CfAsh1 dominates H3K36 methylation and cooperates with CfSet2 to orchestrate chromatin-mediated pathogenicity in Colletotrichum fructicola

Abstract Histone H3 lysine 36 (H3K36) methylation is a crucial chromatin modification that regulates transcription and maintains genome integrity across eukaryotes, but its specific functions in fungal virulence are not fully understood. In this study, we identify and characterize CfAsh1, a nuclear-localized SET-domain methyltransferase in Colletotrichum fructicola , a predominant anthracnose pathogen of Camellia oleifera and other crops. CfAsh1 was identified as the primary enzyme catalyzing H3K36 di- and trimethylation, thereby serving as a central chromatin regulator governing fungal development, stress adaptation, and pathogenicity. Deletion of CfASH1 led to severe defects in vegetative growth, conidiation, appressorium formation, and virulence on both Ca. oleifera and apple. Site-directed mutagenesis revealed that residue I499 is indispensable for CfAsh1 function, while N497 contributes moderately to pathogenicity. CfAsh1 also regulates cell wall integrity, endoplasmic reticulum stress tolerance, and detoxification of host-derived reactive oxygen species (ROS). Western blot analysis of nuclear proteins showed that CfAsh1 and CfSet2 promote H3K36me2 and H3K36me3, while double knockout of CfASH1 and CfSET2 results in the complete loss of H3K36me2/3. It revealed that CfAsh1 is the dominant contributor to H3K36 methylation under normal conditions, while CfSet2 exerts compensatory activity in CfAsh1’s absence. qPCR results indicated that both enzymes jointly regulate key genes involved in melanin synthesis and stress responses. Collectively, our findings establish CfAsh1 as a master epigenetic regulator that controls pathogenic development through H3K36 methylation, functioning in concert with CfSet2. This study advances our understanding of chromatin-based regulation in phytopathogenic fungi and highlights CfAsh1 as a potential target for antifungal intervention strategies.