Leister R Todd, Dahlbeck Douglas, Day Brad, Li Yi, Chesnokova Olga, Staskawicz Brian J
Department of Plant and Microbial Biology, University of California, Berkeley, California 94720-3102, USA.
Plant Cell. 2005 Apr;17(4):1268-78. doi: 10.1105/tpc.104.029637. Epub 2005 Mar 4.
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 tumefaciens-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). Co-immunoprecipitation studies demonstrate that separate epitope-tagged Bs2 domain constructs interact in trans specifically in the plant cell. Co-immunoprecipitation 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 Bs2. 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.
含有Bs2抗性基因的辣椒植株（辣椒）对表达细菌效应蛋白AvrBs2的野油菜黄单胞菌疮痂致病变种（Xcv）菌株具有抗性。AvrBs2通过Xcv的Ⅲ型蛋白分泌系统（TTSS）直接传递到植物细胞中。当表达Bs2基因的植物识别出AvrBs2时，会激活一个信号转导级联反应，从而引发细菌抗病反应。在此，我们描述了一种新型的病理系统，该系统由表达带有表位标签的Bs2的转基因本氏烟草植株和经过工程改造的丁香假单胞菌烟草致病变种菌株组成，这些菌株通过丁香假单胞菌的TTSS传递Xcv AvrBs2蛋白的效应结构域。这种病理系统使我们能够利用本氏烟草作为模式宿主植物，结合根癌农杆菌介导的瞬时蛋白表达与病毒诱导的基因沉默来验证基因，并确定植物宿主抗性表达所需的蛋白相互作用。在本研究中，我们证明两个基因，即NbSGT1和NbNPK1，是Bs2/AvrBs2介导的抗性反应所必需的，但NbRAR1不是。对这些植物的蛋白定位研究表明，全长Bs2主要定位于植物细胞质中。已鉴定出Bs2的三个蛋白结构域：N端、一个中央核苷酸结合位点和一个C端富含亮氨酸重复序列（LRR）。免疫共沉淀研究表明，单独的带有表位标签的Bs2结构域构建体在植物细胞中特异性地反式相互作用。免疫共沉淀研究还表明，Bs2功能需要一种依赖于NbSGT1的分子内相互作用。此外，已证明Bs2通过其LRR结构域与SGT1结合。这些数据表明SGT1在Bs2的正确折叠或形成表达细菌抗病性所需的含Bs2 - SGT1的蛋白复合物中发挥作用。
