Identification and functional characterization of Cladosporium fulvum effectors by genomics, transcriptomics and proteomics approaches

Cladosporium fulvum is a biotrophic fungal pathogen that causes leaf mold of tomato. During infection C. fulvum secretes a number of small proteins into the apoplast of tomato leaves, which are collectively called effectors. So far, ten effector proteins have been characterized that in general show no or limited sequence similarity to other proteins present in public databases. In this study we try to identify and functionally characterize additional C. fulvum effector proteins that are involved in fungal pathogenesis. Recently, the genome of C. fulvum has been sequenced using the 454 technology. This genome sequence enables the identification of all secreted proteins from the fungus, collectively called the secretome. However, for accurate mining and annotation of the effector secretome, gene calling programs need to be first optimized by analyzing high quality expressed sequence tags (ESTs). Therefore, several cDNA libraries of C. fulvum grown under various in vitro and in planta conditions were constructed and are sequenced to support genome annotation. Initial automated annotation of the genome revealed that the fungus contains approximately 13.000 genes, of which approximately 1200 encode putatively secreted proteins. Bioinformatic analyses identified a subset of 300 putative effectors within the predicted secretome, while additional proteomics analysis from apoplastic fluids of tomato leaves infected by C. fulvum, revealed 30 proteins that are specifically produced in the compatible interaction. At this moment we are performing functional profiling of these novel effector proteins by examining their ability to inhibit PAMP triggered Immunity and/or effector-triggered immunity in custom made assays