Le pouvoir pathogène chez Ralstonia solanacearum phylotype II génomique intégrative et paysages transcriptomiques en relation avec l'adaptation à l'hôte

Ralstonia solanacearum is a plant pathogenic bacterium globally distributed with a particularly broad host range. This organism is biologically diverse and is adapted to all types of soil, to planktonic lifestyle and to many plant hosts and natural reservoirs. This bacterium is a species complex and its genetic, phenotypic and host range diversity is a direct consequence of adaptation mechanisms. Phylogenetic analyses have divided this species complex into four distinct phylotypes correlating mostly with strains’ geographical origin. This thesis focuses on using phylotype II strains as an experimental model due to their adaptation to specific hosts: Moko strains pathogenic to banana, ‘Brown rot’ strains adapted to potatoes and emergent pathological variant NPB strains. Our main research topic is the understanding of host adaptation processes. In order to tackle this problematic we sequenced about ten genomes as a starting point of (i) a taxonomic revision of the species complex (ii) a comparative genomic analysis and (iii) an in planta transcriptomic analysis. Together, these complementary approaches allow a more systemic view of this organism’s genetic and phenotypic complexity.Ralstonia solanacearum est une bactérie phytopathogène à la gamme d'hôte exceptionnellement large et à la répartition mondiale. Cet organisme présente une biologie à facettes multiples et s'est adapté à quasiment tous les types de sols, à la vie planctonique, et à de nombreux hôtes et plantes réservoirs. Cette capacité d'adaptation est attestée par une très forte hétérogénéité des souches qui unifient ce complexe d'espèces, aussi bien au plan de la diversité génétique, phénotypique, que de la gamme d'hôte. Des approches phylogénétiques ont montré une structuration de la population mondiale en quatre phylotypes qui correspondent globalement à l'origine géographique des souches. Les travaux de thèse portent sur des souches du phylotype II qui ont valeur de modèle expérimental car épidémiologiquement inféodées à un hôte particulier : souches Moko pathogènes du bananier, souches ‘Brown rot’ adaptées à la pomme de terre et souches émergentes NPB, un variant du pouvoir pathogène. La question de recherche centrale porte sur la compréhension des mécanismes d'adaptation à l'hôte. Pour cela, une dizaine de génomes ont été séquencés dans une perspective (i) de revisiter la taxonomie de ce complexe d'espèce, (ii) d'en faire une analyse génomique comparative et (iii) d'analyser les paysages transcriptomiques produits lors de l'infection (in planta). L'ensemble des ces approches complémentaires permettent ainsi d'intégrer la complexité génétique et phénotypique de l'organisme de manière plus systémique

Genomic and proteomic evidence supporting the division of the plant pathogen Ralstonia solanacearum into three species

Background The increased availability of genome sequences has advanced the development of genomic distance methods to describe bacterial diversity. Results of these fast-evolving methods are highly correlated with those of the historically standard DNA-DNA hybridization technique. However, these genomic-based methods can be done more rapidly and less expensively and are less prone to technical and human error. They are thus a technically accessible replacement for species delineation. Here, we use several genomic comparison methods, supported by our own proteomic analyses and metabolic characterization as well as previously published DNA-DNA hybridization analyses, to differentiate members of the Ralstonia solanacearum species complex into three species. This pathogen group consists of diverse and widespread strains that cause bacterial wilt disease on many different plants. Results We used three different methods to compare the complete genomes of 29 strains from the R. solanacearum species complex. In parallel we profiled the proteomes of 73 strains using Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry (MALDI-TOF-MS). Proteomic profiles together with genomic sequence comparisons consistently and comprehensively described the diversity of the R. solanacearum species complex. In addition, genome-driven functional phenotypic assays excitingly supported an old hypothesis (Hayward et al. (J Appl Bacteriol 69:269–80, 1990)), that closely related members of the R. solanacearum could be identified through a simple assay of anaerobic nitrate metabolism. This assay allowed us to clearly and easily differentiate phylotype II and IV strains from phylotype I and III strains. Further, genomic dissection of the pathway distinguished between proposed subspecies within the current phylotype IV. The assay revealed large scale differences in energy production within the R. solanacearum species complex, indicating coarse evolutionary distance and further supporting a repartitioning of this group into separate species. Conclusions Together, the results of these studies support the proposed division of the R. solanacearum species complex into three species, consistent with recent literature, and demonstrate the utility of proteomic and genomic approaches to delineate bacterial species. (Résumé d'auteur

Degradation of the plant defense signal salicylic acid protects Ralstonia solanacearum from toxicity and enhances virulence on tobacco

