Rôle de la paroi végétale dans l'interaction entre Arabidopsis thaliana et Ralstonia solanacearum : criblage de mutants « paroi » et caractérisation fine de la résistance accrue du mutant walls are thin 1 (wat1)

De part sa localisation à l'interface entre la cellule et son environnement, la paroi végétale joue un rôle clé lors des interactions avec des agents pathogènes. Au cours de ma thèse, l'importance de la paroi végétale lors de l'infection d'Arabidopsis par la bactérie Ralstonia solanacearum a été étudiée. Pour cela, une analyse immunocytologique couplée à une approche bioinformatique a permis d'identifier les modifications pariétales induites en réponse à l'infection et de les corréler à l'arsenal enzymatique existant chez la bactérie. En parallèle, le crible de mutants ''paroi'' d'Arabidopsis testés pour leur sensibilité à différents agents pathogènes a contribué à ouvrir de nouvelles pistes pour mieux comprendre le rôle de la paroi dans les réponses de défense puisque 28 mutants ont une sensibilité modifiée. L'essentiel de mes recherches a porté sur la caractérisation de wat1 (walls are thin 1), un mutant d'Arabidopsis présentant une résistance accrue à R. solanacearum. Par des approches génétique, transcriptomique et métabolomique, nous avons pu définir que wat1 présente une immunité vasculaire et que les mécanismes de résistance impliqueraient une perturbation dans le " crosstalk " entre les métabolismes de l'auxine, des glucosinolates indoliques et de l'acide salicylique au niveau du système racinaire plutôt que des modifications pariétales stricto sensu.Due to its location at the interface between the cell and its environment, the plant cell wall plays a key role in interactions with pathogens. During this project, the importance of plant cell walls during infection of Arabidopsis by the bacterium Ralstonia solanacearum has been studied. First, an immunocytological analysis coupled with a bioinformatic approach allowed us to identify cell wall modifications in response to infection and to correlate them to the cell wall-degrading enzymatic arsenal present in the bacteria. In parallel, the screening of cell wall mutants of Arabidopsis for susceptibility to different pathogens led to the identification of twenty eight mutants with altered sensitivity and, as a result, will open new avenues for understanding the role of the wall in defense responses. Much of my PhD research has focused on the characterization of wat1 (walls are thin 1), an Arabidopsis mutant with increased resistance to R. solanacearum. Through combined genetic, transcriptomic, and metabolomic approaches, we show that wat1 exhibits vascular immunity, most likely resulting from altered crosstalk between auxin, indole glucosinolate and salicylic acid metabolism in roots rather than in cell wall modifications per se

Plant Carbohydrate Scavenging through TonB-Dependent Receptors: A Feature Shared by Phytopathogenic and Aquatic Bacteria

TonB-dependent receptors (TBDRs) are outer membrane proteins mainly known for the active transport of iron siderophore complexes in Gram-negative bacteria. Analysis of the genome of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc), predicts 72 TBDRs. Such an overrepresentation is common in Xanthomonas species but is limited to only a small number of bacteria. Here, we show that one Xcc TBDR transports sucrose with a very high affinity, suggesting that it might be a sucrose scavenger. This TBDR acts with an inner membrane transporter, an amylosucrase and a regulator to utilize sucrose, thus defining a new type of carbohydrate utilization locus, named CUT locus, involving a TBDR for the transport of substrate across the outer membrane. This sucrose CUT locus is required for full pathogenicity on Arabidopsis, showing its importance for the adaptation to host plants. A systematic analysis of Xcc TBDR genes and a genome context survey suggested that several Xcc TBDRs belong to other CUT loci involved in the utilization of various plant carbohydrates. Interestingly, several Xcc TBDRs and CUT loci are conserved in aquatic bacteria such as Caulobacter crescentus, Colwellia psychrerythraea, Saccharophagus degradans, Shewanella spp., Sphingomonas spp. or Pseudoalteromonas spp., which share the ability to degrade a wide variety of complex carbohydrates and display TBDR overrepresentation. We therefore propose that TBDR overrepresentation and the presence of CUT loci designate the ability to scavenge carbohydrates. Thus CUT loci, which seem to participate to the adaptation of phytopathogenic bacteria to their host plants, might also play a very important role in the biogeochemical cycling of plant-derived nutrients in marine environments. Moreover, the TBDRs and CUT loci identified in this study are clearly different from those characterized in the human gut symbiont Bacteroides thetaiotaomicron, which allow glycan foraging, suggesting a convergent evolution of TBDRs in Proteobacteria and Bacteroidetes

