Genomes and transcriptomes of partners in plant-fungal-interactions between canola (Brassica napus) and two Leptosphaeria species

Abstract

Leptosphaeria maculans 'brassicae' is a damaging fungal pathogen of canola (Brassica napus), causing lesions on cotyledons and leaves, and cankers on the lower stem. A related species, L. biglobosa 'canadensis', colonises cotyledons but causes few stem cankers. We describe the complement of genes encoding carbohydrate-active enzymes (CAZys) and peptidases of these fungi, as well as of four related plant pathogens. We also report dual-organism RNA-seq transcriptomes of these two Leptosphaeria species and B. napus during disease. During the first seven days of infection L. biglobosa 'canadensis', a necrotroph, expressed more cell wall degrading genes than L. maculans 'brassicae', a hemi-biotroph. L. maculans 'brassicae' expressed many genes in the Carbohydrate Binding Module class of CAZy, particularly CBM50 genes, with potential roles in the evasion of basal innate immunity in the host plant. At this time, three avirulence genes were amongst the top 20 most highly upregulated L. maculans 'brassicae' genes in planta. The two fungi had a similar number of peptidase genes, and trypsin was transcribed at high levels by both fungi early in infection. L. biglobosa 'canadensis' infection activated the jasmonic acid and salicylic acid defence pathways in B. napus, consistent with defence against necrotrophs. L. maculans 'brassicae' triggered a high level of expression of isochorismate synthase 1, a reporter for salicylic acid signalling. L. biglobosa 'canadensis' infection triggered coordinated shutdown of photosynthesis genes, and a concomitant increase in transcription of cell wall remodelling genes of the host plant. Expression of particular classes of CAZy genes and the triggering of host defence and particular metabolic pathways are consistent with the necrotrophic lifestyle of L. biglobosa 'canadensis', and the hemibiotrophic life style of L. maculans 'brassicae'.

Figure 1. Symptoms on cotyledons of B. napus cv. Westar infected with L. maculans ‘brassicae’ or L. biglobosa ‘canadensis’.

B. napus 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.

Figure 2. Percentages of plant and fungal transcripts in B. napus cotyledons, uninoculated or at 7 and 14 days post-inoculation dpi with L. maculans ‘brassicae’ (Lmb) or L. biglobosa ‘canadensis’ (Lbc).

B. napus 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.

Figure 3. Principal Component Analyses of duplicate sets of RNA-seq data for L. maculans ‘brassicae’ (Lmb), L. biglobosa ‘canadensis’ (Lbc) and B. napus.

Scores plots for principal component analysis of expression values (FPKM) of genes of Lmb IBCN18 (A), Lbc J154 (B), or B. napus (C). Gene expression values during infections of cotyledons are plotted in orange (7 dpi) and green (14 dpi). Gene expression values during growth in vitro 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.

Figure 4. Expression profiles of secreted CAZys of L. maculans ‘brassicae’ (Lmb), L. biglobosa ‘canadensis’ (Lbc) in planta and in vitro.

The top 100 genes expressed across the three treatments (7, 14 dpi and in vitro) 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 in vitro or in planta (Tables 2, 3, 4, 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 in vitro, High expression in planta, Medium expression in planta and in vitro, High expression in planta at 14 dpi, High expression in planta at 7dpi, High expression in planta and in vitro) 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.

Figure 5. Expression of key defence genes of B. napus at 7 or 14 dpi with either L. maculans ‘brassicae’ (Lmb) or L. biglobosa ‘canadensis’ (Lbc).

RNA-seq data for eight key Brassica defence genes were determined at 7 and 14 dpi. Average expression (FPKM) after infection by L. maculans (blue), L. biglobosa (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.

Figure 6. Transcription of B. napus genes involved in metabolic processes including photosynthesis seven days after inoculation with L. maculans ‘brassicae’ (Lmb) or L. biglobosa ‘canadensis’ (Lbc).

RNA-seq gene expression values for a B. napus unigene set were used to calculate a ratio of expression values (log2) for B. napus genes after infection by Lmb or Lbc. Ratios were plotted on major metabolic pathways with Mapman software . A yellow square indicates a B. napus gene that is expressed more highly during Lmb infection, while a blue square indicates a B. napus 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.