The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

Successful pathogens like Pseudomonas syringae translocate type III effector proteins (T3SE) into host cells. Plant hosts react by specifically recognizing these effectors via R proteins that trigger defense responses. The T3SE family HopZ1 has evolved into three allelic forms as a result of diversifying selection. In this thesis, I investigated how virulence and immune-eliciting functions are determined in HopZ1a and HopZ1b in Arabidopsis. Mutational analysis of HopZ1a identified ten residues important for immune elicitation and at least three are involved in virulence functions. These results suggest that distinct key amino acid residues in HopZ1a mediate the two activities. The closely related HopZ1b T3SE elicits an inconsistent immune response in Arabidopsis. We found that HopZ1b-triggered immune response involves a TIR-type R protein and plastid-derived SA. Together, these results highlight an uncharacterized ETI response to the HopZ1 family of T3SEs.

La bactérie phytopathogène du sol, Ralstonia solanacearum cause la maladie du flétrissement bactérien sur un grand nombre de plante hôtes. Un des déterminants clefs de son pouvoir pathogène est le système de sécrétion de type III. Celui-ci permet à la bactérie d'injecter des protéines directement dans les cellules de l'hôte. Dans ma thèse je me suis attaché à décrire et analyser finement la contribution au pouvoir pathogène de la bactérie de certains de ces substrats de l'appareil de sécrétion de type III (RipAX2, RipH2, RipH3 et RipG). Des expériences de double-hybride nous ont permis d'identifier des protéines des plantes hôtes pouvant être ciblées par ces effecteurs de type III. Dans une autre partie de mon travail j'ai contribué à l'étude de RipAX2 qui est induit spécifiquement la résistance dans des lignées d'aubergines porteur d'un locus de résistance. J'ai également travaillé sur l'identification des cellules de plantes soumises à l'injection de type III dans une interaction compatible. Pour cela j'ai utilisé la plante hôte Medicago truncatula, en exprimant dans certaines lignées cellulaires un effecteurs (RipG7) pour lequel nous avions démontré que son expression constitutive das M. truncatula pouvait restaurer l'infection de ces plantes par un mutant bactérien dans ce même effecteur. Enfin, j'ai aussi contribué à l'analyse structure-fonction fine de l'effecteur RipG7 dans sa fonction de contribution au pouvoir pathogène de R. solanacearum sur la plante hôte M. truncatula. Ce travail nous a permis d'identifier les acides aminés de RipG7 qui sont sous sélection positive, et parmi ceux-là, ceux qui contribuent directement à la fonction de RipG7 sur M. truncatula. / The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The type III secretion system (T3SS) and its associated type III effectors (T3Es) are the main virulence determinants of R. solanacearum. In my PhD study, to understand the mode of action of several "core" effectors (RipH2, RipH3, RipG7, RipG6) from R. solanacearum in host cells. We performed yeast two-hybrid screening of plant cDNA library to identify their protein targets. Besides, we also collaborated on the identification and characterization of a specific type III effector RipAX2 which is an avirulence factor that triggers a hypersensitive response in specific Eggplant lines. To understand which plant root cells are actually subjected to type III injection during the compatible interaction, I have generated transiently transformed Medicago truncatula lines (hairy root), expressing a host specificity and core T3E RipG7 in different root cell layers. When the transformed plant expressing RipG7 under 35S promoter, the plant can be infected and colonized by the ripG7 single mutant strain. The study could be refined by using specific root cell layer promoter to identify the root cell layers that are key players in this interaction. We also worked on the characterization of the structure-function of RipG7 from R. solanacearum. Our work revealed the genetic and functional variation of RipG7. Furthermore, positive selection study and mutagenesis analysis enabled us to identify essential functional residues which likely to have been differentially selected during the host-pathogen co-evolution. The potential plant targets of RipG7 were also studies further in our study by differential yeast two-hybrid.

No description available.

