Expression of key defence genes of <i>B. napus</i> at 7 or 14 dpi with either <i>L. maculans</i> ‘brassicae’ (Lmb) or <i>L. biglobosa </i>‘canadensis’ (Lbc).

<p>RNA-seq data for eight key Brassica defence genes were determined at 7 and 14 dpi. Average expression (FPKM) after infection by <i>L. maculans</i> (blue), <i>L. biglobosa</i> (red), and mock inoculum (green) is plotted. Error bars indicate the Cufflinks 95% confidence interval for each FPKM value. The genes assayed were 9-cis-epoxycarotenoid dioxygenase 3 (NCED3), 1-amino-cyclopropane-1-carboxylate synthase 2 (ACS2), chitinase (CHI), hevein-like protein (HEL), isochorismate synthase 1 (ICS1), Pathogenesis related protein 1 (PR-1), WRKY transcription factor 70 (WRKY70), and plant defensin 1 (PDF1.2). Each gene was also classified according to hormone(s) abscisic acid (ABA), ethylene (ET), jasmonic acid (JA) and salicylic acid (SA) that induced higher expression.</p

Principal Component Analyses of duplicate sets of RNA-seq data for <i>L. maculans</i> ‘brassicae’ (Lmb), <i>L. biglobosa </i>‘canadensis’ (Lbc) and <i>B. napus</i>.

<p>Scores plots for principal component analysis of expression values (FPKM) of genes of Lmb IBCN18 (A), Lbc J154 (B), or <i>B. napus</i> (C). Gene expression values during infections of cotyledons are plotted in orange (7 dpi) and green (14 dpi). Gene expression values during growth <i>in vitro</i> are plotted in purple; those in mock-infected cotyledons are plotted in magenta. The percentage of the total variance explained is listed on each axis label.</p

Symptoms on cotyledons of <i>B. napus</i> cv. Westar infected with <i>L. maculans</i> ‘brassicae’ or <i>L. biglobosa</i> ‘canadensis’.

<p><i>B. napus</i> cv. Westar cotyledons were wounded and inoculated with Lmb or Lbc spores and disease allowed to progress for 17 days post inoculation (dpi). Cotyledons were harvested and photographed at 3, 5, 7, 10, 14, and 17 dpi to track lesion development by the two pathogens.</p

Expression profiles of secreted CAZys of <i>L. maculans</i> ‘brassicae’ (Lmb), <i>L. biglobosa </i>‘canadensis’ (Lbc) <i>in planta</i> and <i>in vitro</i>.

<p>The top 100 genes expressed across the three treatments (7, 14 dpi and <i>in vitro</i>) and predicted to be secreted and to contain a CAZy domain (CBM, GH, PL, or AA) were selected. Quantile-normalisation was applied and log10-transformed FPKM values were graphed. The intensity of blue shading is proportional to the expression level. The gene order is based on a dendrogram created from a Euclidean similarity matrix with average group distance. Letters with a triangle point to genes that were amongst the top 20 most highly expressed genes <i>in vitro</i> or <i>in planta</i> (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone-0103098-t002" target="_blank">Tables 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone-0103098-t003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone-0103098-t004" target="_blank">4</a>, or 5). A/Lema_P102640.1 (Lm2LysM); B/Lema_P013700.1 (Glycoside hydrolase family 7); C/Lema_P070100.1 (Lm5LysM); D/Lb_j154_P005286 (pectate lyase); E/Lb_j154_P009204 (cellulase); F/Lb_j154_P001246 (Glycosyl hydrolase family 43); G/Lb_j154_P001652 (cellulase); H/Lb_j154_P009247 (Glycosyl hydrolases family 12); I/Lb_j154_P004093 Glycosyl hydrolases family 39). Categories of gene expression (High expression <i>in vitro</i>, High expression <i>in planta</i>, Medium expression <i>in planta</i> and <i>in vitro</i>, High expression <i>in planta</i> at 14 dpi, High expression <i>in planta</i> at 7dpi, High expression <i>in planta</i> and <i>in vitro</i>) were manually assigned and indicated by coloured polygons linking the genes in each category across Lmb and Lbc; the number of genes in each category is indicated on the vertical sides of the polygon.</p

Top 20 upregulated genes of <i>L. maculans</i> ‘brassicae’ isolate IBCN 18 fourteen days after inoculation of <i>B. napus</i> cv. Westar.

