Colletotrichum trifolii mutants disrupted in the catalytic subunit of cAMP-dependent protein kinase are nonpathogenic

Colletotrichum trifolii is the fungal pathogen of alfalfa that causes anthr acnose disease. For successful plant infection, this fungus must undergo a series of morphological transitions following conidial attachment, includin g germination and subsequent differentiation, resulting in appressorium for mation. Our previous studies with pharmacological effecters of signaling pa thways have suggested the involvement of cyclic AMP (cAMP)-dependent protei n kinase (PKA) during these processes. To more precisely evaluate the role of PKA in C. trifolii morphogenesis, the gene encoding the catalytic (C) su bunit of PKA (Ct-PKAC) was isolated, sequenced, and inactivated by gene rep lacement. Southern blot analysis with C, trifolii genomic DNA suggested tha t Ct-PKAC is a single-copy gene. Northern (RNA) blot analysis with total RN A from different fungal growth stages indicated that the expression of this gene was developmentally regulated. When Ct-PKAC was insertionally inactiv ated by gene replacement, the transformants showed a small reduction in gro wth relative to the wild type and conidiation patterns were altered. Import antly, PKA-deficient strains were unable to infect intact alfalfa (host) pl ants, though only a slight delay was observed in the timing for conidial ge rmination and appressorial formation in the Ct-PKAC disruption mutants. Mor eover, these mutants were able to colonize host tissues following artificia l wounding, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggest that the defect in pathogenicity is likely d ue to a failure in penetration. Our results demonstrate that PKA has an imp ortant role in regulating the transition between vegetative growth and coni diation, and is essential for pathogenic development in C, trifolii.