Table 1.
Descriptive statistics of the different assemblies before and after SSPACE scaffolding.
Fig 1.
The Drechmeria coniospora genome.
The optical maps (omap), potentially corresponding to distinct chromosomes, are depicted using Circos [39] with coloured sectors on the outer layer. As explained in the text, the optical maps 6937 and 49267 are split into two pieces. Scaffold43, corresponding to 23.8kb of mitochondrial DNA, and a further 63 other unanchored scaffolds, including 41 containing at least one predicted gene, and totalling 755,493 bp of genomic sequence, are not printed on the Circos plot. Each layer depicts, from the outside to the inside: (a) Percentage of G+C (red > 0.65, green < 0.45); (b) Percentage of repeat elements (red > 10%); (c and d) CLASS II and CLASS I transposable elements (white and blue blocks, respectively); (e) Members of three superfamilies encoding glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) important for xenobiotic detoxification and oxidative stress resistance in entomopathogenic species [40] are depicted in yellow; (f) TM7 transmembrane proteins (red); (g) ABC proteins (green); (h) groups of genes discussed in the text that encode: putative nonribosomal peptide synthetases (red), diverse proteases (purple) and enterotoxin-like proteins in green (their names without the.t1 suffix are shown); (i) non-coding RNA genes: tRNAs (blue), rRNAs (red), others (green).
Table 2.
Transposable elements in the D. coniospora genome.
Table 3.
Universal single-copy ortholog prediction in D. coniospora and 11 other species.
Table 4.
General characteristics of the D. coniospora genome compared to other species.
Fig 2.
Phylogenetic tree for 12 Pezizomycotina fungi.
Phylogenetic tree for 12 species based on alignments for a concatenation of 97 conserved protein sequences. Branch-lengths are drawn in proportion to the estimated number of substitutions per site. Species known to infect insects (I), nematodes (N) and plants (P) are indicated. All branches are fully supported (100/100 bootstraps).
Fig 3.
Unusual structure of Drechmeria-specific proteins and complex structural relationship between neighbouring proteins.
(A) RADAR analysis [54] reveals the repeated structure in the sequence of g4180.t1, a 471 a.a. protein from OrthoMCL-defined paralogous group 2 (S6 Table). (B) All-against-all dot-plot representation [55] of the alignment of the predicted protein sequences from g4180.t1 and from 5 neighbouring genes on scaffold omap6908, all from the OrthoMCL paralogous group 2. Dots represent regions of sequence similarity (within a 100 a.a. sliding window). The intensity of each dot is proportional to the corresponding alignment score. The “.t1” suffix has been removed from all sequence names.
Table 5.
Families of predicted D. coniospora paralogs.
Fig 4.
Species-specific or atypical protein domain families in the predicted D. coniospora proteome.
Hierarchical clustering of protein domain families present in D. coniospora and not more than 4 of the other fungi, on the basis of the corresponding number of proteins. PFAM domains discussed in the text are highlighted in red. The box highlights 11 families specific to D. coniospora (see S1 Text). The colour code reflects the relative abundance of proteins with each domain, from high (red) to low (blue) across the different species. The serine dehydratase alpha and beta domains (PF03313 and PF03315, respectively), cluster since they occur in a single highly conserved protein (g4699.t1 in D. coniospora).
Fig 5.
Supervised clustering of selected CAZy families in 11 fungal species.
The distribution of the CAZy families involved in complex carbohydrate breakdown (AA, GH and PL classes) across the given species is shown. Clustering of families is based on the number of genes in each family. The colour code reflects the relative abundance of proteins within each family, from high (red) to low (blue) across an individual species.
Table 6.
Number of secondary metabolite backbone genes predicted from the D. coniospora genome compared to other species.
Fig 6.
Predicted secreted proteins in D. coniospora.
The left hand chart shows the distribution of protein predicted to be secreted by 2 different computational methods. For the proteins predicted to be secreted by both, the right hand chart indicates the predicted sub-cellular localisation.
Table 7.
Potential virulence factors among D. coniospora proteins predicted to be secreted and targeted to a host organelle.
Fig 7.
Comparative analysis of the predicted secretome of D. coniospora.
