Interspecific sex in grass smuts and the genetic diversity of their pheromone-receptor system

Abstract

The grass smuts comprise a speciose group of biotrophic plant parasites, so-called Ustilaginaceae, which are specifically adapted to hosts of sweet grasses, the Poaceae family. Mating takes a central role in their life cycle, as it initiates parasitism by a morphological and physiological transition from saprobic yeast cells to pathogenic filaments. As in other fungi, sexual identity is determined by specific genomic regions encoding allelic variants of a pheromone-receptor (PR) system and heterodimerising transcription factors. Both operate in a biphasic mating process that starts with PR-triggered recognition, directed growth of conjugation hyphae, and plasmogamy of compatible mating partners. So far, studies on the PR system of grass smuts revealed diverse interspecific compatibility and mating type determination. However, many questions concerning the specificity and evolutionary origin of the PR system remain unanswered. Combining comparative genetics and biological approaches, we report on the specificity of the PR system and its genetic diversity in 10 species spanning about 100 million years of mating type evolution. We show that three highly syntenic PR alleles are prevalent among members of the Ustilaginaceae, favouring a triallelic determination as the plesiomorphic characteristic of this group. Furthermore, the analysis of PR loci revealed increased genetic diversity of single PR locus genes compared to genes of flanking regions. Performing interspecies sex tests, we detected a high potential for hybridisation that is directly linked to pheromone signalling as known from intraspecies sex. Although the PR system seems to be optimised for intraspecific compatibility, the observed functional plasticity of the PR system increases the potential for interspecific sex, which might allow the hybrid-based genesis of newly combined host specificities.

Figure 1. Multi-gene phylogeny and interspecific sexual compatibility of Ustilaginales.

Concatenated Maximum Likelihood (ML) analysis of 2571 bp of ssu, ITS, lsu rDNA, ef1-α and rpb1. Circles next to branches indicate bootstrap support values and a posteriori probabilities of Bayesian and ML analyses, respectively. Branch lengths correspond to substitutions per site and abbreviated branches indicate longer branches. Connected squares illustrate hybrid filament formation (bold lines) or pheromone response (thin lines). Numbers in squares represent respective a mating types. Coloured boxes depict different phylogenetic clades (see text). Host ages refer to .

Figure 2. Phylogeny of mating type-specific pheromone receptors.

Maximum Likelihood analysis of complete pheromone receptor-coding sequences. Numbers and asterisks next to branches indicate bootstrap (bt) support values and branch lengths correspond to substitutions per site.

Figure 3. Genetic structure of mating type a gene clusters of Ustilaginales.

Shown are three a locus alleles of different Ustilaginales species. Arrows indicate coding regions of respective genes and lines represent non-coding or intron regions. Pheromones and cognate pheromone receptors are depicted in red shades. Homologous border genes are depicted in identical colours or patterns. Strokes represent pheromone response element sites (ACAAAGGGA) with no (black) or one mismatch (grey). Abbreviation signs depict connected regions on respective chromosomes. um and sr gene numbers correspond to gene identifications on MUMDB and MSRDB .

Figure 4. Nucleotide diversity of PR loci-associated and house-keeping genes.

Bars indicate nucleotide diversity (π) estimates divided by the phylogenetic diversity (pd) of respective datasets. Black bars: a locus genes, dark grey bars: a locus-flanking genes, light grey bars: house-keeping genes. Numbers above bars indicate the quantity of analysed sequences.

Figure 5. Multiple alignment of Lga2 homologs and their regulatory regions.

(A) Amino acid alignment of Lga2 sequences from reference species (S. reilianum and U. maydis) and proposed sequences of Ma. eriachnes. Dots indicate predicted mitochondria target signals. Arrowheads indicate positions of introns in the respective genes. Dashes represent alignment indels. Grey shades mark positions with two (light grey) or three (dark grey) identical amino acid residues. Bold letters indicate predicted F-box-like motifs. (B) Nucleotide sequence alignment of the lga2 b-binding site and its flanking regions of U. maydis with 5′ sites of lga2 of S. reilianum and Ma. eriachnes. Grey shades mark sites with two (light grey) or three (dark grey) identical aminoacids and nucleotides, respectively.

Figure 6. Interspecific mating reactions between different species of Ustilaginales.

(A) Differential interference contrast (DIC) images of mating assays in liquid potato dextrose. Images i–iv show yeast cultures of respective species. Images v, vi and vii show confrontations of S. reilianum with S. scitamineum, Us. gigantosporum and U. cynodontis, respectively. All figures are scaled equally. bar: 10 µm, b?: unknown b allele. (B) Filament formation on charcoal-containing potato dextrose media. Images i–vii correspond to sample descriptions in A. Figure width represents 3 mm. (C) SEM images of mating assays of S. reilianum and S. scitamineum (i–iii) and U. cynodontis and U. xerochloae (iv–vi). Single yeast cells (i, iv) form conjugation hyphae that fuse (arrowheads in ii, v), expand and form empty sections by the insertion of basal septa (arrows in iii, vi). bar: 4 µm, dotted bar: 1 µm.

Figure 7. Interspecific induction of mating via pheromone signalling in U. maydis.

(A) Differential interference contrast (DIC) and fluorimetric images from positive pheromone response reactions in liquid potato dextrose. Conjugation hyphae are formed by both mating partners (DIC images). All figures are scaled equally. bar: 10 µm. (B) The diagram illustrates fluorimetric measurements (relative fluorescence units, RFU) from mating assays of U. maydis P_mfa1-egfp strains FB1 (a1b1) and FB2 (a2b2) confronted with different mating types (a1, a2 and a3) of different smut species in water. Black and grey bars refer to RFUs of confrontations with strain FB1 P_mfa1-egfp and strain FB2 P_mfa1-egfp, respectively. U. maydis wild type strains FB6a (a2b1) and FB6b (a1b2) were used as positive controls. The white bar depicts RFU of the mating of FB1 P_mfa1-egfp and FB2 P_mfa1-egfp. Error bars indicate standard deviations of three independent experiments.