The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin
The Caenorhabditis elegans CUB-like-domain containing protein RBT-1 functions as a receptor for Bacillus thuringiensis Cry6Aa toxin
复制标题
含有蛋白 RBT-1 的秀丽隐杆线虫 CUB 样结构域充当苏云金芽孢杆菌 Cry6Aa 毒素的受体。
DOI：
10.1371/journal.ppat.1008501
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发表时间：
2020-05-01
期刊：
影响因子：
6.7
通讯作者：
Sun, Ming
中科院分区：
文献类型：
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作者：
Shi, Jianwei;Peng, Donghai;Sun, Ming
苏云金芽孢杆菌（Bacillus thuringiensis，Bt）晶体蛋白属于致孔毒素（pore-forming toxins，PFTs），通过在细胞膜上形成孔洞而对目标寄主产生毒力。Cry 6A是一种杀线虫的PFT，其表现出独特的蛋白质结构和与另一种杀线虫的PFT Cry 5 B不同的作用模式。然而，对Cry 6A的作用模式知之甚少。虽然报道了Cry 6A的细胞内杀线虫坏死途径，但其细胞外作用模式仍然未知。我们在这里证明，CUB样结构域包含蛋白RBT-1作为Cry 6A，介导肠细胞相互作用和杀线虫活性的毒素的功能性受体。RBT-1代表一类新的晶体蛋白受体。RBT-1对Cry 5 B抗线虫的毒性进行了验证，这与Cry 6A和Cry 5 B具有不同的杀线虫机制一致。我们还发现Cry 6A通过复杂的机制杀死线虫，因为rbt-1突变不影响Cry 6A介导的坏死信号通路。本研究不仅加深了对Bt晶体蛋白-线虫作用机理的认识，而且有利于Cry 6A在线虫防治中的应用。苏云金芽孢杆菌晶体蛋白的两个主要家族Cry 5和Cry 6显示杀线虫活性。先前的工作表明，与中肠受体的结合是Cry毒素作用模式的限制步骤。在Cry 5 Ba的情况下，某些秀丽隐杆线虫糖脂被鉴定为该毒素的受体。然而，Cry 6毒素的受体仍然未知。在本研究中，C.通过亲和层析，鉴定了由磷脂酰肌醇特异性磷脂酶C（PI-PLC）释放的含有秀丽线虫CUB样结构域的蛋白RBT-1为Cry 6Aa结合蛋白。RBT-1含有一个预测的糖基磷脂酰肌醇（GPI）锚位点，并被证明位于中肠细胞表面的脂筏。Western配体印迹分析和ELISA结合分析证实了Cry 6Aa与RBT-1之间的结合作用显示出高亲和力和特异性。此外，rbt-1基因突变降低了C. elegans对Cry 6Aa的反应，而不是Cry 5 Ba。此外，RBT-1介导了C.线虫肠细胞，并显示参与触发孔形成活性，表明RBT-1是Cry 6Aa与线虫中肠细胞相互作用所必需的。这些结果支持RBT-1是Cry 6Aa的功能性受体。
Author summaryBacillus thuringiensis (Bt) crystal proteins belong to pore-forming toxins (PFTs), which display virulence against target hosts by forming holes in the cell membrane. Cry6A is a nematicidal PFT, which exhibits unique protein structure and different mode of action than Cry5B, another nematicidal PFT. However, little is known about the mode of action of Cry6A. Although an intracellular nematicidal necrosis pathway of Cry6A was reported, its extracellular mode of action remains unknown. We here demonstrate that the CUB-like-domain containing protein RBT-1 acts as a functional receptor of Cry6A, which mediates the intestinal cell interaction and nematicidal activity of this toxin. RBT-1 represents a new class of crystal protein receptors. RBT-1 is dispensable for Cry5B toxicity against nematodes, consistent with that Cry6A and Cry5B have different nematicidal mechanisms. We also find that Cry6A kills nematodes by complex mechanism since rbt-1 mutation did not affect Cry6A-mediated necrosis signaling pathway. This work not only enhances the understanding of Bt crystal protein-nematode mechanism, but is also in favor for the application of Cry6A in nematode control.Plant-parasitic nematodes cause huge agricultural economic losses. Two major families of Bacillus thuringiensis crystal proteins, Cry5 and Cry6, show nematicidal activity. Previous work showed that binding to midgut receptors is a limiting step in Cry toxin mode of action. In the case of Cry5Ba, certain Caenorhabditis elegans glycolipids were identified as receptors of this toxin. However, the receptors for Cry6 toxin remain unknown. In this study, the C. elegans CUB-like-domain containing protein RBT-1, released by phosphatidylinositol-specific phospholipase C (PI-PLC), was identified as a Cry6Aa binding protein by affinity chromatography. RBT-1 contained a predicted glycosylphosphatidylinositol (GPI) anchor site and was shown to locate in lipid rafts in the surface of the midgut cells. Western ligand blot assays and ELISA binding analysis confirmed the binding interaction between Cry6Aa and RBT-1 showing high affinity and specificity. In addition, the mutation of rbt-1 gene decreased the susceptibility of C. elegans to Cry6Aa but not that of Cry5Ba. Furthermore, RBT-1 mediated the uptake of Cry6Aa into C. elegans gut cells, and was shown to be involved in triggering pore-formation activity, indicating that RBT-1 is required for the interaction of Cry6Aa with the nematode midgut cells. These results support that RBT-1 is a functional receptor for Cry6Aa.