Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium. (Résumé d'auteur

Variations in type III effector repertoires do not correlate with differences in pathological phenotypes and host range observed for Xanthomonas citri pv. citri pathotypes

Xanthomonas citri pv. citri (Xac) is a quarantine bacterium causing Asiatic citrus canker. Strains of Xac are classified as pathogenic variants i.e. pathotypes, according to their host range: strains of pathotype A infect a wide range of rutaceous species, whereas strains of pathotype A*/Aw infect a restricted host range consisting of Mexican lime (C. aurantifolia) and alemow (C. macrophylla). Based on a collection of 55 strains we investigated the role of type III effectors (T3E) in host specialization. By PCR we screened 56 Xanthomonas T3Es and showed that Xac possesses a repertoire of 28 effectors, 24 of which are shared by all strains, while 4 (xopAI, xopAD, xopAG and xopC1) are present only in some A*/ Aw strains. However, their distribution could not account for host specialization. XopAG is present in all Aw strains, but also in three A* strains genetically distant from Aw , and all xopAG-containing strains induced HR-like reactions on grapefruit and sweet orange. A strains are genetically less diverse, induce identical phenotypic responses, and share exactly the same T3Es. Conversely, A*/ Aw strains exhibited a wider genetic diversity in which clades correlated to geographical origin and T3Es repertoire but not to pathogenicity. A*/Aw strains showed a broad range of reactions on several Citrus, but genetically related strains did not share phenotypic responses. Our results showed that A*/Aw strains are more variable (genetically and pathogenetically) than initially expected and that this variability should not be ignored when trying to describe mechanisms involved in the pathogen evolution and host specialization. (Texte intégral

The Xanthomonas campestris type III effector xopAC triggers vascular immunity in Arabidopsis

Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot on Brassicae and causes disease on crop plants such as cabbage or on the model plant Arabidopsis. The xopAC gene encodes a type III effector which is responsible for avirulence on Arabidopsis ecotype Col-0 exclusively when Xcc is inoculated in the leaf vasculature (1). PCRs and dot-blot hybridizations performed on a large collection of plant pathogenic bacteria revealed that avrAC is specific to Xc. The analysis of more than 50 Xcc strains reveals that avrAC displays a very low allelic diversity and belongs to the Xcc variable effectome. Moreover, the presence of avrAC is tightly correlated with an increase in Xcc aggressiveness on susceptible Arabidopsis. This correlation was experimentally confirmed by reverse genetic in several Xcc strains. In addition, xopAC is responsive for the appearance of necrotic lesions on nonhost pepper plants. We show that the "Leucine-Rich Repeat" (LRR) and "Filamentation induced by cAMP" (Fic) domains of XopAC are both required for avirulence on resistant Arabidopsis and necrosis on pepper. Interestingly, the Fic domains of the VopS and IbpA virulence factors from animal pathogens were recently shown to mediate protein adenylylation, a yet unknown protein posttranslational modification in plants (2,3). Strategies developed to dissect avrAC functions in planta and to study plant vascular immunity will be presented. (Résumé d'auteur

Recherche des cibles végétales de l'effecteur de type-III XOPAC de Xanthomonas Campestris pathovar Capestris (Xcc)

Xanthomonas campestris pv. campestris est une bactérie phytopathogène vasculaire responsable de la pourriture noire des Brassicacées comme le chou, plante d'intérêt économique, ou la plante modèle Arabidopsis thaliana. Xcc possède un système de sécrétion de type III lui permettant d'injecter des protéines de virulence (ou effecteurs) directement à l'intérieur de la cellule végétale. Le gène xopAC code un effecteur de type-III qui est spécifique des Xanthomonas campestris et qui est responsable de l'avirulence sur l'écotype Columbia-0 d'A. thaliana quand Xcc est inoculé directement dans la nervure centrale de la feuille (Xu et al., 2008). L'introduction de xopAC dans la bactérie Ralstonia solanacearum (pathogène vasculaire des solanacées) rend la bactérie avirulente sur Col-0. Ce résultat montre que XopAC est aussi capable d'induire une réponse immunitaire contre un autre pathogène vasculaire. La séquence protéique de XopAC possède un domaine LRR (Répétition Riche en Leucine) en N-terminal et un domaine Fic (Filamentation Induced by cAMP) en Cterminal. Le domaine LRR est un domaine d'interaction avec un ligand. Le domaine Fic issu de deux effecteurs de pathogène d'animaux entraîne la modification posttraductionnelle de thréonines ou tyrosines par adénylylation (rajout d'un groupement AMP; Worby et al., 2009; Yarbrough et al., 2009). Cette modification chez les plantes reste encore à déterminer. La présence des domaines Fic et LRR est essentielle pour la fonction d'avirulence de la protéine XopAC sur l'écotype résistant Col-0 d'A. thaliana, montrant ainsi l'importance biologique de ces deux domaines. En s'appuyant sur ces données récentes nous avons émis l'hypothèse que la protéine XopAC serait impliquée dans une interaction avec une cible végétale via son domaine LRR et dans sa modification posttraductionnelle (par adénylylation) via le domaine Fic. Nous vous présenterons Les expériences menées pour rechercher les cibles végétales de XopAC et étudier l'immunité vasculaire mise en place chez Arabidopsis contre Xcc et d'autres pathogènes vasculaires. (Texte intégral

The complete genome sequence of Xanthomonas albilineans provides new insights into the reductive genome evolution of the xylem-limited Xanthomonadaceae

