EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora
EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora
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DOI：
10.1101/gr.910003
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发表时间：
2003-07-01
期刊：
影响因子：
7
通讯作者：
Kamoun, S
中科院分区：
文献类型：
--
作者：
Torto, TA;Li, SA;Kamoun, S
植物病原微生物具有操纵宿主植物的生化、生理和形态过程的显著能力。这些操纵是通过一系列不同的效应分子来实现的，这些效应分子可以促进感染或触发防御反应。我们描述了一个通用的功能基因组学方法，旨在确定从植物病原微生物的胞外效应蛋白的表达序列标签（ESTs）的数据挖掘与基于病毒的高通量功能表达测定在植物中相结合。Pextrans是一种从EST数据集中自动识别胞外蛋白的算法，并应用于卵菌植物病原体致病疫霉的2147个EST。该程序鉴定了261个EST（12.2%），对应于一组142个非冗余Pex（疫霉胞外蛋白）cDNA。其中，78个（55%）Pex cDNA是新的，在公共数据库中没有显著匹配。使用致病曲霉分泌蛋白的蛋白质组学分析进行Pexlavin的验证。为了鉴定哪些Pex cDNA编码操纵植物过程的效应蛋白，使用携带马铃薯病毒X（PVX）基因组的根癌农杆菌二元载体对63个鉴定的cDNA进行植物中的高通量功能表达测定。这导致了两个新的坏死诱导的cDNA，crn1和crn2，编码细胞外蛋白，属于一个大的和复杂的蛋白质家族在疫霉的发现。对crn基因的进一步表征表明，它们在宿主植物番茄定殖期间在致病疫霉中表达，并且crn2在番茄中诱导防御反应基因。我们的研究结果表明，结合使用Pexplanet的数据挖掘与基于PVX的功能测定可以促进新的病原体效应蛋白的发现。原则上，这种策略可以应用于各种真核植物病原体，包括卵菌，真菌和线虫。
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 A 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.