Abstract
ABSTRACTTwo DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt1 and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. While global changes in DNA methylation have been associated with fungal sectorization and CpDmt1 but not CpDmt2 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 Cryphonectria hypovirus 1 (CHV1-EP713). CpDmt1 is upregulated and CpDmt2 is downregulated by CHV1 infection. Conidium production and response to heat stress are affected only by mutation of CpDmt1, not by CpDmt2 mutation. Significant changes in virulence are observed in opposite directions; i.e., the CpDmt1-null mutant is hypervirulent, while the CpDmt2-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.
Highlights
Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as C. parasitica Dmt1 (CpDmt1) and CpDmt2, which are orthologous to rid and dim-2 of Neurospora crassa, respectively
Sequence analysis revealed the presence of characteristic domains of DNMTases, while phylogenetic analysis of their deduced amino acid sequences indicated that C. parasitica Dmt1 (CpDmt1) and CpDmt2 genes
To analyze how Cryphonectria hypovirus 1 (CHV1) affects the expression of DNMTase genes, we examined the accumulation of transcripts of each of the DNMTase genes in both the wild-type EP155/2 strain and its isogenic CHV1-EP713-infected hypovirulent strain UEP1 under standard liquid-culture conditions using real-time reverse transcription-PCR (RT-PCR) (Fig. 1A and B)
Summary
Two DNA methyltransferase (DNMTase) genes from Cryphonectria parasitica have been previously identified as CpDmt and CpDmt, which are orthologous to rid and dim-2 of Neurospora crassa, respectively. We have demonstrated that DNMTases are important to fungal development and virulence 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 basis for clarifying the epigenetic regulation of fungal virulence, responses to hypovirus infection, and viral clearance. Domain searching followed by phylogenic analysis of DNA methylase domain-containing proteins of C. parasitica draft genome sequence (http://genome.jgi-psf .org/Crypa2/Crypa2.home.html) reveals the presence of putative DIM-2, RID, and DNMT5 [29, 30]
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