Primers used in this study.

In eukaryotic cells, N6-methyladenosine (m6A) is the most prevalent RNA epigenetic modification that plays crucial roles in multiple biological processes. Nevertheless, the functions and regulatory mechanisms of m6A in phytopathogenic fungi are poorly understood. Here, we showed that CpMTA1, an m6A methyltransferase in Cryphonectria parasitica, plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, the acid phosphatase gene CpAphA was implicated to be a target of CpMTA1 by integrated analysis of m6A-seq and RNA-seq, as in vivo RIP assay data confirmed that CpMTA1 directly interacts with CpAphA mRNA. Deletion of CpMTA1 drastically lowered the m6A level of CpAphA and reduced its mRNA expression. Moreover, we found that an m6A reader protein CpYTHDF1 recognizes CpAphA mRNA and increases its stability. Typically, the levels of CpAphA mRNA and protein exhibited a positive correlation with CpMTA1 and CpYTHDF1. Importantly, site-specific mutagenesis demonstrated that the m6A sites, A1306 and A1341, of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica. Together, our findings demonstrate the essential role of the m6A methyltransferase CpMTA1 in C. parasitica, thereby advancing our understanding of fungal gene regulation through m6A modification.