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 in response to pest attack.E-2-hexenal is an important component of VOCs,but it is unclear whether it can induce endog-enous plant resistance to insects.Here,we show that E-2-hexenal activates early signaling events in Ara-bidopsis(Arabidopsis thaliana)mesophyll cells,including an H2O2 burst at the plasma membrane,the directedflow 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 diamond-back moth Plutella xylostella L.,and this phenomenon is largely lost in the wrky46 mutant.Mechanistically,E-2-hexenal induces the expression of WRKY46 and MYC2,and the physical interaction of their encoded proteins was verified by yeast two-hybrid,firefly luciferase complementation imaging,and in vitro pull-down assays.The WRKY46–MYC2 complex directly binds to the promoter of RBOHD to promote its expres-sion,as demonstrated by luciferase reporter,yeast one-hybrid,chromatin immunoprecipitation,and electrophoretic mobility shift assays.This module also positively regulates the expression of E-2-hexenal-induced naringenin biosynthesis genes(TT4 and CHIL)and the accumulation of totalflavonoids,thereby modulating plant tolerance to insects.Together,our results highlight an important role for the WRKY46–MYC2 module in the E-2-hexenal-induced defense response of Arabidopsis,providing new in-sights into the mechanisms by which VOCs trigger plant defense responses.