Figure 1.
Experimental design and sample collection.
A 1×105 sporangia/ml solution of Pseudoperonospora cubensis 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.
Figure 2.
Symptoms and microscopy images of Pseudoperonospora cubensis infected Cucumis sativus cultivar ‘Vlaspik’ of time points used for transcriptome analysis.
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 Ps. cubensis 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.
Figure 3.
Number of total mRNA-Seq reads, reads mapped, and number of genes expressed.
A. Total number of reads, number of reads mapped to the Ps. cubensis genome, and number of genes expressed by Ps. cubensis at different time points are shown. Reads were mapped using Bowtie version 0.12.5 [55] and TopHat version 1.2.0 [55]. Fragments per kilobase pair of exon model per million fragments mapped (FPKM) values were calculated using Cufflinks version 0.9.3 [56]. Genes were considered expressed if the FPKM and 95% confidence interval lower boundary FPKM value was greater than 0.001 and zero, respectively. dpi = days post-inoculation. B. Comparison between number of expressed genes detected and sampling depth. For all time points 5, 10, 15, 20, 25, and 30 million reads were randomly selected from the total pool of reads from different time points. Read mapping and expression abundances are as described in panel 3A.
Figure 4.
Correlation matrix of Pseudoperonospora cubensis expression profiles throughout a time course of Cucumis sativus infection.
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 [56]. Pearson product-moment correlations (PCC) of log2 FPKM values were calculated for all pair-wise combinations using R (http://cran.r-project.org/). PCCs were clustered using hierarchical clustering with a Pearson correlation distance metric and average linkage using Multiple Experiment Viewer Software version 4.5 [57]. The bootstrap support values shown on tree nodes were obtained from 1000 bootstrap replicates. dpi = days post-inoculation.
Table 1.
Number of differentially expressed genes between each combination of time points and sporangia.
Figure 5.
Candidate effectors expressed at different time points.
From inner to outermost circles; 2 dpi, 3 dpi, 4 dpi, 6 dpi and 8 dpi. CRN = Crinkler effectors. dpi = days post-inoculation.
Figure 6.
CAZymes in Pseudoperonospora cubensis expressed during infection on Cucumis sativus.
The CAZymes coding genes in the Ps. cubensis genome were annotated using CAZymes Analysis Toolkit- CAT [66] according to the CAZy database [67] 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.
Figure 7.
Comparison of ribonucleic acid sequencing (mRNA-Seq) and microarray expression patterns.
Microarray expression profiles were obtained from time course analyses of genes expressed in P. infestans (PITG) during infection on S. tuberosum [10]. Single copy orthologous genes between P. infestans and Ps. cubensis (PCU) were identified using OrthoMCL [60], [61]. Log2 transformed expression values of single copy orthologous genes present in both the Ps. cubensis (log2 of fragments per kilobase pair of exon model per million fragments mapped [FPKM]) and P. infestans (log2 intensity) datasets are shown as scatter plots. SCC Spearman correlation coefficient. dpi = days post-inoculation.
Figure 8.
Heat map of the eigengenes representing each gene module.
The columns in the heat map represent time points, and the rows represent eigengenes for each of the six identified co-expression modules [50]. 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 Materials and Methods). dpi = days post-inoculation.