Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea

Abstract

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to <1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Figure 1. Lifecycle of S. sclerotiorum and B. cinerea, with different stages of sexual and asexual development.

Figure 2. Phylogeny of the Sclerotiniaceae (Ascomycota, Leotiomycetes, Helotiales), the sister group Rutstroemiaceae (represented by Lambertella species and “Sclerotinia” homoeocarpa), and the outgroup, Blumeria graminis (Leotiomycetes, Erysiphales).

The topology was estimated using Bayesian inference based on the combined sequence data of five genes. The tree was rooted using B. graminis. Bolded branches represent well-supported nodes with >90% support from 1000 maximum likelihood bootstrapped pseudoreplicates and >0.95 posterior probabilities. Support values for each node are listed in Table S29. Topologies recovered from single genes phylogenetic analyses were congruent with the concatenated gene tree topology. Top row Sclerotinia sclerotiorum, photos by H Lyon (left), LM Kohn (right). Left is apothecium emergent from sclerotium developed in vitro; right are apothecia associated with wild host, Ranunculus ficaria. Bottom row photos by AS Walker. Left is Botryotinia fuckeliana, sexual apothecia emergent from sclerotium developed in vitro; right, conidiophores bearing conidia produced by Botrytis cinerea on grapes.

Figure 3. Genome organization of S. sclerotiorum.

For each putative chromosome of the optical map, alignment of supercontigs is shown in alternating color blocks of black and grey. Syntenic regions with B. cinerea T4 are shown in red. Frequency of repetitive sequences is shown in blue.

Figure 4. Transposable element content of the genomes of S. sclerotiorum and B.cinerea.

Distribution of transposable elements in the genomes of S. sclerotiorum and B. cinerea (T4 and B05.10 isolates) according to the major clades: LTR retro-transposons, Line retro-transposons, TIR DNA transposons, MITE. UNK refers to unclassified transposable elements.

Figure 5. Configuration of the MAT loci in S. sclerotiorum and B. cinerea (strains B05.10 and T4).

S. sclerotiorum is homothallic whereas B. cinerea strains are heterothallic. Strain B05.10 is of MAT1-1 identity whereas strain T4 is of MAT1-2 identity. Orthologous genes are displayed in the same colour and pattern. The entire MAT locus is contained between the genes APN2 (on the left, green) and SLA2 (on the right, yellow stippled). The MAT locus of S. sclerotiorum is displayed on the top line, whereas the MAT loci of both B. cinerea strains are displayed on the bottom line. The truncated fragments of the MAT1-1-2 gene in the MAT1-1 isolate and of the MAT1-1-1 gene in the MAT1-2 isolate are highlighted with a dotted circle. Possible ancestral loci are displayed in the middle. Gene names are indicated above the gene model, the presence of a conserved alpha domain or HMG domain is indicated below the gene model. Two hypothetical inversions are shown, which might convert one configuration into the other. Two separate deletions are shown which may have resulted in the evolution of the MAT1-1 or MAT1-2 locus in B. cinerea.

Figure 6. Number of genes encoding secondary metabolism key enzymes, membrane transporters, and transcription factors in the genomes of S. sclerotiorum and B. cinerea.

S. sclerotiorum/B. cinerea orthologs were determined by BDBH. Secondary metabolism key enzymes (A), membrane transporters (B), and transcription factors (C). In panel C, the numbers in brackets indicate the number of genes lacking orthologs in other fungi.