Effector identification in Pseudocercospora fijiensis

Hypothesis

The infection process of Pseudocercospora fijiensis, the causal agent of Black Leaf Streak disease or Black Sigatoka Disease, involves both biotrophic and necrotrophic phases, during which the pathogen secretes effectors to manipulate host defense mechanisms. Over the past few years, various genomic, transcriptomic, and proteomic data have been generated, leading to the identification of several top effector candidates. The functional validation of these promising effectors is currently being undertaken through various molecular approaches, including gene knockout strategies, protein production in Pichia pastoris, and Nicotiana benthamiana.

Objectives and techniques

• Use CRISPR/Cas9 to precisely edit the genome of P. fijiensis, generating strains lacking specific effector genes and evaluate the influence of these knockouts on fungal virulence by comparing them to wild-type strains.
• Clone the genes encoding the candidate effectors into expression vectors suitable for Pichia pastoris. Transform the yeast with these vectors to produce and purify the effector proteins. Test the necrosis-inducing activity of these proteins by applying them to banana leaf tissues and observing the response.
• Clone the genes encoding the candidate effectors into expression vectors suitable for agroinfiltration in Nicotiana benthamiana. Use Agrobacterium-mediated transformation to transiently express these proteins in the plant. Produce and extract candidate effector proteins in the plant Nicotiana benthamiana. Test the necrosis-inducing activity of these proteins by observing the response in banana tissues.