Ncl1-dependent m5C RNA methylation promotes appressorial autophagy and pathogenicity of the rice blast fungus

Epigenetic regulation, particularly RNA methylation, remains largely unexplored in plant pathogenic fungi. This study investigates the role of 5-methylcytosine (m⁵C) RNA methylation during the infection process of Magnaporthe oryzae, the rice blast fungus, with a focus on its impact on autophagy. We identified Ncl1, an m⁵C RNA methyltransferase, as essential for promoting fungal virulence and appressorium development. Via m⁵C MeRIP-seq (methylated RNA immunoprecipitation sequencing), we mapped the m⁵C landscape of the M. oryzae transcriptome, identifying 9,014 hypomethylated peaks across 5,678 genes in the Δncl1 mutant. RNA-seq analysis revealed that 119 m⁵C-modified genes were upregulated and 199 were downregulated, underscoring Ncl1's critical role in gene expression control. Mechanistically, Ncl1-mediated m⁵C methylation stabilizes ATG5 and ATG16 mRNAs, which are essential for Atg8 lipidation, autophagosome formation, and full pathogenicity. Notably, Ncl1 protein stability is modulated by the Pmk1 signaling pathway via phosphorylation and by Smt3-mediated SUMOylation. Our findings reveal a complex interplay between epigenetic regulation and post-translational modifications in fungi, highlighting the pivotal role of m⁵C RNA methylation in promoting autophagy and pathogenicity in M. oryzae. These insights enhance our understanding of fungal biology and identify potential targets for antifungal strategies.