Embrapa Genetic Resources and Biotechnology, Brasília, DF 70770-917, Brazil; National Institute of Science and Technology, INCT PlantStress Biotech, Embrapa, Brasília, DF 70770-917, Brazil.
Department of Cellular Biology, University of Brasília, Brasília, DF 70910-900, Brazil; Genesilico Biotech, Brasília, DF 71503-508, Brazil.
Plant Sci. 2024 Jul;344:112079. doi: 10.1016/j.plantsci.2024.112079. Epub 2024 Apr 6.
The cotton boll weevil (CBW, Anthonomus grandis) stands as one of the most significant threats to cotton crops (Gossypium hirsutum). Despite substantial efforts, the development of a commercially viable transgenic cotton event for effective open-field control of CBW has remained elusive. This study describes a detailed characterization of the insecticidal toxins Cry23Aa and Cry37Aa against CBW. Our findings reveal that CBW larvae fed on artificial diets supplemented exclusively with Cry23Aa decreased larval survival by roughly by 69%, while supplementation with Cry37Aa alone displayed no statistical difference compared to the control. However, the combined provision of both toxins in the artificial diet led to mortality rates approaching 100% among CBW larvae (LC equal to 0.26 PPM). Additionally, we engineered transgenic cotton plants by introducing cry23Aa and cry37Aa genes under control of the flower bud-specific pGhFS4 and pGhFS1 promoters, respectively. Seven transgenic cotton events expressing high levels of Cry23Aa and Cry37Aa toxins in flower buds were selected for greenhouse bioassays, and the mortality rate of CBW larvae feeding on their T and T generations ranged from 75% to 100%. Our in silico analyses unveiled that Cry23Aa displays all the hallmark characteristics of β-pore-forming toxins (β-PFTs) that bind to sugar moieties in glycoproteins. Intriguingly, we also discovered a distinctive zinc-binding site within Cry23Aa, which appears to be involved in protein-protein interactions. Finally, we discuss the major structural features of Cry23Aa that likely play a role in the toxin's mechanism of action. In view of the low LC for CBW larvae and the significant accumulation of these toxins in the flower buds of both T and T plants, we anticipate that through successive generations of these transgenic lines, cotton plants engineered to overexpress cry23Aa and cry37Aa hold promise for effectively managing CBW infestations in cotton crops.
棉铃象鼻虫（CBW，Anthonomus grandis）是棉花作物（Gossypium hirsutum）的主要威胁之一。尽管付出了巨大努力，但开发出一种商业上可行的转基因棉花事件，以有效控制田间的 CBW 仍难以实现。本研究详细描述了Cry23Aa 和 Cry37Aa 两种昆虫毒素对 CBW 的杀虫作用。我们的研究结果表明，用仅含 Cry23Aa 的人工饲料喂养 CBW 幼虫，幼虫的存活率降低了约 69%，而单独用 Cry37Aa 喂养则与对照组无统计学差异。然而，在人工饲料中同时提供这两种毒素，可使 CBW 幼虫的死亡率接近 100%（LC 等于 0.26 PPM）。此外，我们通过分别在花蕾特异性启动子 pGhFS4 和 pGhFS1 的控制下引入 cry23Aa 和 cry37Aa 基因，构建了转基因棉花植物。选择了七个高水平表达 Cry23Aa 和 Cry37Aa 毒素的转基因棉花事件进行温室生物测定，CBW 幼虫在 T 和 T 代中取食的死亡率在 75%至 100%之间。我们的计算机分析表明，Cry23Aa 具有与糖基化蛋白结合的β-孔形成毒素（β-PFT）的所有特征。有趣的是，我们还在 Cry23Aa 中发现了一个独特的锌结合位点，该位点可能参与了蛋白质-蛋白质相互作用。最后，我们讨论了 Cry23Aa 的主要结构特征，这些特征可能在毒素的作用机制中发挥作用。鉴于 CBW 幼虫的 LC 较低，以及这些毒素在 T 和 T 代棉花花蕾中的大量积累，我们预计通过这些转基因系的连续几代，过表达 cry23Aa 和 cry37Aa 的棉花植物有望有效管理棉花作物中的 CBW 虫害。
