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

革兰氏阴性植物致病菌油菜黄单胞菌。白斑病利用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致病菌的重要致病因子，由跨膜分泌装置组成，该装置与真核质膜上的细胞外毛菌样附属物和转座有关。在过去的融资期内，我们已经从X. campestris pv生成了模块化的T3S系统。利用基于Golden gate的模块化克隆系统MoClo组装启动子和ORF模块的所有遗传元件。所得到的模块化T3S基因簇在油菜X. campestris pv中起作用。Vesicatoria和允许快速和有效的单基因或操作子交换功能研究和报告融合的插入。我们使用模块化T3S基因簇研究了预测的细胞质(C)环组分HrcQ的荧光融合组装，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。本项目的重点是模块化T3S系统的使用和优化，用于T3S系统组件和蛋白质进入植物细胞的功能研究。在第一部分中，我们旨在通过分析细胞质和膜相关成分的荧光报告融合物来进一步表征T3S系统的组装。另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预测T3S系统的细胞质分选平台。在项目的第二部分，我们将重新设计T3S系统操作子，以避免ORF模块中存在重叠序列，从而促进单基因的功能研究。为此，我们将使用改良版的MoClo系统，该系统允许在每个ORF的上游插入核糖体结合位点（RBS），从而优化基因表达。该方法还将有助于优化T3S基因在其他细菌受体中的表达，该项目的第三部分将重点建立模块化T3S系统，作为生物技术方法和基础研究的蛋白质传递工具。

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.