Modularization of the type III secretion system from the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria for functional studies and protein delivery

野油菜黄单胞菌（Xanthomonascampestris pv.）vesicatoria使用III型分泌（T3S）系统将效应蛋白转运到植物细胞中。T3S系统是大多数革兰氏阴性植物和动物病原细菌的基本致病因子，并且由跨膜分泌装置组成，该跨膜分泌装置与细胞外菌毛样附属物和真核细胞质膜中的易位子相关。在过去的资助期间，我们已经从X产生了一个模块化的T3S系统。野油菜致病变种通过使用基于Golden Gate的模块化克隆系统MoClo组装来自启动子和ORF模块的所有遗传元件，由此产生的模块化T3S基因簇在X中是功能性的。野油菜致病变种vesicatoria，并允许快速和有效的交换单基因或操纵子的功能研究和插入报告融合。我们使用模块化T3S基因簇来研究预测的胞质（C）环组分HrcQ的荧光融合的组装，并表明C环的有效组装取决于ATP酶复合物和尚未表征的HrpB4蛋白。 拟议项目的重点是使用和优化模块化T3S系统，用于T3S系统组件和蛋白质递送到植物细胞中的功能研究。在第一部分中，我们的目标是通过分析细胞质和膜相关成分的荧光报告融合进一步表征T3S系统组装。额外的体内和体外相互作用研究将有助于具体分析尚未表征的T3S系统的预测细胞质分选平台。在第二部分，我们将重新设计T3S系统操纵子，以避免ORF模块中存在重叠序列，从而促进单个基因的功能研究。为此，我们将使用MoClo系统的修改版本，其允许在每个ORF的上游插入核糖体结合位点（RBS），从而优化基因表达。这种方法也将有助于优化T3S基因在其他细菌受体中的表达，在拟议项目的第三部分，这将侧重于建立模块化T3S系统作为生物技术方法和基础研究的蛋白质递送工具。

The Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria uses a type III secretion (T3S) system to translocate effector proteins into plant cells. T3S systems are essential pathogenicity factors of most Gram-negative plant- and animal-pathogenic bacteria and consist of a membrane-spanning secretion apparatus, which is associated with an extracellular pilus-like appendage and a translocon in the eukaryotic plasma membrane. In the past funding period, we have generated a modular T3S system from X. campestris pv. vesicatoria by assembling all genetic elements from promoter and ORF modules using the Golden Gate-based modular cloning system MoClo. The resulting modular T3S gene cluster is functional in X. campestris pv. vesicatoria and allows the fast and efficient exchange of single genes or operons for functional studies and the insertion of reporter fusions. We used the modular T3S gene cluster to study the assembly of a fluorescent fusion of the predicted cytoplasmic (C) ring component HrcQ and showed that the efficient assembly of the C ring depends on the ATPase complex and the yet uncharacterized HrpB4 protein. Focus of the proposed project is the use and optimization of the modular T3S system for functional studies of T3S system components and protein delivery into plant cells. In the first part, we aim at the further characterization of the T3S system assembly by the analysis of fluorescent reporter fusions of cytoplasmic and membrane-associated components. Additional in vivo and in vitro interaction studies will help to specifically analyse the yet uncharacterized predicted cytoplasmic sorting platform of the T3S system. In the second part of the project, we will reengineer T3S system operons to avoid the presence of overlapping sequences in ORF modules and thus to facilitate functional studies of single genes. For this, we will use a modified version of the MoClo system which allows the insertion of ribosome binding sites (RBS) upstream of each ORF and thus the optimization of gene expression. This approach will also help to optimize T3S gene expression in other bacterial recipients in the third part of the proposed project, which will focus on the establishment of the modular T3S system as a protein delivery tool for both biotechnological approaches and fundamental research.