Study of the role of the fungal secondary metabolites in plant-pathogen interactions

Les métabolites secondaires sont de petites molécules produites par les plantes et les micro-organismes. Pour la grande majorité d'entre eux, leurs fonctions sont inconnues, mais ils jouent probablement un rôle clé dans l'adaptation des organismes à leur environnement. Chez les champignons phytopathogènes, plusieurs clusters de gènes de biosynthèse (BGC) de métabolites secondaires sont induits spécifiquement pendant l'infection des plantes. Ces métabolites peuvent être des toxines qui aident à l'infection des plantes ou peuvent agir comme des effecteurs pour supprimer l'immunité des plantes. L'objectif de cette thèse est d'étudier le rôle des métabolites fongiques dans les interactions entre les plantes et les agents pathogènes en utilisant deux modèles fongiques : Botrytis cinerea, un champignon nécrotrophe responsable de la pourriture grise, et Colletotrichum higginsianum, l'agent hémibiotrophe causant l'anthracnose des Brassicacées. Le principal obstacle est l'impossibilité d'isoler les métabolites secondaires fongiques produits lors de l'infection de la plante à partir de cultures axéniques. Pour lever ce verrou une approche novatrice, basée sur l'expression hétérologue de BGC chez la levure, a été développée. Cette méthode consiste à introduire dans la levure tous les gènes d'un BGC codant pour les enzymes requises pour synthétiser les métabolites d'intérêt. Dans ce système, tous les gènes du BGC sont mis sous le contrôle d'un même promoteur au sein d'un plasmide polycistronique. Le plasmide est ensuite introduit dans une levure optimisée pour la synthèse de métabolites. Pour valider cette approche, le BGC 16 de C. higginsianum, censé produire les molécules de la famille des colletochlorins, a été exprimé dans le système hétérologue. Les levures exprimant le BGC 16 ont été cultivées en milieu liquide et les métabolites ont été extraits du filtrat et du culot cellulaire et analysés par HPLC-MS, révélant la production de colletochlorins. Ce résultat permet de lier le BGC 16 et la synthèse de colletochlorins et donne de nouveaux éléments sur la voie de biosynthèse de ces molécules. Cet outil permettra d'exprimer des BGC dont le produit est inconnu et facilitera la découverte de nouvelles molécules, de leurs activités biologiques et de leur rôle écologique. Dans une deuxième partie, le rôle du BGC contenant la polykétide synthase BcPKS8 de B. cinerea dans l'infection a été exploré par une approche fonctionnelle. Ce BGC est plus spécifiquement exprimé pendant la phase précoce d'infection des baies de raisin et des tomates. Il est conservé dans toutes les espèces de Botrytis séquencées à ce jour, ainsi que dans Sclerotinia sclerotiorum et dans plusieurs espèces d'ascomycètes interagissant avec les plantes. Néanmoins, le polykétide produit par ce BGC est inconnu. Son rôle dans le processus d'infection a été testé à l'aide de la génétique inverse. Des tests d'infection de mutants nuls sur des baies de tomate ont montré que le polykétide produit est nécessaire pour une virulence complète. Ce mutant possède aussi une sensibilité accrue au stress osmotique indiquant un rôle potentiel du BGC Bcpks8 dans la résistance à ce stress. Pour évaluer la chronologie de l'expression de Bcpks8 avec exactitude, une souche rapportrice a été créée en fusionnant un gène codant une protéine fluorescente au promoteur de BcPKS8. Cette lignée rapportrice révèle une induction de Bcpks8 dans les premiers stades de la formation des coussins d'infection, une structure du champignon formée d'appressoria multiple impliqué dans la pénétration de la plante. L'expression hétérologue de l'ensemble du cluster a aussi été initiée pour déterminer la structure du polykétide. Les travaux de cette thèse apportent des outils qui devraient grandement faciliter l'étude des métabolites secondaires de champignons phytopathogènes. Ils soulignent également le rôle important de ces métabolites dans l'infection à travers l'exemple concret du BGC Bcpks8 de B. cinerea.Secondary metabolites are small molecules produced by plants and micro-organisms such as bacteria and fungi. For the vast majority of these metabolites, their functions are unknown, but they probably play key roles in adaptation of the organism to its environment. In plant pathogenic fungi, a large proportion of biosynthetic gene clusters (BGC) responsible for producing secondary metabolites are induced specifically during plant infection. These metabolites can be toxins that help plant infection (especially in necrotrophic fungi) or may act as effectors to suppress plant immunity (in particular for biotrophic and hemibiotrophic fungi). The aim of this PhD was to study the role of fungal metabolites in plant-pathogen interactions using two fungal models: Botrytis cinerea, a necrotrophic fungus that causes the grey mould disease, and Colletotrichum higginsianum, the hemibiotrophic agent of anthracnose disease of Brassicaceae. The main bottleneck is to produce and isolate fungal secondary metabolites from axenic cultures. To overcome this problem, an innovative approach was used based on the heterologous expression of BGCs in yeast. This method consists of introducing into yeast all the genes of a cluster encoding the enzymes required to synthesize the metabolites of interest. In this system, each gene is put under the control of a single promotor in a polycistronic plasmid. The plasmid is then introduced and expressed in an engineered yeast adapted for polyketide or terpene synthesis, depending on the BGC key gene. To validate this approach, the C. higginsianum BGC16, predicted to produce the colletochlorin family of molecules, was expressed in the heterologous system. Yeast expressing BGC16 were cultivated in liquid medium, and metabolites extracted from the supernatant and cell pellet were analyzed by HPLC-MS, revealing that the colletochlorin metabolites were successfully produced. The findings validate that the enzymes encoded by BGC16 are functional in yeast and are responsable for colletochlorin synthesis, and also give new insights into the biosynthetic pathway. In the future, application of this tool to BGCs for which the product is unknown will facilitate the discovery of new molecules and biological activities, providing a better understanding of their role in interactions with the plant host. In a second part, the role of a BGC containing the polyketide synthase BcPKS8 from B. cinerea was explored using a functional approach. This BGC is specifically expressed during the early stage of infection of grape berries and tomatoes. It is conserved in all Botrytis species sequenced to date and in Sclerotinia sclerotiorum, as well as numerous other plant-interacting ascomycete fungi. However, the polyketide produced by this BGC is unknown. Its role in the infection process was tested using reverse genetics. Infection assays of null mutants on tomato berries showed the polyketide product is required for full virulence. This mutant was also more sensitive to osmotic stress than the wild-type, suggesting a potential role of the BGC in resistance to this stress. To determine the exact chronology of expression of BcPKS8 during host infection, a reporter strain was made by fusing a gene encoding a fluorescent protein to the promoter of BcPKS8. The reporter gene was specifically induced during the early formation of infection cushions, a multiple appressorium-like structure involved in host penetration. The heterologous expression of the whole gene cluster in yeast has been initiated to determine the structure of the polyketide.This PhD provides tools that should greatly facilitate the study of secondary metabolites of phytopathogenic fungi. It also highlights their important role in plant infection through the concrete example of BGC Bcpks8 of B. cinerea

