Auswahl der wissenschaftlichen Literatur zum Thema „Pathogen (Ralstonia solanacearum)“

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<br>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 is a genetically diverse and geographically widespread plant pathogen. It has a wide host range and is a significant pathogen of potato, causing brown rot. Brown rot is caused by a distinct, closely-related, intraspecific group: race 3, biovar 2A. In Europe, infection of potato crops with brown rot primarily occurs via irrigation with contaminated surface water. Brown rot has never been found in Scottish potatoes though the bacterium has been found previously in one Scottish river system, the River Tay, both in water samples and on its secondary host, bittersweet (Solanum dulcamara), growing on the river banks. A molecular strain-typing method principally used in clinical microbiology, multi-locus sequence typing (MLST), was used to study genetic variation within a collection of 106 R. solanacearum isolates, principally race 3 biovar 2A isolates from potato, S. dulcamara and contaminated water sources. Twenty-seven isolates from contaminated water and S. dulcamara from Scotland and other isolates from diverse geographic locations, from a variety of diseased plants and the environment, were resolved into 16 sequence types. A subsequent follow-up to the first experiment was carried out by looking at more variable genes within the race 3, biovar 2A genome and again similar or identical relationships were uncovered. All Scottish isolates were found to be identical and similar to most race 3 biovar 2A isolates tested. Analysis of Variable Number Tandem Repeats (VNTRs) confirmed this observation. After sequencing one of the tandem repeat regions, the results strongly suggest that contamination of the River Tay in Scotland occurred as a single or limited event, since the Scottish isolates had a unique tandem repeat pattern different from the patterns observed for the rest of the race 3 biovar 2A isolates and strains studied. This suggests that the Scottish isolates are clonal and the contamination is a single event and not a multiple contamination.

A murcha bacteriana, causada por Ralstonia solanacearum, afeta principalmente as solanáceas, destacando-se as culturas da batata, berinjela, jiló, pimentão e tomate. No presente trabalho foi conduzida a caracterização molecular de isolados de R. solanacearum e sua possível relação com características relacionadas à morfologia, bioquímica, patogenicidade, agressividade e distribuição geográfica. Foram utilizados 51 isolados pertencentes à biovar 2, sendo 9 provenientes de berinjela e 42 de batata, coletados em diversas regiões brasileiras. A análise molecular permitiu separar os isolados em quatro grupos distintos de padrões de bandas para os iniciadores BOX e ERIC, e em cinco para o iniciador REP. Não foi encontrada relação dos grupos de isolados caracterizados molecularmente com tamanho de colônias, ocorrência de mutantes, produção de melanina, capacidade de colonização do sistema radicular e resistência a antibióticos/fungicidas. A identificação de isolados de batata, como biovar 2-A, e de berinjela, como biovar 2-T, com base em teste bioquímico do uso de trealose, foi confirmadas pela análise molecular. Não houve variação de agressividade entre os isolados inoculados em batata e berinjela, exceção feita ao isolado avirulento CNPH-65. Portanto, isolados das biovares 2-A e 2-T podem infectar estas duas hospedeiras com a mesma intensidade sob altas temperaturas. Para todos os isolados, o desenvolvimento da população bacteriana foi significativamente maior no sistema radicular de plantas das cultivares suscetíveis, tanto para batata como para berinjela. No entanto, dentro de cada cultivar, os isolados se comportaram de maneira semelhante, não sendo possível fazer distinção entre os mesmos. A tentativa de se associar grupos de isolados caracterizados molecularmente com os locais de origem revelou alguns aspectos interessantes. O grupo I agregou somente isolados do Paraná. No grupo II ficaram isolados da Bahia, Distrito Federal e do Paraná. No Grupo III, foram reunidos todos os isolados de berinjela e um único de batata, sendo todos procedentes do Distrito Federal. O grupo IV, de forma semelhante ao grupo II, reuniu isolados de locais diversos como Paraná, Goiás, Rio Grande do Sul e Distrito Federal. Portanto, nos grupos I e III parece haver uma tendência de relação entre grupamento molecular e local de origem, enquanto que para os grupos II e IV, isolados de características genéticas similares são provenientes de locais distintos, apontando considerável diversidade genética do patógeno.