Figure 1.
Symptoms caused by Cladosporium fulvum and Dothistroma septosporum on their host plants.
(A–D) Disease symptoms of Cladosporium fulvum on tomato. A) C. fulvum sporulating on the lower side of a tomato (Solanum lycopersicum) leaf two weeks post inoculation; B) Close-up of C. fulvum sporulating on the lower side of a single leaflet two weeks post inoculation; C) Runner hyphae of C. fulvum (GFP-transgenic strain) at the surface of the leaf; two of them are penetrating a stoma of tomato four days post inoculation; D) Conidiophores of C. fulvum (GFP-transgenic strain) emerging from a stoma at 10 days post inoculation. (E–H) Disease symptoms of Dothistroma septosporum on pine. E) Mortality of mature lodgepole pines (Pinus contorta var. latifolia) in northwest British Columbia, Canada caused by D. septosporum; F) Red band lesions with conidiomata on Pinus radiata needles; G) Penetration of hypha into stoma of P. radiata needle 4 weeks post inoculation; H) Eruption of conidiospores through epidermis of pine needle 8 weeks post inoculation.
Figure 2.
Species phylogeny, amino acid similarity, and repeat content.
A) Maximum likelihood phylogenetic tree based on 51 conserved protein families showing evolutionary relationships of Cladosporium fulvum and Dothistroma septosporum. Branch lengths are indicated by the bar (substitutions/site); bootstrap values are shown as percentage. B) Genome-wide amino acid similarity of homologous proteins between C. fulvum and other sequenced fungal species. A pair of proteins is only reported as homologous when the predicted similarity (blastp) spans at least 70% of their lengths and their length difference is at most 20%. Axis indicates number of homologous proteins. Bar shading indicates similarity: red, 91–100%; orange, 81–90%; light green, 71–80%; medium green, 61–70%; turquoise, 51–60%; light blue, 41–50%; dark blue, 31–40%; and purple, 0–30%. Homologous proteins with high amino acid similarity are likely orthologs, whereas for those with lower similarity this relation cannot be inferred. C) Repeat content of C. fulvum, D. septosporum and other sequenced fungal species. Bar shading indicates repeat class: red, unique non-repeat regions; brown, repeat elements; green, continuous tracts of N characters; blue, duplicated regions; and grey, poorly assembled regions (C. fulvum only). Axis indicates number of nucleotides (Mb).
Table 1.
C. fulvum and D. septosporum genome statistics.
Table 2.
Repetitive elements in the C. fulvum and D. septosporum genomes.
Figure 3.
Organization of repeats and pathogenicity-related genes in the Dothistroma septosporum genome.
The fourteen chromosomes from the D. septosporum genome assembly are shown as GC (dark grey line) and AT (pale grey line) content (%) plots made from a 500-bp sliding window using Geneious (www.geneious.com). All chromosomes have telomere sequence at both ends except chromosomes 2, 11 and 14 which have telomere sequences only at the left end as shown in the figure. Chromosome 1 has been split into two parts in the figure (L, R) because of its length, and the GC/AT content scale is shown beside the right arm of this chromosome. The positions of putative Avr and Ecp effector, secondary metabolite, dothistromin biosynthesis, and mating type genes are shown above the GC/AT content plot, while the positions of repeats (>200-bp) are shown below the plot. Color-coding of the gene and repeat types is indicated in the legend. Most chromosomes have repeat clusters at one or two sites that coincide with regions of high AT content. The chromosome sizes are to scale, as indicated by the vertical pale grey lines, with the values (in kb) shown at the bottom; neither the genes nor the repeats are drawn to scale.
Table 3.
Occurrence of Repeat-Induced Point Mutation (RIP) signatures and repeats in C. fulvum and D. septosporum.
Table 4.
Syntenic and non-syntenic regions between C. fulvum and D. septosporum are unevenly distributed over the C. fulvum scaffolds.
Table 5.
Location of gene models of C. fulvum in regions syntenic or non-syntenic with the D. septosporum genome.
Figure 4.
Recognition of Dothistroma septosporum effectors by tomato Cf receptors.
A) DsEcp2-1, the D. septosporum ortholog of CfEcp2-1, was cloned into pSfinx. Tomato plants were inoculated with Agrobacterium tumefaciens transformants expressing pSfinx::DsEcp2-1. A hypersensitive response (HR) was induced in the tomato line carrying the Cf-Ecp2 resistance gene (MM-Cf-Ecp2). Empty vector was used as a negative control and caused only mosaic symptoms. Pictures were taken at four weeks post inoculation. B) The C. fulvum avirulence gene Avr4 (CfAvr4) and its ortholog in D. septosporum (DsAvr4) were heterologously expressed in Cf-4 transgenic Nicotiana benthamiana using the A. tumefaciens transient transformation assay (ATTA). Expression of CfAvr4 and DsAvr4 results in an HR demonstrating that the tomato Cf-4 receptor recognizes DsAvr4. Picture was taken at six days post inoculation.
Figure 5.
Repetitive regions flanking known effectors of Cladosporium fulvum.
Scaffolds harboring sequenced C. fulvum effector genes with flanking repeats. Repeat regions longer than 200-bp are shown, with different types indicated in different color code. Red arrows depict the effector genes and sizes are in kb. The sizes of scaffolds range from 8 to 213-kb but some are shortened to fit the figure due to differences in size.
Table 6.
Comparison of selected CAZy gene families between C. fulvum, D. septosporum, and five other Ascomycetes.
Figure 6.
Comparative growth profiling of fungi on various carbohydrate substrates.
Growth on different substrates was compared between six fungi on nine media to highlight differences. Cf, C. fulvum; Ds, D. septosporum; Mg, M. graminicola; Sn, S. nodorum; Mo, Magnaporthe oryzae; Ss, Sclerotinia sclerotiorum. D-Glucose, D-xylose, L-arabinose and sucrose were added at a final concentration of 25 mM. Birchwood xylan, apple pectin and lignin were added at a final concentration of 1% (w/v).
Table 7.
Key secondary metabolism genes in Ascomycete genomes.
Figure 7.
Arrangement of predicted dothistromin genes in Dothistroma septosporum and Cladosporium fulvum.
A) Predicted dothistromin genes within the labeled clusters (left to right) are: Ver1, DotC (Ver1 cluster); PksA, CypX, AvfA, MoxY (PksA cluster); AflR, AflJ (AflR/J cluster); OrdB, AvnA, HexB, HexA, HypC, VbsA (VbsA cluster); Nor1, AdhA, VerB (Nor1 cluster). Positions of mini-clusters are approximate and they are not drawn to scale. Dothistromin genes within the published D. septosporum PksA and VbsA clusters [36], [38] and the newly discovered AflR/J and Nor-1 clusters are found in the same order and orientation in C. fulvum. B) Expression of dothistromin biosynthetic genes (Ver1, PksA, VbsA) and regulatory gene (AflR) was determined in D. septosporum by quantitative PCR. Mean expression and standard deviations are shown for at least 3 biological replicates relative to β-tubulin expression. In D. septosporum all genes but DsVbsA are expressed more highly in planta (late-stage sporulating lesions from a forest sample) than in culture (PDB or B5 media) as highlighted by the dashed-grey line. C) Expression of C. fulvum genes is shown as for (B), revealing that expression is not higher during tomato infection than in culture (dashed-grey line). Note the different scales for expression, which reveal a much lower level of transcription both in planta and in PDB medium compared to D. septosporum.