ALKBH1L Is an m6A Demethylase and Mediates PVY Infection in Nicotiana benthamiana Through m6A Modification

N⁶-methyladenosine (m⁶A), the most prevalent internal mRNA modification in eukaryotes, is also present in plants and is known to influence plant-virus interactions. However, its specific role in regulating <i>Potato virus Y</i> (PVY; <i>Potyvirus yituberosi</i>) infection, a major pathogen of potatoes, remains unclear. This study identified 16 potential m⁶A regulator genes in <i>Nicotiana benthamiana</i> through homology screening of <i>Arabidopsis thaliana</i> AlkB family members. Based on expression profiles in leaves at various developmental stages and following PVY infection, NbALKBH1L was selected for further analysis. Enzyme assays confirmed its m⁶A demethylase activity. Experiments with NbALKBH1L mutants, using RT-qPCR and m⁶A-IP-qPCR, demonstrated that it regulates PVY infection via the m⁶A pathway. Further investigation revealed that NbALKBH1L interacts with the PVY-encoded cylindrical inclusion (CI) protein. An interaction network constructed through immunoprecipitation-mass spectrometry (IP-MS) and RNA sequencing (RNA-seq) suggested that NbALKBH1L may serve as a central node in plant antiviral immunity, potentially linking metabolic processes with the regulation of viral infection. In summary, this study advances our understanding of plant m⁶A modifications in antiviral defense and provides valuable insights for future antiviral breeding strategies.