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

野油菜黄单胞菌（Xanthomonascampestris pv.）水疱病是辣椒和番茄植物中细菌性斑点病的病原体。致病性X.野油菜致病变种水疱依赖于III型分泌（T3S）系统，其将效应蛋白易位到真核细胞中。T3S系统由跨膜分泌装置组成，该跨膜分泌装置与真核细胞质膜中的细胞外菌毛样附属物和通道样易位子相关联。在过去的资助期间，我们研究了HrcU和HrcV的功能，它们是X T3 S系统的内膜成分。野油菜致病变种vesicatoria，和预测的细胞质环蛋白HrcQ。突变体的方法，本地化和相互作用的实验结果表明，HrcU，HrcV和HrcQ作为对接网站分泌的蛋白质和HrcU的胞质结构域参与T3S底物特异性的控制。在本项目中，我们提出的保守的HrcR，HrcS和HrcT蛋白，这是T3S输出装置在内膜中的预测组件，是必不可少的T3S的功能的特点。初步实验表明，HrcS形成蛋白质复合物，与细菌膜结合。我们计划研究HrcR，HrcS和HrcT的可能的寡聚化以及与T3S系统的组分和底物的相互作用。此外，我们将通过突变体方法鉴定功能重要的蛋白质区域。在本项目的第二部分中，我们将研究黄单胞菌中保守的HrpB7的功能。但还没有功能上的特征。突变体研究和生物化学方法显示，HrpB7是T3S所必需的，并形成蛋白质复合物，其在T3S系统激活后与细菌膜结合。我们计划确定HrpB7的相互作用伙伴，并分析HrpB7中保守氨基酸基序对蛋白质功能和复合物形成的贡献。

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.