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 Potato virus Y (PVY; Potyvirus yituberosi) infection, a major pathogen of potatoes, remains unclear. This study identified 16 potential m⁶A regulator genes in Nicotiana benthamiana through homology screening of Arabidopsis thaliana 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.