In planta localisation of Leptosphaeria maculans effectors and identification of their plant targets

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.