Functional analysis of the N terminus of the Erwinia amylovora secreted effector DspA/E reveals features required for secretion, translocation, and binding to the chaperone DspB/F.
Functional analysis of the N terminus of the Erwinia amylovora secreted effector DspA/E reveals features required for secretion, translocation, and binding to the chaperone DspB/F.
复制标题
对梨火疫病菌分泌效应子 DspA/E N 末端的功能分析揭示了分泌、易位和与伴侣 DspB/F 结合所需的特征。
DOI：
10.1094/mpmi-22-10-1282
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发表时间：
2009
期刊：
影响因子：
--
通讯作者：
G. Sundin
中科院分区：
文献类型：
--
作者：
L. Triplett;M. Melotto;G. Sundin
DspA/E是苹果和梨病原菌Erwinia amylovora致病所需的III型分泌效应蛋白，DspB/F是参与DspA/E分泌的小分子伴侣蛋白。虽然在人类肠道病原体中的许多III型分泌效应蛋白的分泌和易位信号已被广泛表征，但对植物病原体中的许多效应蛋白（包括大DspE样蛋白）的易位要求知之甚少。在这项研究中，我们报告了DspE的N端的功能分析。分泌，易位和伴侣结合的最低要求进行了表征。使用腺苷酸环化酶（CyaA）报告易位测定表明，前51个氨基酸的DspE是足够的易位和150个氨基酸所需的最佳易位水平。最小易位信号对应于分泌到培养基中的要求。发现氨基酸2至10和31至40中的保守区域的突变影响N-末端DspE-CyaA融合的易位水平。酵母双杂交和体外下拉分析显示，分子伴侣结合位点内的氨基酸51至100的DspE和结合DspF在这一地区被破坏的特定突变。然而，无论是破坏的伴侣结合结构域，也没有删除dspF基因有显着影响易位水平的N-末端DspE-CyaA融合。我们的研究结果表明，DspE的最小易位信号与DspF结合的信号不一致，DspE的N端易位不依赖于N端DspF结合结构域。
DspA/E is a type III secreted effector protein required for pathogenicity in the apple and pear pathogen Erwinia amylovora, and DspB/F is a small chaperone protein involved in DspA/E secretion. While the secretion and translocation signals of many type III secretion effector proteins in human enteric pathogens have been characterized extensively, relatively little is known about the translocation requirements of many effectors in plant pathogens, including large DspE-like proteins. In this study, we report a functional analysis of the N terminus of DspE. The minimal requirements for secretion, translocation, and chaperone binding were characterized. Translocation assays using an adenylate cyclase (CyaA) reporter indicated that the first 51 amino acids of DspE were sufficient for translocation and that 150 amino acids were required for optimal translocation levels. The minimal translocation signal corresponded with the requirements for secretion into culture media. Mutations of conserved regions in amino acids 2 through 10 and 31 through 40 were found to influence translocation levels of an N-terminal DspE-CyaA fusion. Yeast two-hybrid and in-vitro pull-down assays revealed a chaperone-binding site within amino acids 51 through 100 of DspE and binding to DspF in this region was disrupted by specific mutations. However, neither disruption of the chaperone-binding domain nor deletion of the dspF gene had a significant impact on translocation levels of N-terminal DspE-CyaA fusions. Our results indicate that the minimal translocation signal of DspE is not coincident with the signal for DspF binding and that translocation of the N terminus of DspE is not dependent on the N-terminal DspF-binding domain.
影响因子：
16
作者：
Birtalan, SC;Phillips, RM;Ghosh, P
通讯作者：
Ghosh, P
影响因子：
3.6
作者：
Lee, SH;Galán, JE
通讯作者：
Galán, JE
影响因子：
16
作者：
Lilic, M;Vujanac, M;Stebbins, CE
通讯作者：
Stebbins, CE