Etude genetique et moleculaire du determinisme du pouvoir pathogene d'Erwinia amylovora: agent du feu bacterien des Pomoiedees

SIGLEINIST T 75306 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Étude de la toxicité de DspA, protéine essentielle au pouvoir pathogène d'Erwinia amylovora, chez la levure Saccharomyces cerevisiae

La bactérie phytopathogène E. amylovora, est l'agent responsable du Feu bactérien des Spiraeoideae (pommier, poirier, pyracantha), une maladie caractérisée par l'apparition de symptômes nécrotiques des tissus infectés. Le pouvoir pathogène d E. amylovora repose entre autre sur un système de sécrétion de type III (SSTT) qui permet la sécrétion et l'injection d'effecteurs dans la cellule hôte végétale. Parmi les protéines injectées par le T3SS d'E. amylovora, DspA est essentielle au pouvoir pathogène de la bactérie puisqu un mutant dspA est non pathogène sur plante (Gaudriault et al., 1997). Le rôle de DspA est dual, d une part, l expression de dspA est suffisante pour provoquer des symptômes nécrotiques sur plante et une toxicité chez la levure, d autre part, DspA est impliquée dans la suppression des réactions de défense telles que la déposition de callose (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA appartient à la famille des effecteurs AvrE qui sont répandus chez les bactéries phytopathogènes et semblent posséder une fonction similaire. Cependant, peu de connaissance existe sur la structure ainsi que la fonction de DspA. L'objectif de ce travail de thèse était de déterminer les domaines ou motifs importants pour la fonction de DspA. Pour cela nous avons choisi d'effectuer une analyse in silico et fonctionnelle de la protéine DspA. L'analyse in silico révèle la présence d'un domaine bêta-propeller au sein de la protéine DspA ainsi que de tous les homologues analysés. De plus, l'analyse fonctionnelle indique que ce domaine est important pour la structure et la fonction de DspA. Dans un second temps, j'ai étudié le mécanisme d'action de DspA dans la levure Saccharomyces cerevisiae. J'ai pu mettre en évidence que l'expression de dspA chez la levure induit un arrêt de croissance et une forte altération du trafic cellulaire. L'étude de mutants de levure suppresseurs de la toxicité de DspA, effectuée avant mon arrivée au laboratoire, montre que les suppresseurs les plus forts sont affectés dans la voie de biosynthèse des sphingolipides, je me suis donc plus particulièrement intéressée au rôle des sphingolipides dans la toxicité générée par DspA. Nos résultats montrent que DspA inhibe la biosynthèse des sphingolipides indirectement via les régulateurs négatifs de la voie, les protéines Orms.Erwinia amylovora is the causative agent of fire blight of Spiraeoideae (apple, pear, pyracantha), a disease characterized by the apparition of necrotic symptoms on infected tissues. The pathogenicity of E. amylovora relies on a functional type III secretion system (T3SS) that allows secretion and injection of effector proteins into the host plant cell. Among these effector proteins injected by the T3SS of E. amylovora, DspA is essential to the bacteria disease process since a dspA mutant is nonpathogenic on plants (Gaudriault et al., 1997). DspA has a dual role; on the one hand dspA expression is sufficient to induce cell death on plants and toxicity on yeast, on the other hand, DspA is involved on suppression of defense reactions like callose deposition (Degrave et al., 2008; Boureau et al., 2006; Oh et al., 2007; DebRoy et al., 2004). DspA belongs to the AvrE familly of type III effectors which are widespread on phytopathogenic bacteria and likely possess a similar function. However, the structure and function of DspA remain unknown. In the first part of my thesis, I attempted to characterize domains or motifs important for the function of DspA. We performed an in silico and a functional analysis of the DspA protein. In silico analysis predicted a bêta-propeller domain in DspA and all the analysed effectors. In the second part of my thesis, I analysed the mechanism of function of DspA in the yeast Saccharomyces cerevisiae. Results showed that expression of dspA in yeast inhibits cell growth and alters the actin cytoskeleton and endocytosis. Screening of the Euroscarf library for mutants resistant to DspA induced toxicity revealed that mutants impaired in the sphingolipid biosynthetic pathway are the best suppressors. Based on this results, I attempted to determine the role of sphingolipids in the toxicity induced by DspA. Results showed that DspA inhibits indirectly the sphingolipid biosynthetic pathway via the negative regulators, Orm proteins.PARIS11-SCD-Bib. électronique (914719901) / SudocSudocFranceF

Implications des harpines HrpNea et HrpWea, protéines de pathogénie de la bactérie Erwinia amylovora dans les étapes précoces d'interaction avec la cellule végétale

