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      Molecular Profiling of the Phytophthora plurivora Secretome: A Step towards Understanding the Cross-Talk between Plant Pathogenic Oomycetes and Their Hosts

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          Abstract

          The understanding of molecular mechanisms underlying host–pathogen interactions in plant diseases is of crucial importance to gain insights on different virulence strategies of pathogens and unravel their role in plant immunity. Among plant pathogens, Phytophthora species are eliciting a growing interest for their considerable economical and environmental impact. Plant infection by Phytophthora phytopathogens is a complex process coordinated by a plethora of extracellular signals secreted by both host plants and pathogens. The characterization of the repertoire of effectors secreted by oomycetes has become an active area of research for deciphering molecular mechanisms responsible for host plants colonization and infection. Putative secreted proteins by Phytophthora species have been catalogued by applying high-throughput genome-based strategies and bioinformatic approaches. However, a comprehensive analysis of the effective secretome profile of Phytophthora is still lacking. Here, we report the first large-scale profiling of P. plurivora secretome using a shotgun LC-MS/MS strategy. To gain insight on the molecular signals underlying the cross-talk between plant pathogenic oomycetes and their host plants, we also investigate the quantitative changes of secreted protein following interaction of P. plurivora with the root exudate of Fagus sylvatica which is highly susceptible to the root pathogen. We show that besides known effectors, the expression and/or secretion levels of cell-wall-degrading enzymes were altered following the interaction with the host plant root exudate. In addition, a characterization of the F. sylvatica root exudate was performed by NMR and amino acid analysis, allowing the identification of the main released low-molecular weight components, including organic acids and free amino acids. This study provides important insights for deciphering the extracellular network involved in the highly susceptible P. plurivora- F. sylvatica interaction.

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          Phytophthora infestans effector AVR3a is essential for virulence and manipulates plant immunity by stabilizing host E3 ligase CMPG1.

          Fungal and oomycete plant pathogens translocate effector proteins into host cells to establish infection. However, virulence targets and modes of action of their effectors are unknown. Effector AVR3a from potato blight pathogen Phytophthora infestans is translocated into host cells and occurs in two forms: AVR3a(KI), which is detected by potato resistance protein R3a, strongly suppresses infestin 1 (INF1)-triggered cell death (ICD), whereas AVR3a(EM), which evades recognition by R3a, weakly suppresses host ICD. Here we show that AVR3a interacts with and stabilizes host U-box E3 ligase CMPG1, which is required for ICD. In contrast, AVR3a(KI/Y147del), a mutant with a deleted C-terminal tyrosine residue that fails to suppress ICD, cannot interact with or stabilize CMPG1. CMPG1 is stabilized by the inhibitors MG132 and epoxomicin, indicating that it is degraded by the 26S proteasome. CMPG1 is degraded during ICD. However, it is stabilized by mutations in the U-box that prevent its E3 ligase activity. In stabilizing CMPG1, AVR3a thus modifies its normal activity. Remarkably, given the potential for hundreds of effector genes in the P. infestans genome, silencing Avr3a compromises P. infestans pathogenicity, suggesting that AVR3a is essential for virulence. Interestingly, Avr3a silencing can be complemented by in planta expression of Avr3a(KI) or Avr3a(EM) but not the Avr3a(KI/Y147del) mutant. Our data provide genetic evidence that AVR3a is an essential virulence factor that targets and stabilizes the plant E3 ligase CMPG1, potentially to prevent host cell death during the biotrophic phase of infection.
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            The spores of Phytophthora: weapons of the plant destroyer.

            Members of the genus Phytophthora are among the most serious threats to agriculture and food production, causing devastating diseases in hundreds of plant hosts. These fungus-like eukaryotes, which are taxonomically classified as oomycetes, generate asexual and sexual spores with characteristics that greatly contribute to their pathogenic success. The spores include survival and dispersal structures, and potent infectious propagules capable of actively locating hosts. Genetic tools and genomic resources developed over the past decade are now allowing detailed analysis of these important stages in the Phytophthora life cycle.
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              EST mining and functional expression assays identify extracellular effector proteins from the plant pathogen Phytophthora.

              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.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2014
                5 November 2014
                : 9
                : 11
                : e112317
                Affiliations
                [1 ]Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy
                [2 ]Department of Human Protein Science, Geneva University, Geneva, Switzerland
                [3 ]Section Pathology of Woody Plants, Technische Universität München (TUM), Freising-Weihenstephan, Germany
                [4 ]IRCCS Multimedica, Milan, Italy
                Seoul National University, Republic of Korea
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AC WO. Performed the experiments: AC A. Farina VS RJDD FF ADM MS A. Fiorentino. Analyzed the data: AC VS A. Farina. Contributed reagents/materials/analysis tools: AC A. Farina WO AP. Contributed to the writing of the manuscript: AC VS A. Farina FF RJDD ADM MS A. Fiorentino AP WO.

                Article
                PONE-D-14-27454
                10.1371/journal.pone.0112317
                4221288
                25372870
                a6e23c20-c6c8-409d-b0e9-177ec6187ada
                Copyright @ 2014

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 July 2014
                : 5 October 2014
                Page count
                Pages: 11
                Funding
                This work was supported by the German Research Foundation (DFG) and the Technische Universität München within the funding programme Open Access Publishing. The authors acknowledge the European project COST Action FP0801 for supporting researchers cooperation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology and Life Sciences
                Biochemistry
                Proteins
                Proteomics
                Physical Sciences
                Chemistry
                Analytical Chemistry
                Mass Spectrometry
                Research and analysis methods
                Spectrum analysis techniques
                NMR spectroscopy
                Custom metadata
                The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files.

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