基于CRISPR/Cas9技术的PxABCB1基因功能鉴定揭示了其在小菜蛾对Cry1Ac、阿维菌素和甲维盐抗性中的作用

doi:10.1016/j.jia.2023.05.023

Journal of Integrative Agriculture ›› 2023, Vol. 22 ›› Issue (10): 3090-3102.DOI: 10.1016/j.jia.2023.05.023

基于CRISPR/Cas9技术的PxABCB1基因功能鉴定揭示了其在小菜蛾对Cry1Ac、阿维菌素和甲维盐抗性中的作用

收稿日期:2022-12-15 接受日期:2023-02-28 出版日期:2023-10-20 发布日期:2023-10-07

CRISPR/Cas9-based functional characterization of PxABCB1 reveals its roles in the resistance of Plutella xylostella (L.) to Cry1Ac, abamectin and emamectin benzoate

OUYANG Chun-zheng^{1, 2*}, YE Fan^1*, WU Qing-jun¹, WANG Shao-li¹, Neil CRICKMORE³, ZHOU Xu-guo⁴, GUO Zhao-jiang^1#, ZHANG You-jun^1#

¹ State Key Laboratory of Vegetable Biobreeding, Deapartment of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China
²Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642, P.R.China
³ School of Life Sciences, University of Sussex, Brighton BN1 9QG, UK
⁴Department of Entomology, University of Kentucky, Lexington, KY 40546-0091, USA

Received:2022-12-15 Accepted:2023-02-28 Online:2023-10-20 Published:2023-10-07
About author:OUYANG Chun-zheng, E-mail: ouyangchunzheng12@163.com; YE Fan, E-mail: yefan9605@163.com; #Correspondence GUO Zhao-jiang, Tel: +86-10-82109518, E-mail: guozhaojiang@caas.cn; ZHANG You-jun, E-mail: zhangyoujun@caas.cn * These authors contributed equally to this study.
Supported by:
This work was supported by the Laboratory of Lingnan Modern Agriculture Project, China (NT2021003), the National Natural Science Foundation of China (32022074, 32221004 and 32172458), the Beijing Key Laboratory for Pest Control and Sustainable Cultivation of Vegetables, Chinese Academy of Agricultural Sciences, and the Innovation Program of the Chinese Academy of Agricultural Sciences (CAAS-CSCB-202303).

摘要/Abstract

摘要：

鉴定苏云金芽胞杆菌(Bacillus thuringiensis, Bt)杀虫蛋白的中肠功能性受体对于解析昆虫Bt抗性的分子机制至关重要。前期研究发现，PxABCB1基因的表达下调与小菜蛾(Plutella xylostella)对Bt Cry1Ac杀虫蛋白产生抗性密切相关。为了进一步验证PxABCB1是否为小菜蛾Bt Cry1Ac的潜在受体及其在Bt Cry1Ac毒性中的作用，我们利用CRISPR/Cas9技术构建了一个纯合突变种群(ABCB1KO)，该种群小菜蛾个体中PxABCB1基因的第3外显子区域有5个碱基的缺失。ABCB1KO种群小菜蛾个体对Bt Cry1Ac杀虫蛋白的抗性是敏感种群(DBM1Ac-S)的63倍。更有趣的是，ABCB1KO种群小菜蛾个体对阿维菌素和甲维盐的敏感性也显著增加，但未发现其对Bt Cry蛋白或另外多种杀虫剂的敏感性变化。而且，ABCB1KO种群不存在明显的适应度代价。综上所述，我们的研究表明，PxABCB1既可以保护昆虫免受阿维菌素类杀虫剂的伤害，又能促进Bt Cry1Ac杀虫蛋白对昆虫的毒害作用。这项研究有助于提供针对小菜蛾这种破坏性害虫的综合害虫管理方法。

Abstract:

The identification of functional midgut receptors for pesticidal proteins produced by Bacillus thuringiensis (Bt) is critical for deciphering the molecular mechanism of Bt resistance in insects. Reduced expression of the PxABCB1 gene was previously found to be associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). To directly validate the potential receptor role of PxABCB1 and its contribution to Bt Cry1Ac toxicity in P. xylostella, we used CRISPR/Cas9 to generate a homozygous knockout ABCB1KO strain with a 5-bp deletion in exon 3 of its gene. The ABCB1KO strain exhibited a 63-fold resistance to Cry1Ac toxin compared to the parental DBM1Ac-S strain. Intriguingly, the ABCB1KO strain also exhibited significant increases in susceptibility to abamectin and emamectin benzoate. No changes in susceptibility to various other Bt Cry proteins or synthetic insecticides were observed. The knockout strain exhibited no significant fitness costs. Overall, our study indicates that PxABCB1 can protect the insect against avermectin insecticides on one hand, while on the other hand it facilitates the toxic effect of the Bt Cry1Ac toxin. The results of this study will help to inform integrated pest management approaches against this destructive pest.

