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

革兰氏阴性植物病原菌野油菜黄单胞菌泡囊藻使用III型分泌系统(T3S)将效应蛋白转移到植物细胞中。T3S系统是大多数革兰氏阴性动植物病原菌的重要致病因子，由一个跨膜的分泌器组成，该分泌器与真核细胞质膜上的一个胞外毛状附属物和一个转位有关。在过去的资助期间，我们已经从野油菜X.campestris PV中生成了一个模块化的T3S系统。利用基于金门的模块化克隆系统MoClo，通过组装来自启动子和ORF模块的所有遗传元件，实现了对水泡菌的克隆。由此得到的模块化T3S基因簇在野油菜黄单胞菌中具有功能。能够快速有效地交换单个基因或操纵子，用于功能研究和报告融合的插入。我们使用模块化的T3S基因簇来研究预测的细胞质(C)环成分HrcQ的荧光融合的组装，结果表明C环的有效组装依赖于ATPase复合体和尚未鉴定的HrpB4蛋白。拟议项目的重点是使用和优化模块化T3S系统，以研究T3S系统组件的功能和将蛋白质运送到植物细胞中。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来进一步表征T3S系统的组装。更多的体内和体外相互作用研究将有助于具体分析尚未确定的T3S系统的细胞质分选平台。在项目的第二部分，我们将对T3S系统操纵子进行重组，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MoClo系统的修改版本，该系统允许在每个ORF的上游插入核糖体结合位点(RB)，从而优化基因表达。在拟议项目的第三部分，这种方法还将有助于优化其他细菌受体的T3S基因表达，该项目将侧重于建立模块化的T3S系统，作为生物技术方法和基础研究的蛋白质输送工具。

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.