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Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

ABSTRACT: Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expression in a self-fertile strain. Many mating-induced genes were associated with cell wall biosynthesis, which may relate to forming the thick-walled sexual spore (oospore). Several gene families were induced during mating including one encoding histidine, serine, and tyrosine-rich putative wall proteins, and another encoding prolyl hydroxylases which may strengthen the extracellular matrix. The sizes of these families vary >10-fold between Phytophthora species and one exhibits concerted evolution, highlighting two features of genome dynamics within the genus. Proteomic analyses of mature oospores and nonmating hyphae using isobaric tags for quantification identified 835 shared proteins, with 5% showing >2-fold changes in abundance between the tissues. Enriched in oospores were ?-glucanases potentially involved in digesting the oospore wall during germination. Despite being dormant, oospores contained a mostly normal complement of proteins required for core cellular functions. The RNA-seq data generated here and in prior studies were used to identify new housekeeping controls for gene expression studies that are more stable than existing normalization standards. We also observed >2-fold variation in the fraction of polyA+ RNA between life stages, which should be considered when quantifying transcripts and may also be relevant to understanding translational control during development.

SUBMITTER: Niu X

PROVIDER: S-EPMC5999078 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

Niu Xiaofan X Ah-Fong Audrey M V AMV Lopez Lilianna A LA Judelson Howard S HS

PloS one 20180613 6

Sexual reproduction remains an understudied feature of oomycete biology. To expand our knowledge of this process, we used RNA-seq and quantitative proteomics to examine matings in Phytophthora infestans. Exhibiting significant changes in mRNA abundance in three matings between different A1 and A2 strains compared to nonmating controls were 1170 genes, most being mating-induced. Rising by >10-fold in at least one cross were 455 genes, and 182 in all three crosses. Most genes had elevated expressi ...[more]

PMID: 29897992

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Project description:AtGenExpress: A multinational coordinated effort to uncover the transcriptome of the multicellular model organism Arabidopsis thaliana (Hybridisations done at NASC) The activity of genes and their encoded products can be regulated in several ways, but transcription is the primary level, since all other modes of regulation (RNA splicing, RNA and protein stability, etc.) are dependent on a gene being transcribed in the first place. The importance of transcriptional regulation has been underscored by the recent flood of global expression analyses, which have confirmed that transcriptional co-regulation of genes that act together is the norm, not the exception. Moreover, many studies suggest that evolutionary change is driven in large part by modifications of transcriptional programs. An essential first step toward deciphering the transcriptional code is to determine the expression pattern of all genes. With this goal in mind, an international effort to develop a gene expression atlas of Arabidopsis has been underway since fall 2003. This project, dubbed AtGenExpress, is funded by the DFG, and will provide the Arabidopsis community with access to a large set of Affymetrix microarray data. As part of this collaboration, we have generated expression data from 80 biologicaly different samples in triplicate. Series 2: Base line experiment for pathogen infection. Growth and infection conditions: Seeds will be stratified for 3 days at 4°C and sown on soil. Plants are grown at 22°C under a 8/16 hour light/dark regime in Percival growth chambers. All pathogen treatments will be performed on leaves of 5-weeks old plants. Bacterial infiltrations will be performed with 10-8 cfu/ml in 10 mM MgCl2. 5x105 spores of Phytophthora infestans in water will be applied to leaf surfaces. LPS (100 µg/ml), HrpZ (1 µM), NPP1 (2 µM) or flagellin (1 µM flg22) will be applied in water. Experiments should be performed in triplicate at the time points indicated below. Experimenter name = Dierk Scheel , Frederic Brunner , Lore Westphal Experimenter institute = AtGenExpress Keywords: genetic_modification_design; organism_part_comparison_design; development_or_differentiation_design

Project description:BACKGROUND: A critical aspect of plant infection by the majority of pathogens is penetration of the plant cell wall. This process requires the production and secretion of a broad spectrum of pathogen enzymes that target and degrade the many complex polysaccharides in the plant cell wall. As a necessary framework for a study of the expression of cell wall degrading enzymes (CWDEs) produced by the broad host range phytopathogen, Phytophthora parasitica, we have conducted an in-depth bioinformatics analysis of the entire complement of genes encoding CWDEs in this pathogen's genome. RESULTS: Our bioinformatic analysis indicates that 431 (2%) of the 20,825 predicted proteins encoded by the P. parasitica genome, are carbohydrate-active enzymes (CAZymes) involved in the degradation of cell wall polysaccharides. Of the 431 proteins, 337 contain classical N-terminal secretion signals and 67 are predicted to be targeted to the non-classical secretion pathway. Identification of CAZyme catalytic activity based on primary protein sequence is difficult, nevertheless, detailed comparisons with previously characterized enzymes has allowed us to determine likely enzyme activities and targeted substrates for many of the P. parasitica CWDEs. Some proteins (12%) contain more than one CAZyme module but, in most cases, multiple modules are from the same CAZyme family. Only 12 P. parasitica CWDEs contain both catalytically-active (glycosyl hydrolase) and non-catalytic (carbohydrate binding) modules, a situation that contrasts with that in fungal phytopathogens. Other striking differences between the complements of CWDEs in P. parasitica and fungal phytopathogens are seen in the CAZyme families that target cellulose, pectins or ?-1,3-glucans (e.g. callose). About 25% of P. parasitica CAZymes are solely directed towards pectin degradation, with the majority coming from pectin lyase or carbohydrate esterase families. Fungal phytopathogens typically contain less than half the numbers of these CAZymes. The P. parasitica genome, like that of other Oomycetes, is rich in CAZymes that target ?-1,3-glucans. CONCLUSIONS: This detailed analysis of the full complement of P. parasitica cell wall degrading enzymes provides a framework for an in-depth study of patterns of expression of these pathogen genes during plant infection and the induction or repression of expression by selected substrates.

Dataset Information

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

Publications

Transcriptomic and proteomic analysis reveals wall-associated and glucan-degrading proteins with potential roles in Phytophthora infestans sexual spore development.

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