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During infection, plant pathogens secrete an arsenal of molecules collectively known as effectors that circumvent plant innate immunity and trigger infection. The phytopathogenic fungus Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence genes. Fungal effectors mainly correspond to small secreted proteins (SSP) with no known homologs and no predicted functions. Their biological function is therefore difficult to predict, and very little is known about the mode of action of L. maculans effectors during infection.The objective of my thesis was to elucidate the role of L. maculans effectors during infection through their functional characterization which included: i) the determination of their subcellular localization in Nicotiana benthamiana et Arabidopsis thaliana; ii) a search for their plant targets; and iii) the determination of the cellular processes targeted by those effectors through their stable expression in A. thaliana and by testing their ability to suppress cell-death in N. benthamiana. We investigated four effectors in that study: AvrLm10-1, AvrLm10-2, AvrLm4-7 and AvrLm3.AvrLm10-1 and AvrLm10-2 are both necessary to trigger recognition by the Rlm10 resistance gene. We have identified orthologs for AvrLm10-1 and AvrLm10-2 in Dothideomycetes and Sordariomycetes phytopathogens, and several paralogs in L. maculans which are expressed specifically during oilseed rape infection. AvrLm10-1 and AvrLm10-2 both have a nucleo-cytoplasmic localization. AvrLm10-1 and AvrLm10-2 physically interact, and may also interact with a PR1 (Pathogenesis-related 1) protein and a secreted cysteine-protease. AvrLm4-7 is recognized by two resistance genes, Rlm4 and Rlm7, and suppresses recognition of AvrLm3 by Rlm3. AvrLm4-7 suppresses cell-death triggered by general inducers, PAMP-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). AvrLm4-7 has a nucleo-cytoplasmic localization, whether expressed with or without its signal peptide, suggesting an intracellular mode of action. One of the potential plant targets for AvrLm4-7 is a ribosomal protein, just like a Blumeria graminis effector with structural analogy to AvrLm4-7. Transgenic lines of A. thaliana constitutively expressing AvrLm4-7 do not show any morphological phenotypes or any difference in their susceptibility to diverse fungal pathogens. AvrLm3 is an avirulence gene strongly conserved in L. maculans populations. Recognition of AvrLm3 by Rlm3 is suppressed by the presence of AvrLm4-7. AvrLm3 suppresses cell-death triggered by several inducers of PTI and ETI. AvrLm3 is localized in plant apoplasm when expressed with its signal peptide, suggesting an extracellular localization. AvrLm3 potentially interacts with a secreted myrosinase-associated protein implicated in the myrosinase-glucosinolate system, suggesting that AvrLm3 could disturb glucosinolate production, which is a novel mode of action never described for a plant pathogen effector.My thesis allowed us to improve our knowledge on fungal effector function during infection and to propose new strategies for plant diseases management; Pendant l’infection, les agents phytopathogènes sécrètent un arsenal de molécules, appelées effecteurs, éléments clés de la pathogénie qui modulent l’immunité innée de la plante et facilitent l’infection. Leptosphaeria maculans est le champignon responsable de la nécrose du collet du colza. Plus de 650 gènes codant des effecteurs potentiels ont été identifiés dans son génome, dont 7 ont un rôle reconnu dans l’avirulence du champignon. Les effecteurs fongiques correspondent principalement à de petites protéines potentiellement sécrétées (PPS), n’ayant pas d’homologues dans les bases de données et pas de motifs connus. Par conséquent, leur fonction biologique est difficile à prédire, et très peu de choses sont connues sur le mode d’action des effecteurs de L. maculans au cours de l’infection.L’objectif de ma thèse était de caractériser fonctionnellement des effecteurs de L. maculans afin de mieux comprendre leur rôle au cours du processus infectieux. Cette caractérisation fonctionnelle a consisté en : i) la détermination de la localisation subcellulaire de ces effecteurs dans Nicotiana benthamiana et Arabidopsis thaliana ; ii) la recherche de cibles végétales ciblées par ces effecteurs ; et iii) la détermination des processus cellulaires impactés par ces effecteurs par expression stable dans A. thaliana et tests de suppression de mort cellulaire dans N. benthamiana. Quatre effecteurs ont été choisis pour cette étude : AvrLm10-1, AvrLm10-2, AvrLm4-7 et AvrLm3.AvrLm10-1 et AvrLm10-2 sont tous les deux nécessaires pour induire une reconnaissance par le gène de résistance Rlm10. Des orthologues d’AvrLm10-1 et AvrLm10-2 ont été identifiés chez des Dothidéomycètes et des Sordariomycètes phytopathogènes ainsi que plusieurs paralogues exprimés spécifiquement pendant l’infection chez L. maculans. AvrLm10-1 et AvrLm10-2 présentent toutes les deux une localisation nucléo-cytoplasmique. Une interaction physique entre AvrLm10-1 et AvrLm10-2 a été mise en évidence, ainsi qu’une interaction potentielle de ces deux protéines avec une protéine PR1 (Pathogenesis-related 1) et une cystéine-protéase végétale.AvrLm4-7 est reconnu par deux gènes de résistance, Rlm4 et Rlm7, et sa présence empêche la reconnaissance d’AvrLm3 par Rlm3. AvrLm4-7 est capable de supprimer la mort cellulaire provoquée aussi bien par des inducteurs généraux de la mort cellulaire que par des inducteurs de la PAMP-Triggered Immunity (PTI) et de l’Effector-Triggered Immunity (ETI). AvrLm4-7 présente une localisation nucléo-cytoplasmique, qu’il soit exprimé avec ou sans son peptide signal, ce qui suggère un mode d’action intracellulaire. AvrLm4-7 interagit potentiellement avec une protéine ribosomale végétale, de la même manière qu’un effecteur de Blumeria graminis avec lequel il partage des analogies structurales. Cependant, des lignées d’A. thaliana exprimant AvrLm4-7 de façon constitutive ne présentent aucune différence morphologique ou de sensibilité aux maladies comparativement à l’écotype sauvage Col0.AvrLm3 est un gène d’avirulence très conservé dans les populations de L. maculans dont la reconnaissance par le gène de résistance Rlm3 est supprimée en présence d’AvrLm4-7. AvrLm3 est capable de supprimer la mort cellulaire associée à la PTI et à l’ETI. Cet effecteur est localisé dans l’apoplasme des cellules foliaires lorsqu’il est exprimé avec son peptide-signal, suggérant un mode d’action extracellulaire. AvrLm3 interagit potentiellement avec une myrosinase-associated proteine sécrétée impliquée dans le système myrosinase-glucosinolate, suggérant qu’AvrLm3 perturberait la synthèse des glucosinolates, ce qui est un mode d’action inédit pour un effecteur d’agent phytopathogène.Cette thèse a permis de mieux comprendre le mode d’action des effecteurs de L. maculans et de proposer de nouvelles stratégies de contrôle des maladies fongiques.

