Sex-specific homeodomain proteins Sxi1alpha and Sxi2a coordinately regulate sexual development in Cryptococcus neoformans

Abstract

Homeodomain proteins are central regulators of development in eukaryotes. In fungi, homeodomain proteins have been shown to control cell identity and sexual development. Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle that produces spores, the suspected infectious particles. Previously, only a single homeodomain regulatory protein involved in sexual development, Sxi1alpha, had been identified. Here we present the discovery of Sxi2a, a predicted but heretofore elusive cell-type-specific homeodomain protein essential for the regulation of sexual development. Our studies reveal that Sxi2a is necessary for proper sexual development and sufficient to drive this development in otherwise haploid alpha cells. We further show that Sxi1alpha and Sxi2a interact with one another and impart similar expression patterns for two key mating genes. The discovery of Sxi2a and its relationship with Sxi1alpha leads to a new model for how the sexual cycle is controlled in C. neoformans, with implications for virulence.

FIG. 1.

MATa contains a gene encoding the predicted homeodomain protein Sxi2a. (A) The SXI2a gene is located in the MAT locus. A schematic representation of the C. neoformans MAT locus shows MATα on the top and MATa on the bottom. The shaded bars represent the region of nonidentical DNA present in MATα and MATa. Genes within the locus are represented by shaded arrows. Each gene within the locus, with the exception of SXI1α and SXI2a, has a counterpart allele that encodes a similar, but not identical, protein in the opposite mating type. SXI1α and SXI2a are unique to their respective mating types. (B) Sxi2a is a homeodomain protein. The predicted homeodomain region of C. neoformans (Cn) Sxi2a is aligned with known homeodomains of other proteins: S. cerevisiae (Sc) a1, U. maydis (Um) bW2, C. cinereus (Cc) a2-1, S. cerevisiae α2, C. cinereus b1-2, and C. neoformans Sxi1α. A schematic representation of the homeodomain region shows the helices of a classic three-helix bundle found in homeodomains above the sequences. C. neoformans Sxi2a, S. cerevisiae a1, U. maydis bW2, and C. cinereus a2-1 fall into the HD2 class of homeodomains, whereas S. cerevisiae α2, C. cinereus b1-2, and C. neoformans Sxi1α are members of the HD1 family containing a 3-amino-acid insertion between helices 1 and 2. The inverted triangles denote the positions of introns in Sxi2a. Introns 1, 2, and 3, are 65, 66, and 294 bases, respectively. The third intron is conserved in many HD2 homeodomain proteins. (C) SXI2a is linked to MATa. Genomic DNA from segregants of a genetic cross was subjected to PCR analysis with primers to the ORF of SXI2a. Lane 1, marker; lanes 2 and 3, DNA from control a cells and α cells, respectively; lanes 4, 5, 13, and 14, DNA from segregants that mate as a cells; lanes 6 to 12 and 15 to 18, DNA from segregants that mate as α cells.

FIG. 2.

SXI2a is required for sexual development. (A) Sexual development in C. neoformans. When haploid a cells encounter haploid α cells at 25°C in the presence of either an unidentified plant factor in V8 medium or nitrogen limitation, the cells initiate a mating response and fuse with one another. The fused cells adopt a filamentous growth pattern in which the haploid nuclei do not fuse with one another. This dikaryotic filament grows in a polar manner, and adjacent filament cells are linked by fused clamp connections. Ultimately, in response to unknown signals, the terminal filament cell ceases extension and forms a rounded compartment at the distal end of the cell. It is in this basidium that nuclear fusion, meiosis, and sporulation occur. Meiotic products are packaged, and spores are extruded in long chains on the surface of the basidium. (B) sxi2aΔ strains can respond to and fuse with a mating partner but fail to form normal filaments. Wild-type, sxi1αΔ, and sxi2aΔ strains were tested in coculture (top row) (48 h), fusion (middle row) (24 h), and confrontation (bottom row) (48 h) assays to assess the nature of their defects in sexual development. Strain combinations are shown above the top row and indicate the strains used in each cross as well as the strains used in the fusion and confrontation assays. There was no significant difference in the extent of the limited rudimentary filaments produced in crosses by the mutant strains.

