Bradysia odoriphaga is a devastating soil pest of Allium tuberosum (Chinese chive), and current control relies heavily on chemical insecticides. Cry39Ab1 toxins from Bacillus thuringiensis (Bt), which are highly toxic to B. odoriphaga, offer an environmentally friendly alternative. However, its mechanism of action remains unclear. In this study, we identified the involvement of vacuolar H+-ATPase subunit A (V-ATPase A) in Cry39Ab1 insecticidal activities. The full-length cDNA sequences of BoV-ATPase A was contained 1659 bp open reading frame (ORF), encoding a protein of 552 amino acids with a calculated molecular weight of 59 kDa and an isoelectric point of 9.11. Successful expression and purification of BoV-ATPase A (with GST and His tags) and Cry39Ab1 (with GST and His tags) proteins were achieved. GST pull-down assays demonstrated a direct interaction between recombinant BoV-ATPase A and activated Cry39Ab1 toxin in vitro. Heterologous expression of BoV-ATPase A in Cry-insensitive Sf9 cells conferred susceptibility to Cry39Ab1, resulting in a significant increase in cytolysis compared with control cells. Finally, RNAi-mediated knockdown of BoV-ATPase A in larvae significantly decreased their susceptibility to Cry39Ab1, as evidenced by a marked decrease in mortality. This is the first report that BoV-ATPase A is a key protein required for Cry39Ab1 toxicity, revealing its insecticidal mechanism and establishing BoV-ATPase A as a potential target for pest control.
Feng et al. (Fri,) studied this question.