Resistance toBacillus thuringiensisCry1Ac toxin requires mutations in twoPlutella xylostellaATP-binding cassette transporter paralogs

Author summary Bacillus thuringiensis(Bt) foliar sprays and transgenic crops expressing Bt toxins are used extensively to control insect pests, but the evolution of resistance limits their efficacy. Multiple studies have reported that ATP-binding cassette (ABC) transporters are important Bt receptors, and mutations in eitherABCC2orABCC3can lead to Cry1Ac-toxin resistance, although this process is not fully understood. In this study, we applied both forward and reverse genetic analyses to demonstrate that high-level Bt-Cry1Ac resistance inPlutella xylostellarequires concurrent mutations in bothPxABCC2andPxABCC3. We identified inactivating mutations in these two genes from a Cry1Ac-resistant strain (Cry1S1000) ofP.xylostellaand conducted genetic linkage analysis, which supported the role thatPxABCC2andPxABCC3were the causal genes of Cry1Ac resistance. We then knocked outPxABCC2andPxABCC3in aP.xylostellasusceptible reference strain (G88) to confirm that high-level Cry1Ac resistance requires mutation ofPxABCC2andPxABCC3, rather than a mutation of either one gene. This finding expands our understanding of complex Bt resistance processes and may be relevant to Bt-Cry1Ac resistance in other lepidopteran insects. The diamondback moth,Plutella xylostella, is a cosmopolitan pest and the first species to develop field resistance to toxins from the gram-positive bacteriumBacillus thuringiensis(Bt). Although previous work has suggested that mutations of ATP-binding cassette transporter subfamily C2 (ABCC2) or C3 (ABCC3) genes can confer Cry1Ac resistance, here we reveal thatP.xylostellarequires combined mutations in bothPxABCC2andPxABCC3to achieve high-level Cry1Ac resistance, rather than simply a mutation of either gene. We identified natural mutations ofPxABCC2andPxABCC3that concurrently occurred in a Cry1Ac-resistant strain (Cry1S1000) ofP.xylostella, with a mutation (R-A2) causing the mis-splicing ofPxABCC2and another mutation (R-A3) leading to the premature termination of PxABCC3. Genetic linkage analysis showed thatR(A2)andR(A3)were tightly linked to Cry1Ac resistance. Introgression ofR(A2)andR(A3)enabled a susceptible strain (G88) ofP.xylostellato obtain high resistance to Cry1Ac, confirming that these genes confer resistance. To further support the role ofPxABCC2andPxABCC3in Cry1Ac resistance, frameshift mutations were introduced intoPxABCC2andPxABCC3singly and in combination in the G88 strain with CRISPR/Cas9 mediated mutagenesis. Bioassays of CRISPR-based mutant strains, plus genetic complementation tests, demonstrated that the deletion ofPxABCC2orPxABCC3alone provided < 4-fold tolerance to Cry1Ac, while disruption of both genes together conferred >8,000-fold resistance to Cry1Ac, suggesting the redundant/complementary roles of PxABCC2 and PxABCC3. This work advances our understanding of Bt resistance inP.xylostellaby demonstrating mutations within bothPxABCC2andPxABCC3genes are required for high-level Cry1Ac resistance.