A gene‐for‐gene interaction involving a ‘late’ effector contributes to quantitative resistance to the stem canker disease in Brassica napus

International audienceThe control of stem canker disease of Brassica napus (rapeseed), caused by the fungus Leptosphaeria maculans is largely based on plant genetic resistance: single-gene specific resistance (Rlm genes) or quantitative, polygenic, adult-stage resistance. Our working hypothesis was that quantitative resistance partly obeys the gene-for-gene model, with resistance genes "recognizing" fungal effectors expressed during late systemic colonization. Five LmSTEE (stem-expressed effector) genes were selected and placed under the control of the AvrLm4-7 promoter, an effector gene highly expressed at the cotyledon stage of infection, for miniaturized cotyledon inoculation test screening of a gene pool of 204 rapeseed genotypes. We identified a rapeseed genotype, 'Yudal', expressing hypersensitive response to LmSTEE98. The LmSTEE98-RlmSTEE98 interaction was further validated by inactivation of the LmSTEE98 gene with a CRISPR-Cas9 approach. Isolates with mutated versions of LmSTEE98 induced more severe stem symptoms than the wild-type isolate in 'Yudal'. This single-gene resistance was mapped in a 0.6 cM interval of the 'Darmor_bzh' x 'Yudal' genetic map. One typical gene-for-gene interaction contributes partly to quantitative resistance when L. maculans colonizes the stems of rapeseed. With numerous other effectors specific to stem colonization, our study provides a new route for resistance gene discovery, elucidation of quantitative resistance mechanisms, and selection for durable resistance.This article is protected by copyright. All rights reserved

Exploitation of the Leptosphaeria maculans late effector repertoire for diversification of resistances to blackleg in Brassica napus

International audienceLeptosphaeria maculans is a phytopathogenic fungus being responsible for a damaging disease of oilseed rape (Brassica napus): stem canker. The disease is mainly controlled by plant genetic resistance: single-gene specific resistance or quantitative, adult-stage resistance. During its particularly complex and long infectious cycle, L. maculans colonizes asymptomatically the stems of oilseed rape, producing late effectors specific to this colonization stage. In the context of a strong need to identify new sources of disease resistance, we exploited the repertoire of ‘late’ effectors to identify genes in the plant that could contribute to quantitative disease resistance. Our hypothesis was that quantitative resistance partly rely on gene-for-gene interactions, with fungal effectors produced during stem infection being recognized by resistance proteins. Using an innovative strategy of early expression of late effector genes, we validated that the interaction between the late effector LmSTEE98 and the resistance RlmSTEE98 obeys a typical gene-for-gene interaction, occurring during the colonization of oilseed rape stems by L. maculans, that contributes partly to quantitative resistance, in controlled conditions. We then used the same strategy to search for new sources of resistance after having established criteria to select the most relevant late effectors, and chosen ten of these for screening. Our screening approach of 130 diversified genotypes representative of the available diversity of B. napus, allowed us to identify new sources of resistance, displaying diversified interaction phenotypes. The next steps of this project now are further validation of the efficacy of the new sources of resistance in the field and of the validity of the quantitative resistance markers. However, as it stands, our results demonstrate the existence of unsuspected sources of resistance that are potentially more durable than the classic major genes expressed early after penetration in plant tissues