<br>The bacterial wilt disease caused by Ralstonia solonacearum affects mainly the solanaceous species, specially potato, eggplant, peppers, tomato and brazilian gilo (Solanum gilo). This work reports the molecular characterization of R. solanacearum biovar 2 isolates and the possible relationship of this molecular data with other characteristics related to morphology, biochemistry, pathogenicity, aggressiveness and geographical distribution. Fifty-one biovar 2 isolates were studied, 9 isolated from eggplant and 42 from potato, all of them collected from different regions of Brazil. According to the molecular analysis, the isolates were clustered in four different groups, with distinct band patterns to the primers BOX and ERIC, and five groups to the primers REP. There was no relationship between the groups clustered through molecular analyses and phenotypic characteristics, such as colony size, presence of mutants, melanin presence, capability of root system colonization and antibiotic/fungicide resistance. The identification of potato isolates as the biovar 2-A, and the eggplant isolates as biovar 2-T, based on biochemical tests using trealose were confirmed with the molecular analyses. There was no variation of aggressiveness in the isolates inoculated on potato an eggplant, except the avirulent isolate CNPH-65. Consequently, isolates of biovars 2-A and 2-T are able to infect both hosts with the same aggressiveness under high temperatures. The population of all isolates developed in significant levels at the root system of susceptible cultivars of both hosts, potato and eggplant. However, considering each cultivar tested, there was no difference between isolates. Interesting results were observed when the isolates clustered based on molecular data were associated with the geographical region of their collection. The group I clustered only the isolates collected in Paraná. The group II clustered the isolates collected in Bahia, Federal District and some in Paraná. The group III clustered all isolates from eggplant and only one of potato, all of them collected in the Federal District. The group IV, as the group II, clustered isolates from different regions, like Paraná, Goiás, Rio Grande do Sul and Federal District. These results suggest a relationship between the isolates clustered through molecular analysis in the groups II and III and their geographical region of collection. The isolates clustered in the same way, with similar genetic background in the groups II and IV, were however collected in different regions, showing the great genetic variation of this pathogen.

Les lectines sont des protéines capables de reconnaître des glucides complexes de manière spécifique et réversible sur des surfaces des cellules et des membranes cellulaires. Ce travail de thèse est centré sur l’étude des interactions entre des lectines produites par les bactéries pathogèniques burkholderia cenocepacia et ralstonia solanacearum et leurs ligands carbohydrates. Différents outils, tels que la cristallographie, la résonance plasmonique de surface, la microcalorimétrie de titration… ont été utilisés pour caractériser deux lectines. La bactérie ralstonia solanacearum, pathogène de plantes, produit trois lectines solubles, RSI, RS-III et RS20L. Ces lectines ont été précédemment cristallisées mais la caractérisation de RS20L n’est pas complète. Au cours de cette thèse, la production de RS20L recombinante a été optimisée et son comportement physicochimique analysé par 2D SDS-PAGE / MALDI-MS et DSC. Cependant tous les essais pour déterminer la spécificité et l’affinité de cette lectine se sont révélés infructueux et de nouvelles stratégies devront être développées. La deuxième partie de ce travail est consacrée à une lectine de la bactérie opportuniste burkholderia cenocepacia, responsable d’une forte mortalité pour les patients qui souffrent de la mucoviscidose ou de granulomatose chronique. La lectine BC2L-C est une protéine de 28 kDa, composée de deux domaines qui ont été clonés séparément, produits dans E. Coli et caractérisés. Le domaine C-terminal montre des similarités de séquences et de structure avec la lectine PA-IIL de Pseudomonas aeruginosa et a une forte affinité pour les oligosaccharides mannosylés. Le domaine N-terminal a aussi la faculté de lier des glucides avec une spécificité forte pour les oligosaccharides fucosylés tels que déterminants de groupe sanguin de type H ou Lewis. Le domaine N-terminal en complexe avec le selenio-methyl-fucoside cristallise dans une forme trimérique qui n’a pas été encore observée chez les lectines mais qui est très similaire au TNF-α ou au composant C1q du complément. La lectine BC2L-C représente donc une nouvelle superlectine avec deux domaines et leur spécificité<br>Lectins are carbohydrate-binding proteins of non-immune origin that bind specifically to complex carbohydrates. The presented Ph. D. Thesis is approaching the study of molecular mechanisms of interactions between lectins produced by pathogenic bacteria Burkholderia cenocepacia and Ralstonia solanacearum and their carbohydrate ligands. Cristallography, surface plasmon resonance, titration microcalorimetry and other tools were used for the characterisation of two lectins. The plant