Mutation in CpBck1, an ortholog of the cell wall integrity mitogen-activated protein kinase kinase kinase (MAPKKK) of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica resulted in a sporadic sectorization as culture proceeded. The progeny from the sectored area maintained the characteristics of the sector, showing a massive morphogenetic change, including robust mycelial growth without differentiation. Epigenetic changes were investigated as the genetic mechanism underlying this sectorization. Quantification of DNA methylation and whole-genome bisulfite sequencing revealed genome-wide DNA methylation of the wild-type at each nucleotide level and changes in DNA methylation of the sectored progeny. Compared to the wild-type, the sectored progeny exhibited marked genome-wide DNA hypomethylation but increased methylation sites. Expression analysis of two DNA methyltransferases, including two representative types of DNA methyltransferase (DNMTase), demonstrated that both were significantly down-regulated in the sectored progeny. However, functional analysis using mutant phenotypes of corresponding DNMTases demonstrated that a mutant of CpDmt1, an ortholog of RID of Neurospora crassa, resulted in the sectored phenotype but the CpDmt2 mutant did not, suggesting that the genetic basis of fungal sectorization is more complex. The present study revealed that a mutation in a signaling pathway component resulted in sectorization accompanied with changes in genome-wide DNA methylation, which suggests that this signal transduction pathway is important for epigenetic control of sectorization via regulation of genes involved in DNA methylation.

Comprehensive transcriptome analysis was conducted to elucidate the molecular basis of the interaction between chestnut blight fungus, Cryphonectria parasitica, and single-stranded RNA (ssRNA) mycovirus Cryphonectria hypovirus 1 (CHV1), using RNAsequencing (RNA-seq). A total of 1,023 differentially expressed genes (DEGs) were affected by CHV1 infection, of which 753 DEGs were upregulated and 270 DEGs were downregulated. Significant correlations in qRT-PCR analysis of 20 randomly selected DEGs and agreement with previously characterized marker genes validated our RNAseq analysis as representing global transcriptional profiling of virus-free and-infected isogenic strains of C. parasitica. Gene Ontology (GO) analysis of DEGs indicated that "cellular aromatic compound metabolic process" and "transport" were the two most enriched components in the "biological process." In addition, "cytoplasm" was the most enriched term in the "cellular component" and "nucleotide binding" and "cation binding" were the two most enriched terms in the "molecular function" category. These results suggested that altered expression of genes encoding numerous intracellular proteins due to hypoviral infection resulted in changes in specific metabolic processes as well as transport processes. Kyoto Encyclopedia of Genes and Genomes function analysis demonstrated that pathways for "biosynthesis of other secondary metabolites," "amino acid metabolism," "carbohydrate metabolism," and "translation" were enriched among the DEGs in C. parasitica. These results demonstrate that hypoviral infection resulted in massive but specific changes in primary and secondary metabolism, of which antiviral fungal metabolites were highly induced. The results of this study provide further insights into the mechanism of fungal gene regulation by CHV1 at the transcriptome level.

CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.

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We assessed the biological function of CpSlt2, an ortholog of the cell wall integrity (CWI) MAPK of Saccharomyces cerevisiae, in the chestnut blight fungus Cryphonectria parasitica. The CpSlt2-null mutant exhibited marked changes in colonial growth, near absence of conidiation and aerial hyphae, and abnormal pigmentation. In addition, the CpSlt2-null mutant exhibited CWI-related phenotypic defects including hypersensitivity to cell wall-disturbing agents and other stresses. Electron microscopy revealed the presence of abnormal hyphae such as intrahyphal hyphae. In addition, virulence assays indicated that the CpSlt2 gene plays an important role in fungal pathogenesis. As cultivation of the mutant strains progressed, the majority of the colonies showed sporadic sectorization and mycelia from the sectored area stably maintained the sectored phenotype. Although mycelial growth was partially recovered, the sectored progeny had dramatically impaired virulence, confirming the CpSlt2 gene has a role in pathogenicity. Compared to a previous mutant of the CpBck1 gene, a MAPKKK gene in CWI pathway, the CpSlt2-null mutant showed similar, although not identical, phenotypic changes and most phenotypic changes were less severe than those of the CpBck1-null mutant. These results suggest that the unique sectorization is CWI pathway-specific, though the components in the same CWI pathway have common and specific functions.