BACKGROUND: Biopesticides and transgenic crops based on Bacillus thuringiensis (Bt) toxins are extensively used to control insect pests, but the rapid evolution of insect resistance seriously threatens their effectiveness. Bt resistance is often polygenic and complex. Mutations that confer resistance occur in midgut proteins that act as cell surface receptors for the toxin, and it is thought they facilitate its assembly as a membrane-damaging pore. However, the mechanistic details of the action of Bt toxins remain controversial.

RESULTS: We have examined the contribution of two paralogous ABC transporters and two aminopeptidases N to Bt Cry1Ac toxicity in the diamondback moth, Plutella xylostella, using CRISPR/Cas9 to generate a series of homozygous polygenic knockout strains. A double-gene knockout strain, in which the two paralogous ABC transporters ABCC2 and ABCC3 were deleted, exhibited 4482-fold resistance to Cry1A toxin, significantly greater than that previously reported for single-gene knockouts and confirming the mutual functional redundancy of these ABC transporters in acting as toxin receptors in P. xylostella. A double-gene knockout strain in which APN1 and APN3a were deleted exhibited 1425-fold resistance to Cry1Ac toxin, providing the most direct evidence to date for these APN proteins acting as Cry1Ac toxin receptors, while also indicating their functional redundancy. Genetic crosses of the two double-gene knockouts yielded a hybrid strain in which all four receptor genes were deleted and this resulted in a > 34,000-fold resistance, indicating that while both types of receptor need to be present for the toxin to be fully effective, there is a level of functional redundancy between them. The highly resistant quadruple knockout strain was less fit than wild-type moths, but no fitness cost was detected in the double knockout strains.

CONCLUSION: Our results provide direct evidence that APN1 and APN3a are important for Cry1Ac toxicity. They support our overarching hypothesis of a versatile mode of action of Bt toxins, which can compensate for the absence of individual receptors, and are consistent with an interplay among diverse midgut receptors in the toxins’ mechanism of action in a super pest.

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Published in BMC Biology, v. 20, article no. 33.

© 2022 The Author(s)

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This work was supported by the Laboratory of Lingnan Modern Agriculture Project (NT2021003), the National Natural Science Foundation of China (32022074; 32172458), the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables, and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-ASTIP-IVFCAAS).

12915_2022_1226_MOESM1_ESM.docx (2404 kB)
Additional file 1: Figure S1. Diagram of the crossing strategy to obtain homozygous double knockout strains in P. xylostella. Figure S2. Diagram of the crossing strategy used for generation of the genetic hybrid strain. Table S1. Primers used in this study. Table S2. Toxicity to Cry1Ac toxin in larvae from the polygenic knockout strains and their F1 progeny.

12915_2022_1226_MOESM2_ESM.xlsx (1198 kB)
Additional file 2. The raw data of all the figures and statistical analyses in this study.