CAZymes in <i>Pseudoperonospora cubensis</i> expressed during infection on <i>Cucumis sativus</i>.

<p>The CAZymes coding genes in the <i>Ps. cubensis</i> genome were annotated using CAZymes Analysis Toolkit- CAT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#pone.0035796-Park1" target="_blank">[66]</a> according to the CAZy database <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035796#pone.0035796-Cantarel1" target="_blank">[67]</a> in combination with protein family domain analyses. Gene families absent in at least one time point are underlined. CBM = carbohydrate binding module. CE = carbohydrate esterase. GH = glycoside hydrolase. GT = glycosyl transferase. PE = pectin esterase. PL = polysaccharide lyase. dpi = days post-inoculation.</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

Distribution of genes in the maize seedling core and dispensable transcriptomes determined using a semi-qualitative approach.

<p>Reads were mapped to the 5b pseudomolecules (<a href="http://ftp.maizesequence.org/" target="_blank">http://ftp.maizesequence.org/</a>) using Bowtie version 0.12.7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Langmead1" target="_blank">[50]</a> and TopHat version 1.2.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Trapnell1" target="_blank">[51]</a>, and fragments per kilobase of exon model per million fragments mapped (FPKM) were determined with Cufflinks version 0.9.3 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Trapnell2" target="_blank">[56]</a> and the 5b annotation (<a href="http://ftp.maizesequence.org/" target="_blank">http://ftp.maizesequence.org/</a>).For each gene, a line was considered not expressed if the low confidence FPKM value was equal to zero, low expressed if the low confidence interval was greater than zero and the FPKM value was less than 5, medium expressed if the low confidence interval was greater than zero and the FPKM value was greater than or equal to 5 and less than or equal to 200, and high expressed if the low confidence interval was greater than zero and the FPKM value was greater than 200.</p

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

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

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

Distribution of the number of single nucleotide polymorphisms (SNPs) and SNP density per gene.

<p>Reads were mapped against the 5b pseudomolecules (<a href="http://ftp.maizesequence.org/" target="_blank">http://ftp.maizesequence.org/</a>) with Bowtie version 0.12.7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Langmead1" target="_blank">[50]</a> and TopHat version 1.2.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Trapnell1" target="_blank">[51]</a> requiring a unique hit for the SNP mapping. Gene assignment was determined based on the 5b annotation (<a href="http://ftp.maizesequence.org/" target="_blank">http://ftp.maizesequence.org/</a>), and not all SNPs identified were assigned to a gene model. (A) Distribution of the number of SNPs per gene. (B) Distribution of the average number of SNPs per 100 bp window per gene.</p

Neighbor-Joining tree of 21 diverse maize lines based on 351,710 single nucleotide polymorphisms (SNPs).

<p>Frequency based distances were calculated as pair-wise Rogers distances <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Rogers1" target="_blank">[53]</a>. PowerMarker version 3.25 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Liu2" target="_blank">[54]</a> was used to construct the tree.</p

Read mapping, expression, and single nucleotide polymorphism (SNP) summary for 21 diverse maize lines.

<p>Reads were mapped requiring a unique hit for the SNPs and multiple hits for fragments per kilobase of exon model per million fragments mapped (FPKM) and mapping to the pseudomolecules plus the Velvet and Oases <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Zerbino1" target="_blank">[45]</a> assembled transcripts. FPKM values were calculated using Cufflinks <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Trapnell2" target="_blank">[56]</a>. Genes with a FPKM 95% confidence interval lower boundary greater than zero were considered expressed. For each inbred line, a gene was considered to have SNP coverage if there was at least one polymorphic locus with coverage in the gene. SSS = Stiff Stalk Synthetic, NSS = Non-Stiff Stalk Synthetic, NA = Not applicable.</p

Frequency of novel <i>de novo</i> assembled transcripts across lines and heterotic groups.

<p>Reads were mapped to the 5b pseudomolecules plus assembled novel transcripts with Bowtie version 0.12.7 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Langmead1" target="_blank">[50]</a> and TopHat version 1.2.0 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Trapnell1" target="_blank">[51]</a>. Novel transcripts from unmapped reads were assembled using Velvet version 1.0.17 and Oases version 0.1.18 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033071#pone.0033071-Zerbino1" target="_blank">[45]</a>. (A) Distribution of the number of inbred lines with read support for each novel <i>de novo</i> assembled transcript requiring unique alignments and allowing for multiple mapping. (B) Venn diagram of shared and group specific novel <i>de novo</i> assembled transcripts. (C) Venn diagram of shared and group specific novel <i>de novo</i> assembled transcripts and transcripts from the 5b annotated genes (<a href="http://ftp.maizesequence.org/" target="_blank">http://ftp.maizesequence.org/</a>).</p