Torto Trudy A, Li Shuang, Styer Allison, Huitema Edgar, Testa Antonino, Gow Neil A R, van West Pieter, Kamoun Sophien
Department of Plant Pathology, The Ohio State University, Ohio Agricultural Research and Development Center, Wooster, Ohio 44691, USA.
Genome Res. 2003 Jul;13(7):1675-85. doi: 10.1101/gr.910003.
Plant pathogenic microbes have the remarkable ability to manipulate biochemical, physiological, and morphological processes in their host plants. These manipulations are achieved through a diverse array of effector molecules that can either promote infection or trigger defense responses. We describe a general functional genomics approach aimed at identifying extracellular effector proteins from plant pathogenic microorganisms by combining data mining of expressed sequence tags (ESTs) with virus-based high-throughput functional expression assays in plants. PexFinder, an algorithm for automated identification of extracellular proteins from EST data sets, was developed and applied to 2147 ESTs from the oomycete plant pathogen Phytophthora infestans. The program identified 261 ESTs (12.2%) corresponding to a set of 142 nonredundant Pex (Phytophthora extracellular protein) cDNAs. Of these, 78 (55%) Pex cDNAs were novel with no significant matches in public databases. Validation of PexFinder was performed using proteomic analysis of secreted protein of P. infestans. To identify which of the Pex cDNAs encode effector proteins that manipulate plant processes, high-throughput functional expression assays in plants were performed on 63 of the identified cDNAs using an Agrobacterium tumefaciens binary vector carrying the potato virus X (PVX) genome. This led to the discovery of two novel necrosis-inducing cDNAs, crn1 and crn2, encoding extracellular proteins that belong to a large and complex protein family in Phytophthora. Further characterization of the crn genes indicated that they are both expressed in P. infestans during colonization of the host plant tomato and that crn2 induced defense-response genes in tomato. Our results indicate that combining data mining using PexFinder with PVX-based functional assays can facilitate the discovery of novel pathogen effector proteins. In principle, this strategy can be applied to a variety of eukaryotic plant pathogens, including oomycetes, fungi, and nematodes.
植物病原微生物具有显著能力来操控其寄主植物的生化、生理和形态过程。这些操控是通过各种各样的效应分子实现的，这些效应分子既可以促进感染，也可以触发防御反应。我们描述了一种通用的功能基因组学方法，旨在通过将表达序列标签（EST）的数据挖掘与基于病毒的植物高通量功能表达分析相结合，从植物病原微生物中鉴定细胞外效应蛋白。开发了一种名为PexFinder的算法，用于从EST数据集中自动鉴定细胞外蛋白，并将其应用于卵菌植物病原菌致病疫霉的2147条EST。该程序鉴定出261条EST（12.2%），对应于一组142个非冗余的Pex（致病疫霉细胞外蛋白）cDNA。其中，78个（55%）Pex cDNA是新的，在公共数据库中没有显著匹配项。使用致病疫霉分泌蛋白的蛋白质组分析对PexFinder进行了验证。为了确定哪些Pex cDNA编码操控植物过程的效应蛋白，使用携带马铃薯X病毒（PVX）基因组的根癌农杆菌二元载体，对63个已鉴定的cDNA进行了植物高通量功能表达分析。这导致发现了两个新的坏死诱导cDNA，crn1和crn2，它们编码属于致病疫霉中一个庞大而复杂蛋白家族的细胞外蛋白。对crn基因的进一步表征表明，它们在致病疫霉侵染寄主植物番茄的过程中均有表达，并且crn2在番茄中诱导防御反应基因。我们的结果表明，将使用PexFinder的数据挖掘与基于PVX的功能分析相结合，可以促进新型病原体效应蛋白的发现。原则上，这种策略可以应用于多种真核植物病原体，包括卵菌、真菌和线虫。
