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.

A DnaJ-like gene, Cpdj1, a molecular chaperone and regulator of Hsp70 in Cryphonectria parasitica, was characterized. The protein product of Cpdj1 gene consists of 379 amino acids with a predicted molecular mass of 40.6 kDa and a pI of 7.79. The deduced protein sequence revealed preservation of the conserved hall-mark J-region and exhibited high homolo y to all known DnaJ-like proteins. Disruption of the Cpdj1 gene resulted in slow growth and produced colonies characterized by retarded growth and deep orange color. Accordingly, reduced virulence of the Cpdj1-null mutant was observed. This reduced growth rate was magnified when the Cpdj1-null mutant was cultured under heat-stress conditions. Reduced conidiation was also observed in the Cpdj1-null mutant, indicating that Cpdj1 gene, although not essential for cell viability, is required for appropriate cellular processes including growth and sporulation. Northern analysis showed that Cpdj1 was constitutively expressed, and when the culture was subject to high temperature, a strong induction of the transcript was observed. No significant difference in the expression and induction pattern of Cpdj1 was observed between virus-free EP155/2 and virus-infected hypovirulent UEP1 strains. However, further severe defects in mycelia growth and conidiation were observed in the hypovirus-infected Cpdj1-null mutant suggesting that the presence of Cpdj1 is required for mycelia growth and sporulation of the hypovirus-infected strain.