Functional analysis of the epsilon glutathione S-transferases in the adaptation of Spodoptera litura to xanthotoxin

Through long-term coevolution with host plants, insects have evolved sophisticated detoxification systems to counteract plant secondary metabolites (PSMs). However, the precise mechanisms underlying these adaptations remain incompletely characterized. Our previous research identified epsilon glutathione S-transferases (GSTes) as critical mediators of xanthotoxin adaptation in Spodoptera litura, a model linear furanocoumarin. This study reveals that heterologous overexpression of five xanthotoxin-responsive GSTes in Escherichia coli significantly enhances bacterial tolerance to this PSM. Disk diffusion assays and metabolic studies demonstrated that both GSTe1 and GSTe16 mediate xanthotoxin adaptation via dual mechanisms involving antioxidant activity and catalytic metabolism. Fluorescent competitive binding experiments indicated that all five GSTes exhibit non- catalytic xanthotoxin sequestration capabilities. These in vitro observations were complemented by in vivo genetic manipulation of GSTe16, which exhibited the most potent defense activity against xanthotoxin. CRISPR/ Cas9-mediated GSTe16 knockout in S. litura significantly increased larval susceptibility to xanthotoxin, while transgenic Drosophila melanogaster overexpressing GSTe16 showed enhanced tolerance to xanthotoxin. Furthermore, the endogenous biosynthesis of 20-hydroxyecdysone (20E) was provoked upon exposure to xanthotoxin, and 20E application enhanced the larval tolerance to xanthotoxin as well as the expression levels of GSTe16. Dual-luciferase reporter assays identified two functional cis-regulatory elements in the GSTe16 promoter that facilitate transcriptional activation by the ecdysone receptor (EcR)/ultraspiracle (USP) heterodimer. Overall, this study elucidates the biochemical defense characteristics and transcriptional responses of GSTes to xanthotoxin in S. litura, providing novel insights into the counter-defense mechanisms of herbivorous insects against host plants.