Xiao Tianxiang, Deng Mengqing, Huang Xiaodan, Wang Wenxiu, Xu Xiyue, Zhao Xinyu, Li Jun, Jiang Yingjie, Pan Bo, He Ziyu, Yang Zhiming, Sun Zhongxiang, Lu Kai
Key Laboratory of Agri-products Quality and Biosafety (Ministry of Education), Anhui Province Key Laboratory of Crop Integrated Pest Management, School of Plant Protection, Anhui Agricultural University, Hefei, 230036, China.
State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Plant Protection, Yunnan Agricultural University, Kunming, 650201, China.
Insect Biochem Mol Biol. 2025 May;180:104299. doi: 10.1016/j.ibmb.2025.104299. Epub 2025 Mar 10.
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.
通过与寄主植物的长期共同进化，昆虫进化出了复杂的解毒系统来对抗植物次生代谢产物（PSMs）。然而，这些适应性背后的确切机制仍未完全明确。我们之前的研究确定了ε-谷胱甘肽S-转移酶（GSTes）是斜纹夜蛾对花椒毒素适应的关键介质，花椒毒素是一种典型的线性呋喃香豆素。本研究表明，在大肠杆菌中异源过表达五种对花椒毒素有反应的GSTes可显著增强细菌对这种PSM的耐受性。纸片扩散法和代谢研究表明，GSTe1和GSTe16通过涉及抗氧化活性和催化代谢的双重机制介导对花椒毒素的适应。荧光竞争性结合实验表明，所有五种GSTes都具有非催化性的花椒毒素螯合能力。这些体外观察结果通过对GSTe16的体内基因操作得到了补充，GSTe16对花椒毒素表现出最强的防御活性。CRISPR/Cas9介导的斜纹夜蛾GSTe16基因敲除显著增加了幼虫对花椒毒素的易感性，而过量表达GSTe16的转基因黑腹果蝇对花椒毒素的耐受性增强。此外，接触花椒毒素会激发20-羟基蜕皮激素（20E）的内源性生物合成，施用20E可增强幼虫对花椒毒素的耐受性以及GSTe16的表达水平。双荧光素酶报告基因检测在GSTe16启动子中鉴定出两个功能性顺式调控元件，它们可促进蜕皮激素受体（EcR）/超气门蛋白（USP）异二聚体的转录激活。总体而言，本研究阐明了斜纹夜蛾中GSTes对花椒毒素的生化防御特性和转录反应，为食草昆虫对抗寄主植物的防御机制提供了新的见解。
