Tomato gray mold, caused by Botrytis cinerea (B. cinerea), poses a major challenge to tomato production. In plants, B. cinerea resistance is positively regulated by melatonin
however, the regulatory mechanism of melatonin biosynthesis during B. cinerea infection is not known. Here, we established the working model of jasmonic acid (JA) and nitric oxide (NO) on melatonin biosynthesis in the state of B. cinerea infection. NO responded to B. cinerea infection earlier than JA. In the early stage of infection, tomato caffeic acid O-methyltransferase 2 (SlCOMT2) was S-nitrosylated by NO at Cys344, enhancing the stability of SlCOMT2 and preventing its degradation via the 26S proteasome. In the late stage of infection, JA-triggered SlMYC2 bound to SlCOMT1 and SlCOMT2 promoters for their transcription. NO and JA synergistically enhanced COMT-mediated melatonin biosynthesis during B. cinerea infection via post-translational modification and transcriptional activation. The accumulation of melatonin in tomato leaves inhibited cell death by scavenging reactive oxygen species (ROS), thereby preventing B. cinerea from establishing infection sites. We propose that SlCOMT2Cys344 is a genetic manipulation site or biological breeding target that can be used to enhance melatonin synthesis and B. cinerea resistance in tomato.