Identification of mating type genes in the bipolar basidiomycetous yeast Rhodosporidium toruloides: first insight into the MAT locus structure of the Sporidiobolales

Abstract

Rhodosporidium toruloides is a heterothallic, bipolar, red yeast that belongs to the Sporidiobolales, an order within a major lineage of basidiomycetes, the Pucciniomycotina. In contrast to other basidiomycetes, considerably less is known about the nature of the mating type (MAT) loci that control sexual reproduction in this lineage. Three genes (RHA1, RHA2, and RHA3) encoding precursors of the MAT A1 pheromone (rhodotorucine A) were previously identified and formed the basis for a genome walking approach that led to the identification of additional MAT genes in complementary mating strains of R. toruloides. Two mating type-specific alleles encoding a p21-activated kinase (PAK; Ste20 homolog) were found between the RHA2 and RHA3 genes, and identification in MAT A2 strains of a gene encoding a presumptive pheromone precursor enabled prediction of the structure of rhodotorucine a. In addition, a putative pheromone receptor gene (STE3 homolog) was identified upstream of RHA1. Analyses of genomic data from two closely related species, Sporobolomyces roseus and Sporidiobolus salmonicolor, identified syntenic regions that contain homologs of all the above-mentioned genes. Notably, six novel pheromone precursor genes were uncovered, which encoded, similarly to the RHA genes, multiple tandem copies of the peptide moiety. This suggests that this structure, which is unique among fungal lipopeptide pheromones, seems to be prevalent in red yeasts. Species comparisons provided evidence for a large, multigenic MAT locus structure in the Sporidiobolales, but no putative homeodomain transcription factor genes (which are present in all basidiomycetous MAT loci characterized thus far) could be found in any of the three species in the vicinity of the MAT genes identified.

FIG. 1.

Structure of the STE20 region of the MAT A1 and MAT A2 loci of R. toruloides. Coding regions are shown as gray arrows, indicating the direction of transcription, and the transcribed regions identified by Akada et al. (2) are shown as black boxes. Genomic DNA fragments 1 to 6 were obtained either by genome walking or by PCR with gene-specific primers. The primers used in each case are indicated by arrowheads (see Table S1 in the supplemental material).

FIG. 2.

Nucleotide sequence of the genomic region encoding the RHA2.A2 gene, showing the predicted pheromone precursor sequence. CT-rich regions possibly involved in transcription initiation are underlined. The two repeats proposed to represent the peptide moiety of the mature pheromone are shadowed. The sequences resembling the CAAX prenylation motif are enclosed in boxes.

FIG. 3.

Alignment of the predicted pheromone precursor peptides from the three sporidiobolaceous species, as deduced from the sequence of the respective genes: RHA (R. toruloides), SsRHA (S. salmonicolor), and SrRHA (S. roseus). Sequences representing the mature pheromones are shadowed, and the sequences resembling the CAAX prenylation motif are underlined in the C terminus of each precursor.

FIG. 4.

Structure of a putative MAT locus in S. roseus and comparison with homologous genomic regions in R. toruloides and S. salmonicolor. The top panel shows the gene content and organization in a 40-kb region around the putative pheromone precursor (box A) and pheromone receptor (box B) genes in S. roseus. Additional genes identified in this region are depicted as white boxes and are listed in Table S3 of the supplemental material. The lower panels show the gene content and organization of the regions around the pheromone precursor (A) and the pheromone receptor (B) homologs in the three species. Unknown sequences are shown in light gray. The relative position and orientation of the genes marked “?” are unknown. All the remaining features are represented as in Fig. 1.

FIG. 5.

Phylogenetic tree showing the relationships between PAK kinases from different fungi, based on alignment of the respective protein sequences. Kinases lacking the PH domain are located in the gray branch of the tree. The sequences used and respective accession numbers are listed in Table S4 of the supplemental material, and the alignment of the three conserved domains in a subset of the proteins is shown in Fig. S1 of the supplemental material. The evolutionary distances were computed using Protdist (JTT matrix-based method) (12). Neighbor-joining (12) and MEGA4 (36) were used to construct and visualize the phylogenetic tree. Numbers on branches are bootstrap values inferred from 10,000 replicates (values below 50% are not shown).