Scavenger receptor-C acts as a receptor for Bacillus thuringiensis vegetative insecticidal protein Vip3Aa and mediates the internalization of Vip3Aa via endocytosis

The vegetative insecticidal proteins (Vip), secreted by many Bacillus thuringiensis strains during their vegetative growth stage, are genetically distinct from known insecticidal crystal proteins (ICPs) and represent the second-generation insecticidal toxins. Compared with ICPs, the insecticidal mechanisms of Vip toxins are poorly understood. In particular, there has been no report of a definite receptor of Vip toxins to date. In the present study, we identified the scavenger receptor class C like protein (Sf-SR-C) from the Spodoptera frugiperda (Sf9) cells membrane proteins that bind to the biotin labeled Vip3Aa, via the affinity magnetic bead method coupled with HPLC-MS/MS. We then certified Vip3Aa protoxin could interact with Sf-SR-C in vitro and ex vivo. In addition, downregulation of SR-C expression in Sf9 cells and Spodoptera exigua larvae midgut reduced the toxicity of Vip3Aa to them. Coincidently, heterologous expression of Sf-SR-C in transgenic Drosophila midgut significantly enhanced the virulence of Vip3Aa to the Drosophila larvae. Moreover, the complement control protein domain and MAM domain of Sf-SR-C are involved in the interaction with Vip3Aa protoxin. Furthermore, endocytosis of Vip3Aa mediated by Sf-SR-C correlates with its insecticidal activity. Our results confirmed for the first time that Sf-SR-C acts as a receptor for Vip3Aa protoxin and provides an insight into the mode of action of Vip3Aa that will significantly facilitate the study of its insecticidal mechanism and application.多种苏云金芽孢杆菌（Bacillus thuringiensis）菌株在营养生长阶段分泌的营养期杀虫蛋白（vegetative insecticidal proteins, Vip），与已知的杀虫晶体蛋白（insecticidal crystal proteins, ICPs）在遗传上存在显著差异，属于第二代杀虫毒素。与ICPs相比，目前对Vip毒素的杀虫机制尚不清楚，尤其迄今尚未见Vip毒素确定性受体的相关报道。本研究通过亲和磁珠法结合高效液相色谱-串联质谱（HPLC-MS/MS），从结合生物素标记Vip3Aa的草地贪夜蛾（Spodoptera frugiperda, Sf9）细胞膜蛋白中，鉴定得到清道夫受体C类样蛋白（Sf-SR-C）。随后通过体外（in vitro）及离体（ex vivo）实验证实，Vip3Aa原毒素可与Sf-SR-C发生相互作用。此外，在Sf9细胞及甜菜夜蛾（Spodoptera exigua）幼虫中肠内下调SR-C的表达，会降低Vip3Aa对二者的毒性。与之相符的是，在转基因果蝇中肠中异源表达Sf-SR-C，可显著增强Vip3Aa对果蝇幼虫的毒力。进一步研究发现，Sf-SR-C的补体控制蛋白结构域（complement control protein domain）与MAM结构域参与了与Vip3Aa原毒素的结合。此外，Sf-SR-C介导的Vip3Aa内吞作用与其杀虫活性密切相关。本研究首次证实Sf-SR-C可作为Vip3Aa原毒素的受体，为阐明Vip3Aa的作用模式提供了新视角，将有力推动其杀虫机制研究与实际应用。