Functional redundancy in the toxic pathway of Bt protein Cry1Ab, but not Cry1Fa, against the Asian corn borer

Xingliang Wang, Yujin Yue, Yuqian Zhai, Falong Wang, Xuna Zhuang, Shuwen Wu, Yihua Yang, Bruce E. Tabashnik, and Yidong Wu

PNAS April 17 2025; 122 (16) e2503674122; https://doi.org/10.1073/pnas.2503674122

Significance

Crops genetically engineered to produce insect-killing proteins from the bacterium Bacillus thuringiensis (Bt) control some major pests and reduce use of insecticide sprays. However, evolution of resistance to Bt crops has diminished these benefits. Understanding of the genetic basis of resistance is needed to improve the ability to detect and counter pest resistance. We used gene editing to disrupt singly and in pairs three larval midgut proteins that mediate toxicity of Bt proteins in a major caterpillar pest, the Asian corn borer. The results show that toxicity occurs via two independent pathways for Bt protein Cry1Ab but only one for Cry1Fa. The functional redundancy of Cry1Ab could prolong its efficacy and may represent a natural strategy for delaying resistance.

Abstract

Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests, but their benefits decrease when pests evolve resistance. Better understanding of the genetic basis of resistance is needed to effectively monitor, manage, and counter pest resistance to Bt crops. Resistance to Bt proteins in at least 11 species of Lepidoptera, including many important crop pests, is associated with naturally occurring mutations that disrupt one or more of three larval midgut proteins: cadherin and ATP-binding cassette proteins ABCC2 and ABCC3. Here, we determined how CRISPR/Cas9-mediated mutations disrupting cadherin, ABCC2, and ABCC3 singly and in pairs affect resistance to Bt proteins Cry1Ab and Cry1Fa in the Asian corn borer (Ostrinia furnacalis), which is the most damaging pest of corn in Asia and is closely related to the European corn borer (Ostrinia nubilalis), a major pest in Europe and North America. The results from bioassays of six knockout strains and their parent susceptible strain support a model in which Cry1Ab can kill larvae via one path requiring ABCC2 or another path requiring cadherin and ABCC3, whereas Cry1Fa uses only the first path. The model’s predictions are generally supported by results from genetic linkage analyses and responses to Cry1Ab and Cry1Fa of Sf9 cells and Xenopus oocytes modified to produce cadherin, ABCC2, and ABCC3 singly or in pairs. The functional redundancy identified here for Cry1Ab could sustain its efficacy against O. furnacalis and may exemplify a widespread natural strategy for delaying resistance.

See https://www.pnas.org/doi/10.1073/pnas.2503674122

Figure 1. CRISPR/Cas9-mediated knockout of the genes OfABCC2, OfABCC3, or both on chromosome 15 of O. furnacalis. (A) Genomic structure of OfABCC2 and OfABCC3, each with 25 exons (blue and green boxes, respectively). Red lines and scissors show sgRNA target sites for knockouts: C2-sgRNA1 and C2-sgRNA2 for OfABCC2 (C2–KO), C3-sgRNA1 and C3-sgRNA2 for OfABCC3 (C3–KO), and C2-sgRNA1 and C3-sgRNA3 for both (C2+C3–KO). (B) Sequences for each knockout: wild-type (Top) and representative histograms from direct sequencing of PCR products after knockout (Bottom). In the wild-type sequences, the red letters show the protospacer adjacent motif (PAM) and the blue letters show the protospacer sequence. The paired dotted lines indicate where each deletion starts and ends in the wild-type sequence and where the deletion occurs in the knockout sequence.