# Methylation of mycovirus DNA is mediated by the RNAi machinery in vegetative hyphae of Fusarium graminearum

## TL;DR

This study shows that DNA methylation in fungal hyphae helps defend against mycoviruses by silencing viral genes.

## Contribution

The study reveals RNAi-mediated DNA methylation in vegetative hyphae of fungi as a novel antiviral defense mechanism.

## Key findings

- DNA methylation of FgGMTV1 occurs in promoter regions, with up to 55.87% methylation in DNA-C fragment MeC5.
- RNAi machinery components Ago and Dicer are essential for directing methylation of viral DNA and foreign promoters.
- Deletion of RNAi genes leads to increased virus accumulation and impaired fungal growth and infection.

## 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.

Graphical Abstract