Mating-type locus of Cryptococcus neoformans: a step in the evolution of sex chromosomes

Abstract

The sexual development and virulence of the fungal pathogen Cryptococcus neoformans is controlled by a bipolar mating system determined by a single locus that exists in two alleles, alpha and a. The alpha and a mating-type alleles from two divergent varieties were cloned and sequenced. The C. neoformans mating-type locus is unique, spans >100 kb, and contains more than 20 genes. MAT-encoded products include homologs of regulators of sexual development in other fungi, pheromone and pheromone receptors, divergent components of a MAP kinase cascade, and other proteins with no obvious function in mating. The alpha and a alleles of the mating-type locus have extensively rearranged during evolution and strain divergence but are stable during genetic crosses and in the population. The C. neoformans mating-type locus is strikingly different from the other known fungal mating-type loci, sharing features with the self-incompatibility systems and sex chromosomes of algae, plants, and animals. Our study establishes a new paradigm for mating-type loci in fungi with implications for the evolution of cell identity and self/nonself recognition.

FIG. 1.

Structures of the serotype D (MATa of JEC20 and MATα of JEC21) and serotype A (MATa of 125.91 and MATα of H99) α and a mating-type alleles and adjacent genomic regions. The mating-type-specific regions are shown as thick bold lines, and flanking regions are shown as thinner black lines. Sequences were analyzed using BLASTX, and identified genes are shown as arrows in the direction of transcription. Genes encoding pheromone response pathway elements are shown as black arrows, locus-specific genes are shown as white arrows, and all other genes are shown as grey arrows. Bars above the mating-type alleles represent the BAC clones, genomic fragments, and PCR products analyzed.

FIG. 2.

Mapping of the serotype D (A) and serotype A (B) MATα loci by hybridization. The serotype D and serotype A MATα BAC libraries (strains JEC21 and H99) were screened with probes specific for several MAT-specific genes, the right end (RE) of the locus, and the flanking gene APG9. BAC clones that hybridized to these probes were analyzed by dot blot hybridizations using probes to the underlined genes. Additional hybridizations were conducted with a high-density BAC clone filter. These hybridization data were used to generate a linkage map and establish the gene order of the MAT locus. For all BAC clones depicted here, end sequences from the University of British Columbia database were incorporated to define endpoints that lie between hybridization probes. BACs sequenced to completion are marked with asterisks. The sizes of the BAC clones and BAC contigs depicted were determined at the University of British Columbia Genome Center. Clones used to span a gap in the H99 MAT locus are depicted as short grey lines above the locus. For additional details, see the legend to Fig. 1.

FIG. 3.

Mapping of the ends of the mating-type locus by sequence comparison. Twenty kilobases of the sequences surrounding the proposed junctions between the MATα and MATa mating-type alleles and flanking DNA and surrounding the RPO41 mitochondrial RNA polymerase genes of the serotype D MATα and MATa strains JEC21 and JEC20 were compared using the DNA Strider program. Corresponding sequences were subjected to a pairwise comparison using a window size of 11 bp. In the graphical outputs, sequence identity is indicated by dots and stretches of sequence identity appear as diagonal lines. For additional details, see the legend to Fig. 1.

FIG. 4.

Mating-type locus is stable through several genetic crosses. The strains and backcrossing scheme used during the construction of the congenic pair of serotype D strains JEC21 and JEC20 are shown on the left. The strains used in the analysis are indicated in boldface type. Mating type is indicated as α or a. Strains in boldface type were subjected to restriction enzyme digestion (BamHI, HindIII, and PstI) and Southern blotting using the mating-type-specific and nonspecific probes indicated. No differences were apparent between JEC21 and JEC20 and the ancestral strains NIH12, NIH433, B3501, and B3502. The relative positions of the probes used are indicated below in the corresponding mating-type locus.

FIG. 5.

Multiple transposon remnants and repetitive sequences are embedded in the MAT locus. Transposable element-related sequences are depicted for the four alleles of the MAT locus. Complete element copies are indicated in a larger font size and boldface type. In addition, local sequence repeats were identified and annotated for each allele.

FIG. 6.

Recombination between the MAT alleles is suppressed. Twenty-four progeny from two defined crosses were tested by PCR for recombination events between mating-type alleles. Primer pairs were either mating-type specific (pairs 3, 4, 5, and 6) or strain specific (pairs 1, 2, 7, and 8). Fragments amplified within the mating-type region by the different primer pairs are indicated as thick black bars. No recombination events were observed, and the PCR-amplified fragments all cosegregated with the corresponding mating types (α or a), as determined by backcrosses. The control strains were the serotype D strains JEC20 (MATa) and JEC21 (MATα), indicated by a* and α*, respectively. The meiotic progeny were also tested for segregation of parental markers. Ten of 24 strains showed a recombinant pattern (r), whereas the remaining 14 strains exhibited a parental genotype (p), which is indicated only for the control strains.

FIG. 7.

The MATα mating-type locus is conserved in the population. Using one primer combination, overlapping fragments (3 to 10 kb) spanning part of the mating-type locus were PCR amplified from (lanes from left to right in panel 1) the control strain JEC21 and the unrelated serotype D clinical isolates CDC92-18, CDC92-27, and MMRL760. Fragments of identical sizes were amplified from all four strains with nine primer combinations (panels 1 to 4 and 6 to 10). For descriptions of the 10 primer pairs, which are also represented by numbered bars indicating their positions on the MAT locus, see Table 1. One primer pair yielded a larger PCR product for strain MMRL760 (panel 5, lane 4), indicative of an ∼4-kb insertion between the STE11α and MFα1 genes. DNA sequence analysis revealed that the insertion of a novel mariner-related transposable element resulted in the addition of 4,006 bp relative to the JEC21 serotype D α allele and the creation of a TA target site duplication.

FIG. 8.

Structural comparison of the C. neoformans mating-type alleles. (A) Comparison of the α and a mating-type alleles of serotype D for analysis of the relative positions of the genes found within and adjacent to the MAT locus. Vertical and diagonal lines connect diverged gene alleles present in both alleles and illustrate substantial gene rearrangements within the MAT locus, whereas gene order outside the MAT locus has been conserved. In addition, a few genes that are present in only one of the two MAT alleles were identified (white arrows). (B) Comparison of the α mating-type alleles between serotypes A and D. Similar to what occurred in the α and a alleles from serotype D, significant rearrangements of gene order have occurred in the α allele during strain divergence (vertical and diagonal lines), and some genes are unique to one serotype (white arrows). Interestingly, the IKS1 gene flanks the MAT locus in serotype D but is located within the locus in serotype A. For additional details, see the legend to Fig. 1.