Structure-function relationship of the type III-secreted protein NopE1 of Bradyrhizobium japonicum

日本慢生根瘤菌Ⅲ型分泌蛋白NopE1的结构与功能关系

• 批准号: 162945840
• 负责人: Dr. Susanne Zehner
• 金额: --
• 依托单位: Professur für Molekulargenetik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2009
• 资助国家: 德国
• 起止时间: 2008-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/162945840?language=en
• 关键词: Structure; function; relationship; type; III

Previous studies showed that the type III secretion system of Bradyrhizobium japonicum affects symbiosis with different host plants. We identified two highly similar secreted proteins, NopE1 and NopE2, which are responsible for a nodulation-promoting effect on soybean and Macroptilium atropurpureum. Interestingly, both proteins exhibit self-cleavage. Because nodulation assays and in vitro analysis showed that both proteins have the same or a very similar function, we will concentrate on the in vivo activity of NopE1. For localization of NopE1 in nodule cells, reporter gene fusions with the calmodulin-dependent adenylate cyclase domain of the Bordetella pertussis CyaA protein, differential fractionation and immunological techniques will be used. This will also reveal if the fusion protein is present as fulllength protein or in cleaved form. To further examine if autocleavage is necessary for in vivo activity, a NopE1-NopE2 double-mutant will be complemented with the wildtype gene and a mutant derivative, which encodes a non-cleavable NopE1 protein. For autocleavage of NopE1 calcium ions are required. Because calcium is a wellstudied signalling molecule in plants, the calcium-binding activity of NopE1 and its part in protein function is of interest. Therefore, the putative calcium-binding sites, which form EF hand-like calcium-binding domains, will be mutagenised. The calciumbinding capacity of wild-type and modified proteins will be compared. Derivatives that are unable to bind calcium will be used for complementation of the nopE1-nopE2 double-mutant. These data will provide a new insight into the function of the novel effector protein NopE1.

以往的研究表明，慢生根瘤菌的III型分泌系统影响与不同寄主植物的共生关系。我们确定了两个高度相似的分泌蛋白，NopE 1和NopE 2，这是负责对大豆和Macroptilium atropurpureum的结瘤促进作用。有趣的是，这两种蛋白质都表现出自我切割。由于体外分析和诱导试验表明两种蛋白质具有相同或非常相似的功能，因此我们将集中研究NopE 1的体内活性。对于NopE 1在结节细胞中的定位，将使用报告基因与百日咳博德特氏菌CyaA蛋白的钙调蛋白依赖性腺苷酸环化酶结构域的融合、差异分级分离和免疫学技术。这也将揭示融合蛋白是否作为全长蛋白或以裂解形式存在。为了进一步检查自身切割是否是体内活性所必需的，NopE 1-NopE 2双突变体将与野生型基因和突变体衍生物互补，所述突变体衍生物编码不可切割的NopE 1蛋白。对于NopE 1的自裂解，需要钙离子。由于钙是植物中一种被广泛研究的信号分子，因此NopE 1的钙结合活性及其在蛋白质功能中的作用引起了人们的兴趣。因此，形成EF手样钙结合结构域的推定钙结合位点将被诱变。将比较野生型和修饰蛋白的钙结合能力。不能结合钙的衍生物将用于nopE 1-nopE 2双突变体的互补。这些数据将为新型效应蛋白NopE 1的功能提供新的见解。