Arabidopsiscell wall composition determines disease resistance specificity and fitness

[EN] Plant cell walls are complex structures subject to dynamic remodeling in response to developmental and environmental cues and play essential functions in disease resistance responses. We tested the specific contribution of plant cell walls to immunity by determining the susceptibility of a set of Arabidopsis cell wall mutants (cwm) to pathogens with different parasitic styles: a vascular bacterium, a necrotrophic fungus, and a biotrophic oomycete. Remarkably, most cwm mutants tested (29/34; 85.3%) showed alterations in their resistance responses to at least one of these pathogens in comparison to wild-type plants, illustrating the relevance of wall composition in determining disease-resistance phenotypes. We found that the enhanced resistance of cwm plants to the necrotrophic and vascular pathogens negatively impacted cwm fitness traits, such as biomass and seed yield. Enhanced resistance of cwm plants is not only mediated by canonical immune pathways, like those modulated by phytohormones or microbeassociated molecular patterns, which are not deregulated in the cwm tested. Pectin-enriched wall fractions isolated from cwm plants triggered immune responses in wild-type plants, suggesting that wall-mediated defensive pathways might contribute to cwm resistance. Cell walls of cwm plants show a high diversity of composition alterations as revealed by glycome profiling that detect specific wall carbohydrate moieties. Mathematical analysis of glycome profiling data identified correlations between the amounts of specific wall carbohydrate moieties and disease resistance phenotypes of cwm plants. These data support the relevant and specific function of plant wall composition in plant immune response modulation and in balancing disease resistance/development trade-offs.SIThis work has been also financially supported by the Severo Ochoa Program for Centers of Excellence in R&D from the Agencia Estatal de Investigación of Spain (Grant SEV-2016-0672 (2017-2021) to the Centro de Biotecnología y Genómica de Plantas). In the frame of this program, H.M. was a postdoctoral fellow. H.M. was also supported by an Individual Fellowship grant (SignWALLINg-624721) from the European Union. E.M. was a Juan de la Cierva Postdoctoral Fellow from MINECO, and L.B. was a Formacion Personal Investigador fellow of MICIU. The generation of the CCRC-series of plant cell glycan-directed monoclonal antibodies used in this work was supported by the US NSF (DBI-0421683 and IOS 0923992) to M.G.H

Identification of genetic relationships and subspecies signatures in Xylella fastidiosa

Abstract Background The phytopathogenic bacterium Xylella fastidiosa was thought to be restricted to the Americas where it infects and kills numerous hosts. Its detection worldwide has been blooming since 2013 in Europe and Asia. Genetically diverse, this species is divided into six subspecies but genetic traits governing this classification are poorly understood. Results SkIf (Specific k-mers Identification) was designed and exploited for comparative genomics on a dataset of 46 X. fastidiosa genomes, including seven newly sequenced individuals. It was helpful to quickly check the synonymy between strains from different collections. SkIf identified specific SNPs within 16S rRNA sequences that can be employed for predicting the distribution of Xylella through data mining. Applied to inter- and intra-subspecies analyses, it identified specific k-mers in genes affiliated to differential gene ontologies. Chemotaxis-related genes more prevalently possess specific k-mers in genomes from subspecies fastidiosa, morus and sandyi taken as a whole group. In the subspecies pauca increased abundance of specific k-mers was found in genes associated with the bacterial cell wall/envelope/plasma membrane. Most often, the k-mer specificity occurred in core genes with non-synonymous SNPs in their sequences in genomes of the other subspecies, suggesting putative impact in the protein functions. The presence of two integrative and conjugative elements (ICEs) was identified, one chromosomic and an entire plasmid in a single strain of X. fastidiosa subsp. pauca. Finally, a revised taxonomy of X. fastidiosa into three major clades defined by the subspecies pauca (clade I), multiplex (clade II) and the combination of fastidiosa, morus and sandyi (clade III) was strongly supported by k-mers specifically associated with these subspecies. Conclusions SkIf is a robust and rapid software, freely available, that can be dedicated to the comparison of sequence datasets and is applicable to any field of research. Applied to X. fastidiosa, an emerging pathogen in Europe, it provided an important resource to mine for identifying genetic markers of subspecies to optimize the strategies attempted to limit the pathogen dissemination in novel areas

