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

T. A. Torto, Shuang Li, A. Styer, E. Huitema, A. Testa, Neil Andrew Robert Gow, Pieter Van West, S. 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 pages10
JournalGenome Research
Volume13
Issue number7
DOIs
Publication statusPublished - Jul 2003

Keywords

  • POTATO-VIRUS-X
  • AVIRULENCE GENE
  • PHYTOPATHOGENIC BACTERIA
  • POLYACRYLAMIDE-GELS
  • INNATE IMMUNITY
  • RESISTANCE GENE
  • SIGNAL PEPTIDES
  • INFESTANS
  • RECOGNITION
  • SEQUENCES

Cite this

EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora. / Torto, T. A.; Li, Shuang; Styer, A.; Huitema, E.; Testa, A.; Gow, Neil Andrew Robert; Van West, Pieter; Kamoun, S.

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

Research output: Contribution to journalArticle

Torto, T. A. ; Li, Shuang ; Styer, A. ; Huitema, E. ; Testa, A. ; Gow, Neil Andrew Robert ; Van West, Pieter ; Kamoun, S. / 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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T1 - EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora

AU - Torto, T. A.

AU - Li, Shuang

AU - Styer, A.

AU - Huitema, E.

AU - Testa, A.

AU - Gow, Neil Andrew Robert

AU - Van West, Pieter

AU - Kamoun, S.

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AB - 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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KW - INNATE IMMUNITY

KW - RESISTANCE GENE

KW - SIGNAL PEPTIDES

KW - INFESTANS

KW - RECOGNITION

KW - SEQUENCES

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JO - Genome Research

JF - Genome Research

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