Grapholita molesta V-ATPase D acts as a susceptibility determinant of larvae to Bt Cry2Ab toxin

Grapholita molesta is a globally significant and destructive pest, particularly notorious for its impact on the fruit industry. Cry2Ab is a crucial alternative Bt protein used to manage the resistance to Cry1A toxins, and it is effective against a range of Lepidoptera pests. Previous studies have confirmed that subunits A, B, C, and E of vacuolar H⁺-ATPase are involved in the insecticidal efficacy of Bt. Based on the previous LC-MS/MS results, V-ATPase D was selected in this study to clarify its role in the toxic effect of Cry2Ab on G. molesta. Exposure of G. molesta larvae to Cry2Ab resulted in a marked reduction in GmolV-ATPase D transcript levels was observed. Further experiments validated the binding of GmolV-ATPase D to the Cry2Ab toxin, as evidenced by homologous and heterologous competition assays. The susceptibility of Sf9 cells to Cry2Ab was enhanced by the heterologous expression of GmolV-ATPase D, whereas Cry2Ab-induced lethality in G. molesta larvae was diminished by the knockdown of GmolV-ATPase D using dsRNA. Moreover, in vivo bioassays demonstrated that G. molesta larvae consuming a diet containing E.coli-expressed GmolV-ATPase D protein and Cry2Ab protoxin experienced notably elevated mortality compared to those exposed to Cry2Ab alone. These findings reveal that V-ATPase D serves as a susceptibility determinant for Cry2Ab and plays an essential role in mediating Cry2Ab toxicity in G. molesta. These findings shed light on the insecticidal mechanism of Cry2Ab and offer valuable guidance for the developing protein-targeting biopesticides.