m⁶A modification of plant virus enables host recognition by NMD factors in plants

N⁶-methyladenosine (m⁶A) is the most abundant eukaryotic mRNA modification and is involved in various biological processes. Increasing evidence has implicated that m⁶A modification is an important anti-viral defense mechanism in mammals and plants, but it is largely unknown how m⁶A regulates viral infection in plants. Here we report the dynamic changes and functional anatomy of m⁶A in Nicotiana benthamiana and Solanum lycopersicum during Pepino mosaic virus (PepMV) infection. m⁶A modification in the PepMV RNA genome is conserved in these two species. Overexpression of the m⁶A writers, mRNA adenosine methylase A (MTA), and HAKAI inhibit the PepMV RNA accumulation accompanied by increased viral m⁶A modifications, whereas deficiency of these writers decreases the viral RNA m⁶A levels but enhances virus infection. Further study reveals that the cytoplasmic YTH-domain family protein NbECT2A/2B/2C as m⁶A readers are involved in anti-viral immunity. Protein-protein interactions indicate that NbECT2A/2B/2C interact with nonsense-mediated mRNA decay (NMD)-related proteins, including NbUPF3 and NbSMG7, but not with NbUPF1. m⁶A modification-mediated restriction to PepMV infection is dependent on NMD-related factors. These findings provide new insights into the functionality of m⁶A anti-viral activity and reveal a distinct immune response that NMD factors recognize the m⁶A readers-viral m⁶A RNA complex for viral RNA degradation to limit virus infection in plants.