Vencato Monica, Tian Fang, Alfano James R, Buell C Robin, Cartinhour Samuel, DeClerck Genevieve A, Guttman David S, Stavrinides John, Joardar Vinita, Lindeberg Magdalen, Bronstein Philip A, Mansfield John W, Myers Christopher R, Collmer Alan, Schneider David J
Department of Plant Pathology, Cornell University, Ithaca, NY 14853, USA.
Mol Plant Microbe Interact. 2006 Nov;19(11):1193-206. doi: 10.1094/MPMI-19-1193.
The ability of Pseudomonas syringae pv. phaseolicola to cause halo blight of bean is dependent on its ability to translocate effector proteins into host cells via the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS). To identify genes encoding type III effectors and other potential virulence factors that are regulated by the HrpL alternative sigma factor, we used a hidden Markov model, weight matrix model, and type III targeting-associated patterns to search the genome of P. syringae pv. phaseolicola 1448A, which recently was sequenced to completion. We identified 44 high-probability putative Hrp promoters upstream of genes encoding the core T3SS machinery, 27 candidate effectors and related T3SS substrates, and 10 factors unrelated to the Hrp system. The expression of 13 of these candidate HrpL regulon genes was analyzed by real-time polymerase chain reaction, and all were found to be upregulated by HrpL. Six of the candidate type III effectors were assayed for T3SS-dependent translocation into plant cells using the Bordetella pertussis calmodulin-dependent adenylate cyclase (Cya) translocation reporter, and all were translocated. PSPPH1855 (ApbE-family protein) and PSPPH3759 (alcohol dehydrogenase) have no apparent T3SS-related function; however, they do have homologs in the model strain P. syringae pv. tomato DC3000 (PSPTO2105 and PSPTO0834, respectively) that are similarly upregulated by HrpL. Mutations were constructed in the DC3000 homologs and found to reduce bacterial growth in host Arabidopsis leaves. These results establish the utility of the bioinformatic or candidate gene approach to identifying effectors and other genes relevant to pathogenesis in P. syringae genomes.
菜豆丁香假单胞菌致病变种引起菜豆晕疫病的能力取决于其通过过敏反应和致病性（Hrp）III型分泌系统（T3SS）将效应蛋白转运到宿主细胞中的能力。为了鉴定由HrpL替代σ因子调控的III型效应蛋白及其他潜在毒力因子的编码基因，我们使用隐马尔可夫模型、权重矩阵模型和III型靶向相关模式搜索了菜豆丁香假单胞菌致病变种1448A的基因组，该菌株最近已完成测序。我们在编码核心T3SS机制的基因上游鉴定出44个高概率假定Hrp启动子、27个候选效应蛋白及相关T3SS底物，以及10个与Hrp系统无关的因子。通过实时聚合酶链反应分析了这些候选HrpL调控子基因中的13个的表达，发现它们均被HrpL上调。使用百日咳博德特氏菌钙调蛋白依赖性腺苷酸环化酶（Cya）转运报告系统检测了6个候选III型效应蛋白向植物细胞的T3SS依赖性转运，发现它们均可被转运。PSPPH1855（ApbE家族蛋白）和PSPPH3759（乙醇脱氢酶）没有明显的与T3SS相关的功能；然而，它们在模式菌株番茄丁香假单胞菌致病变种DC3000中分别有同源物（分别为PSPTO2105和PSPTO0834），且同样被HrpL上调。在DC3000同源物中构建了突变体，发现其在宿主拟南芥叶片中的细菌生长减少。这些结果确立了生物信息学或候选基因方法在鉴定丁香假单胞菌基因组中与致病相关的效应蛋白和其他基因方面的实用性。
