Bioinformatic characterisation of genes encoding cell wall degrading enzymes in the Phytophthora parasitica genome

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 β-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 β-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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-785) contains supplementary material, which is available to authorized users

A Vernalization Response in a Winter Safflower (Carthamus tinctorius) Involves the Upregulation of Homologs of FT, FUL, and MAF

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Summary of expression profiles of <i>P</i>. <i>parasitica</i> CWDEs during the infection of lupin roots.

<p>The top row shows representations of the six main expression profiles displayed by <i>P</i>. <i>parasitica</i> CWDEs. CAZyme families have been assigned to putative polysaccharide category according to homology with characterised CAZymes. Within each major wall polysaccharide category, the NRPK values of CAZyme family members whose expression peaks at the same time (the number of genes is shown in the column labelled #) have been summed and the total peak NRPK value shown in the columns labelled NRPK. CAZyme families whose members act on multiple substrates are indicated by superscripts. These CAZyme families have be allocated to a selected substrate according to recent literature on their most likely function. The superscripts indicate their possible targets: ⁱpectin and glycoproteins; ⁱⁱpectin, hemicellulose and glycoproteins; ⁱⁱⁱcellulose and β-1,3-glucans; ^ivcellulose, hemicellulose and glycoproteins; ^vhemicellulose and glycoproteins; ^vihemicellulose and β-1,3-glucans.</p

Representation of the percentage of NRPK counts for each CWDE family during early (green, 30–36 hpi), middle (yellow, 42–48 hpi) and late (red, 54–60 hpi) infection of lupin roots as a percentage of the total number of NRPK counts from all CWDEs predicted to target a particular substrate.

<p>The total number of NRPK counts for each substrate are shown below the columns. The inset at the top shows the relative heights (transcript abundance) for the different substrate categories. HG: homogalacturonan; RGI: rhamnogalacturonan I.</p

CWDEs in the top 200 differentially expressed genes between 30 hpi and 60 hpi.

<p>The rank, log2 ratio, CAZyme family, predicted function, and transcript numbers are shown.</p><p>CWDEs in the top 200 differentially expressed genes between 30 hpi and 60 hpi.</p

Infection of lupin roots by <i>P</i>. <i>parasitica</i>.

<p><b>(A)</b> Percentage of roots with visible lesions during the infection time course, 0 hpi to 60 hpi. (n = 12). <b>(B)</b> Transverse hand section of a lupin root inoculated with <i>P</i>. <i>parasitica</i> zoospores. By 27 hpi, <i>P</i>. <i>parasitica</i> hyphae grow both intercellularly (arrowheads) and intracellularly (arrows) during colonisation of the root cortex. Scale bar = 50 μm. <b>(C)</b> Ratio of <i>P</i>. <i>parasitica</i> DNA:lupin DNA as determined by qPCR measurement of the levels of the <i>P</i>. <i>parasitica WS041</i> gene and the lupin nitrilase gene 4A (<i>NIT4A</i>). Data are from the three biological replicates chosen for RNA-Seq transcriptome analysis. <i>P</i>. <i>parasitica</i> DNA was not detected in mock-inoculated or 12 hpi or 18 hpi samples. Error bars indicate the standard error of the mean.</p

Total NRPKs from the multiple location data set for genes targeting major categories of wall components.

<p>Because many genes act on cellulose and hemicelluloses, counts for these two substrates have been combined. <b>(A)</b> Total NRPK counts show the relative transcript abundance for each substrate category. <b>(B)</b> Total NRPK counts expressed as a percentage of the maximum counts show trends in the expression profiles over time. Genes whose products target pectins have been assigned to the first and second halves of the 60-h infection time-course.</p

A reconfigured Kennedy pathway which promotes efficient accumulation of medium-chain fatty acids in leaf oils

Medium-chain fatty acids (MCFA, C6-14 fatty acids) are an ideal feedstock for biodiesel and broader oleochemicals. In recent decades, several studies have used transgenic engineering to produce MCFA in seeds oils, although these modifications result in unbalance membrane lipid profiles that impair oil yields and agronomic performance. Given the ability to engineer nonseed organs to produce oils, we have previously demonstrated that MCFA profiles can be produced in leaves, but this also results in unbalanced membrane lipid profiles and undesirable chlorosis and cell death. Here we demonstrate that the introduction of a diacylglycerol acyltransferase from oil palm, EgDGAT1, was necessary to channel nascent MCFA directly into leaf oils and therefore bypassing MCFA residing in membrane lipids. This pathway resulted in increased flux towards MCFA rich leaf oils, reduced MCFA in leaf membrane lipids and, crucially, the alleviation of chlorosis. Deep sequencing of African oil palm (Elaeis guineensis) and coconut palm (Cocos nucifera) generated candidate genes of interest, which were then tested for their ability to improve oil accumulation. Thioesterases were explored for the production of lauric acid (C12:0) and myristic (C14:0). The thioesterases from Umbellularia californica and Cinnamomum camphora produced a total of 52% C12:0 and 40% C14:0, respectively, in transient leaf assays. This study demonstrated that the introduction of a complete acyl-CoA-dependent pathway for the synthesis of MFCA-rich oils avoided disturbing membrane homoeostasis and cell death phenotypes. This study outlines a transgenic strategy for the engineering of biomass crops with high levels of MCFA rich leaf oils