Data_Sheet_1_The Contribution of the Predicted Sorting Platform Component HrcQ to Type III Secretion in Xanthomonas campestris pv. vesicatoria Depends on an Internal Translation Start Site.pdf

Pathogenicity of the Gram-negative bacterium Xanthomonas campestris pv. vesicatoria depends on a type III secretion (T3S) system which translocates effector proteins into plant cells. T3S systems are conserved in plant- and animal-pathogenic bacteria and consist of at least nine structural core components, which are designated Sct (secretion and cellular translocation) in animal-pathogenic bacteria. Sct proteins are involved in the assembly of the membrane-spanning secretion apparatus which is associated with an extracellular needle structure and a cytoplasmic sorting platform. Components of the sorting platform include the ATPase SctN, its regulator SctL, and pod-like structures at the periphery of the sorting platform consisting of SctQ proteins. Members of the SctQ family form a complex with the C-terminal protein domain, SctQC, which is translated as separate protein and likely acts either as a structural component of the sorting platform or as a chaperone for SctQ. The sorting platform has been intensively studied in animal-pathogenic bacteria but has not yet been visualized in plant pathogens. We previously showed that the SctQ homolog HrcQ from X. campestris pv. vesicatoria assembles into complexes which associate with the T3S system and interact with components of the ATPase complex. Here, we report the presence of an internal alternative translation start site in hrcQ leading to the separate synthesis of the C-terminal protein region (HrcQC). The analysis of genomic hrcQ mutants showed that HrcQC is essential for pathogenicity and T3S. Increased expression levels of hrcQ or the T3S genes, however, compensated the lack of HrcQC. Interaction studies and protein analyses suggest that HrcQC forms a complex with HrcQ and promotes HrcQ stability. Furthermore, HrcQC colocalizes with HrcQ as was shown by fluorescence microscopy, suggesting that it is part of the predicted cytoplasmic sorting platform. In agreement with this finding, HrcQC interacts with the inner membrane ring protein HrcD and the SctK-like linker protein HrpB4 which contributes to the docking of the HrcQ complex to the membrane-spanning T3S apparatus. Taken together, our data suggest that HrcQC acts as a chaperone for HrcQ and as a structural component of the predicted sorting platform.野油菜黄单胞菌辣椒斑点病致病型（Xanthomonas campestris pv. vesicatoria）作为一种革兰氏阴性菌（Gram-negative bacterium），其致病性依赖于Ⅲ型分泌系统（type III secretion system，T3S），该系统可将效应蛋白转运至植物细胞内。Ⅲ型分泌系统在植物及动物致病细菌中保守存在，至少包含9种结构核心组分，在动物致病细菌中被命名为Sct（secretion and cellular translocation，分泌与细胞转运）。Sct蛋白参与跨膜分泌装置的组装，该装置与细胞外针状结构及细胞质分选平台相关联。分选平台的组分包括ATP酶SctN、其调节因子SctL，以及位于分选平台外周、由SctQ蛋白构成的囊状小体结构。SctQ家族成员可与C端蛋白结构域SctQC形成复合物；SctQC作为独立翻译的蛋白，可能作为分选平台的结构组分或SctQ的分子伴侣发挥功能。目前针对动物致病细菌的分选平台已开展了深入研究，但在植物病原菌中尚未观测到该结构的存在。我们前期研究表明，野油菜黄单胞菌辣椒斑点病致病型中的SctQ同源蛋白HrcQ可组装形成与T3S系统结合的复合物，并能与ATP酶复合物的组分发生相互作用。本研究发现hrcQ基因中存在一个内部可变翻译起始位点，可独立合成其C端蛋白区域（HrcQC）。对hrcQ基因组突变体的分析显示，HrcQC对于病原菌的致病性及T3S功能是必需的。然而，提高hrcQ或T3S相关基因的表达水平，可弥补HrcQC缺失所带来的缺陷。互作研究与蛋白分析结果表明，HrcQC可与HrcQ形成复合物，并促进HrcQ的稳定性。此外，通过荧光显微镜观测证实，HrcQC与HrcQ存在共定位现象，提示HrcQC属于预测的细胞质分选平台的组成部分。与此发现一致的是，HrcQC可与内膜环蛋白HrcD以及类似SctK的连接蛋白HrpB4发生相互作用；后者参与HrcQ复合物与跨膜T3S分泌装置的对接过程。综上，本研究数据表明，HrcQC可作为HrcQ的分子伴侣，并作为预测的分选平台的结构组分发挥功能。