Functional studies ofcAMP-dependent protein kinase in development and pathogenicity of Colletotrichum trifolii

Colletotrichum trifolii Bain and Essary is a fungal pathogen causing alfalfa anthracnose. Early developmental switches including conidial germination and appressorial development are crucial for disease initiation. These processes are poorly understood at the molecular level. Our research goal is to understand the signaling events during prepenetration development and disease initiation in C. trifolii. Such information is likely to facilitate the design of novel and durable strategies for controlling alfalfa anthracnose. An experimental approach to address this subject is to isolate and analyze potentially important signaling molecules expressed during development and disease of animal pathogens, based on the assumption that similar signal transduction pathways exist between C. trifolii and other organisms. In this study, cAMP-dependent protein kinase (PKA) was functionally investigated during C. trifolii development and morphogenesis. Initial studies with pharmacological reagents and biochemical assays indicated the involvement of cAMP and PKA during conidial germination and appressorial development. PKA activities as well as endogenous cAMP levels fluctuated during distinct growth stages. A potent PKA inhibitor blocked both conidial germination and appressorial formation. Chemicals that increase endogenous cAMP levels induced appressorial development in conditions where normally appressoria do not develop. For direct examination of PKA function, the genes encoding both PKA catalytic subunit and regulatory subunit were isolated and cloned. Southern analyses showed that both genes exist in single copy. Northern blot analysis with total RNA from tissues during development revealed that the expression of the two genes were coordinately regulated during morphogenesis. The catalytic subunit gene was found to be functional in Schizosaccharomyces pombe by complementing a S. pombe PKA mutant. Further more, when the catalytic subunit gene was disrupted by one-step gene replacement, the mutants were affected in normal growth and reproduction. More importantly, the disruption mutants made appressoria but failed to infect intact host plants. However, these disruptants were able to colonize wounded leaves, suggesting the loss of pathogenicity might be due to a failure in penetration. These results indicated that PKA plays pivotal roles in morphogenesis and pathogenicity of C. trifolii