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Hypovirus infection of the chestnut blight fungus Cryphonectria parasitica is known to downregulate the fungal laccase1 (lac1), the modulation of which is tightly governed by the inositol triphosphate (IP(3)) and calcium second messenger system in a virus-free strain. We cloned the gene cplc1 encoding a phosphatidyl inositol-specific phospholipase C (PLC), to investigate the regulation of lac1 expression and to better characterize fungal gene regulation by hypovirus. Sequence analysis of the cplc1 gene indicated that the protein product contained both the X and Y domains, which are the two conserved regions found in all known PLCs, with a 133 amino acid extension between the 2nd beta-strand and the alpha-helix in the X domain. In addition, the gene organization appeared to be highly similar to that of a delta-type PLC. Disruption of the cplc1 gene resulted in slow growth and produced colonies characterized by little aerial mycelia and deep orange in color. Accordingly, reduced virulence of the cplc1-null mutant as compared to the wild-type was observed, which can be ascribed to the growth defect. However, other PLC-associated characteristics including temperature sensitivity and osmosensitivity did not differ from the wild-type strain. Northern blot analysis revealed no accumulation of the lac1 gene transcript due to the disruption of the cplc1 gene. Functional complementation of the cplc1-null mutant with the PLC1 gene from Saccharomyces cerevisiae restored lac1 expression, which suggests that the cloned gene encodes PLC activity. The present study indicates that the cplc1 gene is required for normal mycelial growth rate and colony morphology, and that it regulates the lac1 expression, which is also modulated by the hypovirus. Although several PLC genes have been identified in various simple eukaryotic organisms, the deletion analysis of the cplc1 gene in this study appears to be the first report on the functional analysis of PLC in filamentous fungi.

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.