Turnip Mosaic Virus Co-Opts Host RNA Methylation Machinery to Orchestrate Plant Infection

Abstract N6-methyladenosine (m 6 A) is a key RNA modification that regulates transcript stability and translation. However, its function in plant viruses remains largely unclear. Here, we show that the positive-sense single-stranded +ssRNA Turnip mosaic virus (TuMV) relies on the host m 6 A machinery to support efficient infection. Our findings uncover a previously unrecognized nuclear phase in the TuMV life cycle, during which viral RNA undergoes extensive methylation by host enzymes. We identify a complex and non-canonical methylation landscape on the TuMV genome, where canonical DRACH motifs are embedded within clusters of additional virus-specific non-canonical m 6 A sites. Notably, we also detect the presence of another RNA modification, m 5 C (5-methylcytosine), in close proximity to m 6 A-marked regions. This coordinated methylation landscape appears to be critical for efficient viral polyprotein synthesis. In its absence, the virus displays aberrant methylation and reduced infectivity as observed in m 6 A writer and reader mutants. Based on this, we propose a “seeding” model in which initial m 6 A deposition at canonical sites nucleates the formation of a broader network of m 6 A and m 5 C marks, likely guided by RNA structure rather than sequence motifs alone.