Experimental design and sample collection.

<p>A 1×10⁵ sporangia/ml solution of <i>Pseudoperonospora cubensis</i> was used to inoculate the abaxial leaf surface of cucumber cultivar ‘Vlaspik’. Samples were collected using a #3 cork borer to minimize uninfected tissue (black circles) at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Leaf disks were used for microscopic analysis of infection stages or pooled for RNA extraction. mRNA-Seq libraries were made for each time point from 2 biological replicates. Within a biological replicate, libraries were barcoded and sequenced in multiple lanes. The sporangia-only library (SP) was not barcoded and was sequenced on its own.</p

Distribution of various carbohydrate-active enzymes (CAZymes) in stramenopile genomes.

<p>The CAZymes coding genes were annotated using the CAZymes Analysis Toolkit- CAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075072#pone.0075072-Park1" target="_blank">[62]</a> according to the CAZy database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075072#pone.0075072-Cantarel1" target="_blank">[61]</a> in combination with protein family domain analyses. Gene families absent in at least 2 species are underlined. Comparison of total CAZymes from different classes is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075072#pone.0075072.s007" target="_blank">Table S6</a>. CE, carbohydrate esterase; GH, glycoside hydrolase; GT, glycosyl transferase; PL, polysaccharide lyase; Pap, <i>Pythium aphanidermatum</i>; Par, <i>Pythium arrhenomanes</i>; Pir, <i>Pythium irregulare</i>; Piw, <i>Pythium iwayamai</i>; Pult, <i>Pythium ultimum</i> var. <i>ultimum</i>; Puls, <i>Pythium ultimum</i> var. <i>sporangiiferum</i>; Pve, <i>Pythium vexans</i>; Phin, <i>Phytophthora infestans</i>; Phrm, <i>Phytophthora ramorum</i>; Phsj, <i>Phytophthora sojae</i>; Hpa, <i>Hyaloperonospora arabidopsidis</i>; Thps, <i>Thalassiosira pseudonana</i>; Phtr, <i>Phaeodactylum tricornutum</i>.</p

Phylogenetic relationship among predicted xyloglucan-β-1,4-D-endoglucanases (GH12) of oomycetes.

<p>Bayesian analysis was performed for 300,000 generations using Blosum model of evolution. Bayesian probabilities are shown next to each branch. An endoglucanase of <i>Aspergillus clavatus</i> (XP_001269687) was used as outgroup. Leaves indicate the locus number of predicted proteins in the genomes of each species (as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072572#pone-0072572-g001" target="_blank">Figure 1</a>).</p

Heat map of the eigengenes representing each gene module.

<p>The columns in the heat map represent time points, and the rows represent eigengenes for each of the six identified co-expression modules <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#pone.0035796-Langfelder2" target="_blank">[50]</a>. The numbers of genes in each module are given in parentheses. The cells in the heat map show eigengene values between 0 and 1, indicators of relative expression levels of all genes in the module (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#s3" target="_blank">Materials and Methods</a>). dpi = days post-inoculation.</p

Pythium irregulare DAOM BR486 Genome Assembly and Annotation

Pythium irregulare DAOM BR486 Genome Assembly and Annotation Contents: pir_functional_annotation.txt; pir.maker.proteins.fasta; pir.maker.transcripts.fasta; pir_contigs_asm.fasta; pir_contigs_asm.maker.gff

Carbohydrate-Active Enzymes in <i>Pythium</i> and Their Role in Plant Cell Wall and Storage Polysaccharide Degradation

