Figure 1.
Phylogenetic analysis of Cladosporium fulvum PKS and NRPS enzymes.
Alignments of (A) full-length protein sequences of non-reducing polyketide synthases (PKSs) and (B) A-domains of non-ribosomal peptide synthetases (NRPSs) were used to construct maximum likelihood phylogenetic trees. The third A domain of ClafuNps9 was not included because of poor alignment. CYCLO: cyclosporine synthetases; SID: siderophore synthetases; EAS: Euascomycete clade synthetases. Only bootstrap values over 70 are shown. C. fulvum secondary metabolism enzymes are indicated in bold. The chemical structures of secondary metabolites potentially produced by C. fulvum are shown. Accession numbers are given in Table S1 and Table S4 in File S1.
Figure 2.
Synteny and rearrangements of conserved secondary metabolism gene clusters in Cladosporium fulvum.
The organization of gene clusters conserved in C. fulvum was compared to the previously described clusters involved in the biosynthesis of (A) elsinochrome, (B) monodictyphenone, (C) cercosporin, (D) ferricrocin and (E) ferrichrome in other fungi. Genes are represented as arrows, indicating their orientation. Representation of genes is not to scale.
Figure 3.
Expression profile of Cladosporium fulvum secondary metabolism functional core genes.
Expression profiles were measured by RT-qrtPCR using RNA isolated from mycelium grown in diverse in vitro growth conditions: PDB, B5 with different pHs, B5 without carbon source (B5-C), B5 without nitrogen source (B5-N), B5 without FeCl3 (B5-Fe), stationary phase (B5-12days) and autoclaved tomato leaves; and from tomato plants inoculated with the sequenced C. fulvum race 0WU strain from 0 to 16 days post-inoculation (dpi). Results are shown relative to the actin gene expression according to the E−ΔCt method, where E is the efficiency of a given primer pair. Tubulin gene was used as a control for calibration and the effector genes Avr4 and Avr9 were used as positive controls for the tomato infection experiment. The grey dotted line indicates the tubulin expression level. Values are the mean of three biological repeats and the error bars represent standard deviations (SD). (A) Only six genes show expression during in vitro growth, while (B) two genes are down-regulated during colonization of tomato. Pictures of tomato infected by a GFP-tagged C. fulvum strain are shown below to indicate the development of the fungus at the different time points of infection. Expression in each in vitro condition was compared to that in B5 pH4 medium using multiple t-tests, not assuming consistent SD, correcting for multiple comparisons with the Holm-Sidak method. For each gene, each in planta time point was compared to the previous one using a Two-way ANOVA followed by a multiple comparison test corrected with the Holm-Sidak method. All statistical tests were performed at the alpha significance threshold of 0.05. Red asterisks indicate significant differences only (* p<0.01; ** p<0.001; *** p<0.0001).
Figure 4.
Cladosporium fulvum secondary metabolite profiling.
Secondary metabolites (SMs) were extracted from in vitro liquid cultures in PDB and B5 media. Non-inoculated media served as negative controls. Culture filtrate and mycelium were separated by filtration prior to SM extraction and LC-MS analysis. Cladofulvin (retention time ca. 8.6 min) was the only SM detected in these samples. The peak at ca. 11.6 min corresponds to the fatty acid linoleic acid.
Figure 5.
Disruption of Cladosporium fulvum gene clusters by transposable elements.
(A) The HPS1 and (B) NPS1 genes in C. fulvum are orthologs of core genes that belong to conserved gene clusters in related fungal species. Genes are represented as arrows, indicating their orientation. Black arrows are genes with conserved orientation. Dark grey arrows are genes in the gene clusters that have undergone rearrangements. Light grey arrows are genes that border the gene cluster. Triangles represent transposable elements. Representation of genes is not to scale.