Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi

Abstract

The class Dothideomycetes is one of the largest groups of fungi with a high level of ecological diversity including many plant pathogens infecting a broad range of hosts. Here, we compare genome features of 18 members of this class, including 6 necrotrophs, 9 (hemi)biotrophs and 3 saprotrophs, to analyze genome structure, evolution, and the diverse strategies of pathogenesis. The Dothideomycetes most likely evolved from a common ancestor more than 280 million years ago. The 18 genome sequences differ dramatically in size due to variation in repetitive content, but show much less variation in number of (core) genes. Gene order appears to have been rearranged mostly within chromosomal boundaries by multiple inversions, in extant genomes frequently demarcated by adjacent simple repeats. Several Dothideomycetes contain one or more gene-poor, transposable element (TE)-rich putatively dispensable chromosomes of unknown function. The 18 Dothideomycetes offer an extensive catalogue of genes involved in cellulose degradation, proteolysis, secondary metabolism, and cysteine-rich small secreted proteins. Ancestors of the two major orders of plant pathogens in the Dothideomycetes, the Capnodiales and Pleosporales, may have had different modes of pathogenesis, with the former having fewer of these genes than the latter. Many of these genes are enriched in proximity to transposable elements, suggesting faster evolution because of the effects of repeat induced point (RIP) mutations. A syntenic block of genes, including oxidoreductases, is conserved in most Dothideomycetes and upregulated during infection in L. maculans, suggesting a possible function in response to oxidative stress.

Figure 1. Estimated phylogeny and divergence times of Dothideomycetes, based on sequences of three protein-coding genes.

Species with a sequenced genome that are included in this study are highlighted in dark blue. Vertical lines in blue and green indicate minimum and maximum ages for specific nodes, respectively. The age ranges for highlighted taxa are indicated by blocks with different shades of gray. Horizontal green lines indicate bootstrap recovery for specific nodes – thickened branches represent more than 70%, normal branches, 50–70% and less than 50% are indicated with dashed lines. In some cases relevant horizontal lines were stylistically extended to highlight node labels. Only families with multiple genomes are indicated. Orders, suborders and families that contain important plant-pathogenic species are colored brown and those containing majority lichenized species are green. Brown squares indicate plant pathogenic and green triangles lichenized species. Saprotrophs and fungi with other nutritional modes are not labeled.

Figure 2. Phylogeny and genome characteristics of the 18 studied Dothideomycetes.

A. Genome-based phylogenetic tree of 18 Dothideomycetes computed using 51 conserved protein families. Bootstrap values are indicated on the branches. Lifestyles and strategies of pathogenesis (green circle for necrotrophs, orange circle for saprotrophs and blue circle for [hemi]biotrophs) are indicated. Aspergillus nidulans was used as an outgroup and its branch on the tree is not drawn to scale. B. Genome size and repeat content. Repeat content varies widely among Dothideomycetes, but in general the largest part consists of long terminal repeats. Asterisks indicate genomes that were sequenced exclusively with Illumina technology. Repeat content in these genomes is likely an underestimate. C. Number of predicted genes, broken down by level of conservation. D. Gene counts of classes that have been implicated in plant pathogenesis. Members of Capnodiales have fewer genes in these classes than Pleosporales and Hysteriales (with the exception of Cladosporium fulvum). This trend is also illustrated by the estimated gene counts for the last common ancestors of the indicated taxa (below the x-axis), which correspond to the taxa in (A). See also Figure S3. Bars on all graphs (B, C, and D) correspond to the organisms on the tree in (A).

Figure 3. Whole-genome DNA comparison of Cochliobolus heterostrophus C5 to progressively distantly related organisms reveals the process leading from macrosynteny to mesosynteny.

A. Strains C4 and C5 of C. heterostrophus are progeny of C. heterostrophus backcrosses and show clear macrosynteny. B. When C. heterostrophus C5 is compared to C. sativus, macrosynteny is observed. However, intra-chromosomal inversions are observed in several comparisons of scaffold pairs. C. Numerous intra-chromosomal inversions have occurred in all scaffolds when compared to Setosphaeria turcica. D. A pattern of mesosynteny is observed when compared to Stagonospora nodorum. Syntenic regions are short and spread across the scaffold pairs. Scaffolds in this figure are not drawn to scale and only a subset of the scaffolds is depicted.

Figure 4. Simulation of chromosome evolution leading to mesosynteny.

A. Two identical sequences show perfect macrosynteny. B. This is also the case for scaffold_1 of Cochliobolus heterostrophus C4 and scaffold_2 of C. heterostrophus C5, reflecting their close relationship as progeny. C. The two sequences from (A) have each undergone one random inversion. D. Scaffold_4 of C. heterostrophus C5 and scaffold_9 of C. sativus show a very similar pattern as in (C). E. The two sequences in (A) have each undergone 25 random inversions. F. Scaffold_8 of Setosphaeria turcica and part of scaffold_10 of C. heterostrophus C5 show a pattern of syntenic regions progressively spreading across the scaffolds similar to that in (E) G. The two sequences from (A) have each undergone 500 random inversions. Syntenic regions are short and spread homogeneously across the two scaffolds. H. Scaffold_1 of Dothistroma septosporum and scaffold_1 of Mycosphaerella populorum show a very similar pattern as in (G). Scaffolds in this figure are not drawn to scale.

Figure 5. The full and core proteomes of the 18 Dothideomycetes.

A. The full proteome of the Dothideomycetes contains 215,225 proteins and for the majority of these the function according to KOG is unknown or poorly characterized. B. The core proteome contains the 66,761 proteins from multi-gene families that had at least one member in each Dothideomycete. Relative to (A), this set of proteins has more KOG annotations than the full proteome. In particular genes involved in metabolism are over-represented.

Figure 6. Heat map of CAZY families in the Dothideomycetes.

Both the CAZY families and the organisms are hierarchically clustered. The clustering of organisms largely follows the phylogeny in Figure 2A. Notable exceptions are the observation that the biotroph C. fulvum clusters as an outgroup to the hemibiotrophs and saprotroph within the Capnodiales, and the observation that the two pathogens of Brassica spp. (L. maculans and A. brassicicola) cluster together.

Figure 7. The RIP index (TpA/ApT) of genes as a function of the distance from a transposable element.

The RIP index is highest near the transposable elements and levels off after approximately 2000 bp, signifying that these regions are subjected to repeat induced point mutations.