Key words: Bacillus thuringiensis , Plutella xylostella , CRISPR/Cas9 , ABCB1 , bioinsecticide resistance

OUYANG Chun-zheng, YE Fan, WU Qing-jun, WANG Shao-li, Neil CRICKMORE, ZHOU Xu-guo, GUO Zhao-jiang, ZHANG You-jun. 基于CRISPR/Cas9技术的PxABCB1基因功能鉴定揭示了其在小菜蛾对Cry1Ac、阿维菌素和甲维盐抗性中的作用[J]. Journal of Integrative Agriculture, 2023, 22(10): 3090-3102.

OUYANG Chun-zheng, YE Fan, WU Qing-jun, WANG Shao-li, Neil CRICKMORE, ZHOU Xu-guo, GUO Zhao-jiang, ZHANG You-jun. CRISPR/Cas9-based functional characterization of PxABCB1 reveals its roles in the resistance of Plutella xylostella (L.) to Cry1Ac, abamectin and emamectin benzoate[J]. Journal of Integrative Agriculture, 2023, 22(10): 3090-3102.

参考文献

Adang M J, Crickmore N, Jurat-Fuentes J L. 2014. Diversity of Bacillus thuringiensis crystal toxins and mechanism of action. Advances in Insect Physiology, 47, 39–87.

Adegawa S, Nakama Y, Endo H, Shinkawa N, Kikuta S, Sato R. 2017. The domain II loops of Bacillus thuringiensis Cry1Aa form an overlapping interaction site for two Bombyx mori larvae functional receptors, ABC transporter C2 and cadherin-like receptor. Biochimica et Biophysica Acta, 1865, 220–231.

Aurade R M, Jayalakshmi S K, Sreeramulu K. 2010. P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function. Biochimica et Biophysica Acta, 1798, 1135–1143.

Bravo A, Likitvivatanavong S, Gill S S, Soberón M. 2011. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 41, 423–431.

Chen L P, Wang P, Sun Y J, Wu Y J. 2016. Direct interaction of avermectin with epidermal growth factor receptor mediates the penetration resistance in Drosophila larvae. Open Biology, 6, 150231.

Dean M, Moitra K, Allikmets R. 2022. The human ATP-binding cassette (ABC) transporter superfamily. Human Mutation, 43, 1162–1182.

Endo H. 2022. Molecular and kinetic models for pore formation of Bacillus thuringiensis Cry toxin. Toxins, 14, 433.

Epis S, Porretta D, Mastrantonio V, Urbanelli S, Sassera D, De Marco L, Mereghetti V, Montagna M, Ricci I, Favia G, Bandi C. 2014. Temporal dynamics of the ABC transporter response to insecticide treatment: insights from the malaria vector Anopheles stephensi. Scientific Reports, 4, 7435.

Figueira-Mansur J, Ferreira-Pereira A, Mansur J F, Franco T A, Alvarenga E S L, Sorgine M H F, Neves B C, Melo A C A, Leal W S, Masuda H, Moreira M F. 2013. Silencing of P-glycoprotein increases mortality in temephos-treated Aedes aegypti larvae. Insect Molecular Biology, 22, 648–658.

Furlong M J, Wright D J, Dosdall L M. 2013. Diamondback moth ecology and management: problems, progress, and prospects. Annual Review of Entomology, 58, 517–541.

Gong L, Kang S, Zhou J, Sun D, Guo L, Qin J, Zhu L, Bai Y, Ye F, Akami M, Wu Q, Wang S, Xu B, Yang Z, Bravo A, Soberon M, Guo Z, Wen L, Zhang Y. 2020. Reduced expression of a novel midgut trypsin gene involved in protoxin activation correlates with Cry1Ac resistance in a laboratory-selected strain of Plutella xylostella (L.). Toxins, 12, 15.

Gott R C, Kunkel G R, Zobel E S, Lovett B R, Hawthorne D J. 2017. Implicating ABC transporters in insecticide resistance: Research strategies and a decision framework. Journal of Economic Entomology, 110, 667–677.

Güney G, Cedden D, Hänniger S, Heckel D G, Coutu C, Hegedus D D, Mutlu D A, Suludere Z, Sezen K, Güney E, Toprak U. 2021. Silencing of an ABC transporter, but not a cadherin, decreases the susceptibility of Colorado potato beetle larvae to Bacillus thuringiensis ssp. tenebrionis Cry3Aa toxin. Archives of Insect Biochemistry and Physiology, 108, e21834.

