Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana
Molecular genetic evidence for the role of SGT1 in the intramolecular complementation of Bs2 protein activity in Nicotiana benthamiana
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DOI：
10.1105/tpc.104.029637
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发表时间：
2005-04-01
期刊：
影响因子：
11.6
通讯作者：
Staskawicz, BJ
中科院分区：
文献类型：
--
作者：
Leister, RT;Dahlbeck, D;Staskawicz, BJ
含有Bs2抗性基因的辣椒对表达细菌效应蛋白AvrBs2的油菜黄单胞菌（Xcv）具有抗性。AvrBs2通过Xcv的III型蛋白分泌系统（TTSS）直接传递到植物细胞。当表达Bs2基因的植物识别AvrBs2后，一个信号转导级联被激活，导致细菌抗病反应。在这里，我们描述了一个新的病理系统，该系统由表位标记的表达bs2的转基因benthamiana植物和通过丁香假单胞菌的TTSS传递Xcv AvrBs2蛋白效应域的烟草工程菌株组成。这一病理系统使我们能够利用benthamiana作为模式寄主植物，利用农杆菌介导的瞬时蛋白表达与病毒诱导的基因沉默相结合来验证基因，并确定植物寄主抗性表达所需的蛋白相互作用。在这项研究中，我们证明了两个基因，NbSGT1和NbNPK1，是Bs2/ avrbs2介导的耐药应答所必需的，但NbRAR1不是。在这些植物中的蛋白定位研究表明，全长Bs2主要定位于植物细胞质中。Bs2的三个蛋白结构域已被确定：N端、一个中心核苷酸结合位点和一个c端富亮氨酸重复序列（LRR）。共免疫沉淀研究表明，不同的表位标记的Bs2结构域构建物在植物细胞中以反式特异性相互作用。共免疫沉淀研究也表明，Bs2的功能需要依赖于nbsgt1的分子内相互作用。此外，Bs2已被证明通过w的LRR结构域与SGT1结合，这些数据表明SGT1在Bs2的适当折叠或Bs2-SGT1蛋白复合物的形成中发挥作用，这是细菌抗病表达所必需的。
Pepper plants (Capsicum annuum) containing the Bs2 resistance gene are resistant to strains of Xanthomonas campestris pv vesicatoria (Xcv) expressing the bacterial effector protein AvrBs2. AvrBs2 is delivered directly to the plant cell via the type III protein secretion system (TTSS) of Xcv. Upon recognition of AvrBs2 by plants expressing the Bs2 gene, a signal transduction cascade is activated leading to a bacterial disease resistance response. Here, we describe a novel pathosystem that consists of epitope-tagged Bs2-expressing transgenic Nicotiana benthamiana plants and engineered strains of Pseudomonas syringae pv tabaci that deliver the effector domain of the Xcv AvrBs2 protein via the TTSS of P. syringae. This pathosystem has allowed us to exploit N. benthamiana as a model host plant to use Agrobacterium tumetaciens-mediated transient protein expression in conjunction with virus-induced gene silencing to validate genes and to identify protein interactions required for the expression of plant host resistance. In this study, we demonstrate that two genes, NbSGT1 and NbNPK1, are required for the Bs2/AvrBs2-mediated resistance responses but that NbRAR1 is not. Protein localization studies in these plants indicate that full-length Bs2 is primarily localized in the plant cytoplasm. Three protein domains of Bs2 have been identified: the N terminus, a central nucleotide binding site, and a C-terminal Leu-rich repeat (LRR). Coimmunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Coimmunoprecipitation studies also demonstrate that an NbSGT1-dependent intramolecular interaction is required for Bs2 function. Additionally, Bs2 has been shown to associate with SGT1 via the LRR domain of W. These data suggest a role for SGT1 in the proper folding of Bs2 or the formation of a Bs2-SGT1-containing protein complex that is required for the expression of bacterial disease resistance.