Source  Nucleic Acids Research

Published  JUN 24 2025

DOI: 10.1093/nar/gkaf478

IF  13.1

Abstract  DNA cytosine methylation is an important epigenetic mechanism for transposon silencing and gene regulation in fungi, particularly during sexual reproduction. However, its occurrence in vegetative hyphae and role in defense against mycoviruses is unclear. In this study, we demonstrated that genomic-wide cytosine methylation of the tripartite genomovirus FgGMTV1 occurs in the hyphae of Fusarium graminearum, a destructive pathogen of wheat and barley worldwide. Elevated methylation levels were predominantly observed in the promoter regions of FgGMTV1, with the highest level reaching 55.87% in the DNA-C fragment MeC5. Methylation of the Rep promoter in DNA-A was showed to be mediated by DNA methyltransferase DIM2 and lead to its transcriptional activity suppression, resulting in a significant reduction in virus accumulation. Furthermore, we uncovered that small RNAs (sRNAs) derived from FgGMTV1 direct the methylation of viral DNA and integrated foreign promoters, which requires the core components of the RNAi machinery, including the Ago and Dicer genes. Deletion of dcl1/2 or ago1/2 in FgGMTV1-infected strains resulted in an increase in virus accumulation and defects in hyphal growth, stress response, and plant infection. Taken together, our findings reveal that RNAi-mediated DNA methylation occurs in vegetative hyphae and plays a crucial role in antiviral defense mechanisms in fungi.

This study reveals that fungi use DNA methylation-a chemical modification-as a defense against viruses during their vegetative growth phase. In Fusarium graminearum, a harmful wheat/barley pathogen, widespread cytosine methylation targets the tripartite virus FgGMTV1, particularly silencing its promoter regions and suppressing viral replication. This process relies on the DNA methyltransferase DIM2 and is guided by small RNAs (sRNAs) derived from the virus via RNA interference (RNAi). Deleting key RNAi genes dcl1/2 or ago1/2 increased viral levels and impaired fungal growth, stress tolerance, and plant infection. These findings highlight RNAi-directed DNA methylation as a critical antiviral mechanism in fungi, even outside reproductive stages, offering insights into fungal epigenetics and host-virus interactions.