bacterial pathogen Ralstonia solanacearum produces three soluble lectins RSL, RS-IIL and RS20L. All of them have been previously studied but the characterisation of RS20L was not complete. During this thesis, the production of the RS20L lectin was optimised and its physicochemical behaviour analysed by 2D SDS-PAGE / MALDI-MS analysis and DSC. However, different assays for determining carbohydrate specificity and affinity were not successful and different strategies have to be designed. The second part of the thesis is devoted to a lectin from the human opportunistic pathogen Burkholderia cenocepacia, responsible of high mortality in patients with cystic fibrosis or chronic granulomatous diseases. The BC2L-C lectin is a 28 kDa protein composed of two distinct domains that were separately cloned and produced in E. Coli and characterised. The C-terminal domain shows sequence and structure similarity to the Pseudomonas aeruginosa lectin (PA-IIL) and recognises with high affinity D-mannosylated glycans. The N-terminal domain also displays sugar-binding ability with a strong preference for L-fucosylated oligosaccharides such as H-type and Lewis histo-blood group determinants. The N-terminal domain complexed with selenio-methyl-fucoside crystallises as a trimeric assembly that has not been observed for lectins earlier but that is highly similar to the TNF- α or C1q complement structures. The BC2L-C lectin is therefore a new superlectin with two different carbohydrate-biding domains and specificities

Several recent studies have shown that bacteria which are non pathogenic to in vivo plants become pathogenic to in vitro plants. Bacteria can occur in the internal tissue of in vivo plants, often remaining in a latent state and be retained within the explant during the process of in vitro culture. The reasons why non pathogenic bacteria often become pathogenic to plants under in vitro conditions are unknown. In this study plant~bacterial complexes were established to measure the interactions between plant and bacteria in axenic plants grown under in vitro and photoautotrophic conditions. The plant~bacterial complexes were developed using Solanum tuberosum cv. Atlantic with a plant pathogen Pseudomonas solanacearum,(virulent and avirulent forms), and a plant saprophyte Pseudomonas fluorescens.These complexes were used to study interactions that would correlate with known plant defence mechanisms. Interactions of the plant~bacterial complexes were investigated by determining the following aspects: the activity of the enzyme peroxidase; the number of colony forming units mL-1 in infected tissue; attachment and envelopment of bacteria by the plant cell wall using transmission electron microscopy; attachment of bacteria to external surfaces of roots using scanning electron microscopy; the anatomical changes in infected roots and stems using light microscopy; and points of entry of bacterial cells into the root system using light microscopy and scanning electron microscopy.Peroxidase activity in uninfected plants grown under in vitro conditions was significantly higher than in plants grown in photoautotrophic axenic conditions. The factors of low light and sucrose were shown to increase the peroxidase levels in the stem of uninfected plants.

Ralstonia solanacearum, agent du flétrissement bactérien, affecte près de 200 espèces végétales. Les gènes RRS1-R confèrent à l'écotype d'A. Thaliana Nd-1 une résistance à différentes souches de R. Solanacearum. RRS1-R code une protéine de structure modulaire associant les domaines typiques de nombreuses protéines de résistance TIR-NBS-LRR et un domaine signature de facteurs de transcription WRKY. Dans l'écotype sensible Col-0, le gène RRS1-S code pour une protéine qui présente une structure très semblable. Au cours de ce travail, nous avons montré que les gènes RRS1-R et RRS1-S s'expriment essentiellement dans les cellules du péricycle et les cellules de passage de l'endoderme des racines matures et de la base de l'hypocotyle, cellules qui correspondent aux sites de pénétration des bactéries dans le système vasculaire où elles se multiplient. Nous avons montré que les deux domaines WRKY des protéines codées par ces gènes se fixent spécifiquement aux boites W, reconnues par les facteurs de transcription de la famille WRKY. Nous avons par la suite développé une approche DamID (DNA adenine methyltransferase IDentification) visant à identifier les gènes cibles des protéines RRS1-R et RRS1-S in vivo. L'analyse a été focalisée sur l'identification des gènes cibles de RRS1-R, dans le fond génétique résistant Nd-1 exprimant, ou pas, la protéine d'avirulence PopP2 sous contrôle d'un promoteur inductible. Dans chacun des cas le séquençage d'une centaine de FARMs (Fragments Associated to RRS1-driven Methylation) a permis de proposer des cibles potentielles et un modèle de fonctionnement de RRS1-R comme régulateur transcriptionel. Ce travail se poursuit par une analyse globale au niveau