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

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