Instituto Universitario de Biotecnología y Biomedicina (BIOTECMED), Department of Genetics, Universitat de València, Burjassot, Spain.
Appl Environ Microbiol. 2021 Nov 24;87(24):e0178721. doi: 10.1128/AEM.01787-21. Epub 2021 Sep 29.
Vegetative insecticidal proteins (Vip3) from Bacillus thuringiensis have been used, in combination with Cry proteins, to better control insect pests and as a strategy to delay the evolution of resistance to Cry proteins in Bt crops (crops protected from insect attack by the expression of proteins from B. thuringiensis). In this study, we have set up the conditions to analyze the specific binding of I-Vip3Af to Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). Heterologous competition binding experiments revealed that Vip3Aa shares the same binding sites with Vip3Af, but Vip3Ca does not recognize all of them. As expected, Cry1Ac and Cry1F did not compete for Vip3Af binding sites. By trypsin treatment of selected alanine mutants, we were able to generate truncated versions of Vip3Af. Their use as competitors with I-Vip3Af indicated that only those molecules containing domains I to III (DI-III and DI-IV) were able to compete with the trypsin-activated Vip3Af protein for binding and that molecules only containing either domain IV or domains IV and V (DIV and DIV-V) were unable to compete with Vip3Af. These results were further confirmed with competition binding experiments using I-DI-III. In addition, the truncated protein I-DI-III also bound specifically to Sf21 cells. Cell viability assays showed that the truncated proteins DI-III and DI-IV were as toxic to Sf21 cells as the activated Vip3Af, suggesting that domains IV and V are not necessary for the toxicity to Sf21 cells, in contrast to their requirement This study shows that Vip3Af binding sites are fully shared with Vip3Aa, only partially shared with Vip3Ca, and not shared with Cry1Ac and Cry1F in two spp. Truncated versions of Vip3Af revealed that only domains I to III were necessary for the specific binding, most likely because they can form the functional tetrameric oligomer and because domain III is supposed to contain the binding epitopes. In contrast to results obtained (bioassays against larvae), domains IV and V are not necessary for toxicity to Sf21 cells.
苏云金芽孢杆菌的杀虫晶体蛋白（Cry）与植物源杀虫蛋白 VIP3 联合使用，可更好地控制害虫，并作为延缓抗 Cry 蛋白作物中抗虫性进化的策略（通过表达苏云金芽孢杆菌蛋白而免受昆虫侵害的作物）。在这项研究中，我们建立了分析 VIP3Af 与斜纹夜蛾和草地贪夜蛾刷状缘膜囊泡（BBMV）特异性结合的条件。异源竞争结合实验表明，Vip3Aa 与 Vip3Af 共享相同的结合位点，但 Vip3Ca 并不能识别所有这些结合位点。不出所料，Cry1Ac 和 Cry1F 不与 Vip3Af 竞争结合位点。通过对选定的丙氨酸突变体进行胰蛋白酶处理，我们能够生成 VIP3Af 的截断版本。用 VIP3Af 作为竞争物，发现仅包含结构域 I 到 III（DI-III 和 DI-IV）的分子能够与经胰蛋白酶激活的 VIP3Af 蛋白竞争结合，而仅包含结构域 IV 或结构域 IV 和 V（DIV 和 DIV-V）的分子则无法与 VIP3Af 竞争。使用 I-DI-III 进行竞争结合实验进一步证实了这一结果。此外，截短蛋白 I-DI-III 也特异性地与 Sf21 细胞结合。细胞活力测定表明，截短蛋白 DI-III 和 DI-IV 与激活的 VIP3Af 一样对 Sf21 细胞有毒性，这表明与对 Sf21 细胞的毒性要求相反，结构域 IV 和 V 不是必需的。这项研究表明，在两种 spp. 中，VIP3Af 的结合位点与 Vip3Aa 完全共享，与 Vip3Ca 部分共享，与 Cry1Ac 和 Cry1F 不共享。VIP3Af 的截断版本表明，只有结构域 I 到 III 是特异性结合所必需的，这很可能是因为它们可以形成功能性四聚体寡聚物，并且结构域 III 应该包含结合表位。与幼虫生物测定获得的结果相反，结构域 IV 和 V 对 Sf21 细胞的毒性不是必需的。