Plants use the signaling molecule salicylic acid (SA) to trigger defenses against diverse pathogens, including the bacterial wilt pathogen Ralstonia solanacearum. SA can also inhibit microbial growth. Most sequenced strains of the heterogeneous R. solanacearum species complex can degrade SA via gentisic acid to pyruvate and fumarate. R. solanacearum strain GMI1000 expresses this SA degradation pathway during tomato pathogenesis. Transcriptional analysis revealed that subinhibitory SA levels induced expression of the SA degradation pathway, toxin efflux pumps, and some general stress responses. Interestingly, SA treatment repressed expression of virulence factors, including the type III secretion system, suggesting that this pathogen may suppress virulence functions when stressed. A GMI1000 mutant lacking SA degradation activity was much more susceptible to SA toxicity but retained the wild-type colonization ability and virulence on tomato. This may be because SA is less important than gentisic acid in tomato defense signaling. However, another host, tobacco, responds strongly to SA. To test the hypothesis that SA degradation contributes to virulence on tobacco, we measured the effect of adding this pathway to the tobacco-pathogenic R. solanacearum strain K60, which lacks SA degradation genes. Ectopic addition of the GMI1000 SA degradation locus, including adjacent genes encoding two porins and a LysR-type transcriptional regulator, significantly increased the virulence of strain K60 on tobacco. Together, these results suggest that R. solanacearum degrades plant SA to protect itself from inhibitory levels of this compound and also to enhance its virulence on plant hosts like tobacco that use SA as a defense signal molecule. (Résumé d'auteur

Pathogenicity of Ralstonia solanacearum phylotype : integrative genomics and transcriptomic landscapes associated with host specificity

Ralstonia solanacearum est une bactérie phytopathogène à la gamme d'hôte exceptionnellement large et à la répartition mondiale. Cet organisme présente une biologie à facettes multiples et s'est adapté à quasiment tous les types de sols, à la vie planctonique, et à de nombreux hôtes et plantes réservoirs. Cette capacité d'adaptation est attestée par une très forte hétérogénéité des souches qui unifient ce complexe d'espèces, aussi bien au plan de la diversité génétique, phénotypique, que de la gamme d'hôte. Des approches phylogénétiques ont montré une structuration de la population mondiale en quatre phylotypes qui correspondent globalement à l'origine géographique des souches. Les travaux de thèse portent sur des souches du phylotype II qui ont valeur de modèle expérimental car épidémiologiquement inféodées à un hôte particulier : souches Moko pathogènes du bananier, souches ‘Brown rot’ adaptées à la pomme de terre et souches émergentes NPB, un variant du pouvoir pathogène. La question de recherche centrale porte sur la compréhension des mécanismes d'adaptation à l'hôte. Pour cela, une dizaine de génomes ont été séquencés dans une perspective (i) de revisiter la taxonomie de ce complexe d'espèce, (ii) d'en faire une analyse génomique comparative et (iii) d'analyser les paysages transcriptomiques produits lors de l'infection (in planta). L'ensemble des ces approches complémentaires permettent ainsi d'intégrer la complexité génétique et phénotypique de l'organisme de manière plus systémique.Ralstonia solanacearum is a plant pathogenic bacterium globally distributed with a particularly broad host range. This organism is biologically diverse and is adapted to all types of soil, to planktonic lifestyle and to many plant hosts and natural reservoirs. This bacterium is a species complex and its genetic, phenotypic and host range diversity is a direct consequence of adaptation mechanisms. Phylogenetic analyses have divided this species complex into four distinct phylotypes correlating mostly with strains’ geographical origin. This thesis focuses on using phylotype II strains as an experimental model due to their adaptation to specific hosts: Moko strains pathogenic to banana, ‘Brown rot’ strains adapted to potatoes and emergent pathological variant NPB strains. Our main research topic is the understanding of host adaptation processes. In order to tackle this problematic we sequenced about ten genomes as a starting point of (i) a taxonomic revision of the species complex (ii) a comparative genomic analysis and (iii) an in planta transcriptomic analysis. Together, these complementary approaches allow a more systemic view of this organism’s genetic and phenotypic complexity

Hydroxycinnamic Acid Degradation, a Broadly Conserved Trait, Protects Ralstonia solanacearum from Chemical Plant Defenses and Contributes to Root Colonization and Virulence.

Plants produce hydroxycinnamic acid (HCA) defense compounds to combat pathogens, such as the bacterium Ralstonia solanacearum. We showed that an HCA degradation pathway is genetically and functionally conserved across diverse R. solanacearum strains. Further, a feruloyl-CoA synthetase (Δfcs) mutant that cannot degrade HCA was less virulent on tomato plants. To understand the role of HCA degradation in bacterial wilt disease, we tested the following hypotheses: HCA degradation helps the pathogen i) grow, as a carbon source; ii) spread, by reducing HCA-derived physical barriers; and iii) survive plant antimicrobial compounds. Although HCA degradation enabled R. solanacearum growth on HCA in vitro, HCA degradation was dispensable for growth in xylem sap and root exudate, suggesting that HCA are not significant carbon sources in planta. Acetyl-bromide quantification of lignin demonstrated that R. solanacearum infections did not affect the gross quantity or distribution of stem lignin. However, the Δfcs mutant was significantly more susceptible to inhibition by two HCA, namely, caffeate and p-coumarate. Finally, plant colonization assays suggested that HCA degradation facilitates early stages of infection and root colonization. Together, these results indicated that ability to degrade HCA contributes to bacterial wilt virulence by facilitating root entry and by protecting the pathogen from HCA toxicity