Les harpines sont des effecteurs protéiques sécrétés par le système de sécrétion de type III de certaines bactéries phytopathogènes. L'objectif des travaux présentés dans cette thèse fut d'étudier l'implication des harpines HrpNea et HrpWea dans l'interaction entre Erwinia amylovora et la cellule végétale. Dans un premier temps, nous avons travaillé avec des cellules en suspension d'Arabidopsis thaliana, plante non-hôte d'E. amylovora (situation incompatible). Sur ce modèle, l'inhibition des canaux anioniques est un événement précoce déterminant conduisant à la mort cellulaire en réponse à HrpNea. Nous nous sommes ensuite intéressés aux effets de HrpWea, l'autre harpine d'E. amylovora. Nos résultats montrent que HrpWea est une harpine dont les effets sur la mort cellulaire sont doubles. A 200 nM, HrpWea induit la mort tandis qu'à 0,2 nM, HrpWea est capable d'inhiber la mort cellulaire induite par la harpine HrpNea. La modulation des canaux anioniques apparaît aussi comme un événement déterminant de l'effet " protecteur " de HrpWea. Plus largement, ces résultats suggèrent que HrpWea peut être considérée comme un inhibiteur des réactions de défense de la cellule végétale. Enfin, nous avons étudié les effets de HrpNea chez un modèle cellulaire hôte (situation compatible), les cellules en suspension de pommier. Nous avons montré que les réponses induites par HrpNea en contexte hôte et en contexte non-hôte sont différentes, ce qui explique vraisemblablement le fait qu'HrpNea n'induit pas la mort chez ses plantes hôtes.Considérées dans leur ensemble, ces données ont permis d'améliorer la compréhension de l'implication des harpines d'E. amylovora dans la pathogénie et la réponse hypersensible induites par cette bactérie. D'une façon plus générale, ils ont aussi permis d'apporter de nouvelles informations quant au rôle des canaux ioniques dans les phénomènes de mort cellulaire programmée.Harpins are protean effectors secreted by type III secretion system of phytopathogenic bacteria. The aim of the present work was to investigate the involvement of HrpNea and HrpWea harpins from Erwinia amylovora during the interaction between this bacteria and the plant cell. We first worked with suspension cells from the non-host plant Arabidopsis thaliana (incompatible situation). On this model, we showed that anion channel inhibition is a determinant event leading to cell death triggered by HrpNea. We then studied the effects of HrpWea. Our results showed that HrpWea has different effects depending on its concentration. At high concentration , HrpWea induced cell death, whereas at low concentration , HrpWea was able to inhibit HrpNea induced cell death. Anion channel modulation also appeared to be a determinant event of cell death inhibition. More widely, these results suggested that HrpWea could be a plant cell defense mechanism inhibitor. Finally, we studied the effects of HrpNea on the host apple cells (compatible situation). We showed differential responses between host and non-host plant cells, likely explaining that HrpNea did not trigger cell death in host plants.Taken together, these data allowed us to improve our knowledge concerning involvement of HrpNea and HrpWea in E. amylovora hypersensitive response and pathogenicity. Moreover, these results bring new data concerning the role of ion channels during cell death.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Gram-negative phytopathogenic bacteria, all hemibiotrophs after all?

International audienceIrrespective of the bacteria considered, after an initial biotrophic phase, in which the bacteria avoid the elicitation of or suppress plant defences, a switch to necrotrophic stage, in which the bacteria actively kill plant cells, is observed. Both phases are required to induce disease although the length of time of each phase will vary between bacteria. The length of each phase will also vary for the same bacteria between experimental conditions, as the inoculum concentration, age of the plant and the inoculation procedure will influence the outcome of the interaction. Another difficulty arises because the switch between the initial biotrophic phase and the later necrotrophic phase cannot be visualised through microscopic examination, as it has been performed with fungi. This makes the necrotroph/biotroph classification highly hazardous for phytopathogenic bacteria. Furthermore, all phytopathogenic bacteria possess weapons to supress or avoid premature elicitation of plants defences, indicating they initially develop on living hosts, and weapons involved in cell death induction, probably resulting in nutrient acquisition. Therefore, phytopathogenic bacteria should always be considered as hemibiotrophs. Although the biotrophic phase has been thoroughly described for type III-dependent phytobacteria, it has been overlooked for type II-dependent soft rot pectinolytic bacteria, probably because the asymptomatic biotrophic phase is highly dependent on environmental conditions and is difficult to reproduce in laboratory conditions. Moreover, because the zig-zag model has been so powerful in understanding the biotrophic phase of type III-dependent phytobacteria, their necrotrophic phase is often underestimated. To achieve a better comprehension of plant-bacterial interactions, the hemibiotrophic nature of these interactions should be recognized

Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear

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Draft genomes of “Pectobacterium peruviense” strains isolated from fresh water in France

International audienceBacteria belonging to the genus Pectobacterium are responsible for soft rot disease on a wide range of cultivated crops. The "Pectobacterium peruviense" specie, recently proposed inside the Pectobacterium genus, gathers strains isolated from potato tubers cultivated in Peru at high altitude. Here we report the draft genome sequence of two strains belonging to "P. peruviense" isolated from river water in France indicating that the geographic distribution of this specie is likely to be larger than previously anticipated. We compared these genomes with the one published from the "P. peruviense" specie type strain isolated in Peru

The AvrE superfamily: ancestral type III effectors involved in suppression of pathogen-associated molecular pattern-triggered immunity

International audienceThe AvrE superfamily of type III effectors (T3Es) is widespread among type III-dependent phytobacteria and plays a crucial role during bacterial pathogenesis. Members of the AvrE superfamily are vertically inherited core effectors, indicating an ancestral acquisition of these effectors in bacterial plant pathogens. AvrE-T3Es contribute significantly to virulence by suppressing pathogen-associated molecular pattern (PAMP)-triggered immunity. They inhibit salicylic acid-mediated plant defences, interfere with vesicular trafficking and promote bacterial growth in planta. AvrE-T3Es elicit cell death in both host and non-host plants independent of any known plant resistance protein, suggesting an original interaction with the plant immune system. Recent studies in yeast have indicated that they activate protein phosphatase 2A and inhibit serine palmitoyl transferase, the first enzyme of the sphingolipid biosynthesis pathway. In this review, we describe the current picture that has emerged from studies of the different members of this fascinating large family.</p

Pectobacterium aquaticum sp. nov., isolated from waterways

International audienceThis work aimed to establish the taxonomic status of six strains (A212-S19-A16T, A127-S21-F16, A105-S21-F16, A104-S21-F16, A101-S19-F16 and A35-S23-M15) isolated from three different waterways in 2015 and 2016 in south-east France. Amplification and sequencing of the gapA housekeeping gene clustered these six strains together inside the genus Pectobacterium outside of already described or proposed Pectobacterium species and supspecies. Phenotypic analysis, using GENIII Biolog plates performed with strains A212-S19-A16T, A105-S21-F16, A101-S19-F16 and the closely related Pectobacterium polaris (CFBP 1403), Pectobacterium carotovorum subsp. odoriferum (CFBP 1878T), ‘ Pectobacterium carotovorum subsp. actinidiae’ (CFBP 7370), Pectobacterium carotovorum subsp. carotovorum (CFBP 2046T), ‘ Pectobacterium carotovorum subsp. brasiliense ’ (CFBP 6617) or the most distantly related Pectobacterium aroidearum (CFBP 8168T) failed to identify specific compounds metabolized by these three strains, but weak activity was specifically observed at pH 5 with these three strains. Illumina sequencing was used to sequence these six strains. Based on phylogenetic data, average nucleotide identity values and in silico DNA–DNA hybridization results, strains A212-S19-A16T, A127-S21-F16, A105-S21-F16, A101-S19-F16, A35-S23-M15 and A104-S21-F16 are suggested to represent a novel species of the genus Pectobacterium , for which the name Pectobacterium aquaticum sp. nov. is proposed. The type strain is A212-S19-A16 T (=CFBP 8637T=NCPPB 4640T)

A survey of soft rot pectobacteriaceae along the anthropogenic gradient of the Durance river

International audienceBackground: Soft rot Pectobacteriaceae (SRP: Pectobacterium spp. and Dickeya spp.) infect a large number of plant species worldwide, including economically important plants. While the diversity of SRP observed on plants is fairly well described, the presence and diversity of SRP outside the plant context is not known in detail.Objectives: To anticipate disease emergence from environmental reservoirs and to propose regulatory guidelines and good practices for crop health management it is important to gain knowledge of SRP ecology outside of agronomic contexts.Methods: SRP sampling was performed all along the Durance river catchment in winter, spring, summer and autumn 2016 and 2017. This river catchment is interesting because it links alpine streams above the Serre-Ponçon lake to the Mediterranean agricultural basin of Avignon along an anthropogenic gradient. SRP sampling was coupled to detailed analysis of water physicochemical characteristics. Diversity of isolated SRP was further characterized through sequencing of one house-keeping gene.Conclusion: SRP distribution along the river stream was uneven, subjected to seasonal variation, land use and water physicochemical characteristics. This study further revealed a large previously unrecognized SRP diversity and the different ecological behaviours of Pectobacterium spp. and Dickeya spp. The potential virulence on various crops of the isolated SRP is currently under investigation