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Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.

ABSTRACT: Ostrinia furnacalis is an important borer on maize. Long-term and large-scale planting of transgenic corn has led O. furnacalis evolving resistance and reducing the control effect. Recently, high levels of resistance to Bt Cry1 toxins have been reported to be genetically linked to the mutation or down-regulation of ABC transporter subfamily G gene ABCG4 in O. furnacalis. In order to further determine the relationship between ABCG4 gene and the resistance to Cry1 toxins in O. furnacalis, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct ABCG4-KO knockout homozygous strain. Bioassay results indicated that an ABCG4-KO strain had a higher resistance to Cry1 proteins compared with a susceptible strain (ACB-BtS). The result indicates that the ABCG4 gene may act as a receptor of the Bt Cry1 toxin in O. furnacalis. Furthermore, the development time was significantly changed in the early stage ABCG4-KO larvae, and the population parameters were also significantly changed. In summary, our CRISPR/Cas9-mediated genome editing study presents evidence that ABCG4 gene is a functional receptor for Bt Cry1 toxins, laying the foundation for further clarification of the Bt resistance mechanism.

SUBMITTER: Gao Q

PROVIDER: S-EPMC8780026 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:The adoption of transgenic crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins has reduced insecticide application, increased yields, and contributed to food safety worldwide. However, the efficacy of transgenic Bt crops is put at risk by the adaptive resistance evolution of target pests. Previous studies indicate that resistance to Bacillus thuringiensis Cry1A and Cry1F toxins was genetically linked with mutations of ATP-binding cassette (ABC) transporter subfamily C gene ABCC2 in at least seven lepidopteran insects. Several strains selected in the laboratory of the Asian corn borer, Ostrinia furnacalis, a destructive pest of corn in Asian Western Pacific countries, developed high levels of resistance to Cry1A and Cry1F toxins. The causality between the O. furnacalisABCC2 (OfABCC2) gene and resistance to Cry1A and Cry1F toxins remains unknown. Here, we successfully generated a homozygous strain (OfC2-KO) of O. furnacalis with an 8-bp deletion mutation of ABCC2 by the CRISPR/Cas9 approach. The 8-bp deletion mutation results in a frame shift in the open reading frame of transcripts, which produced a predicted protein truncated in the TM4-TM5 loop region. The knockout strain OfC2-KO showed much more than a 300-fold resistance to Cry1Fa, and low levels of resistance to Cry1Ab and Cry1Ac (<10-fold), but no significant effects on the toxicities of Cry1Aa and two chemical insecticides (abamectin and chlorantraniliprole), compared to the background NJ-S strain. Furthermore, we found that the Cry1Fa resistance was autosomal, recessive, and significantly linked with the 8-bp deletion mutation of OfABCC2 in the OfC2-KO strain. In conclusion, in vivo functional investigation demonstrates the causality of the OfABCC2 truncating mutation with high-level resistance to the Cry1Fa toxin in O. furnacalis. Our results suggest that the OfABCC2 protein might be a functional receptor for Cry1Fa and reinforces the association of this gene to the mode of action of the Cry1Fa toxin.

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Knockout of ABC Transporter ABCG4 Gene Confers Resistance to Cry1 Proteins in Ostrinia furnacalis.

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