During plant infection, pathogens secrete an arsenal of effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Fungal effector genes typically encode small proteins, predicted to be secreted, with no homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. The phytopathogenic ascomycete Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence proteins. We develop a project aiming at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. Our strategy is illustrated here with AvrLm4-7, a 143 amino-acid long secreted protein important for fungal fitness and recognized by two oilseed rape resistance proteins, Rlm4 and Rlm7. One single amino-acid change is sufficient to lose recognition by Rlm4 while maintaining recognition by Rlm7. 3D-structure of an isoform of AvrLm4-7 only recognized by Rlm7 was previously determined, allowing us to define regions implicated in recognition by Rlm7 and translocation into plant cell. We recently determined the 3D-structure of another isoform of AvrLm4-7 recognized both by Rlm4 and Rlm7, showing that the amino-acid change allowing to escape Rlm4-recognition was located on an external loop and did not change the overall structure of the protein. AvrLm4-7 was also recently shown to suppress recognition of another L. maculans avirulence gene, AvrLm3, by its cognate resistance gene Rlm3, leading us to hypothesize a suppression of Effector-Triggered Immunity (ETI) by AvrLm4-7. In order to test that hypothesis, we transiently expressed AvrLm4-7 and several cell-death inducers in Nicotiana benthamiana epidermal cells: AvrLm4-7 was able to suppress cell death induced by BAX and AvrPto. We also generated transgenic lines of Arabidopsis thaliana constitutively expressing AvrLm4-7 and are currently characterizing the lines for their susceptibility to pathogens with contrasted lifestyles and for their ability to suppress recognition of Pseudomonas syringae avirulence proteins. A better understanding of the role of an effector implicated in the masking of another effector will allow us to develop alternative strategies to genetically control stem canker disease.

International audience; Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted, with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. On these bases, the StructuraLEP project aims at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. We are investigating six L. maculans effectors chosen for their biological significance (involvement in fungal fitness, cognate R gene identified) or because they may represent novel modes of interaction with their plant target (two AVR genes have to be recognized by a specific R gene). We present here the strategies developed within the project to answer two questions: (i) “Where do L. maculans effectors act during plant infection?” and (ii) “Which proteins interact with L. maculans effectors?”. In order to localise L. maculans effectors into plant cells and to identify their plant targets, we will transiently express effectors with a fluorescent tag into tobacco leaf epidermal cells and observe effector localisation by confocal microscopy. Tobacco leaves expressing effectors will be used to perform pull-down assays and tobacco proteins interacting with effectors will be identified through mass spectrometry. A more exploratory but biologically relevant strategy will also be tested. L. maculans transformants stably expressing effectors with HA-tag will be used to infect oilseed rape leaves. Localisation of effectors in oilseed rape leaves will be performed by immunocytolocalisation using antibodies against the HA-tag. The infected oilseed rape leaves will also be used to perform pull-down assays in order to identify plant proteins targeted by L. maculans effectors.

