Roden Julie Anne, Belt Brandis, Ross Jason Barzel, Tachibana Thomas, Vargas Joe, Mudgett Mary Beth
Department of Biological Sciences, Stanford University, Stanford, CA 94305, USA.
Proc Natl Acad Sci U S A. 2004 Nov 23;101(47):16624-9. doi: 10.1073/pnas.0407383101. Epub 2004 Nov 15.
The bacterial pathogen Xanthomonas campestris pv. vesicatoria (Xcv) uses a type III secretion system (TTSS) to translocate effector proteins into host plant cells. The TTSS is required for Xcv colonization, yet the identity of many proteins translocated through this apparatus is not known. We used a genetic screen to functionally identify Xcv TTSS effectors. A transposon 5 (Tn5)-based transposon construct including the coding sequence for the Xcv AvrBs2 effector devoid of its TTSS signal was randomly inserted into the Xcv genome. Insertion of the avrBs2 reporter gene into Xcv genes coding for proteins containing a functional TTSS signal peptide resulted in the creation of chimeric TTSS effector::AvrBs2 fusion proteins. Xcv strains containing these fusions translocated the AvrBs2 reporter in a TTSS-dependent manner into resistant BS2 pepper cells during infection, activating the avrBs2-dependent hypersensitive response (HR). We isolated seven chimeric fusion proteins and designated the identified TTSS effectors as Xanthomonas outer proteins (Xops). Translocation of each Xop was confirmed by using the calmodulin-dependent adenylate cydase reporter assay. Three xop genes are Xanthomonas spp.-specific, whereas homologs for the rest are found in other phytopathogenic bacteria. XopF1 and XopF2 define an effector gene family in Xcv. XopN contains a eukaryotic protein fold repeat and is required for full Xcv pathogenicity in pepper and tomato. The translocated effectors identified in this work expand our knowledge of the diversity of proteins that Xcv uses to manipulate its hosts.
细菌性病原菌野油菜黄单胞菌疮痂致病变种（Xcv）利用Ⅲ型分泌系统（TTSS）将效应蛋白转运到宿主植物细胞中。TTSS是Xcv定殖所必需的，但通过该装置转运的许多蛋白的身份尚不清楚。我们通过遗传筛选从功能上鉴定Xcv TTSS效应蛋白。一个基于转座子5（Tn5）的转座子构建体，包含去除了TTSS信号的Xcv AvrBs2效应蛋白的编码序列，被随机插入到Xcv基因组中。将avrBs2报告基因插入编码含有功能性TTSS信号肽的蛋白的Xcv基因中，导致产生嵌合的TTSS效应蛋白::AvrBs2融合蛋白。含有这些融合蛋白的Xcv菌株在感染期间以TTSS依赖的方式将AvrBs2报告基因转运到抗性BS2辣椒细胞中，激活了avrBs2依赖的过敏反应（HR）。我们分离出了7种嵌合融合蛋白，并将鉴定出的TTSS效应蛋白命名为黄单胞菌外蛋白（Xops）。通过使用钙调蛋白依赖性腺苷酸环化酶报告基因检测法确认了每个Xop的转运。3个xop基因是黄单胞菌属特有的，而其余基因的同源物则在其他植物致病细菌中发现。XopF1和XopF2在Xcv中定义了一个效应蛋白基因家族。XopN包含一个真核蛋白折叠重复序列，是Xcv在辣椒和番茄中完全致病所必需的。在这项工作中鉴定出的转运效应蛋白扩展了我们对Xcv用于操纵其宿主的蛋白多样性的认识。