Pepper is a popular and important vegetable crop grown and consumed worldwide. However, pepper production is threatened by the gram-negative bacterium Xanthomonas euvesicatoria (Xe) which causes bacterial spot (BS) disease, one of the most common and destructive diseases on pepper. Due to limited genetic resistance resources in host species, a promising strategy for controlling BS disease is to transfer nonhost disease resistance (R) genes from other plant species into pepper plants to confer broad-spectrum and durable resistance. A maize R gene Rxo1 has been functionally transferred to rice plants and confers nonhost resistance to rice pathogen Xanthomonas oryzae pv. oryzicola (Xoc) carrying a type III effector (T3E) AvrRxo1. Most Xe strains carry a T3E Xe4428, a homolog of AvrRxo1. Therefore, Rxo1 could be potentially employed to develop Xe-resistant pepper. In addition, a better understanding of the virulence function of Xe4428 may provide insights into the pathogenesis of Xe and new strategies for crop improvement. In this dissertation, we transformed Rxo1 into the far-related dicot species Nicotiana benthamiana and pepper, and characterized the Rxo1-mediated disease resistance against Xe strains carrying AvrRxo1 or Xe4428. In addition, we explored the virulence function and mechanism of Xe4428. In the Rxo1-transgenic N. benthamiana, we demonstrated that Rxo1 could condition resistance to Xe harboring AvrRxo1 but not Xe4428. We revealed that AvrRxo1 could directly interact with the nucleotide-binding domain of Rxo1 in vivo and in vitro. We further demonstrated that the nucleus localization of AvrRxo1 was required for its avirulence and virulence functions. In addition, the cytosol localization of Rxo1 was also necessary to confer disease resistance. The downstream signaling component NbNDR1 was demonstrated to be involved in Rxo1/AvrRxo1-mediated disease resistance. By RNAseq-based gene expression profiling, we identified six candidate genes of interest up-regulated by the Rxo1-AvrRxo1 recognition. Through virus-induced gene silencing screening, a gene encoding phenylalanine ammonia-lyase 4 was demonstrated to be critical for Rxo1/AvrRxo1-mediated disease resistance in N. benthamiana. Rxo1-transgenic pepper plants were resistant to the Xe strain with the complementary Xoc effector AvrRxo1 but not the wild-type Xe strain that carries Xe4428. A Xe4428 mutant with only one nucleotide substitution could trigger the Rxo1-mediated disease resistance in pepper. Both wild-type and mutant Xe4428 had significant virulence functions that could promote the Xe bacterial proliferation on wild-type pepper plants. In addition, the mutant Xe4428 had a higher expression level than wild-type Xe4428 in Xe bacterial cells, which might explain why the mutant Xe4428 but not wild-type Xe4428, could trigger the Rxo1-mediated disease resistance in pepper. We identified 14 pepper cystatin genes (CaCys), among which two genes (CaCys1 and CaCys13) could be induced, and two genes (CaCys3 and CaCys5) were suppressed by Xe4428. Ectopically expressing one of the induced genes CaCys1 in N. benthamiana increased the stomatal opening and promoted the Xe growth in N. benthamiana plants. Thus, we illuminate one possible mechanism of Xe4428's virulence function is to regulate the stomata apertures in N. benthamiana. Bacterial fruit blotch (BFB) caused by the gram-negative bacterial pathogen Acidovorax citrulli (A. citrulli) is one of the most destructive diseases in cucurbit crops, including melon and watermelon. A better understanding of the virulence and avirulence functions of T3Es in A. citrulli helps breeders engineer crop resistance to BFB. To this end, a clean genetic background of A. citrulli with multiple effector genes deleted is desired. Here, we optimized a marker-exchange-based method for sequential effector deletion and generated an AAC00-1 mutant with five effector genes (Aave2166, Aave3626, Aave1548, Aave2938, Aave2708) deleted (AAC00-15). AAC00-15 was less virulent in watermelon but more virulent in N. benthamiana. Through complementation, we characterized the function of individual effectors and identified a promising R gene, Roq1, that could be used to control BFB disease. / Doctor of Philosophy / As an essential ingredient in almost all cuisines, pepper is grown and consumed worldwide, providing human beings with favorable flavor and nutrients. However, pepper production is threatened by the destructive bacterial spot (BS) disease caused by the bacterial pathogen Xanthomonas euvesicatoria (Xe). Due to limited genetic resistance resources in host species, nonhost resistance (R) genes from other plant species are desired to confer broad-spectrum and durable resistance to the pepper pathogen Xe. Previously, a maize (corn) R gene called Rxo1 was transferred to rice plants. This gene helped these rice plants resist a rice bacterial pathogen that causes leaf streak disease on rice. This rice pathogen has an effector (a virulent protein produced by bacteria to infect plants) that is required for the disease resistance. The pepper pathogen carries a similar effector, so transferring the maize R gene Rxo1 to pepper plants might similarly benefit peppers and help fight against the bacterial spot disease. In this dissertation, we successfully transferred the maize R gene Rxo1 into Nicotiana benthamiana and pepper plants. Our results indicate that this gene can help control disease caused by the pepper pathogen harboring the effector of the rice pathogen but not its native effector. We also illuminate how the disease resistance conferred by this maize gene happens in Nicotiana benthamiana plants. In addition, we explain how the corresponding effector helps infect plants. This research provides insights into the application of R gene transfer between far-related plant species and new tools to improve crop disease resistance.

Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells. / Doctor of Philosophy / Peppers and tomatoes are two of the most important vegetables grown worldwide, providing humans with high quality of flavor and aroma, vitamins, and antioxidants. The pepper and tomato production is frequently threatened by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on peppers and tomatoes. The BS disease can be easily identified due to the appearance of the dark, irregular, water-soaked areas on the leaf, which can cause approximately 10% loss of the total yield of peppers and tomatoes. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most critical breeding goals. A better understanding of the virulence mechanism of Xe could help breeders to design new strategies for resistance breeding. In my seminar, I will discuss the virulence and avirulence functions of Xe type three secretion (T3S) effectors: Xe XopN, Xe XopQ, and Xe XopX. In my study, I identified Xe XopN is a key factor that regulates the development of the water-soaking symptom on pepper plants infected with Xe. In addition, we revealed Xe XopN interacts with a transcription factor NbVOZ to regulate the expression of NbNPR1 and PR1 genes expression, which may also contribute to the development of water-soaking phenotype. In addition, I identified that Xe XopN could interact with a transcription factor, NbVOZ, and represses the expression of NbNPR1, a key component of the basal defense, and the pathogenesis-related gene PR1. Therefore, Xe XopN has a role in regulating a water-affluent environment to promote bacterial proliferation in the infected plant tissue. Xe XopQ is a Xe T3S effector that functions as a determinant of host specificity. In my study, I identified another T3S effector Xe XopX that could interact with Xe XopQ to trigger the defense response in Nicotiana benthamiana. I also confirmed Xe XopQ physically interacts with Xe XopX inside of plant cells by using bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, Xe XopX could also interact with multiple Xe T3Es including AvrBS2 in a co-IP assay. The virulence and avirulent functions of Xe XopQ and AvrBS2 are compromised in the absence of Xe XopX.

