EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora

Trudy A. Torto, Shuang Li, Allison Styer, Edgar Huitema, Antonino Testa, Neil A. R. Gow, Pieter van West, Sophien Kamoun

    Research output: Contribution to journalArticle

    253 Citations (Scopus)

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)1675-1685
    Number of pages11
    JournalGenome Research
    Volume13
    Issue number7
    DOIs
    Publication statusPublished - Jul 2003

    Cite this

    Torto, Trudy A. ; Li, Shuang ; Styer, Allison ; Huitema, Edgar ; Testa, Antonino ; Gow, Neil A. R. ; van West, Pieter ; Kamoun, Sophien. / EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. In: Genome Research. 2003 ; Vol. 13, No. 7. pp. 1675-1685.
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    abstract = "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.",
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    EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. / Torto, Trudy A.; Li, Shuang; Styer, Allison; Huitema, Edgar; Testa, Antonino; Gow, Neil A. R.; van West, Pieter; Kamoun, Sophien.

    In: Genome Research, Vol. 13, No. 7, 07.2003, p. 1675-1685.

    Research output: Contribution to journalArticle

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