FIG. 3.

SXI2a induces sexual development in α cells. (A) Expression of SXI2a in haploid α cells results in filamentation and spore formation. Panels show the amount of filamentation achieved under mating conditions for each of the strain combinations indicated (from left to right): a wild-type cross between haploid a and α strains (a × α), a haploid α strain (α), and a haploid α strain expressing the a-specific gene SXI2a (α+SXI2a) (B) Schematic representation of filament formation in a/a diploid cells expressing SXI1α and a table showing analysis of spore products. The “parent genotype” is that of the homozygous a/a diploid used in the experiment. The “spore phenotypes” are those combinations expected to result from sporulation of the parent diploid. The frequencies presented result from an analysis of 39 random spores that were micromanipulated, germinated, and analyzed from the a/a+SXI1α strain under filamentation conditions. n/a, not applicable. The structure on the side of the filament cells represents an unfused clamp connection.

FIG. 4.

Sxi1α and Sxi2a regulate pheromone gene expression. A Northern blot showing the levels of selected transcripts in wild-type, sxi1αΔ, and sxi2aΔ strains after growth on V8 medium in three different contexts, indicated below the lane numbers: haploid strains alone, mixed mating cocultures, and diploid strains alone. The top two panels show RNA probed with the MFa and MFα ORFs. The third panel shows the same blot probed with portions of the SXI1α and SXI2a ORFs. The bottom panel shows the same blot probed with a portion of the GPD1 ORF. Lane 1, wild-type haploid a; lane 2, wild-type haploid α; lane 3, haploid sxi1αΔ; lane 4, haploid sxi2aΔ; lane 5, wild-type a cocultured with wild-type α; lane 6, wild-type a cocultured with sxi1αΔ; lane 7, sxi2aΔ cocultured with a wild-type α; lane 8, sxi2aΔ cocultured with a sxi1αΔ; lane 9, a/α diploid; lane 10, a/sxi1αΔ diploid; lane 11, sxi2aΔ/α diploid; lane 12, sxi2aΔ/sxi1αΔ diploid.

FIG. 5.

Sxi1α and Sxi2a interact with one other. The top panel in each set (−leu −trp) confirms the presence of each two-hybrid plasmid under selection. The middle and bottom panels indicate the presence or absence of activation of a biosynthetic reporter gene (either ADE2 or HIS3). The presence or absence of indicated constructs is represented with + or − over each panel. The fusion proteins tested are indicated in schematic form: the hatched box BD represents the Gal4 DNA BD, the speckled box AD represents the Gal4 AD, and the black box represents the Sxi2a homeodomain region. Growth in a test spot indicates activation of the reporter. (A) Lanes 1 to 4 reveal a transactivating property of Sxi2a in the absence of Sxi1α when fused to a DNA BD. (B) Lanes 5 to 11 confirm an interaction between Sxi1α and Sxi2a and define the region of Sxi2a necessary for interaction. The results of liquid β-galactosidase assays in each column are shown in Miller units.

FIG. 6.

Model for the regulation of sexual development in C. neoformans. Model for the role of Sxi1α and Sxi2a in controlling sexual development. Large ovals represent cells. A solid circle represents the a nucleus, and a hatched blue circle represents the α nucleus. The yellow oval X and shaded blue square Y represent unknown a- and α-specific factors, respectively, that lead to the induction of SXI1α and SXI2a. The blue shaded oval represents Sxi1α, and the yellow star represents Sxi2a. (Top) SXI1α and SXI2a are expressed at low levels in haploid cells. (Middle) Following cell fusion, SXI1α and SXI2a expression is dependent upon and induced by factors from both a and α cells (X and Y). (Bottom) After induction, Sxi1α and Sxi2a form a heterodimeric complex that establishes the dikaryotic state and induces sexual development through the repression of mating genes.