La bactériose vasculaire du riz (BLB) et bactériose non-vasculaire du riz (BLS), causées respectivement par Xanthomonas oryzae pv. oryzae (Xoo) et X. oryzae pv. oryzicola (Xoc), sont les deux plus importantes maladies bactériennes du riz. Ces maladies limitent le rendement de la production de riz dans les zones rizicoles en Asie et dans certaines régions d'Afrique. L'infection et la multiplication bactérienne dans les tissus de l'hôte dépendent souvent des facteurs de virulence de ces bactéries dont le type le système de sécrétion de type III (T3SS) et ses substrats. Dans cette thèse, nous avons identifié neuf effecteurs non-TAL (transcription Transcription activator-like) sécrétés par des effecteurs de type III de la souche chinoise 13751 de Xoo en utilisant le domaine d'induction HR de la protéine avirulente AvrBs1 comme gène reporter. Parmi eux, XopAE13751 a été expérimentalement confirmé pour la première fois comme étant un effecteur non-TAL. Ensuite, par l'analyse mutationnelle de ces gènes effecteurs identifiés dans Xoo, nous avons constaté que l'effecteur non-TAL XopR13751 était nécessaire pour la virulence de la souche chinoise de Xoo sur le riz hybride Teyou63. En parallèle, nous avons démontré que le gène rsmA (repressor of secondary metabolism) - comme le gène rsmAXoo de l'espèce chinoise Xoo 13751- régule positivement l'expression des gènes associés aux facteurs de virulence, tels que le système de sécrétion de type III, les enzymes extracellulaires et le DSF (diffusible signal factor). De plus, le gène effecteur non-TAL xopO s'est avéré être peu répandu chez les Xanthomonas puisqu'il est présent uniquement chez X. euvesicatoria (Xe) et Xoc mais est absent chez Xoo. En considérant les deux pathovars de X. oryzae, avec deux modes d'infection différents, xopO a été examiné comme un facteur de la spécificité du tissu par l'inactivation mutationnelle du gène dans Xoc et par l'expression du gène dans Xoo. Les résultats ont montré que xopO n'est pas la cause déterminante de la spécificité de tissu chez Xoc. Enfin, nous avons étudié les VNTRs (Variable Number of Tandem Repeats) comme outil de typage moléculaire rapide, fiable et rentable, pour améliorer le contrôle des épidémies et pour évaluer la structure de population des souches de Xoc. 28 loci candidats VNTR ont été prédits par le criblage de trois génomes de Xoc (souche philippine BLS256, souche chinoise GX01 et souche malienne MAI10). Des paires d'amorces pour l'amplification de PCR de chacun des 28 loci ont été conçues et testées à un pannel de 20 souches de Xoc provenant de l'Asie et de l'Afrique. Le séquençage des amplicons de PCR a confirmé 25 loci VNTR robustes et polymorphes communs entre les souches Xoc asiatiques et africaines. Un dendrogramme, construit à partir de la combinaison des 25 loci de VNTR (MLVA-25), a montré que la plupart des souches asiatiques sont clairement distinguables des souches africaines. Cependant, en accord avec de précédents rapports, une souche Malienne se distingue et semble être liée aux souches asiatiques, suggérant une introduction possible de souches sur le continent africain. Ce nouvel outil de typage basé sur les VNTR sera utile pour l'étude de structures de populations et pour la surveillance épidémiologique de Xoc. / Bacterial leaf blight (BLB) and Bacterial leaf streak (BLS), caused respectively by Xanthomonas oryzae pv. oryzae (Xoo) and X. oryzae pv. oryzicola (Xoc), are two most important bacterial diseases on rice, constraining severely the rice yield in the rice-growing areas in Asia and in parts of Africa. Successful infection and bacterial multiplication in host tissue often depend on virulence factors from these bacteria including type Ⅲ secretion system (T3SS) and its substrates. In this thesis, we identified nine type Ⅲ secreted non-TAL (Transcription activator-like) effectors of Xoo Chinese strain 13751 using HR-inducing domain of avirulence protein AvrBs1 as the reporter, among them, XopAE13751 was first found experimentally to be non-TAL effector. Subsequently, through mutational analysis of these identified effector genes in Xoo, we showed non-TAL effector XopR13751 was found to be required for full virulence of Xoo Chinese strain in hybrid rice Teyou63. In parallel, we demonstrated that rsmA (repressor of secondary metabolism)-like gene rsmAXoo of Xoo Chinese strain 13751 positively regulated the expression of genes associated with virulence factors such as type Ⅲ secretion system, extracellular enzymes and diffusible signal factor (DSF). Furthermore, non-TAL effector gene xopO was found to be narrowly distributed in Xanthomonas, which was only present in X. euvesicatoria (Xe) and Xoc, but not in Xoo. Based on the consideration of two X. oryzae pathovars carrying two different infection ways, xopO was tested in host and tissue specificity by analysis of mutational analysis of the gene in Xoc and expression of the gene in Xoo. The results showed that xopO of Xoc did not function as a determinant in host and tissue specificity. Finally, we explored Variable Number of Tandem Repeats (VNTRs) as a fast, reliable and cost-effective molecular typing tool, to better monitoring epidemics and assess the population structure of Xoc strains. 28 candidate VNTR loci were predicted by screening of three Xoc genome sequences (Philippine strain BLS256, Chinese strain GX01 and Malian strain MAI10). Primer pairs for PCR amplification of all 28 loci were designed and applied to a panel of 20 Xoc strains originating from Asia and Africa. Sequencing of PCR amplicons revealed 25 robust and polymorphic VNTR loci which are shared among Asian and African strains of Xoc. A dendrogram was constructed from 25 VNTR loci-combinating data (MLVA-25), indicating that most Asian strains were clearly discriminated from African strains. However, in agreement with previous reports, one strain from Mali appeared to be related to Asian strains, pointing to a possible introduction of strains to the African continent. A detailed analysis of the evolutionary relationships among a larger set of Xoc strains from China will be presented, considering different spatial scales. In conclusion, a new VNTR-based tool useful for studies of population structures and epidemiological monitoring of Xoc was successfully established.