The Xanthomonadaceae includes two xylem-limited plant pathogenic bacteria, Xanthomonas albilineans and Xylella fastidiosa. The complete genome of X. albilineans was sequenced, providing not only strong clues to identify new pathogenicity factors in this pathogen causing sugarcane leaf scald disease, but also new insights into the evolution of Xanthomonadaceae. Previous phylogenetic analysis suggested that the Xanthomonas and Stenotrophomonas genera of Xanthomonadaceae form a coherent group excluding X. fastidiosa. Surprisingly, phylogenetic analysis using X. albilineans genomic sequences resulted in a different tree in which X. fastidiosa belongs to the Xanthomonas group. Based on this latter tree, X. albilineans and X. fastidiosa are derived from the progenitor of the Xanthomonas genus which itself is derived from the progenitor of Stenotrophomonas. Comparative genomic analysis identified 551 ancestral genes which are present in both Xanthomonas axonopodis pv. vesicatoria and Stenotrophomonas but absent in both X. fastidiosa and X. albilineans, revealing that these two latter species experienced a similar reductive genome evolution during their descent from the progenitor of the Xanthomonas genus. This degenerative evolution is probably driven by adaptation to the nutrient-poor xylem elements and to the cloistered environmental niche of xylem vessels. Comparable genomic erosion is observed among intracellular animal bacteria and is associated with a mutualistic life style. Adaptation of X. albilineans and X. fastidiosa to a xylem-limited life style is also illustrated by the unique characteristics of the enzymes involved in cellulose degradation, and the absence of a type III secretion system of the Hrp1 and Hrp2 injectisome families. (Texte intégral

Genome sequencing of Xanthomonas axonopodis pv. phaseoli CFBP4834-R reveals that flagellar motility is not a general feature of xanthomonads.

Xanthomonads are plant-associated bacteria that establish neutral, commensal or pathogenic relationships with plants. The list of common characteristics shared by all members of the genus Xanthomonas is now well established based on the entire genome sequences that are currently available and that represent various species, numerous pathovars of X. axonopodis (sensu Vauterin et al., 2000), X. oryzae and X. campestris, and many strains within some pathovars. These ?-proteobacteria are motile by a single polar flagellum. Motility is an important feature involved in biofilm formation, plant colonization and hence considered as a pathogenicity factor. X. axonopodis pv. phaseoli var. fuscans (Xapf) is one of the causal agents of common bacterial blight of bean and 4834-R is a highly aggressive strain of this pathogen that was isolated from a seed-borne epidemic in France in 1998. We obtained a high quality assembled sequence of the genome of this strain with 454-Solexa and 2X Sanger sequencing. Housekeeping functions are conserved in this genome that shares core characteristics with genomes of other xanthomonads: the six secretion systems which have been described so far in Gram negative bacteria are all present, as well as their ubiquitous substrates or effectors and a rather usual number of mobile elements. Elements devoted to the adaptation to the environment constitute an important part of the genome with a chemotaxis island and dispersed MCPs, numerous two-component systems, and numerous TonB dependent transporters. Furthermore, numerous multidrug efflux systems and functions dedicated to biofilm formation that confer resistance to stresses are also present. An intriguing feature revealed by genome analysis is a long deletion of 35 genes (33 kbp) involved in flagellar biosynthesis. This deletion is replaced by an insertion sequence called ISXapf2. Genes such as flgB to flgL and fliC to fleQ which are involved in the flagellar structure (rod, P- and L-ring, hook, cap and filament) are absent in the genome of strain 4834-R that is not motile. Primers were designed to detect this deletion by PCR in a collection of more than 300 strains representing different species and pathovars of Xanthomonas, and less than 5% of the tested xanthomonads strains were found nonmotile because of a deletion in the flagellum gene cluster. We observed that half of the Xapf strains isolated from the same epidemic than strain 4834-R was non-motile and that this ratio was conserved in the strains colonizing the next bean seed generation. Isolation of such variants in a natural epidemic reveals that either flagellar motility is not a key function for fitness or that some complementation occurs within the bacterial population. (Résumé d'auteur

Etudes des mécanismes de résistance d'Arabidopsis thaliana induits par l'effecteur de type Ill XopAC de Xanthomonas campestris pv. campestris

Xanthomonas campestris pv. campestris (Xcc) est un phytopathogène vasculaire responsable de la pourriture noire des crucifères d'intérêt agronomique comme le chou-fleur, le chou, le navet et le radis ainsi que la plante modèle Arabidopsis thaliana. L'effecteur de type JII XopAC/AvrAC confère l'avirulence à Xcc chez l'ecotype Col-0 d'Arabidopsis. XopAC interagit avec plusieurs RLCK (Receptor-Like Cytoplasmic Kinase) de la famille VII importantes pour la PTI (PAMP-triggered immunity) et de I'ETI (Effector-triggered immunity) et les inhibe par uridylylation: BIKl (Bottytis-lnduced Kinase 1) et RIPK (RlN4-interacting receptor-like protein kinase). Au sein de l'équipe, nous avons pu montrer que XopAC interagit avec d'autres RLCK de la famille Vll. De manière particulièrement intéressante, les mutants RLCK ripk et pbl2 (PBS1-like 2) perdent leur resistance dépendante de xopAC. Parallèlement, xopAC exprime dans des plantes transgéniques provoque un arrêt de croissance au stade cotyledon ainsi qu'une chlorose. Des mutations sxc (suppressors of xopAC) supprimant ce phénotype confèrent également la sensibilité à Xcc exprimant xopAC. Le clonage positionnel de ces mutations par séquençage haut débit a permis l'identification de deux gènes dont un code une protéine CC-NB-LRR requise pour la reconnaissance de xopAC chez Arabidopsis thaliana. L'état d'avancement de ces différents travaux sera presenté. (Résumé d'auteur