Sun Dan, Xu Qiuchen, Guo Le, Bai Yang, Shentu Xuping, Yu Xiaoping, Crickmore Neil, Zhou Xuguo, Bravo Alejandra, Soberón Mario, Zhang Youjun, Guo Zhaojiang
Zhejiang Provincial Key Laboratory of Biometrology and Inspection & Quarantine, College of Life Science, China Jiliang University, Hangzhou 310018, China.
State Key Laboratory of Vegetable Biobreeding, Department of Plant Protection, Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Fundam Res. 2024 May 27;5(2):674-682. doi: 10.1016/j.fmre.2024.05.007. eCollection 2025 Mar.
The insecticidal Cry proteins produced by the bacterium (Bt) are extensively used for pest control in formulated sprays and in genetically modified crops, but resistance to Bt toxins threatens their sustainable use in agriculture. Understanding the molecular mechanisms involved in Bt pathogenesis is crucial for the development of effective resistance management strategies. Previously, we showed a strong correlation between Cry1Ac resistance in (L.) and down-regulation of the glycosylphosphatidylinositol (GPI)-anchored membrane-bound alkaline phosphatase (mALP) and aminopeptidase (APN) and members of the ATP-binding cassette (ABC) transporter subfamily C (ABCC), but we do not yet have a clear understanding of the relative contribution of each midgut receptor type. Here, a strain homozygous for the gene knockout was generated using CRISPR/Cas9 and the results showed that this strain had a 294-fold resistance to Cry1Ac toxin and 394-fold cross-resistance to Cry1Ab. Moreover, a triple knockout strain lacking , and exhibited 9,660-fold resistance to Cry1Ac and 5,662-fold cross-resistance to Cry1Ab. These resistance levels surpassed those observed in the previously described double and knockout mutant, revealing a functional redundancy between ABC transporters and PxmALP In addition, the activity of Cry1A toxins against Sf9 cells expressing PxmALP, PxABCC2 or PxABCC3 confirmed that each of these can act as a functional receptor. Our findings are crucial for unraveling the relative role of multiple receptors and the molecular mechanisms underlying Bt resistance in insects.
由苏云金芽孢杆菌（Bt）产生的杀虫Cry蛋白广泛用于配制喷雾剂和转基因作物的害虫防治，但对Bt毒素的抗性威胁到它们在农业中的可持续利用。了解Bt致病过程中涉及的分子机制对于制定有效的抗性管理策略至关重要。此前，我们发现棉铃虫（L.）对Cry1Ac的抗性与糖基磷脂酰肌醇（GPI）锚定的膜结合碱性磷酸酶（mALP）、氨肽酶（APN）以及ATP结合盒（ABC）转运蛋白亚家族C（ABCC）成员的下调之间存在很强的相关性，但我们尚未清楚每种中肠受体类型的相对贡献。在此，利用CRISPR/Cas9技术构建了PxABCC2基因敲除纯合的棉铃虫品系，结果表明该品系对Cry1Ac毒素具有294倍的抗性，对Cry1Ab具有394倍的交叉抗性。此外，缺失PxABCC1、PxABCC2和PxABCC3的三基因敲除品系对Cry1Ac表现出9660倍的抗性，对Cry1Ab表现出5662倍的交叉抗性。这些抗性水平超过了先前描述的PxABCC2和PxABCC3双基因敲除突变体所观察到的抗性水平，揭示了ABC转运蛋白和PxmALP之间的功能冗余。此外，Cry1A毒素对表达PxmALP、PxABCC2或PxABCC3的Sf9细胞的活性证实，它们均可作为功能性受体。我们的研究结果对于阐明多种受体的相对作用以及昆虫对Bt抗性的分子机制至关重要。