Ralstonia solanacearum, une béta-proteobactérie d'origine tellurique, est l'une des phytobactérioses les plus nuisibles au niveau mondial. Cette bactérie est capable d'infecter plus de 250 espèces différentes dont certaines présentent un intérêt économique majeur (tomate, pomme de terre, tabac). R. solanacearum est divisée en 4 phylotypes distincts présentant des origines géographiques différentes : I (asiatique), IIA et IIB (américain), III (africain), IV (indonésien). Parmi ces phylotypes, le phylotype I est en expansion démographique, hautement recombinogène, réparti mondialement et possède une large gamme d'hôtes. Il possède donc un fort potentiel évolutif (sensu McDonald et Linde, 2002). Afin de contrôler cette bactérie, la lutte génétique reste la méthode la plus prometteuse : elle consiste à déployer des cultivars possédant différents sources de résistance (i.e., des gènes de résistance). La variété d'aubergine AG91-25 (E6) possède un gène majeur de résistance (ERs1) lui permettant de contrôler certaines souches de R. solanacearum de phylotype I. Cependant, la gestion de cette résistance requiert d'étudier au préalable sa durabilité afin d'en éviter le contournement. Cette durabilité peut être estimée en étudiant le potentiel évolutif d'un agent pathogène face à cette source de résistance, ainsi qu'en décryptant les mécanismes moléculaires de l'interaction entre l'hôte (gène R) et le pathogène (effecteur de types trois). Afin d'étudier la dynamique évolutive de R. solanacearum sous une pression de sélection exercée par la variété résistante E6, nous avons mis en place un essai d'évolution expérimentale au champ. Cet essai est composé de trois couples de microparcelles d'aubergines résistantes E6 et d'aubergines sensibles E8, implantées deux fois par an, pendant trois ans (soit 5 cycles). Un schéma MLVA (« Multi-Locus VNTR Analysis ») composé de 8 loci minisatellites a été développé afin de caractériser les souches extraites de ces cycles de cultures. Ces VNTR sont spécifiques aux souches de R. solanacearum de phylotype I, hautement polymorphes et discriminants à toutes les échelles : mondiale, régionale et locale. Nos résultats démontrent une absence de contournement de la résistance d'E6 par les populations parcellaires de R. solanacearum, confirmant le caractère durable de cette résistance. Cette variété aurait fortement réduit les populations bactériennes du sol, ne leur permettant plus d'infecter l'hôte résistant. Parallèlement, 100% des plants d'E8 sont morts à partir du cycle 2. La maladie au sein des microparcelles semble progresser selon une dynamique de « plante-à-plante ». Une baisse de la diversité génétique a aussi été observée au cours des cycles de culture répétés d'E8, associée à l'augmentation en fréquence de deux haplotypes. Cependant, aucune structuration génétique claire n'a été observée à l'échelle de la parcelle entière ou de la microparcelle. En revanche, les données d'isolement par la distance semblent indiquer qu'une structure spatiale semble être en cours d'établissement. L'ensemble de nos résultats suggère une structure épidémique clonale de nos populations parcellaires. Nous nous sommes aussi intéressés à l'implication de 10 ET3 dans l'interaction R. solanacearum vs aubergine résistante (E6). La distribution des 10 ET3 candidats est variable au sein d'une collection de souches phylogénétiquement diverses (91 souches) : ripAJ et ripE1 sont les ET3 les plus partagés alors que ripP1 et ripP2 sont les moins fréquemment. Certains ET3 présentent peu (ripAJ) voire pas (ripE1 et ripP2) de polymorphisme de taille, alors que d'autres (ripAU) sont extrêmement polymorphes. Cependant la composition en effecteurs d'une souche ne semble pas être corrélée à un phénotype sur aubergine E6. Nous avons identifié le gène d'effecteur ripAX2 comme ayant une fonction d'avirulence sur aubergine résistante E6. Sa reconnaissance par E6 semble s'opérer au niveau de la zone hypocotylaire. / Ralstonia