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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.).
Insect Biochem Mol Biol. 2019 Apr; 107:31-38.IB

Abstract

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 homozygous 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 ABCC2KO (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.

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Publisher Full Text (DOI)

Authors+Show Affiliations

Guo Z
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: guozhaojiang@caas.cn.
Sun D
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: sun13804560684@163.com.
Kang S
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: kangshi0718@163.com.
Zhou J
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: zhoujunlei2006@126.com.
Gong L
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: gonglijun025@163.com.
Qin J
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: qinjianying0203@163.com.
Guo L
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: guole930323@163.com.
Zhu L
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: liuhongzhu1992@126.com.
Bai Y
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: baiyang15765533722@163.com.
Luo L
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: luoliang10090419@163.com.
Zhang Y
Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing, 100081, China. Electronic address: zhangyoujun@caas.cn.

MeSH

AnimalsBacillus thuringiensisBacillus thuringiensis ToxinsBacterial ProteinsBase SequenceCRISPR-Cas SystemsEndotoxinsGene Knockout TechniquesHemolysin ProteinsInsect ProteinsInsecticide ResistanceInsecticidesLarvaMothsMultidrug Resistance-Associated Protein 2Multidrug Resistance-Associated Proteins

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30710623

Citation

Guo, Zhaojiang, et al. "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.)." Insect Biochemistry and Molecular Biology, vol. 107, 2019, pp. 31-38.
Guo Z, Sun D, Kang S, et al. 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.). Insect Biochem Mol Biol. 2019;107:31-38.
Guo, Z., Sun, D., Kang, S., Zhou, J., Gong, L., Qin, J., Guo, L., Zhu, L., Bai, Y., Luo, L., & Zhang, Y. (2019). 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.). Insect Biochemistry and Molecular Biology, 107, 31-38. https://doi.org/10.1016/j.ibmb.2019.01.009
Guo Z, et al. 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.). Insect Biochem Mol Biol. 2019;107:31-38. PubMed PMID: 30710623.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - 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.). AU - Guo,Zhaojiang, AU - Sun,Dan, AU - Kang,Shi, AU - Zhou,Junlei, AU - Gong,Lijun, AU - Qin,Jianying, AU - Guo,Le, AU - Zhu,Liuhong, AU - Bai,Yang, AU - Luo,Liang, AU - Zhang,Youjun, Y1 - 2019/01/30/ PY - 2018/11/23/received PY - 2018/12/18/revised PY - 2019/1/29/accepted PY - 2019/2/3/pubmed PY - 2019/12/18/medline PY - 2019/2/3/entrez KW - ABC transporter KW - Bacillus thuringiensis KW - CRISPR/Cas9 KW - Cry1Ac resistance KW - Plutella xylostella SP - 31 EP - 38 JF - Insect biochemistry and molecular biology JO - Insect Biochem Mol Biol VL - 107 N2 - 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 homozygous 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 ABCC2KO (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. SN - 1879-0240 UR - https://www.unboundmedicine.com/medline/citation/30710623/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___ DB - PRIME DP - Unbound Medicine ER -