ATP synthase subunit γ mediates Cry1Ac binding and toxicity in Grapholita molesta.

The insect midgut peritrophic membrane (PM) plays important roles in insect-microbe interactions. Bacillus thuringiensis (Bt) and its proteinaceous toxins are widely used for insect control. To understand the role of PM in insects against Bt toxins, this study selected Grapholita molesta Busck (Lepidoptera: Tortricidae), a worldwide pest infesting fruit trees, as the research subject. Ligand blotting coupled with mass spectrometry confirmed the binding of this protein to Cry1Ac on the PM. RT-qPCR analysis revealed that GmolATPs-γ was primarily expressed in the 4th instar larvae, with significantly reduced transcript levels following Cry1Ac exposure. Ligand blotting combined with homologous and heterologous competition assays confirmed specific, physical binding between GmolATPs-γ and activated Cry1Ac toxin. Heterologous expression of GmolATPs-γ in Sf9 cells conferred increased susceptibility to Cry1Ac. The expression of the GmolATPs-γ gene was significantly decreased after RNAi. Concurrently, larval mortality decreased significantly when larvae were treated with Cry1Ac after RNAi. In vivo experiments demonstrated that co-administration of Cry1Ac with GmolATPs-γ protein enhanced larval mortality compared to Cry1Ac alone. These results indicate that GmolATPs-γ binds Cry1Ac and modulates its toxicity, providing theoretical foundations for the future use of Bt in controlling G. molesta in the field.