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BIOlogie et GEstion des Risques en agriculture - Champignons Pathogènes des Plantes

Bourse de recherches Jean & Marie-Louise DUFRENOY de l’Académie d’Agriculture de France

Bourse Dufresnoy
Two PhD of Bioger receive funding from the French Academy of Agriculture and the Genetic Society of America to attend the 2017 Fungal Genetics Conference in Asilomar, California.

Two PhD of Bioger, Julie Gervais, working on dissection of waves of fungal effector expression in late stages of tissue colonisation,and Yohann Petit, working on functional analysis of fungal effectors including identification of their plant targets, received a ‘Jean & Marie-Louise DUFRENOY’ grant from the French Academy of Agriculture and funding from the GSA to attend the 29th Fungal Genetics Conference to be held on March 14-19, 2017, in Asilomar, California.
Julie will present a communication entitled "Leptosphaeria maculans effectors involved in the oilseed rape systemic colonization". Yohann will present a communication entitled "Structural and functional characterization of Leptosphaeria maculans effectors: the example of AvrLm4-7".

Abstracts of the communications :

Leptosphaeria maculans effectors involved in the oilseed rape systemic colonization

Gervais Julie1, Plissonneau Clémence1, Linglin Juliette1, Meyer Michel1, Labadie Karine2, Cruaud Corinne2, Ollivier Bénédicte1, Fudal Isabelle1, Rouxel Thierry1, Balesdent Marie-Hélène1
1INRA, UMR 1290 BIOGER INRA-AgroParisTech, 78850 Thiverval-Grignon, France.
2CEA-Institut de Génomique, GENOSCOPE, Centre National de Séquençage, Evry Cedex, France

The stem canker disease, caused by Leptosphaeria maculans, is one of the most devastating diseases of oilseed rape (canola). It colonizes the plant in two stages: a short and early colonisation stage corresponding to cotyledon or leaf colonisation, and a late colonisation stage during which the fungus colonises systemically and symptomlessly the plant during several months before stem canker appears. To date, determinants of the late colonisation stage remain poorly understood.
By a transcriptomic approach, we previously identified two waves of effector candidate expression during the early and late colonisation stages (Gervais et al, 2016). The late effector candidates are located in gene-rich genomic regions, whereas the early effector genes are located in gene-poor regions of the genome.
Among the late effector candidates identified, we selected 6 genes for further characterization. We created mutants silenced for these effector candidates. For one of these genes, its expression level correlated negatively with the size of the necrosis observed in the stem.
The identification of new effector genes would contribute to the identification of new resistance genes specific to these effectors. To easily identify matching resistance genes in oilseed rape, we created transgenic isolates expressing these 6 late effectors at the early steps of infection to provide medium-throughput strategies to screen more efficiently different cultivars. Preliminary results indicate that some cultivars with adult resistance were more resistant to these transgenic isolates in cotyledon assays. With this approach, we also identified a cultivar carrying a specific resistance to one these 6 effector candidates.

Reference: Gervais, J., Plissonneau, C., Linglin, J., Meyer, M., Labadie, K., Cruaud, C., Fudal, I., Rouxel, T. and Balesdent, M.H. (2016) Different waves of effector genes with contrasted genomic location are expressed by Leptosphaeria maculans during cotyledon and stem colonization of oilseed rape. Mol. Plant Pathol.


Structural and functional characterization of Leptosphaeria maculans effectors: the example of AvrLm4-7

Yohann Petit-Houdenot1, Françoise Blaise1, Clémence Plissonneau1, Thierry Rouxel1, Marie-Hélène Balesdent1, Karine Blondeau2, Noureddine Lazar2, Inès Gallay2, Théo Le Moigne2, Herman van Tilbeurgh2 and Isabelle Fudal1,
1UMR BIOGER, INRA/AgroParisTech/Université Paris-Saclay, Thiverval-Grignon, France.
2UMR I2BC Université Paris-Sud / CNRS / CEA, Orsay, France

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.