Scheibner Felix, Schulz Steve, Hausner Jens, Marillonnet Sylvestre, Büttner Daniela
Institute of Biology, Department of Genetics, Martin Luther University Halle-Wittenberg, Halle (Saale), Germany.
Leibniz Institute of Plant Biochemistry, Halle (Saale), Germany.
Appl Environ Microbiol. 2016 May 16;82(11):3331-3347. doi: 10.1128/AEM.00537-16. Print 2016 Jun 1.
The plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria employs a type III secretion (T3S) system to translocate effector proteins into plant cells. The T3S apparatus spans both bacterial membranes and is associated with an extracellular pilus and a channel-like translocon in the host plasma membrane. T3S is controlled by the switch protein HpaC, which suppresses secretion and translocation of the predicted inner rod protein HrpB2 and promotes secretion of translocon and effector proteins. We previously reported that HrpB2 interacts with HpaC and the cytoplasmic domain of the inner membrane protein HrcU (C. Lorenz, S. Schulz, T. Wolsch, O. Rossier, U. Bonas, and D. Büttner, PLoS Pathog 4:e1000094, 2008, http://dx.doi.org/10.1371/journal.ppat.1000094). However, the molecular mechanisms underlying the control of HrpB2 secretion are not yet understood. Here, we located a T3S and translocation signal in the N-terminal 40 amino acids of HrpB2. The results of complementation experiments with HrpB2 deletion derivatives revealed that the T3S signal of HrpB2 is essential for protein function. Furthermore, interaction studies showed that the N-terminal region of HrpB2 interacts with the cytoplasmic domain of HrcU, suggesting that the T3S signal of HrpB2 contributes to substrate docking. Translocation of HrpB2 is suppressed not only by HpaC but also by the T3S chaperone HpaB and its secreted regulator, HpaA. Deletion of hpaA, hpaB, and hpaC leads to a loss of pathogenicity but allows the translocation of fusion proteins between the HrpB2 T3S signal and effector proteins into leaves of host and non-host plants.
The T3S system of the plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria is essential for pathogenicity and delivers effector proteins into plant cells. T3S depends on HrpB2, which is a component of the predicted periplasmic inner rod structure of the secretion apparatus. HrpB2 is secreted during the early stages of the secretion process and interacts with the cytoplasmic domain of the inner membrane protein HrcU. Here, we localized the secretion and translocation signal of HrpB2 in the N-terminal 40 amino acids and show that this region is sufficient for the interaction with the cytoplasmic domain of HrcU. Our results suggest that the T3S signal of HrpB2 is required for the docking of HrpB2 to the secretion apparatus. Furthermore, we provide experimental evidence that the N-terminal region of HrpB2 is sufficient to target effector proteins for translocation in a nonpathogenic X. campestris pv. vesicatoria strain.
植物致病细菌野油菜黄单胞菌疮痂致病变种利用III型分泌（T3S）系统将效应蛋白转运到植物细胞中。T3S装置跨越细菌的两层膜，并与细胞外菌毛以及宿主质膜中的通道样转运体相关联。T3S由开关蛋白HpaC控制，HpaC抑制预测的内杆蛋白HrpB2的分泌和转运，并促进转运体和效应蛋白的分泌。我们之前报道过HrpB2与HpaC以及内膜蛋白HrcU的细胞质结构域相互作用（C. 洛伦茨、S. 舒尔茨、T. 沃尔施、O. 罗西耶、U. 博纳斯和D. 比特纳，《公共科学图书馆·病原体》4：e1000094，2008，http://dx.doi.org/10.1371/journal.ppat.1000094）。然而，控制HrpB2分泌的分子机制尚不清楚。在此，我们在HrpB2的N端40个氨基酸中定位到了一个T3S和转运信号。用HrpB2缺失衍生物进行的互补实验结果表明，HrpB2的T3S信号对蛋白质功能至关重要。此外，相互作用研究表明，HrpB2的N端区域与HrcU的细胞质结构域相互作用，这表明HrpB2的T3S信号有助于底物对接。HrpB2的转运不仅受到HpaC的抑制，还受到T3S伴侣蛋白HpaB及其分泌的调节蛋白HpaA的抑制。缺失hpaA、hpaB和hpaC会导致致病性丧失，但允许HrpB2 T3S信号与效应蛋白之间的融合蛋白转运到宿主植物和非宿主植物的叶片中。
植物致病细菌野油菜黄单胞菌疮痂致病变种的T3S系统对致病性至关重要，并将效应蛋白输送到植物细胞中。T3S依赖于HrpB2，HrpB2是分泌装置预测的周质内杆结构的一个组成部分。HrpB2在分泌过程的早期阶段被分泌，并与内膜蛋白HrcU的细胞质结构域相互作用。在此，我们将HrpB2的分泌和转运信号定位在N端40个氨基酸中，并表明该区域足以与HrcU的细胞质结构域相互作用。我们的结果表明，HrpB2的T3S信号是HrpB2对接分泌装置所必需的。此外，我们提供了实验证据，表明HrpB2的N端区域足以将效应蛋白靶向转运到非致病性野油菜黄单胞菌疮痂致病变种菌株中。
