Hypovirus-Induced Phosphorylation of CpIre1 Modulates Unfolded Protein Response and Virulence in Cryphonectria parasitica.

The chestnut blight fungus Cryphonectria parasitica and its hypovirus constitute a valuable model for investigating fungal pathogenesis and cross-kingdom virus-host interplay. To investigate how hypovirus regulates protein function at the phosphorylation level in C. parasitica, we performed a comparative phosphoproteomic analysis in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Comparative profiling between the wild-type (EP155) and hypovirus-infected (EP155/CHV1-EP713) strains revealed 700 differentially phosphorylated sites (174 upregulated, 526 downregulated). Among these, the serine 896 and 897 sites on the endoplasmic reticulum (ER) stress-sensing protein CpIre1 drew our particular attention, as hypovirus-induced phosphorylation targets. Western blot analysis showed that virus-encoded p29, p40, and p48 proteins could promote CpIre1 phosphorylation. Site-specific mutagenesis revealed that Ser-896 and Ser-897 are essential for CpIre1 phosphorylation, which regulates fungal phenotypic traits, virulence, and stress tolerance in C. parasitica. Reverse-transcription-quantitative PCR analysis of the ER stress marker genes CpHac1 and CpBip1 confirmed that CpIre1 and its phosphorylation are essential for a functional ER stress response. Notably, hypovirus replication was significantly impaired in phospho-deficient CpIre1 mutants, showing about 40% reduction in viral RNA accumulation, whereas phospho-mimic mutants maintained wild-type levels of viral RNA. This indicates that efficient hypovirus accumulation requires functional phosphorylation of CpIre1. Our findings demonstrate that hypovirus-induced phosphorylation of CpIre1 modulates fungal ER homeostasis, pathogenicity, and viral RNA accumulation, thereby revealing a mechanism through which the virus reprogrammes its host via targeted post-translational modification.