Updated project metadata. Kac, a reversible PTM, plays essential roles in various biological processes, including those involving metabolic pathways, pathogen resistance and transcription, in both prokaryotes and eukaryotes. TMV, the major factor that causes poor quality of Solanaceae crops worldwide, directly alters many metabolic processes in tobacco. However, the extent and function of Kac during TMV infection have not been determined. Here, using LC−MS/MS in conjunction with highly sensitive immune-affinity purification, we comprehensively analyzed the changes in the proteome and acetylome of TMV-infected tobacco (Nicotiana benthamiana) seedlings. In total, 2082 lysine-acetylated sites on 1319 proteins differentially expressed in response to TMV infection were identified. Extensive bioinformatic studies disclosed changes in acetylation of proteins engaged in cellular metabolism and biological processes. The vital influence of Kac in fatty acid degradation and alpha-linolenic acid metabolism was also revealed in TMV-infected seedlings. This study first revealed Kac information in N. benthamiana under TMV infection and expands upon the existing landscape of acetylation in pathogen infection.