Deciphering the functions of type III secretion system components from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria

革兰氏阴性植物病原菌野油菜黄单胞菌水泡菌是辣椒和番茄上细菌性斑点病的病原菌。野油菜黄单胞菌的致病性。膀胱依赖于III型分泌(T3S)系统，该系统将效应蛋白转移到真核细胞中。T3S系统由一个跨膜的分泌器组成，它与真核细胞质膜上的一个胞外毛状附属物和一个通道样转位蛋白相联系。在过去的资助期间，我们研究了野油菜黄单胞菌T3S系统的内膜成分HrcU和HrcV的功能。以及预测的胞质环蛋白HrcQ。突变方法、定位和相互作用实验的结果表明，HrcU、HrcV和HrcQ是分泌蛋白的对接部位，HrcU的细胞质结构域参与了T3S底物特异性的控制。在本项目中，我们建议对保守的HRCR、HRCS和HRCT蛋白的功能进行鉴定，这些蛋白是内膜中T3S输出装置的预测成分，对T3S是必不可少的。初步实验表明，HRCS形成与细菌膜结合的蛋白质复合体。我们计划研究HRCR、HRCS和HRCT可能的寡聚以及与T3S系统的成分和底物的相互作用。此外，我们将通过突变的方法来确定重要的蛋白质功能区域。在本项目的第二部分，我们将研究HrpB7的功能，它在Xanthomonas spp中是保守的。但尚未确定其功能特征。突变研究和生化方法表明，HrpB7对T3S是必不可少的，并形成蛋白质复合体，当T3S系统激活时，蛋白质复合体与细菌膜结合。我们计划寻找HrpB7的相互作用伙伴，并分析HrpB7中保守的氨基酸基序对蛋白质功能和复合体形成的贡献。

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of X. campestris pv. vesicatoria depends on a type III secretion (T3S) system, which translocates effector proteins into eukaryotic cells. T3S systems consist of a membrane-spanning secretion apparatus, which is associated with an extracellular pilus-like appendage and a channel-like translocon in the eukaryotic plasma membrane. In the past funding period, we investigated the functions of HrcU and HrcV, which are inner membrane components of the T3S system from X. campestris pv. vesicatoria, and the predicted cytoplasmic ring protein HrcQ. The results of mutant approaches, localization and interaction experiments suggest that HrcU, HrcV and HrcQ serve as docking sites for secreted proteins and that the cytoplasmic domain of HrcU is involved in the control of T3S substrate specificity. In the present project, we propose to characterize the functions of the conserved HrcR, HrcS and HrcT proteins, which are predicted components of the T3S export apparatus in the inner membrane and are essential for T3S. Preliminary experiments revealed that HrcS forms protein complexes, which associate with the bacterial membranes. We plan to investigate a possible oligomerization of HrcR, HrcS and HrcT as well as interactions with components and substrates of the T3S system. Furthermore, we will identify functionally important protein regions by mutant approaches. In the second part of this project, we will investigate the function of HrpB7, which is conserved in Xanthomonas spp. but has not yet been functionally characterized. Mutant studies and biochemical approaches revealed that HrpB7 is essential for T3S and forms protein complexes, which associate with the bacterial membranes upon activation of the T3S system. We plan to identify HrpB7 interaction partners and to analyse the contribution of conserved amino acid motifs in HrpB7 to protein function and complex formation.