Deciphering the route of Ralstonia solanacearum colonization in Arabidopsis thaliana roots during a compatible interaction: focus at the plant cell wall

International audienceThe compatible interaction between the model plant, Arabidopsis thaliana, and the GMI1000 strain of the phytopathogenic bacterium, Ralstonia solanacearum, was investigated in an in vitro pathosystem. We describe the progression of the bacteria in the root from penetration at the root surface to the xylem vessels and the cell type-specific, cell wall-associated modifications that accompanies bacterial colonization. Within 6 days post inoculation, R. solanacearum provoked a rapid plasmolysis of the epidermal, cortical, and endodermal cells, including those not directly in contact with the bacteria. Plasmolysis was accompanied by a global degradation of pectic homogalacturonanes as shown by the loss of JIM7 and JIM5 antibody signal in the cell wall of these cell types. As indicated by immunolabeling with Rsol-I antibodies that specifically recognize R. solanacearum, the bacteria progresses through the root in a highly directed, centripetal manner to the xylem poles, without extensive multiplication in the intercellular spaces along its path. Entry into the vascular cylinder was facilitated by cell collapse of the two pericycle cells located at the xylem poles. Once the bacteria reached the xylem vessels, they multiplied abundantly and moved from vessel to vessel by digesting the pit membrane between adjacent vessels. The degradation of the secondary walls of xylem vessels was not a prerequisite for vessel colonization as LM10 antibodies strongly labeled xylem cell walls, even at very late stages in disease development. Finally, the capacity of R. solanacearum to specifically degrade certain cell wall components and not others could be correlated with the arsenal of cell wall hydrolytic enzymes identified in the bacterial genome

Arabidopsis Response Regulator 6 (ARR6) Modulates Plant Cell-Wall Composition and Disease Resistance

International audienceThe cytokinin signaling pathway, which is mediated by Arabidopsis response regulator (ARR) proteins, has been involved in the modulation of some disease-resistance responses. Here, we describe novel functions of ARR6 in the control of plant disease-resistance and cell-wall composition. Plants impaired in ARR6 function (arr6) were more resistant and susceptible, respectively, to the necrotrophic fungus Plectosphaerella cucumerina and to the vascular bacterium Ralstonia solanacearum, whereas Arabidopsis plants that overexpress ARR6 showed the opposite phenotypes, which further support a role of ARR6 in the modulation of disease-resistance responses against these pathogens. Transcriptomics and metabolomics analyses revealed that, in arr6 plants, canonical disease-resistance pathways, like those activated by defensive phytohormones, were not altered, whereas immune responses triggered by microbe-associated molecular patterns were slightly enhanced. Cell-wall composition of arr6 plants was found to be severely altered compared with that of wild-type plants. Remarkably, pectin-enriched cell-wall fractions extracted from arr6 walls triggered more intense immune responses than those activated by similar wall fractions from wild-type plants, suggesting that arr6 pectin fraction is enriched in wall-related damage-associated molecular patterns, which trigger immune responses. This work supports a novel function of ARR6 in the control of cell-wall composition and disease resistance and reinforces the role of the plant cell wall in the modulation of specific immune responses

Comparative genomics and transcriptomics of #Xanthomonas campestris# : Session 3- Physiologie, génétique et génomique des bactéries

International audienceThe Xanthomonas campestris species causes different diseases on a wide range of Brassicas and is composed of at least four pathovars (campestris, incanae. raphani. unnamed). This species encompasses both vascular and non-vascular pathogens. A comparative genomic analysis of X campestris diversity was performed at the intraspecific and intrapathovar levels by sequencing more than 40 strains. Structural genome annotation was performed and benefited from deep sequencing of small and large RNAs. This approach allowed the exact determination of transcriptional start sites and the identification of small noncoding RNAs. We also used RNA sequencing to characterize the type III regulon in several strains and could identify novel type 111 effectomes. Core and variable genomes and type 111 effectomes were determined. These results evidenced an unsuspected genomic diversity in this species. The latest progress in this project will be presented. (Texte intégral