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

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。我们鉴定了两个高度相似的分泌蛋白，NopE1和NopE2，它们对大豆和紫毛藻具有促进结瘤的作用。有趣的是，这两种蛋白质都表现出自我裂解。由于结瘤实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，所以我们将集中在NopE1的体内活性上。对于NopE1在结节细胞中的定位，将使用报告基因与百日咳杆菌CyaA蛋白的钙调蛋白依赖的腺苷环化酶结构域融合、差异分离和免疫学技术。这也将揭示融合蛋白是以全长蛋白质形式存在还是以切割形式存在。为了进一步研究体内活性是否需要自动切割，将用野生型基因和突变衍生物补充NopE1-NopE2双突变，后者编码不可切割的NopE1蛋白。对于NOPE1的自动裂解，需要钙离子。因为钙是植物中一种被广泛研究的信号分子，所以NopE1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手型钙结合结构域的推定的钙结合部位将被突变。对野生型和修饰蛋白的钙结合能力进行比较。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器蛋白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.