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

已有研究表明，日本慢生根瘤菌III型分泌系统影响其与不同寄主植物的共生关系。我们发现了两种高度相似的分泌蛋白NopE1和NopE2，它们对大豆和atropurpureum的结瘤促进作用负责。有趣的是，这两种蛋白质都表现出自裂。由于结瘤试验和体外分析表明这两种蛋白具有相同或非常相似的功能，因此我们将集中研究NopE1的体内活性。为了在结节细胞中定位NopE1，将使用报告基因与百日咳杆菌CyaA蛋白的钙调素依赖性腺苷酸环化酶结构域融合，差异分离和免疫学技术。这也将揭示融合蛋白是作为全长蛋白还是以裂解形式存在。为了进一步研究自裂对体内活性是否必要，将一个NopE1- nope2双突变体与野生型基因和一个突变衍生物互补，该突变衍生物编码一个不可切割的NopE1蛋白。对于NopE1的自裂解需要钙离子。由于钙在植物中是一种被广泛研究的信号分子，因此NopE1的钙结合活性及其在蛋白质功能中的作用备受关注。因此，形成EF手状钙结合结构域的假定钙结合位点将被诱变。将比较野生型和修饰型蛋白的钙结合能力。不能结合钙的衍生物将用于补充nopE1-nopE2双突变体。这些数据将为新的效应蛋白NopE1的功能提供新的见解。

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.