V-ATPase B mediates Bt Cry1Ac binding and toxicity in Grapholita molesta

Grapholita molesta is a worldwide pest. Cry1Ac is a significant alternative Bacillus thuringiensis (Bt) protein that exhibits substantial toxicity towards Lepidoptera insects. This investigation employed ligand blot, SDS-PAGE and liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques to identify the proteins potentially binding Cry1Ac on the peritrophic membrane (PM) of G. molesta. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis revealed the highest expression of GmolV-ATPase B in the midgut of 4th instar larvae. Following the ingestion of Cry1Ac protoxin by G. molesta larvae, a notable reduction in the expression level of GmolV-ATPase B was observed. The interaction between GmolV-ATPase B and activated Cry1Ac toxin was confirmed through ligand blot and homologous and heterologous competition experiments. Overexpression of GmolV-ATPase B in Sf9 cells led to an increase in Cry1Ac cytotoxicity, while RNAi targeting GmolV-ATPase B resulted in reduced mortality. In vivo bioassays demonstrated that the combined action of GmolV-ATPase B protein and Cry1Ac protoxin significantly enhanced the toxicity of Cry1Ac towards G. molesta larvae compared to Cry1Ac alone. These findings shed light on the binding of Cry1Ac to PM of G. molesta and its insecticidal mechanism, offering a valuable important reference for the development of biopesticides targeting midgut PM proteins.