Distinct Roles of Two DNA Methyltransferases from Cryphonectria parasitica in Fungal Virulence, Responses to Hypovirus Infection, and Viral Clearance

Two DNA methyltransferase (DNMTase) genes from have been previously identified as and , which are orthologous to and of , respectively. While global changes in DNA methylation have been associated with fungal sectorization and but not has been implicated in the sporadic sectorization, the present study continues to investigate the biological functions of both DNMTase genes. Transcription of both DNMTases is regulated in response to infection with the hypovirus 1 (CHV1-EP713). is upregulated and is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of , not by mutation. Significant changes in virulence are observed in opposite directions; i.e., the -null mutant is hypervirulent, while the -null mutant is hypovirulent. Compared to the CHV1-infected wild type, CHV1-transferred single and double mutants show severe growth retardation: the colony size is less than 10% that of the parental virus-free null mutants, and their titers of transferred CHV1 are higher than that of the wild type, implying that no defect in viral replication occurs. However, as cultivation proceeds, spontaneous viral clearance is observed in hypovirus-infected colonies of the null mutants, which has never been reported in this fungus-virus interaction. This study demonstrates that both DNMTases are significant factors in fungal development and virulence. Each fungal DNMTase affects fungal biology in both common and separate ways. In addition, both genes are essential to the antiviral responses, including viral clearance which depends on their mutations. Although relatively few in number, studies of DNA methylation have shown that fungal DNA methylation is implicated in development, genome integrity, and genome defense. While fungal DNMTase has been suggested as playing a role in genome defense, studies of the biological function of fungal DNMTase have been very limited. In this study, we have shown distinct biological functions of two DNA methyltransferases from the chestnut blight fungus We have demonstrated that DNMTases are important to fungal development and virulence. In addition, these genes are shown to play an important role in the fungal response to hypoviral CHV1 infection, including severely retarded colonial growth, and in viral clearance, which has never been previously observed in mycovirus infection. These findings provide a better understanding of the biological functions of fungal DNA methyltransferase and a bas