Synergistic resistance of Helicoverpa armigera to Bt toxins linked to cadherin and ABC transporters mutations

Abstract

Resistance evolution of target pests reduces efficacy of Bacillus thuringiensis Cry toxins used in insect-pest control. Mutations in Cadherin (CAD) or ATP-binding cassette (ABC) transporters genes are linked to Cry resistance in different pests. Also, it has been shown that ABCC2 and CAD have synergistic interaction on Cry toxicity when co-express in cell lines, which we confirmed here by Helicoverpa armigera HaABCC2 and HaCAD expression in Hi5 cells. To confirm that CAD and ABC transporters interact in vivo, we constructed nearly H. armigera isogenic lines such as LFC2 and 96CAD strains, linked to HaABCC2 and HaCAD mutations that showed 512- and 396-fold Cry1Ac resistance-ratios, respectively. Interestingly, Fusion-1 strain linked to both HaABCC2 and HaCAD mutations, showed 6273-fold resistance-ratio, significantly higher than the single mutant strains. To confirm the interaction of HaABCC2 and CAD in Cry1Ac resistance, we analyzed the Cry1Ac susceptibility in CRISPR/Cas9 knockdown strains, C2-KO (ABCC2-gene knockout-strain) and CAD-KO (CAD-gene knockout-strain), that showed 112- and 531-fold Cry1Ac resistance-ratios, respectively. However, the resistance-ratio of Fusion-2 strain obtained from crossing C2-KO and CAD-KO strains, was only 816-fold. The analysis of HaABCC3 gene transcript levels showed nearly 4-fold lower expression in LFC2 and Fusion-1 strains compared to the susceptible strain, suggesting that additional mutations in these strains resulted in low HaABCC3 expression, which contribute to their enhanced Cry1Ac resistance. Our data show that the CAD and ABCC2/ABCC3 interact synergistically to induce high Cry1Ac resistance in H. armigera. These results can be helpful for Bt resistance monitoring and pest management.

Keywords: ABC transporters; Bt resistance; Cadherin; Helicoverpa armigera.