P-362 - Functional study of ferric reductase CpFre1 in chestnut blight fungus, Cryphonectria parasitica related to hypovirus infection and sectorization

The chestnut blight fungus, Cryphonectria parasitica, and its interaction with hypovirus, Cryphonectria parasitica hypovirus 1 (CHV1), is known as a model to study the mechanism of hypovirulence. In our previous study, the function of CpBck1 and CpSlt2, a MAPKKK and MAPK gene in the cell wall integrity pathway respectively, was affected by CHV1 infection. The unique phenotype, sporadic sectorization, was observed in CpBck1- and CpSlt2-null mutant strains. Transcriptome analysis revealed that 22 DEGs were found to be sectorization-specific and CHV1-specific. Among these 22 DEGs, the putative ferric reductase gene (CpFre1) showing the highest similarity to the known Saccharomyces cerevisiae ferric reductase 7 (Fre7) was further analyzed. The CpFre1 gene was predicted to encode a membrane protein with the function of reducing ferric (Fe³⁺) to ferrous (Fe²⁺) for cytosol up-take. Three knock-out mutants of CpFre1 were obtained. Compared to the wild type EP155/2, the CpFre1-null mutants showed severe growth inhibition and abnormally intense pigmentation. The retarded growth of the mutants was restored on PDAmb plate containing ferrous indicating that the cloned gene encoded a ferric reductase. We observed sporadic sectorization in the CpFre1-null mutant, which exhibited no pigmentation and a normal hyphal shape. Compared to the the wild-type strain, the pathogenicity of the mutants was reduced on chestnut. Thus, CpFre1 plays an important role in colony morphology, pigmentation, and virulence.