The WRKY46–MYC2 module plays a critical role in E-2-hexenal-induced anti-herbivore responses by promoting flavonoid accumulation

Volatile organic compounds (VOCs) play key roles in plant–plant communication, especially under attack by pests. E-2-hexenal is an important component of VOCs, and whether it can induce endogenous insect resistance in plants is not clear. Here, we show that E-2-hexenal activates early signaling events in Arabidopsis (Arabidopsis thaliana) mesophyll cells, including an H2O2 burst at the plasma membrane, the directed flow of calcium ions, and an increase in cytosolic calcium concentration. Treatment of wild-type Arabidopsis plants with E-2-hexenal increases their resistance when challenged with the diamondback moth Plutella xylostella L., while the phenomenon is largely lost in wrky46 mutant. Mechanically, E-2-hexenal could induce the expression of WRKY46 and MYC2, their physical interaction was verified using yeast two-hybrid, firefly luciferase complementation imaging, and in vitro pull-down assays. In turn, the WRKY46-MYC2 complex directly binds the promoter of RBOHD to promote its expression through LUC reporter assays, yeast one-hybrid, chromatin immunoprecipitation and electrophoretic mobility shift assays, which also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes (TT4 and CHIL) and the accumulation of total flavonoids, eventually modulates plant tolerance to insects. Together, our results highlight an important role for WRKY46-MYC2 module in the E-2-hexenal-induced defense response in Arabidopsis, providing new insights into how VOCs trigger defense responses in plants.