<p>The top 100 <i>in planta</i> up regulated genes are listed in Table S5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone.0103098.s002" target="_blank">file S2</a>.</p

Transcription of <i>B. napus</i> genes involved in metabolic processes including photosynthesis seven days after inoculation with <i>L. maculans</i> ‘brassicae’ (Lmb) or <i>L. biglobosa </i>‘canadensis’ (Lbc).

<p>RNA-seq gene expression values for a <i>B. napus</i> unigene set <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone.0103098-Love1" target="_blank">[27]</a> were used to calculate a ratio of expression values (log2) for <i>B. napus</i> genes after infection by Lmb or Lbc. Ratios were plotted on major metabolic pathways with Mapman software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone.0103098-Thimm1" target="_blank">[39]</a>. A yellow square indicates a <i>B. napus</i> gene that is expressed more highly during Lmb infection, while a blue square indicates a <i>B. napus</i> gene with higher expression during Lbc infection. An expression ratio close to zero is shown with a white square and indicates equivalent expression during infection by either pathogen. Only genes with expression values greater than 10 FPKM were included. Abbreviations: LDH, lactate dehydrogenase; ADH, Alcohol dehydrogenases, TCA, tricarboxylic acid cycle; raff, raffinose; Treh, trehalose; PSI, photosystem one; PSII, photosystem two; ABA, abscisic acid; ET, Ethylene; SA, salicylic acid; JA, jasmonic acid.</p

<i>B. napus</i> defence genes analysed [50].

<p>Defence signalling pathways indicate regulatory pathway to which the gene belongs. RRES unigenes are listed from the Brassica exon array <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone.0103098-Love1" target="_blank">[27]</a>, to which RNA-seq reads were aligned. Corresponding GenBank accession numbers are given for each gene. The response of each gene (with the exception of PDF1.2) to its corresponding signalling pathway was confirmed by Sasek et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103098#pone.0103098-aek1" target="_blank">[50]</a>.</p

Distribution of peptidase domains in six Dothideomycete genomes.

<p>Peptidase domains are classified according to the catalytic type, or inhibitor activity. Peptidase types are Aspartic (A), Cysteine (C), Glutamic (G), Inhibitor, Mettallo-(M), Serine (S), Threonine (T), Unknown (U).</p

Distribution of CAZy domains in predicted proteins of six Dothideomycetes.

<p>The classes of CAZy domains are Auxiliary Activities (AA) Carbohydrate Binding Modules (CBM), Carbohydrate Esterases (CE), Glycosyl Hydrolases (GH), Glycosyl Tranferases (GT) Polysaccharide lyases (PL). Some genes contain multiple CAZy domains.</p

Percentages of plant and fungal transcripts in <i>B. napus</i> cotyledons, uninoculated or at 7 and 14 days post-inoculation dpi with <i>L. maculans</i> ‘brassicae’ (Lmb) or <i>L. biglobosa</i> ‘canadensis’ (Lbc).

<p><i>B. napus</i> cotyledons were infected with Lmb, Lbc or water (control mock inoculum). Total RNA was extracted from lesion tissue at 7 and 14 dpi for Illumina RNA-seq sequencing. The percentage of reads aligned to the Brassica exon array unigene set (green) or the reference genomes for Lmb or Lbc (blue) is presented, as well as a photo of each representative infection. All of the aligned sequence reads were deposited at the NCBI Sequence Read Archive (SRA), accessible under bioproject accession SRP035525.</p