National audience; Fungal effector genes are very diverse and typically encode small proteins, predicted to be secreted, with no or low homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. On these bases, the StructuraLEP project aims at elucidating the involvement of L. maculans effectors in pathogenicity through the structural and functional characterization of a few major effector proteins and the determination of their interactants. We are investigating six L. maculans effectors chosen for their biological significance (involvement in fungal fitness, cognate R gene identified) or because they may represent novel modes of interaction with their plant target (two AVR genes have to be recognized by a specific R gene). We present here the strategies developed within the project to answer two questions: (i) “Where do L. maculans effectors act during plant infection?” and (ii) “Which proteins interact with L. maculans effectors?”. In order to localise L. maculans effectors into plant cells and to identify their plant targets, we will transiently express effectors with a fluorescent tag into tobacco leaf epidermal cells and observe effector localisation by confocal microscopy. Tobacco leaves expressing effectors will be used to perform pull-down assays and tobacco proteins interacting with effectors will be identified through mass spectrometry. A more exploratory but biologically relevant strategy will also be tested. L. maculans transformants stably expressing effectors with HA-tag will be used to infect oilseed rape leaves. Localisation of effectors in oilseed rape leaves will be performed by immunocytolocalisation using antibodies against the HA-tag. The infected oilseed rape leaves will also be used to perform pull-down assays in order to identify plant proteins targeted by L. maculans effectors.

International audience; Leptosphaeria maculans is a hemibiotophic ascomycete which causes stem canker of oilseed rape. That phytopathogenic fungus interacts with its host (Brassica napus) according to the gene-for-gene concept. The most economically and environment friendly method of control of stem canker is the genetic control by using host resistance. Single gene resistance is extremely efficient, but races of the pathogen virulent towards a resistance gene can appear in a few years and necessitates continuously new breeding programs. Moreover, specific resistances are rare in oilseed rape, and a lot of efforts are made to find other resistance genes in other Brassica species. To date, 11 interactions were genetically characterized between L. maculans avirulence genes and corresponding resistance genes in Brassica, and 5 of those avirulence genes were cloned. Recently, the avirulence gene AvrLm10 which is recognized by the resistance gene Rlm10 of the black mustard (Brassica nigra) has been cloned. AvrLm10 corresponds in fact to two avirulence genes AvrLm10_1 and AvrLm10_2 which are located in the same AT-rich genomic region. They encore for small secreted proteins (SSP), are co-regulated and over-expressed 7 days post-infection. Each of them is necessary but not sufficient to induce resistance towards Rlm10. Silencing of one of those genes is sufficient to abolish recognition by Rlm10. Silencing by RNA interference of AvrLm10-1 induces an increase of lesion size on oilseed rape leaves while silencing of AvrLm10-2 has no major effect on aggressiveness of the fungus. That interaction of two avirulence genes against one resistance gene is therefore different from the classical gene-for-gene concept. It suggests that AvrLm10_1 and AvrLm10_2 could directly interact and / or that they could target the same plant protein. A Y2H screen suggested a direct interaction between AvrLm10-1 and AvrLm10-2. This interaction was confirmed with Bimolecular Fluorescence Complementation (BiFC) experiments. Coimmunoprecipitation experiments are also in progress to confirm this interaction.

National audience

National audience; Leptosphaeria maculans is a hemibiotophic ascomycete which causes stem canker of oilseed rape. That phytopathogenic fungus interacts with its host (Brassica napus) according to the gene-for-gene concept. The most economically and environment friendly method of control of stem canker is the genetic control by using host resistance. Single gene resistance is extremely efficient, but races of the pathogen virulent towards a resistance gene can appear in a few years and necessitates continuously new breeding programs. Moreover, specific resistances are rare in oilseed rape, and a lot of efforts are made to find other resistance genes in other Brassica species. To date, 11 interactions were genetically characterized between L. maculans avirulence genes and corresponding resistance genes in Brassica, and 5 of those avirulence genes were cloned. Recently, the avirulence gene AvrLm10 which is recognized by the resistance gene Rlm10 of the black mustard (Brassica nigra) has been cloned. AvrLm10 corresponds in fact to two avirulence genes AvrLm10_1 and AvrLm10_2 which are located in the same AT-rich genomic region. They encore for small secreted proteins (SSP), are coregulated and over-expressed 7 days post-infection. Each of them is necessary but not sufficient to induce resistance towards Rlm10. Silencing of one of those genes is sufficient to abolish recognition by Rlm10. Silencing by RNA interference of AvrLm10-1 induces an increase of lesion size on oilseed rape leaves while silencing of AvrLm10-2 has no major effect on aggressiveness of the fungus. That interaction of two avirulence genes against one resistance gene is therefore different from the classical gene-for-gene concept. It suggests that AvrLm10_1 and AvrLm10_2 could directly interact and / or that they could target the same plant protein. A Y2H screen suggested a direct interaction between AvrLm10-1 and AvrLm10-2. This interaction was confirmed with Bimolecular Fluorescence Complementation (BiFC) experiments. Coimmunoprecipitation experiments are also in progress to confirm this interaction.