Screening for proteins interacting with the perilipin-like protein CAP20 by a yeast two-hybrid system and identification of a protein kinase a catalytic subunit as an interacting protein in Colletotrichum siamense

CAP20 is a lipid droplet-coating protein perilipin homolog that plays a key role in Colletotrichum functional appressorium development and virulence. To obtain proteins interacting with CAP20 in Colletotrichum, the bait protein expression plasmid pGBKT7-Cap20 and the cDNA library of Colletotrichum siamense (the major causative species of rubber tree anthracnose) was constructed. By yeast two hybrid system, sixteen proteins, including a cAMP-dependent protein kinase catalytic subunit (PKAC1), protein kinase, acetate kinase, hydrophobin, and hypersensitive response-inducing protein, may interacting with CAP20 were identified after sequencing and bioinformatics analysis. Furthermore, the interaction between CAP20 and the catalytic subunit of protein kinase A (PKAC1) was validated by GST pull-down analysis in vitro and co-immunoprecipitation (co-IP) assays in vivo. qPCR revealed a positive correlation between the expression of PkaC1 and Cap20 in Colletotrichum treated with PKA activators or inhibitors. This research identified candidate proteins by the yeast two-hybrid system and confirmed an interaction between the pathogenicity-related protein CAP20 and PKAC1. The findings lay the foundation for further studies of the function and regulation mechanism of CAP20.