CAM5, WRKY53, and TGA5 regulate defense gene expression mediated by the volatile organic compound ethyl vinyl ketone in Arabidopsis

Plants produce ethyl vinyl ketone (evk) in response to biotic stress; however, the downstream defense responses triggered by evk remain unclear. Our research utilizing reverse-transcription quantitative PCR showed that evk treatment antagonized the effects of salicylic acid (SA) and jasmonic acid (JA) and that CALMODULIN 5 (CAM5) positively regulated WRKY53 expression, which, in turn, positively regulated the expression of SA-related genes. Additionally, the GST-pulldown assay, yeast two-hybrid assays, and luciferase complementation test all suggested that CAM5 and WRKY53 physically interacted and formed a complex that was unlocked by Ca²⁺ to release WRKY53. Electrophoretic mobility shift and dual-luciferase reporter assays demonstrated that WRKY53 enhanced the binding of TGA5 to the PATHOGENESIS-RELATED 1 (PR1) promoter, thereby enhancing the expression of the defense gene PR1. Conversely, WRKY53 diminishes the binding of TGA5 to the PLANT DEFENSIN 1.2 (PDF1.2) promoter and inhibits PDF1.2 expression. As the expression of WRKY53 decreases in later stages, this inhibitory effect weakens, allowing TGA5 to positively activate PDF1.2 expression. In this study, we propose a framework in which evk, as a resistance-inducing substance, facilitates the plant’s ‘rescue' through the regulation of defense-related gene expressions in Arabidopsis.