Guo L, Cheng Z, Qin J, Sun D, Wang S, Wu Q, Crickmore N, Zhou X, Bravo A, Soberón M, Guo Z, Zhang Y. 2022. MAPK-mediated transcription factor GATAd contributes to Cry1Ac resistance in diamondback moth by reducing PxmALP expression. PLoS Genetics, 18, e1010037.

Guo Z, Guo L, Bai Y, Kang S, Sun D, Qin J, Ye F, Wang S, Wu Q, Xie W, Yang X, Crickmore N, Zhou X, Zhang Y. 2023. Retrotransposon-mediated evolutionary rewiring of a pathogen response orchestrates a resistance phenotype in an insect host. Proceedings of the National Academy of Sciences of the United States of America, 120, e2300439120.

Guo Z, Gong L, Kang S, Zhou J, Sun D, Qin J, Guo L, Zhu L, Bai Y, Bravo A, Soberón M, Zhang Y. 2020a. Comprehensive analysis of Cry1Ac protoxin activation mediated by midgut proteases in susceptible and resistant Plutella xylostella (L.). Pesticide Biochemistry and Physiology, 163, 23–30.

Guo Z, Guo L, Qin J, Ye F, Sun D, Wu Q, Wang S, Crickmore N, Zhou X, Bravo A, Soberón M, Zhang Y. 2022. A single transcription factor facilitates an insect host combating Bacillus thuringiensis infection while maintaining fitness. Nature Communications, 13, 6024.

Guo Z, Kang S, Chen D, Wu Q, Wang S, Xie W, Zhu X, Baxter S W, Zhou X, Jurat-Fuentes J L, Zhang Y. 2015a. MAPK signaling pathway alters expression of midgut ALP and ABCC genes and causes resistance to Bacillus thuringiensis Cry1Ac toxin in diamondback moth. PLoS Genetics, 11, e1005124.

Guo Z, Kang S, Sun D, Gong L, Zhou J, Qin J, Guo L, Zhu L, Bai Y, Ye F, Wu Q, Wang S, Crickmore N, Zhou X, Zhang Y. 2020b. MAPK-dependent hormonal signaling plasticity contributes to overcoming Bacillus thuringiensis toxin action in an insect host. Nature Communications, 11, 3003.

Guo Z, Kang S, Wu Q, Wang S, Crickmore N, Zhou X, Bravo A, Soberón M, Zhang Y. 2021. The regulation landscape of MAPK signaling cascade for thwarting Bacillus thuringiensis infection in an insect host. PLoS Pathogens, 17, e1009917.

Guo Z, Kang S, Zhu X, Wu Q, Wang S, Xie W, Zhang Y. 2015b. The midgut cadherin-like gene is not associated with resistance to Bacillus thuringiensis toxin Cry1Ac in Plutella xylostella (L.). Journal of Invertebrate Pathology, 126, 21–30.

Guo Z, Kang S, Zhu X, Xia J, Wu Q, Wang S, Xie W, Zhang Y. 2015c. Down-regulation of a novel ABC transporter gene (Pxwhite) is associated with Cry1Ac resistance in the diamondback moth, Plutella xylostella (L.). Insect Biochemistry and Molecular Biology, 59, 30–40.

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.

Hou W, Jiang C, Zhou X, Qian K, Wang L, Shen Y, Zhao Y. 2016. Increased expression of P-glycoprotein is associated with chlorpyrifos resistance in the German cockroach (Blattodea: Blattellidae). Journal of Economic Entomology, 109, 2500–2505.

Igboeli O O, Fast M D, Heumann J, Burka J F. 2012. Role of P-glycoprotein in emamectin benzoate (SLICE®) resistance in sea lice, Lepeophtheirus salmonis. Aquaculture, 344–349, 40–47.

ISAAA (International Service for the Acquisition of Agri-biotech Applications). 201 9. Global Status of Commercialized Biotech/GM Crops in 2019. Brief 55. ISAAA, Ithaca, NY, USA.

Jurat-Fuentes J L, Crickmore N. 2017. Specificity determinants for Cry insecticidal proteins: Insights from their mode of action. Journal of Invertebrate Pathology, 142, 5–10.

Jurat-Fuentes J L, Heckel D G, Ferré J. 2021. Mechanisms of resistance to insecticidal proteins from Bacillus thuringiensis. Annual Review of Entomology, 66, 121–140.

