Structure-Based Screening Combined with Functional Validation Identifies BtGSTs6-Mediated Neonicotinoid Detoxification in Bemisia tabaci

Abstract Neonicotinoid resistance in the whitefly Bemisia tabaci causes substantial agricultural and economic losses worldwide. Although glutathione S-transferases (GSTs) are key phase II detoxification enzymes, their contribution to neonicotinoid resistance in this pest remains poorly defined. Here, we screened 23 GST genes against neonicotinoids using a “static screening + dynamic validation” strategy. Molecular docking and molecular dynamics simulations were used for candidate prioritization, followed by validation with RT-qPCR, RNAi, transgenic Drosophila, and UPLC-MS/MS. BtGSTs6 emerged as the principal candidate and was overexpressed in resistant populations. RNAi-mediated knockdown of BtGSTs6 increased susceptibility, whereas transgenic flies showed enhanced resistance to imidacloprid, thiamethoxam, and clothianidin. UPLC-MS/MS further confirmed that BtGSTs6 depleted the three insecticides to different extents, indicating substrate-dependent detoxification. These findings establish BtGSTs6 as a key mediator of neonicotinoid resistance and a promising target for resistance management and sustainable whitefly control.