Hirt, H.; Almeida-Trapp, M.; Nawaz, K.; Secco, N.; Sheikh, A.

N6-methyladenosine (m6A) 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 m6A 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 m6A sites. Notably, we also detect the presence of another RNA modification, m5C (5-methylcytosine), in close proximity to m6A-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 m6A writer and reader mutants. Based on this, we propose a "seeding" model in which initial m6A deposition at canonical sites nucleates the formation of a broader network of m6A and m5C marks, likely guided by RNA structure rather than sequence motifs alone.