A deviation from the bipolar-tetrapolar mating paradigm in an early diverged basidiomycete

Abstract

In fungi, sexual identity is determined by specialized genomic regions called MAT loci which are the equivalent to sex chromosomes in some animals and plants. Usually, only two sexes or mating types exist, which are determined by two alternate sets of genes (or alleles) at the MAT locus (bipolar system). However, in the phylum Basidiomycota, a unique tetrapolar system emerged in which four different mating types are generated per meiosis. This occurs because two functionally distinct molecular recognition systems, each encoded by one MAT region, constrain the selection of sexual partners. Heterozygosity at both MAT regions is a pre-requisite for mating in both bipolar and tetrapolar basidiomycetes. Tetrapolar mating behaviour results from the absence of genetic linkage between the two regions bringing forth up to thousands of mating types. The subphylum Pucciniomycotina, an early diverged lineage of basidiomycetes encompassing important plant pathogens such as the rusts and saprobes like Rhodosporidium and Sporidiobolus, has been so far poorly explored concerning the content and organization of MAT loci. Here we show that the red yeast Sporidiobolus salmonicolor has a mating system unlike any previously described because occasional disruptions of the genetic cohesion of the bipolar MAT locus originate new mating types. We confirmed that mating is normally bipolar and that heterozygosity at both MAT regions is required for mating. However, a laboratory cross showed that meiotic recombination may occur within the bipolar MAT locus, explaining tetrapolar features like increased allele number and evolution rates of some MAT genes. This pseudo-bipolar system deviates from the classical bipolar-tetrapolar paradigm and, to our knowledge, has never been observed before. We propose a model for MAT evolution in the Basidiomycota in which the pseudo-bipolar system may represent a hitherto unforeseen gradual form of transition from an ancestral tetrapolar system to bipolarity.

Figure 1. Diversity and phylogeny of MAT gene alleles in S. salmonicolor.

(A) Phylogeny of HD1/HD2 genes. Mating types A1, A2, and asexual strains are designated as A1, A2 and AS, respectively. Boxes designate the various HD1/HD2 alleles with numerals after the mating type designation. Circles after strain numbers depict the type of pheromone receptor gene (yellow, STE3.A1; blue, STE3.A2). Asterisks indicate the two instances where common ancestry of HD1/HD2 alleles associated with opposite mating types is best supported. (B) Comparison of the number and distribution of MAT gene alleles in emblematic species representing the different mating systems (HD, homeodomain region; PR, pheromone receptor region; the continuous or discontinuous line between the HD and PR boxes denotes genetic linkage or its absence, respectively).

Figure 2. Evolution rate of the HD1 gene in S. salmonicolor.

Synonymous (dS), non-synonymous (dN) and dN/dS average values for all pairwise comparisons between nine alleles of the HD1 gene. Y axis – evolution rates; X axis – base pairs of HD1 coding region. Average values are given for the highly variable N-terminal domain which is involved in non-self recognition, and for the more conserved homeodomain (HD) and C-terminal domains.

Figure 3. Segregation analysis of the STE3 and HD1/HD2 genes after meiosis.

(A) T1 to T8 refer to 8 individually germinated teliospores obtained by micromanipulation from the cross between strains CBS 6832 (A2–15) and ML 2241 (A1–3). The result of the diagnostic PCRs for the presence of the alternate STE3.A1 and STE3.A2 genes and the identification of HD1/HD2 amplicons by Rsa I digestion is shown. Screening of the parental origin of the DMC1 allele by PCR and digestion with Msp I show clonality of the various colonies examined for each teliospore. For each germinated teliospore, three to six colonies were studied. With the exception of teliospore T1, only one mating type was recovered per meiosis presumably due to asynchronous germination of basidiospores (see Figure S5C). Teliospore T8 yielded a strain which is apparently diploid, and the new mating type was recovered from teliospore T7.

Figure 4. Sexual proficiency of T7.1, the new mating type of S. salmonicolor.

For each cross the top section indicates the type of cross and the strains involved. Micrographs of crosses (4 days of incubation at 25°C on corn meal agar) are shown in the middle section (inserts depict details in higher magnification to show the presence or absence of teliospores). The bottom section shows the STE3 and HD genotypes of each strain and the outcome of the cross evaluated by the presence (+) or absence (−) of mycelium with clamp connections and teliospores.

Figure 5. Analysis of meiotic progeny.

(A) Genomic regions surrounding the HD1/HD2 and pheromone receptor genes in S. salmonicolor MAT A1. Genes in the immediate vicinity of STE3.A1 are depicted as block arrows to denote the direction of transcription. The same genes are highlighted with an orange box. Synteny with the homologous genomic region of S. roseus was confirmed for all genes except those shown as empty arrows (depicted in the arrangement present in S. roseus). Outside the highlighted region, genes present in the same S. roseus scaffold at different distances (indicated by the arrows) from STE3.A1 were selected. Four genes were similarly selected from the HD1/HD2 scaffold, assuming synteny with S. roseus. For eight independent teliospores (T1–T8) the parental origin of the genes is indicated. Genes from strain ML 2241 (A1–3) are shown in green and those from strain CBS 6832 (A2–15), in red. Asterisks mark sites were crossovers were detected. Possible gene conversion events are marked with “C”. The two scaffolds are depicted in the most likely orientation. The region shadowed in light yellow encompasses the putative pseudo-bipolar MAT locus in S. salmonicolor MAT A1. (B) Segregation patterns and recombination frequencies (RF) of nine autosomal markers located in four different scaffolds. The position and relative distances between the genes are depicted as in S. roseus genome. T8 is a diploid strain since it contains both parental alleles for all genes tested.

Figure 6. The pseudo-bipolar system and the evolution of MAT loci in Basidiomycota.

The ancestral basidiomycete mating system may have been similar to the pseudo-bipolar system found in S. salmonicolor. The classic tetrapolar system emerged in the common ancestor of the Ustilaginomycotina and Agaricomycotina through a chromosomal translocation that placed the two MAT regions on different chromosomes. In U. hordei and C. neoformans a second translocation may have triggered a precipitous transition to bipolarity, as previously proposed , . In the mushroom Co. disseminatus, the pheromone receptor (white box) ceased to be associated to MAT leading to bipolarity. Mating systems in the Pucciniomycotina may have evolved to extant systems exhibiting partial (S. salmonicolor) or complete (M. violaceum) suppression of recombination between the two MAT regions. Tetrapolar systems in this lineage might be present in Leucosporidium scottii and the rusts (Uredinales) but have not been characterized at the molecular level yet. In these systems, MAT regions may be distantly located on the same chromosome or on different chromosomes. The number of MAT gene alleles identified in each species is shown next to the boxes representing the homeodomain (HD) and pheromone receptor (PR) regions. Stripes between the HD and PR regions denote reduced or suppressed recombination.