Chang Jeff H, Urbach Jonathan M, Law Terry F, Arnold Larry W, Hu An, Gombar Saurabh, Grant Sarah R, Ausubel Frederick M, Dangl Jeffery L
Department of Biology CB#3280, Carolina Center for Genome Sciences, University of North Carolina, Chapel Hill, NC 27599, USA.
Proc Natl Acad Sci U S A. 2005 Feb 15;102(7):2549-54. doi: 10.1073/pnas.0409660102. Epub 2005 Feb 8.
Pseudomonas syringae strains deliver variable numbers of type III effector proteins into plant cells during infection. These proteins are required for virulence, because strains incapable of delivering them are nonpathogenic. We implemented a whole-genome, high-throughput screen for identifying P. syringae type III effector genes. The screen relied on FACS and an arabinose-inducible hrpL sigma factor to automate the identification and cloning of HrpL-regulated genes. We determined whether candidate genes encode type III effector proteins by creating and testing full-length protein fusions to a reporter called Delta79AvrRpt2 that, when fused to known type III effector proteins, is translocated and elicits a hypersensitive response in leaves of Arabidopsis thaliana expressing the RPS2 plant disease resistance protein. Delta79AvrRpt2 is thus a marker for type III secretion system-dependent translocation, the most critical criterion for defining type III effector proteins. We describe our screen and the collection of type III effector proteins from two pathovars of P. syringae. This stringent functional criteria defined 29 type III proteins from P. syringae pv. tomato, and 19 from P. syringae pv. phaseolicola race 6. Our data provide full functional annotation of the hrpL-dependent type III effector suites from two sequenced P. syringae pathovars and show that type III effector protein suites are highly variable in this pathogen, presumably reflecting the evolutionary selection imposed by the various host plants.
丁香假单胞菌菌株在感染植物细胞的过程中会向植物细胞输送数量不等的III型效应蛋白。这些蛋白是致病所必需的，因为无法输送这些蛋白的菌株没有致病性。我们实施了一项全基因组高通量筛选，以鉴定丁香假单胞菌的III型效应基因。该筛选依赖于荧光激活细胞分选技术（FACS）和一种阿拉伯糖诱导型hrpL σ因子，以自动鉴定和克隆受HrpL调控的基因。我们通过构建并测试与一种名为Delta79AvrRpt2的报告蛋白的全长蛋白融合体，来确定候选基因是否编码III型效应蛋白。当Delta79AvrRpt2与已知的III型效应蛋白融合时，它会被转运，并在表达RPS2植物抗病蛋白的拟南芥叶片中引发超敏反应。因此，Delta79AvrRpt2是III型分泌系统依赖性转运的一个标志物，这是定义III型效应蛋白的最关键标准。我们描述了我们的筛选方法以及从丁香假单胞菌的两个致病变种中收集的III型效应蛋白。这种严格的功能标准确定了来自丁香假单胞菌番茄致病变种的29种III型蛋白，以及来自丁香假单胞菌菜豆致病变种6号小种的19种III型蛋白。我们的数据提供了来自两个已测序的丁香假单胞菌致病变种中依赖hrpL的III型效应蛋白组的完整功能注释，并表明在这种病原体中III型效应蛋白组高度可变，这大概反映了各种寄主植物施加的进化选择。