<div><p>Carbohydrate-active enzymes (CAZymes) are involved in the metabolism of glycoconjugates, oligosaccharides, and polysaccharides and, in the case of plant pathogens, in the degradation of the host cell wall and storage compounds. We performed an <i>in silico</i> analysis of CAZymes predicted from the genomes of seven <i>Pythium</i> species (<i>Py. aphanidermatum</i>, <i>Py. arrhenomanes</i>, <i>Py. irregulare</i>, <i>Py. iwayamai</i>, <i>Py. ultimum</i> var. <i>ultimum</i>, <i>Py. ultimum</i> var. <i>sporangiiferum</i> and <i>Py. vexans</i>) using the “CAZymes Analysis Toolkit” and “Database for Automated Carbohydrate-active Enzyme Annotation” and compared them to previously published oomycete genomes. Growth of <i>Pythium</i> spp. was assessed in a minimal medium containing selected carbon sources that are usually present in plants. The <i>in silico</i> analyses, coupled with our <i>in vitro</i> growth assays, suggest that most of the predicted CAZymes are involved in the metabolism of the oomycete cell wall with starch and sucrose serving as the main carbohydrate sources for growth of these plant pathogens. The genomes of <i>Pythium</i> spp. also encode pectinases and cellulases that facilitate degradation of the plant cell wall and are important in hyphal penetration; however, the species examined in this study lack the requisite genes for the complete saccharification of these carbohydrates for use as a carbon source. Genes encoding for xylan, xyloglucan, (galacto)(gluco)mannan and cutin degradation were absent or infrequent in <i>Pythium</i> spp.. Comparative analyses of predicted CAZymes in oomycetes indicated distinct evolutionary histories. Furthermore, CAZyme gene families among <i>Pythium</i> spp. were not uniformly distributed in the genomes, suggesting independent gene loss events, reflective of the polyphyletic relationships among some of the species.</p></div

Glycoside hydrolase (GH) families associated with cellulose metabolism.

<p>Families GH1, GH3 and GH5 are cellulase candidates, <i>i.e.</i>, they may or may not be related to cellulose metabolism. Genes belonging to GH6 and GH7 encode enzymes that are strictly related to cellulose metabolism, either to the oomycete cell wall (membrane attached) or to the plant cellulose catabolism (extracellular directed). Species abbreviations are as defined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072572#pone-0072572-g001" target="_blank">Figure 1</a>.</p

Symptoms and microscopy images of <i>Pseudoperonospora cubensis</i> infected <i>Cucumis sativus</i> cultivar ‘Vlaspik’ of time points used for transcriptome analysis.

<p>Symptom images were collected of the adaxial (top row) and abaxial (middle row) at 1, 2, 3, 4, 6, and 8 days post-inoculation (dpi). Microscopy (bottom row) to assess stages of <i>Ps. cubensis</i> invasion were collected from the same time points using ethanol-cleared, trypan blue stained samples. Scale bars at 1–4 dpi are 25 µm. Scale bars at 6 and 8 dpi are 50 µm. Dotted lines represent position of stomata relative to the pathogen structure. e = encysted zoospore. s = stomate. h = haustorium.</p

Proportional growth of <i>Pythium</i> species on a minimal medium (MM) containing various carbon sources to its growth on V8 juice agar.

*<p>Clades denominations are based on Lévesque & de Cock [1414].</p><p>Species: <i>Pyap</i> = <i>Pythium aphanidermatum</i>, <i>Pyar</i> = <i>Py. arrhenomanes</i>, <i>Pyir</i> = <i>Py. irregulare</i>, <i>Pyiw</i> = <i>Py. iwayamai</i>, <i>Pyuu</i> = <i>Py. ultimum</i> var. <i>ultimum</i>, <i>Pyus</i> = <i>Py. ultimum</i> var. <i>sporangiiferum</i>, and <i>Pyve</i> = <i>Py. vexans</i>.</p>**<p>Values represent proportional diameter growth means of each isolate on carbon sources relative to its growth on V8 juice agar (± standard error).</p

Correlation matrix of <i>Pseudoperonospora cubensis</i> expression profiles throughout a time course of <i>Cucumis sativus</i> infection.

<p>Normalized transcript abundances for 7,821 genes were calculated in fragments per kilobase pair of exon model per million fragments mapped (FPKM) with Cufflinks version 0.9.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#pone.0035796-Trapnell1" target="_blank">[56]</a>. Pearson product-moment correlations (PCC) of log2 FPKM values were calculated for all pair-wise combinations using R (<a href="http://cran.r-project.org/" target="_blank">http://cran.r-project.org/</a>). PCCs were clustered using hierarchical clustering with a Pearson correlation distance metric and average linkage using Multiple Experiment Viewer Software version 4.5 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#pone.0035796-Saeed1" target="_blank">[57]</a>. The bootstrap support values shown on tree nodes were obtained from 1000 bootstrap replicates. dpi = days post-inoculation.</p