(A) Distribution of sequence-based protein clusters across five nematopathogenic fungal genomes. Only clusters containing at least one secreted protein are shown. Except for empty sectors, in each sector, there are 3 numbers: total number of clusters/number of clusters with members only in nematophagous fungi/number of clusters with at least one member that matches a PHI base entry with an annotation of reduced virulence or loss of pathogenicity. (B) A correspondence analysis of sequence-based clusters of secreted proteins from five nematopathogenic fungi. The first two dimensions are shown. Crosses represent the position of the individual fungal species and circles represent protein clusters. Circles are sized according the number of constituent proteins as indicated. When clusters have identical coordinates, the size of the circle represents the sum of the number of proteins in each cluster. For example, the circle at (2.33, 3.6) corresponds to 19 clusters of proteins, in this case unique to D. coniospora, including Cluster01087. The proximity of each circle to the species’ apices is a measure of the contribution of the species to that cluster’s content. The distance between the circles is a measure of the similarity of their content (number of proteins from each species). (C) Multiple sequence alignment of proteins from the D. coniospora-specific cluster Cluster01087. Only 3 of the 6 proteins are predicted to be secreted (g2506.t1, g2508.t1, g2511.t1; S11 Table).
Table 8.
Selection of D. coniospora genes expressed during the infection of C. elegans.
Fig 8.
Transformation of D. coniospora.
(A) A recombinant strain of D. coniospora, expressing GFP under the control of a ß-tubulin promoter, viewed by fluorescence microscopy (upper panel) combined with differential interference contrast microscopy (lower panel; scale bar, 20 μm). The fusion of 2 mycelia is highlighted by an arrow. (B) Higher magnification view of fused mycelia (scale bar, 5 μm). (C-E) The Dso mutant has a defect in anastomosis. (C) As highlighted with the arrow, in culture, mycelia are seen to grow across one another but never fuse. The mutant strain was engineered to express GFP constitutively. Fungal mycelia growing in living animals, visualized using a stereo fluorescence dissecting microscope (D), or confocal fluorescence microscope (E; the shape of the worm is traced by white lines), were not observed to fuse. Worms had been infected overnight (D) or for 60 h (A, B, E) before images were taken. Scale bars in C, D, E: 20, 50, 40 μm. (F) The growth of the fungus can also be followed using the Profiler of the COPAS Biosort. The graphs show fluorescence profiles for green and red channels for an uninfected worm (top); a worm infected at the head and vulva (peak in green signal on the left and in the middle, respectively in the middle graph) and analysed after 24 h; another worm infected at the vulva (peak in green signal in the middle, bottom graph) and analysed after 36 h. Fluorescence and length are measured in arbitrary but constant units.
Fig 9.
Saposin A-domain protein expression during infection of C. elegans and its in vitro interaction with SPP-5.
(A) A strain of D. coniospora engineered to express GFP constitutively and a SapA::dsRed chimeric protein under the control of the sapA promoter, visualized using confocal fluorescence microscope (right panel) combined with differential interference contrast microscopy (left panel), during the infection of C. elegans. In both panels, a bright red fluorescent spore can be seen on the left. In the centre, a mycelium that is starting to exit the worm shows bright red fluorescence at its tip. On the right, 2 adjacent mycelia that have emerged can be seen. At the point where they leave the epidermis, a ring of SapA::dsRed can be seen, marked by asterisks in the right-hand panel. A general, less concentrated, signal can also be seen in the infected tissue. Worms had been infected for 60 h before images were taken. Scale bar = 5 μm. (B) Physical interaction between SPP-5 and SapA::dsRed. Recombinant His-tagged SPP-1, SPP-5 or SPP-12 was mixed with a protein extract from fungi expressing SapA::dsRed. The mix was analysed by Western blot probed with an anti-His-tag antibody before (left hand panel) or after (right hand panel) immunoprecipitation with anti-dsRed antibody-coated beads. In the sample before immunoprecipitation, in addition to the band at the expected size (11.9 kDa) marked by an asterisk, higher molecule weight species were detected, corresponding to the previously described oligomerization [86]. SPP-5 was co-immunoprecipitated with SapA::dsRed, principally in its monomeric form, but not if incubated with blocked beads (lanes marked with an asterisk; a control for non-specific binding). Neither SPP-1 nor SPP-12 gave any indication of co-immunoprecipitating with SapA::dsRed, even if SPP-1 was more abundant in the sample before immunoprecipitation.