du génome, grâce au séquençage haut débit des FARMS et par l'étude de la fonction dans la réponse de la plante et de la régulation transcriptionelle de quelques cibles d'intérêt. Les résultats de ce travail illustrent dans leur ensemble l'importance de RRS1-R pour réguler la réponse des plantes à R. Solanacearum<br>In nature, plants are constantly exposed to microbial pathogens and have evolved an effective and dynamic immune system in order to survive. R. Solanacearum, the causing agent of wilt disease, is a soil-borne bacteria pathogenic on more than 200 plant species. Bacteria enter roots, invade xylem vessels and then spread rapidly to aerial parts of the plant through the vasculature. In A. Thaliana Nd-1 plants, RRS1-R, with its partner RPS4 allows resistance to strains of R. Solanacearum that deliver PopP2, a type III effector, into the plant cells. Previous studies showed that RRS1 and RPS4 are two NBS-LRR receptor proteins involved in the perception of the effector. Interestingly, RRS1 also harbors a WRKY transcription factor domain in its C-terminal end. In a susceptible Arabidopsis ecotype Col 0, RRS1-S is an allelic gene of RRS1-R, which encodes a similar structure. The recognition of bacterial and plant proteins leads to RRS1 protein accumulation in the nucleus, triggering possibly transcriptional gene regulation. Important genomic reprogramming of the infected plant cells has indeed been shown. My work shows that the RRS1-S and RRS1-R genes are expressed mainly in mature roots and basal hypocotyls, in pericycle cells and passage cells from the endoderm. These cells correspond to entry sites of the invading R. Solanacearum bacteria within the vascular tissues. We also demonstrated the binding of WRKY domain of RRS1-R and RRS1-S, in vitro, to W boxes which are cis-regulatory elements recognized by WRKY transcription factors. In order to identify the in vivo target sequences of RRS1-R and RRS1-S, a DamID (DNA adenine methyltransferase IDentification) approach, detecting transitory DNA-protein associations was developed. DamID is based on the fusion of a protein of interest to a DNA Adenine Methyl-transferase from E. Coli, which will methylate DNA in the vicinity of the binding sites of this protein. The fingerprints can be further mapped by DNA restriction with methylation sensitive enzymes, and cloned or directly sequenced. Analysis was focused on RRS1-R, by cloning FARMs (Fragment Associated to RRS1 driven Methylation) from Nd-1 plants expressing or not an inducible PopP2 gene. This allowed the identification of several putative targets of RRS1-R and led us to propose a model for its function as a transcription factor. High throughput sequencing was then initiated at a whole genome scale analysis. The function and transcriptional regulation of a putative RRS1 target gene was evaluated. Taken together, the results of this study illustrate the important role of RRS1-R in the regulation of the plant response to R. Solanacearum

Ralstonia solanacearum a dans son répertoire d’effecteurs de type III une famille de gènes codant pour des protéines nommées GALA. Ces protéines possèdent une structure caractéristique des protéines à F-box eucaryotes qui entrent dans la composition des complexes SCF impliqués dans l’ubiquitination des protéines. Nous avons démontré l’action collective des protéines GALA dans la virulence de la bactérie. Nous avons identifié au sein de cette famille le premier effecteur contrôlant la virulence de la bactérie sur une plante hôte. Il est apparu que les effecteurs GALA se comportent comme des protéines à F-box de plante et pourraient ainsi contrôler l’ubiquitination de protéines cibles dans la cellule végétale. Une cible candidate a été identifiée. Nous avons mis en évidence une protéine végétale susceptible d’être impliquée dans une voie de signalisation contrôlant la mise en place du Flétrissement bactérien. Nous proposons une hypothèse sur l’activité moléculaire des effecteurs GALA<br>Ralstonia solanacearum type III effector candidate repertory contains a gene family coding for proteins designated GALA. The GALA proteins possess a protein structure characteristic of eukaryotic F-box proteins. F-box proteins are subunits of SCF complexes involved in protein ubiquitination; a process controlling eukaryotic cellular homeostasis. In the course of this work, we demonstrated that GALA proteins are genuine effectors, and that they collectively play a role in R. Solanacearum virulence. Within the family, we identified the first effector from this bacteria controlling virulence on a specific host. Our studies revealed that GALA effectors behave as plant F-box proteins and that they could mediate ubiquitination of target proteins in the host cell. A target has been identified. We identified for the first time a plant protein potentially involved in a signaling pathway controlling Bacterial wilt establishment. We propose a model for the molecular activity of GALA effectors