CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to Bacillus thuringiensis Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.)
CRISPR/Cas9-mediated knockout of both the PxABCC2 and PxABCC3 genes confers high-level resistance to Bacillus thuringiensis Cry1Ac toxin in the diamondback moth, Plutella xylostella (L.)
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CRISPR/Cas9 介导的 PxABCC2 和 PxABCC3 基因敲除赋予小菜蛾 Plutella xylostella (L.) 对苏云金芽孢杆菌 Cry1Ac 毒素的高水平抗性
DOI：
10.1016/j.ibmb.2019.01.009
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发表时间：
2019-04-01
影响因子：
3.8
通讯作者：
Zhang, Youjun
中科院分区：
文献类型：
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作者：
Guo, Zhaojiang;Sun, Dan;Zhang, Youjun
害虫抗药性的快速进化严重危害了生物农药和转基因作物的可持续利用，这些作物产生源自昆虫病原细菌苏云金芽孢杆菌（Bt）的杀虫晶体蛋白。最近，据报道，包括小菜蛾 (L.) 在内的 7 种鳞翅目昆虫对 Bt Cry1 毒素的高水平抗性与 ABC 转运蛋白亚家族 C 基因 ABCC2 和 ABCC3 的突变或下调有关。为了进一步确定小菜蛾PxABCC2和PxABCC3基因的改变与Cry1Ac抗性之间的因果关系，利用新型CRISPR/Cas9基因组工程系统成功构建了两个敲除菌株：ABCC2KO菌株为PxABCC2基因外显子3 4 bp缺失的纯合菌株，ABCC3KO菌株为外显子5 bp缺失的纯合菌株。 PxABCC3 基因的 3 个，两者都只能产生截短的 ABCC 蛋白。生物测定结果表明，与原始易感 DBM1Ac-S 菌株相比，ABCC2K0（724 倍）和 ABCC3KO（413 倍）菌株对 Cry1Ac 原毒素具有高水平的抗性。随后，显性度和遗传互补测试表明，两个敲除菌株中的Cry1Ac抗性都是不完全隐性的，Cry1Ac抗性等位基因位于包含PxABCC2和PxABCC3基因的经典BtR-1抗性位点，与近等基因抗性NIL-R菌株相似。此外，定性毒素结合测定表明，与易感 DBM1Ac-S 菌株相比，两种敲除菌株中 Cry1Ac 毒素与中肠刷状缘膜囊泡 (BBMV) 的结合显着减少。总之，我们的 CRISPR/Cas9 介导的基因组编辑研究首次提供了 ABCC2 和 ABCC3 蛋白作为昆虫 Bt Cry1 毒素中肠功能受体的体内反向遗传学证据，这为了解 ABCC2 和 ABCC3 蛋白在昆虫抗 Bt Cry1 毒素的复杂分子机制中的关键作用提供了新的见解。
Rapid evolution of resistance by insect pests severely jeopardizes the sustainable utilization of biopesticides and transgenic crops that produce insecticidal crystal proteins derived from the entomopathogenic bacterium Bacillus thuringiensis (Bt). 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 C genes ABCC2 and ABCC3 in seven lepidopteran insects, including Plutella xylostella (L.). To further determine the causal relationship between alterations in the PxABCC2 and PxABCC3 genes and Cry1Ac resistance in P. xylostella, the novel CRISPR/Cas9 genome engineering system was utilized to successfully construct two knockout strains: the ABCC2KO strain is homo-zygous for a 4-bp deletion in exon 3 of the PxABCC2 gene, and the ABCC3KO strain is homozygous for a 5-bp deletion in exon 3 of the PxABCC3 gene, both of which can produce only truncated ABCC proteins. Bioassay results indicated that high levels of resistance to the Cry1Ac protoxin were observed in both the ABCC2K0 (724 fold) and ABCC3KO (413-fold) strains compared to the original susceptible DBM1Ac-S strain. Subsequently, dominance degree and genetic complementation tests demonstrated that Cry1Ac resistance in both the knockout strains was incompletely recessive, and Cry1Ac resistance alleles were located in the classic BtR-1 resistance locus that harbored the PxABCC2 and PxABCC3 genes, similar to the near-isogenic resistant NIL-R strain. Moreover, qualitative toxin binding assays revealed that the binding of the Cry1Ac toxin to midgut brush border membrane vesicles (BBMVs) in both knockout strains was dramatically reduced compared to that in the susceptible DBM1Ac-S strain. In summary, our CRISPR/Cas9-mediated genome editing study presents, for the first time, in vivo reverse genetics evidence for both the ABCC2 and ABCC3 proteins as midgut functional receptors for Bt Cry1 toxins in insects, which provides new insight into the pivotal roles of both the ABCC2 and ABCC3 proteins in the complex molecular mechanism of insect resistance to Bt Cry1 toxins.