Deletion of a hypoviral-regulated cppk1 gene in a chestnut blight fungus, Cryphonectria parasitica, results in microcolonies

Abstract

The cppk1 gene encodes a Ser/Thr protein kinase of Cryphonectria parasitica and is transcriptionally up-regulated by the presence of hypovirus CHV1-EP713. A cppk1-null mutant was constructed to determine the function of cppk1. The cppk1-null mutant was initially isolated as a heterokaryotic form containing both wild-type and cppk1-deleted nuclei. The pure cppk1-null homokaryon was obtained by the single spore isolation of the heterokaryon. While the parental heterokaryon appeared normal, the pure cppk1-null mutant exhibited dramatic changes in colony morphology. It showed characteristics of microcolonial growth. The functional complementation, restoration of filamentous growth, of the cppk1-null mutant using a wild-type cppk1 gene indicated that the phenotypic changes were due to the disruption of cppk1. Neither sporulation nor hyphal differentiation into feeding hyphae, a mycelial mat, or aerial hyphae was observed in the cppk1-null mutant. Instead of bright yellow, dark brown pigmentation appeared as the culture grew. Hyphae were shortened and hyperbranched with globose to bulbose cells. Electron microscopy revealed the presence of intrahyphal hyphae, the most striking ultrastructural change. Subtle changes in the expression of CpPK1 resulted in abnormalities in colony morphology and pigmentation, which indicated that cppk1 is important for coordinating growth with development and maintaining cell wall integrity.