Kang S, Sun D, Qin J, Guo L, Zhu L, Bai Y, Wu Q, Wang S, Zhou X, Guo Z, Zhang Y. 2022a. Fused: a promising molecular target for an RNAi-based strategy to manage Bt resistance in Plutella xylostella (L.). Journal of Pest Science, 95, 101–114.

Kang S, Zhu X, Wu Q, Wang S, Crickmore N, Zhang Y, Guo Z. 2022b. Critical analysis of multi-omic data from a strain of Plutella xylostella resistant to Bacillus thuringiensis Cry1Ac toxin. Journal of Agricultural and Food Chemistry, 70, 11419–11428.

Li Q, Jin M, Yu S, Cheng Y, Shan Y, Wang P, Yuan H, Xiao Y. 2022. Knockout of the ABCB1 gene increases susceptibility to emamectin benzoate, beta-cypermethrin and chlorantraniliprole in Spodoptera frugiperda. Insects, 13, 137.

Li Z, Feng X, Liu S S, You M, Furlong M J. 2016. Biology, ecology, and management of the diamondback moth in China. Annual Review of Entomology, 61, 277–296.

Liu L, Chen Z, Yang Y, Xiao Y, Liu C, Ma Y, Soberón M, Bravo A, Yang Y, Liu K. 2018. A single amino acid polymorphism in ABCC2 loop 1 is responsible for differential toxicity of Bacillus thuringiensis Cry1Ac toxin in different Spodoptera (Noctuidae) species. Insect Biochemistry and Molecular Biology, 100, 59–65.

Merzendorfer H. 2014. ABC transporters and their role in protecting insects from pesticides and their metabolites. Advances in Insect Physiology, 46, 1–72.

Niu X, Kassa A, Hasler J, Griffin S, Perez-Ortega C, Procyk L, Zhang J, Kapka-Kitzman D M, Nelson M E, Lu A. 2020. Functional validation of DvABCB1 as a receptor of Cry3 toxins in western corn rootworm, Diabrotica virgifera virgifera. Scientific Reports, 10, 15830.

Pauchet Y, Bretschneider A, Augustin S, Heckel D G. 2016. A P-glycoprotein is linked to resistance to the Bacillus thuringiensis Cry3Aa toxin in a leaf beetle. Toxins, 8, 362.

Peterson B, Bezuidenhout C C, Van den Berg J. 2017. An overview of mechanisms of Cry toxin resistance in lepidopteran insects. Journal of Economic Entomology, 110, 362–377.

Pinos D, Andrés-Garrido A, Ferré J, Hernández-Martínez P. 2021. Response mechanisms of invertebrates to Bacillus thuringiensis and its pesticidal proteins. Microbiology and Molecular Biology Reviews, 85, e00007-20.

Pohl P C, Klafke G M, Carvalho D D, Martins J R, Daffre S, Vaz I S, Masuda A. 2011. ABC transporter efflux pumps: A defense mechanism against ivermectin in Rhipicephalus (Boophilus) microplus. International Journal for Parasitology, 41, 1323–1333.

Porretta D, Gargani M, Bellini R, Medici A, Punelli F, Urbanelli S. 2008. Defence mechanisms against insecticides temephos and diflubenzuron in the mosquito Aedes caspius: the P-glycoprotein efflux pumps. Medical and Veterinary Entomology, 22, 48–54.

Qin J, Guo L, Ye F, Kang S, Sun D, Zhu L, Bai Y, Cheng Z, Xu L, Ouyang C, Xiao L, Wang S, Wu Q, Zhou X, Crickmore N, Zhou X, Guo Z, Zhang Y. 2021a. MAPK-activated transcription factor PxJun suppresses PxABCB1 expression and confers resistance to Bacillus thuringiensis Cry1Ac toxin in Plutella xylostella (L.). Applied and Environmental Microbiology, 87, e00466-21.

Qin J, Ye F, Xu L, Zhou X, Crickmore N, Zhou X, Zhang Y, Guo Z. 2021b. A cis-acting mutation in the PxABCG1 promoter is associated with Cry1Ac resistance in Plutella xylostella (L.). International Journal of Molecular Sciences, 22, 6106.

Rees D, Johnson E, Lewinson O. 2009. ABC transporters: The power to change. Nature Reviews Molecular Cell Biology, 10, 218–227.

Sato R, Adegawa S, Li X, Tanaka S, Endo H. 2019. Function and role of ATP-binding cassette transporters as receptors for 3D-Cry toxins. Toxins, 11, 124.

Silva R, Vilas-Boas V, Carmo H, Dinis-Oliveira R J, Carvalho F, Bastos M L, Remião F. 2015. Modulation of P-glycoprotein efflux pump: Induction and activation as a therapeutic strategy. Pharmacology & Therapeutics, 149, 1–123.

