Functional redundancy of two ABC transporter proteins in mediating toxicity of Bacillus thuringiensis to cotton bollworm

Abstract

Evolution of pest resistance reduces the efficacy of insecticidal proteins from the gram-positive bacterium Bacillus thuringiensis (Bt) used widely in sprays and transgenic crops. Better understanding of the genetic basis of resistance is needed to more effectively monitor, manage, and counter pest resistance to Bt toxins. Here we used CRISPR/Cas9 gene editing to clarify the genetics of Bt resistance and the associated effects on susceptibility to other microbial insecticides in one of the world’s most damaging pests, the cotton bollworm ( Helicoverpa armigera). We discovered that CRISPR-mediated knockouts of ATP-binding cassette (ABC) transporter genes HaABCC2 and HaABCC3 together caused >15,000-fold resistance to Bt toxin Cry1Ac, whereas knocking out either HaABCC2 or HaABCC3 alone had little or no effect. Inheritance of resistance was autosomal and recessive. Bioassays of progeny from interstrain crosses revealed that one wild type allele of either HaABCC2 or HaABCC3 is sufficient to sustain substantial susceptibility to Cry1Ac. In contrast with previous results, susceptibility to two insecticides derived from bacteria other than Bt (abamectin and spinetoram), was not affected by knocking out HaABCC2, HaABCC3, or both. The results here provide the first evidence that either HaABCC2 or HaABCC3 protein is sufficient to confer substantial susceptibility to Cry1Ac. The functional redundancy of these two proteins in toxicity of Cry1Ac to H. armigera is expected to reduce the likelihood of field-evolved resistance relative to disruption of a toxic process where mutations affecting a single protein can confer resistance.

Author summary

Insect-killing proteins from the soil bacterium Bacillus thuringiensis (Bt) are increasingly important as environmentally friendly alternatives to conventional insecticides. However, widespread planting for the past two decades of crops genetically engineered to produce Bt proteins has spurred rapid evolution of resistance in pests. Better understanding of the genetic basis of pest resistance to Bt toxins is urgently needed to address this problem. We used CRISPR gene editing to clarify the genetics of resistance to Bt toxins in the cotton bollworm, one of the world’s most damaging pests. We discovered that CRISPR-mediated inactivation of two larval midgut transporter proteins that act as receptors for Bt toxins caused a 15,000-fold increase in the concentration of Bt toxin needed to kill fifty per cent of larvae. By contrast, inactivating each of these transporter proteins alone had little or no effect. These results provide the first evidence that either of these two transporter proteins can confer susceptibility to Bt toxins. Because resistance to Bt toxins requires mutations in each of the two genes encoding these two proteins, pest resistance is less likely to evolve via this route relative to interference with toxic processes where a single mutation can confer resistance.