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

Abstract

Plants produce ethyl vinyl ketone (evk) in response to biotic stress, but the evk’s identification and downstream defense response remain unclear. In this paper, it is predicted by docking for the first time that evk can be recognized by RBOH protein and assist the electron transfer of RBOHD/RBOHF by binding to its FAD or NADPH binding site. Here, we show that evk treatment increased H₂O₂ and intracellular calcium concentrations in Arabidopsis thaliana mesophyll cells, as observed by confocal laser scanning microscopy and non-invasive micro-test technology, and that H₂O₂ signaling functioned upstream of Ca²⁺ signaling. Yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays demonstrated that the ACA8 (AUTOINHIBITED Ca²⁺-ATPASE, ISOFORM 8)–CML8 (CALMODULIN-LIKE 8) interaction promoted Ca²⁺ efflux to return Ca²⁺ levels to the resting state. Evk treatment led to the antagonism of salicylic acid (SA) and jasmonic acid (JA). CALMODULIN 5 (CAM5) positively regulates WRKY53 expression, and CAM5 and WRKY53 positively regulate SA-related gene expression. These proteins physically interact and form a complex that is unlocked by Ca²⁺ to release WRKY53. An electrophoretic mobility shift assay and dual-luciferase reporter assay demonstrated that WRKY53 and TGA5 cooperate to enhance the expression of the defense gene PATHOGENESIS-REALTED 1 (PR1) and that WRKY53 enhances the binding of TGA5 to the PR1 promoter. This paper proposes a framework that evk, as a RES substance, can achieve plant’s ‘REScue’ through complete defense response.