Solanacearum is a soilborn beta-proteobacterium responsible of bacterial wilt on Solanaceaous crops. This bacterium is considered as one of the most harmful plant disease worldwide. This bacterium possesses the ability to infect more than 250 different species, including crops with major economic importance (tomato, potato, tobacco, eucalyptus…). R. solanacearum is divided into four phylotypes originated from different areas: I (Asian), IIA and IIB (American), III (African), IV (Indonesian). Among these phylotype, phylotype I is currently in demographic expansion, is highly recombinogenic and has a wide hosts range. Thus, altogether, these characteristics demonstrated that this phylotype has a high evolutionary potential (sensu McDonald and Linde, 2002). In order to control this bacterium, genetic plant resistance seems to be the most promising method. This method consists in using cultivars with different source of resistance such as resistance genes and/or resistant QTLs. The AG91-25 (E6), an eggplant cultivar possessing a major resistance gene (ERs1), is capable to control some of phylotype I strains of R. solanacearum. However, in order to optimize the management of this resistance and to avoid its fast breakdown, we need to deeply investigate the durability of this resistant gene. Durability can be estimated by studying the evolutionary potential of our pathogen faced to E6 source of resistance and by understanding the molecular mechanisms underlying the interaction between the host (R gene) and its pathogene (Type III Effector – T3E). In order to study R. solanacearum evolutionary dynamics under selective pressure from E6 resistant cultivar, we set up an experimental evolution trial in the field. This trial consisted of three couples of resistant (E6) and susceptible eggplants (E8) microplots, implanted twice a year during three years, hence consisting of 5 cycles. A Multi-Locus VNTR Analysis (MLVA) scheme, consisting of 8 minisatellite loci, was developed in order to characterize the strains extracted from these crop cycles. These VNTRs were specific to R. solanacearum phylotype I strains, they were highly polymorphic and discriminatory at different scale: globally, regionally and locally.Our results showed no breakdown of E6 resistance by R. solanacearum populations, which confirms that this resistance is durable. It seemed that this cultivar reduced the soil bacterial population, preventing bacterial population to infest the resistant host. At the same time, 100% of the E8 plants have died, starting at cycle 2. Bacterial wilt seemed to spread with a “plant-to-plant” dynamics within each microplot. Genetic diversity reduction was also observed during the successive cycle of susceptible eggplant, associated with the increase of frequency of two main haplotypes. However, we failed to identify a clear genetic structuration, neither at the plot scale nor at the microplot scale. Nevertheless, isolation-by-distance data seemed to show that a spatial structure is currently establishing. Altogether, our results suggested that our plot populations appeared to have a clonal epidemic structure.We also looked into 10 T3Es' involvement in the interaction between R. solanacearum and the resistant eggplant (E6). Their distribution was completely different within a collection of phylogenetically diverse strains (91 strains): ripAJ and ripE1 are the most shared T3Es whereas ripP1 and ripP2 were the less common T3E whithin our collection of strains. Some T3Es showed few (ripAJ) or no length polymorphism at all (ripE1 and ripP2) whereas some other (ripAU) are extremely polymorphic. Nevertheless, the T3E effector repertoire did not seemed to be correlated to a specific phenotype on E6 eggplant. Its recognition by E6 seemed to occur in the hypocotyle region rather than in the mesophyll, highlighting a possible organ-specificity of the interaction between ERs1 and ripAX2.

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012