College of Life Sciences, Henan Agricultural University, Zhengzhou 450046, China; State key Laboratory of Wheat and Maize Crop Science/College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, China.
State key Laboratory of Wheat and Maize Crop Science/College of Plant Protection, Henan Agricultural University, Zhengzhou 450046, China.
Pestic Biochem Physiol. 2024 Jan;198:105744. doi: 10.1016/j.pestbp.2023.105744. Epub 2023 Dec 13.
Cry2Ab is one of the important alternative Bt proteins that can be used to manage insect pests resistant to Cry1A toxins and to expand the insecticidal spectrum of pyramided Bt crops. Previous studies have showed that vacuolar H-ATPase subunits A and B (V-ATPase A and B) may be involved in Bt insecticidal activities. The present study investigated the role of V-ATPases subunit E in the toxicity of Cry2Ab in Helicoverpa amigera. RT-PCR analysis revealed that oral exposure of H. amigera larvae to Cry2Ab led to a significant reduction in the expression of H. armigera V-ATPase E (HaV-ATPase E). Ligand blot, homologous and heterologous competition experiments confirmed that HaV-ATPases E physically and specifically bound to activated Cry2Ab toxin. Heterologous expressing of HaV-ATPase E in Sf9 cells made the cell line more susceptible to Cry2Ab, whereas knockdown of the endogenous V-ATPase E in H. zea midgut cells decreased Cry2Ab's cytotoxicity against this cell line. Further in vivo bioassay showed that H. armigera larvae fed a diet overlaid with both Cry2Ab and E. coli-expressed HaV-ATPase E protein suffered significantly higher mortality than those fed Cry2Ab alone. These results support that V-ATPases E is a putative receptor of Cry2Ab and can be used to improve Cry2Ab toxicity and manage Cry2Ab resistance at least in H. armigera.
Cry2Ab 是一种重要的可替代 Bt 蛋白，可用于防治对 Cry1A 毒素产生抗性的昆虫，以及扩大含 Cry1A 毒素的 Bt 作物的杀虫谱。先前的研究表明液泡型 H+-ATP 酶亚基 A 和 B（V-ATPase A 和 B）可能参与 Bt 杀虫活性。本研究调查了 V-ATPase 亚基 E 在 Cry2Ab 对棉铃虫毒性中的作用。RT-PCR 分析表明，Cry2Ab 经棉铃虫幼虫口服暴露后，H. armigera V-ATPase E（HaV-ATPase E）的表达显著降低。配体印迹、同源和异源竞争实验证实 HaV-ATPase E 与激活的 Cry2Ab 毒素物理结合并具有特异性。HaV-ATPase E 在 Sf9 细胞中的异源表达使该细胞系对 Cry2Ab 更敏感，而在家蚕中肠细胞中敲低内源性 V-ATPase E 则降低了 Cry2Ab 对该细胞系的细胞毒性。进一步的体内生物测定表明，喂食同时含有 Cry2Ab 和表达于大肠杆菌的 HaV-ATPase E 蛋白的饲料的棉铃虫幼虫死亡率明显高于单独喂食 Cry2Ab 的幼虫。这些结果支持 V-ATPase E 是 Cry2Ab 的一个假定受体，可用于提高 Cry2Ab 的毒性，并至少在棉铃虫中管理 Cry2Ab 抗性。
