Drehkopf Sabine, Otten Christian, Büttner Daniela
Institute for Biology, Department of Genetics, Martin Luther University Halle-Wittenberg, Halle (Saale), Saxony-Anhalt, Germany.
Front Plant Sci. 2022 Jul 28;13:955776. doi: 10.3389/fpls.2022.955776. eCollection 2022.
The Gram-negative plant-pathogenic bacterium is the causal agent of bacterial spot disease in pepper and tomato plants. Pathogenicity of depends on a type III secretion () system which translocates effector proteins into plant cells and is associated with an extracellular pilus and a translocon in the plant plasma membrane. Effector protein translocation is activated by the cytoplasmic chaperone HpaB which presumably targets effectors to the system. We previously reported that HpaB is controlled by the translocated regulator HpaA which binds to and inactivates HpaB during the assembly of the system. In the present study, we show that translocation of HpaA depends on the substrate specificity switch protein HpaC and likely occurs after pilus and translocon assembly. Translocation of HpaA requires the presence of a translocation motif (TrM) in the N-terminal region. The TrM consists of an arginine-and proline-rich amino acid sequence and is also essential for the function of HpaA. Mutation of the TrM allowed the translocation of HpaA in mutant strains but not in the wild-type strain, suggesting that the recognition of the TrM depends on HpaB. Strikingly, the contribution of HpaB to the TrM-dependent translocation of HpaA was independent of the presence of the C-terminal HpaB-binding site in HpaA. We propose that HpaB generates a recognition site for the TrM at the system and thus restricts the access to the secretion channel to effector proteins. Possible docking sites for HpaA at the system were identified by and interaction studies and include the ATPase HrcN and components of the predicted cytoplasmic sorting platform of the system. Notably, the TrM interfered with the efficient interaction of HpaA with several system components, suggesting that it prevents premature binding of HpaA. Taken together, our data highlight a yet unknown contribution of the TrM and HpaB to substrate recognition and suggest that the TrM increases the binding specificity between HpaA and system components.
革兰氏阴性植物病原菌是辣椒和番茄植株细菌性斑点病的致病因子。该病原菌的致病性取决于III型分泌（T3S）系统，该系统将效应蛋白转运到植物细胞中，并与细胞外菌毛和植物质膜中的转位体相关。效应蛋白的转运由细胞质伴侣蛋白HpaB激活，HpaB可能将效应蛋白靶向T3S系统。我们之前报道过，HpaB受转运调节因子HpaA的控制，HpaA在T3S系统组装过程中与HpaB结合并使其失活。在本研究中，我们表明HpaA的转运取决于T3S底物特异性开关蛋白HpaC，并且可能发生在菌毛和转位体组装之后。HpaA的转运需要在其N端区域存在一个转运基序（TrM）。TrM由富含精氨酸和脯氨酸的氨基酸序列组成，对HpaA的T3S功能也至关重要。TrM的突变使得HpaA能够在T3S突变菌株中转运，但在野生型菌株中则不能，这表明TrM的识别依赖于HpaB。令人惊讶的是，HpaB对HpaA依赖TrM的转运的作用与HpaA中C端HpaB结合位点的存在无关。我们提出，HpaB在T3S系统上为TrM生成一个识别位点，从而限制效应蛋白进入分泌通道。通过亲和和相互作用研究确定了HpaA在T3S系统上可能的对接位点，包括ATP酶HrcN和T3S系统预测的细胞质分选平台的组分。值得注意的是，TrM干扰了HpaA与几个T3S系统组分的有效相互作用，这表明它可防止HpaA过早结合。综上所述，我们的数据突出了TrM和HpaB在底物识别方面尚未明确的作用，并表明TrM增加了HpaA与T3S系统组分之间的结合特异性。