Sun D, Guo Z, Liu Y, Zhang Y. 2017. Progress and prospects of CRISPR/Cas systems in insects and other arthropods. Frontiers in Physiology, 8, 608.

Sun D, Zhu L, Guo L, Wang S, Wu Q, Crickmore N, Zhou X, Bravo A, Soberón M, Guo Z, Zhang Y. 2022. A versatile contribution of both aminopeptidases N and ABC transporters to Bt Cry1Ac toxicity in the diamondback moth. BMC Biology, 20, 33.

Sun H, Pu J, Chen F, Wang J, Han Z. 2017. Multiple ATP-binding cassette transporters are involved in insecticide resistance in the small brown planthopper, Laodelphax striatellus. Insect Molecular Biology, 26, 343–355.

Tabashnik B E, Carrière Y. 2017. Surge in insect resistance to transgenic crops and prospects for sustainability. Nature Biotechnology, 35, 926–935.

Tanaka S, Endo H, Adegawa S, Iizuka A, Imamura K, Kikuta S, Sato R. 2017. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin. Insect Biochemistry and Molecular Biology, 91, 44–54.

Tanaka S, Miyamoto K, Noda H, Endo H, Kikuta S, Sato R. 2016. Single amino acid insertions in extracellular loop 2 of Bombyx mori ABCC2 disrupt its receptor function for Bacillus thuringiensis Cry1Ab and Cry1Ac but not Cry1Aa toxins. Peptides, 78, 99–108.

Tian L, Yang J, Hou W, Xu B, Xie W, Wang S, Zhang Y, Zhou X, Wu Q. 2013. Molecular cloning and characterization of a P-glycoprotein from the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae). International Journal of Molecular Sciences, 14, 22891–22905.

Wu C, Chakrabarty S, Jin M, Liu K, Xiao Y. 2019. Insect ATP-binding cassette (ABC) transporters: roles in xenobiotic detoxification and Bt insecticidal activity. International Journal of Molecular Sciences, 20, 2829.

Xiang M, Zhang L, Lu Y, Tang Q, Liang P, Shi X, Song D, Gao X. 2017. A P-glycoprotein gene serves as a component of the protective mechanisms against 2-tridecanone and abamectin in Helicoverpa armigera. Gene, 627, 63–71.

Xiao Y, Liu K, Zhang D, Gong L, He F, Soberón M, Bravo A, Tabashnik B E, Wu K. 2016. Resistance to Bacillus thuringiensis mediated by an ABC transporter mutation increases susceptibility to toxins from other bacteria in an invasive insect. PLoS Pathogens, 12, e1005450.

Xiao Y, Wu K. 2019. Recent progress on the interaction between insects and Bacillus thuringiensis crops. Philosophical Transactions of the Royal Society (B: Biological Sciences), 374, 20180316.

Xu L, Qin J, Fu W, Wang S, Wu Q, Zhou X, Crickmore N, Guo Z, Zhang Y. 2022. MAP4K4 controlled transcription factor POUM1 regulates PxABCG1 expression influencing Cry1Ac resistance in Plutella xylostella (L.). Pesticide Biochemistry and Physiology, 182, 105053.

Xu Z, Shi L, Peng J, Shen G, Wei P, Wu Q, He L. 2016. Analysis of the relationship between P-glycoprotein and abamectin resistance in Tetranychus cinnabarinus (Boisduval). Pesticide Biochemistry and Physiology, 129, 75–82.

Zhou J, Guo Z, Kang S, Qin J, Gong L, Sun D, Guo L, Zhu L, Bai Y, Zhang Z, Zhou X, Zhang Y. 2020. Reduced expression of the P-glycoprotein gene PxABCB1 is linked to resistance to Bacillus thuringiensis Cry1Ac toxin in Plutella xylostella (L.). Pest Management Science, 76, 712–720.

Zuo Y Y, Huang J L, Wang J, Feng Y, Han T T, Wu Y D, Yang Y H. 2018. Knockout of a P-glycoprotein gene increases susceptibility to abamectin and emamectin benzoate in Spodoptera exigua. Insect Molecular Biology, 27, 36–45.

基于CRISPR/Cas9技术的PxABCB1基因功能鉴定揭示了其在小菜蛾对Cry1Ac、阿维菌素和甲维盐抗性中的作用

CRISPR/Cas9-based functional characterization of PxABCB1 reveals its roles in the resistance of Plutella xylostella (L.) to Cry1Ac, abamectin and emamectin benzoate

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