Shenzhen Branch, Guangdong Laboratory for Lingnan Modern Agriculture, Genome Analysis Laboratory of the Ministry of Agriculture, Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen, China.
College of Life Sciences, Central China Normal Universitygrid.411407.7, Wuhan, China.
Appl Environ Microbiol. 2022 Apr 12;88(7):e0250521. doi: 10.1128/aem.02505-21. Epub 2022 Mar 9.
Bacillus thuringiensis Cry proteins are used worldwide for insect control. It was proposed that Cry-protoxins must be converted into activated toxin by proteases to bind midgut cell proteins to kill insects. However, Cry-protoxins also bind to midgut proteins and kill insects that have evolved resistance to activated toxins suggesting an independent toxicity pathway. Cadherin (CAD) and ABCC transporters are recognized as important receptors for Cry proteins. Here we constructed different Helicoverpa armigera mutations in these receptors by CRISPR/Cas9. HaCAD-KO mutant showed much higher resistance to Cry1Ac activated toxin than to Cry1Ac protoxin. In contrast, the HaABCC2-M and HaABCC3-M mutants showed higher resistance to Cry1Ac-protoxin than to activated toxin. However, in the double HaABCC2/3-KO mutant, very high levels of resistance were observed to both Cry1Ac protoxin and activated toxin, supporting that both ABC transporters have redundant functions for these two proteins. In addition, Hi5 cells transfected with HaCAD were susceptible only to the activated toxin but not to protoxin. In contrast, both forms of Cry1Ac were similarly toxic to Hi5 cells expressing HaABCC2 or HaABCC3. Co-expression of HaCAD with HaABCC2 or HaABCC3 revealed a more important synergistic effect for activated toxin compared to protoxin. Overall, our results show that toxicity of Cry1Ac activated toxin involves synergistic interplay of HaCAD with ABCC transporters, while the Cry1Ac protoxin toxicity is mainly mediated by ABCC transporters with little participation of HaCAD. These data help to understand the mode of action of Cry proteins that will be relevant to enhance efficacy and durability of Bt-crops. Better understanding of the mode of action of Bacillus thuringiensis toxins is beneficial for the sustainable application of Bt crops. It is generally accepted that Cry-protoxins need to be activated by proteases to bind with midgut cell proteins and exert toxicity against insects. Here, we provide new insights into the toxic pathway of Cry proteins in the cotton bollworm. First, our results demonstrate that Cry1Ac protoxin is able to exert cytotoxicity against the insect cells expressing ABCC transporters. Second, we reveal that CAD plays a critical role in the different toxicity of protoxin and toxin by facilitating a synergistic interplay with ABCC transporters. Our results provide and experimental evidence supporting that Cry1Ac protoxin exerts toxicity against via different steps from that of toxin. These new findings on the mode of action of Cry proteins could be beneficial for efficacy enhancement and durability of Bt-crops.
苏云金芽孢杆菌 Cry 蛋白被广泛用于昆虫防治。据推测，Cry-原毒素必须被蛋白酶转化为活性毒素，才能与中肠细胞蛋白结合杀死昆虫。然而，Cry-原毒素也与中肠蛋白结合，并杀死对活性毒素产生抗性的昆虫，这表明存在独立的毒性途径。钙粘蛋白 (CAD) 和 ABCC 转运体被认为是 Cry 蛋白的重要受体。在这里，我们通过 CRISPR/Cas9 构建了这些受体在棉铃虫中的不同突变。HaCAD-KO 突变体对 Cry1Ac 激活毒素的抗性明显高于 Cry1Ac 原毒素。相比之下，HaABCC2-M 和 HaABCC3-M 突变体对 Cry1Ac-原毒素的抗性高于激活毒素。然而，在双 HaABCC2/3-KO 突变体中，对两种蛋白的原毒素和激活毒素都表现出非常高的抗性，表明这两种 ABC 转运体对这两种蛋白具有冗余功能。此外，转染了 HaCAD 的 Hi5 细胞仅对激活毒素敏感，而对原毒素不敏感。相反，Cry1Ac 的两种形式对表达 HaABCC2 或 HaABCC3 的 Hi5 细胞都具有类似的毒性。HaCAD 与 HaABCC2 或 HaABCC3 的共表达显示，与原毒素相比，激活毒素的协同作用更为重要。总的来说，我们的结果表明，Cry1Ac 激活毒素的毒性涉及 HaCAD 与 ABCC 转运体的协同相互作用，而 Cry1Ac 原毒素的毒性主要由 ABCC 转运体介导，HaCAD 的参与较少。这些数据有助于了解 Cry 蛋白的作用模式，这将有助于提高 Bt 作物的功效和持久性。 更好地了解苏云金芽孢杆菌毒素的作用模式有利于 Bt 作物的可持续应用。通常认为，Cry-原毒素需要被蛋白酶激活，才能与中肠细胞蛋白结合并对昆虫产生毒性。在这里，我们为棉铃虫中 Cry 蛋白的毒性途径提供了新的见解。首先，我们的结果表明 Cry1Ac 原毒素能够对表达 ABCC 转运体的昆虫细胞产生细胞毒性。其次，我们揭示了 CAD 通过与 ABCC 转运体的协同相互作用，在原毒素和毒素的不同毒性中起着关键作用。我们的结果提供了 和 实验证据，支持 Cry1Ac 原毒素通过不同于毒素的步骤对 发挥毒性作用。这些关于 Cry 蛋白作用模式的新发现可能有助于提高 Bt 作物的功效和持久性。
