Structural andTranscriptional Plasticity of OBP3Confers Insecticide Adaptation in Spodoptera litura

This study reveals a dual defense role of odorant-binding protein OBP3 in Spodoptera litura, combing direct insecticide sequestration and stress-responsive transcriptional regulation. We found stage-specific OBP3 enrichment in larval chemosensory tissues (head/integument), with 2.51 to 16.56-fold upregulation following exposure to pyrethroids and GABAergics. Functional validation showed RNAi suppression increased insecticide susceptibility by 20–30.67%, while heterologous bacterial expression boosted tolerance over 3-fold. Fluorescence competitive binding assays demonstrated preferential affinity for fipronil/indoxacarb (<i>K</i>_i = 7.58–9.27 μM), over pyrethroids (<i>K</i>_i = 19.79–28.91 μM). Structural analyses uncovered OBP3′s multispecific binding architecture, employing conserved aromatic (Phe114/Phe127) and polar (Arg75) residues to sequester GABAergics. Transcriptional regulation studies identified CncC/MafK and AhR/ARNT as key drivers of insecticide-responsive <i>OBP3</i> expression, elevating promoter activity by 1.84- and 1.71-fold, respectively. These findings establish olfactory proteins as frontline resistance mediators and propose targeted pest control through disrupting OBP3′s ligand-binding pocket or inhibiting defense regulatory network.