., 2006).

CHAPTER IV Functional analyses of RGA4-RGA5 interaction

The interaction between RGA4 and RGA5 appears not to be disrupted by AVR-Pia

To investigate whether the interaction between RGA4 and RGA5 is disrupted by AVR-Pia, we co-expressed RGA5 and RGA4 with and without AVR-Pia. For this, we used the tagged proteins RGA4-GFP, YFP-RGA5, HA-RGA5, HA-AVR-Pia and YFP and we analyzed the complex formation by co-IP experiments.

All tagged proteins were well expressed and proteins fused to either YFP or GFP were in all cases efficiently immuneprecipitated. HA-RGA5 was well co-precipitated by RGA4-GFP in the presence and absence of HA-AVR-Pia (Figure 34) indicating that AVR-Pia does not abolish RGA4-RGA5 interaction. In addition, HA-AVR-Pia was not co-precipitated by RGA4-GFP suggesting that no stable trimeric RGA4-RGA5-AVR-Pia complex is formed. However, HA-AVR-Pia was co-precipitated by YFP-RGA5 (Figure 34).

We then used a non-tagged AVR-Pia protein to confirm that the interaction between YFP-RGA5 and RGA4-HA is not affected by this effector. RGA4-HA was co-precipitated by RGA5 in presence of AVR-Pia which supports the idea that AVR-Pia does not abolish the interaction between RGA5 and RGA4 (Figure 35). However, whether AVR-Pia reduces the amount of coprecipitated RGA4-HA cannot be determined from this experiment since the immunoprecipitation of RGA5 failed for the YFP-RGA5-RGA4-HA sample where AVR-Pia is absent.

Taken together these results suggest that RGA4 and RGA5 interaction is not abolished by AVR-Pia, however whether RGA5 and RGA4 form a stable three-protein-complex with AVRremains to be elucidated.

SUMMARY POINTS (CHAPTER IV)

 CC domains are dispensable in the intermolecular interaction between RGA5-RGA4.  CC, NB and LRR domains of RGA5 are involved in the intermolecular interaction with their corresponding domains in RGA4.  RGA4 and RGA5 form hetero-complexes in presence or absence of AVR-Pia. pGBKT7-53 and activation domain (AD) fusions of AVR-Pia, AVR1-CO39 and empty vector as well as RGA5 variants in pGADT7, were transformed in Gold and Y187 yeast strain respectively. Interactions assays were performed according to the Matchmaker Gold yeast two-hybrid system protocol (Clontech).

Entry clone

Destination vectors yeast Destination vectors in N. bentamiana

*AVR-Pia without tag pSC95 Plant diseases caused by fungi constitute a worldwide threat to food security and disease resistance (R) genes that allow to breed resistant crops are key elements for efficient disease control. The vast majority of R genes code for NLR multi domain proteins characterized by nucleotide-binding and leucine-rich repeat domains and acting as intracellular immune receptors for pathogen-secreted virulence factors termed effectors. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins.

The first question I addressed in my PhD thesis is: how is the tremendous diversity of fungal effectors generated? A structural analysis had identified the family of sequence-unrelated but structurally conserved MAX-effectors (Magnaporthe Avrs and ToxB like) that has expanded specifically in Magnaporthe oryzae the causal agent of rice blast disease. By expression analysis, I confirmed that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during host colonization. MAX effectors are the first structurally defined family of effectors in fungi and this study gives therefore news clues for the identification of candidate effectors in fungi and constitutes a crucial step towards a better understanding of effector evolution. In fact, the scenario observed for MAX-effectors leads to the hypothesis that the enormous number of sequence-unrelated fungal effectors belong in fact to a restricted set of structurally conserved effector families.

The second question I investigated in my PhD thesis is: what are the molecular mechanisms of effector recognition by NLR immune receptors? I addressed this question by studying recognition of the M. oryzae effector AVR-Pia by the rice NLR pair RGA4/RGA5. Previous work has shown that RGA5 acts as a receptor that binds directly to AVR-Pia while RGA4 acts as a constitutively active signaling protein that is, in the absence of pathogen, repressed by RGA5. This functional interaction involves formation of an RGA4/RGA5 receptor complex. By protein-protein interaction studies, I showed that complex formation involves interactions between the RA4 and RGA5 NB-ARC and LRR domains, in addition to previously identified interactions between the coiled-coil domains. AVR-Pia recognition seems not to induce dissociation of the RGA4/RGA5 complex but a ternary RGA4/RGA5/AVR-Pia complex could also not be detected consistently. How effector recognition is translated into receptor complex activation remains therefore to be elucidated in more detail in the future.

Previous work has shown that a domain of RGA5 normally not present in NLRs and related to the copper chaperone ATX1 (RATX1 domain) interacts physically with AVR-Pia and may be crucial for effector recognition. The RATX1 domain was hypothesized to mimic the true host targets of AVR-Pia leading to the development of the 'integrated decoy' model that states