Sexual Differentiation Is Coordinately Regulated by Cryptococcus neoformans CRK1 and GAT1

Abstract

The heterothallic basidiomycetous fungus Cryptococcus neoformans has two mating types, MATa and MATα. Morphological progression of bisexual reproduction in C. neoformans is as follows: yeast to hyphal transition, filament extension, basidium formation, meiosis, and sporulation. C. neoformans Cdk-related kinase 1 (CRK1) is a negative regulator of bisexual mating. In this study, we characterized the morphological features of mating structures in the crk1 mutant and determined the genetic interaction of CRK1 in the regulatory networks of sexual differentiation. In the bilateral crk1 mutant cross, despite shorter length of filaments than in the wild-type cross, dikaryotic filaments and other structures still remained intact during bisexual mating, but the timing of basidium formation was approximately 18 h earlier than in the cross between wild type strains. Furthermore, gene expression analyses revealed that CRK1 modulated the expression of genes involved in the progression of hyphal elongation, basidium formation, karyogamy and meiosis. Phenotypic results showed that, although deletion of C. neoformans CRK1 gene increased the efficiency of bisexual mating, filamentation in the crk1 mutant was blocked by MAT2 or ZNF2 mutation. A bioinformatics survey predicted the C. neoformans GATA transcriptional factor Gat1 as a potential substrate of Crk1 kinase. Our genetic and phenotypic findings revealed that C. neoformansGAT1 and CRK1 formed a regulatory circuit to negatively regulate MAT2 to control filamentation progression and transition during bisexual mating.

Keywords: CRK1; Cryptococcus neoformans; GAT1; bisexual mating; filament differentiation.

Figure 1

Nuclear distribution in the mating structures of the wild-type and bilateral crk1 mutant crosses during bisexual mating process. Mating of the wild-type (A) and bilateral crk1 mutant (B) crosses was conducted on V8 solid medium and incubated at 26 °C. Nuclei were visualized by Gfp-H2b fusion protein in the MATa wild-type and MATa crk1 mutant cells. Bright-field (BF) and fluorescent (GFP) images of dikaryotic filaments, basidia and long chains of basidiospores were photographed at 400× magnification. Photos taken from 16 to 72 h were shown and merged photos were created with ImageJ. White star indicates clamp cell, white triangle indicates nucleus, and white arrow indicates basidia. Scale bar = 5 µm.

Figure 1

Nuclear distribution in the mating structures of the wild-type and bilateral crk1 mutant crosses during bisexual mating process. Mating of the wild-type (A) and bilateral crk1 mutant (B) crosses was conducted on V8 solid medium and incubated at 26 °C. Nuclei were visualized by Gfp-H2b fusion protein in the MATa wild-type and MATa crk1 mutant cells. Bright-field (BF) and fluorescent (GFP) images of dikaryotic filaments, basidia and long chains of basidiospores were photographed at 400× magnification. Photos taken from 16 to 72 h were shown and merged photos were created with ImageJ. White star indicates clamp cell, white triangle indicates nucleus, and white arrow indicates basidia. Scale bar = 5 µm.

Figure 2

Morphology of dikaryotic filaments in the wild-type and bilateral crk1 mutant crosses. Mating mixtures of the wild-type (A) and bilateral crk1 mutant (B) crosses were incubated on SLAD medium containing 25 µg/mL calcofluor white and kept at 26 °C under dark condition. Nuclei were labeled by Gfp-H2b fusion protein. Cell wall was stained with calcofluor white. Dikaryotic filaments were photographed at 13 and 17 h post-incubation at 60× magnification. White star indicates clamp cell and white triangle marks nucleus. White double-headed arrow indicates the range of dikaryotic filament measurement. Scale bar = 15 µm.

Figure 3

The expression of mating-, hyphal extension-and sporulation-related genes was elevated at 18 h in the bilateral crk1 mutant cross. Bilateral crosses involved the MATa and MATα wild-type and crk1 mutants were conducted on V8 agar plates and incubated at 26 °C in the dark. Samples were collected at 0, 12, 14, 16, 18, 20, 22 and 24 h post-incubation. The expression of (A) CRK1, (B) MAT2, (C) ZNF2, (D) PUM1, (E) KAR7, and (F) DMC1 was examined by real-time qRT-PCR analysis. Triplicate reactions for each sample were conducted. Error bar represents the standard deviation from the mean of three replicates. The results were normalized to C. neoformans GPD1 expression. (** indicates p < 0.005).

Figure 4

Sexual differentiation in the crk1 mutant was blocked by the mutation of MAT2 and CRK1 expression was reduced in the bilateral mat2 mutant cross. (A) C. neoformans MATa and MATα strains were crossed as indicated. Mating was conducted on V8 agar plates and incubated at 26 °C in the dark. Photos were taken 24 h post-incubation at 100× magnification. Bilateral crosses involved the MATa and MATα wild-type and crk1, mat2, and mat2crk1 double mutants were conducted. Samples were collected at 0, 6, 18 and 24 h post-incubation and subjected to RNA extraction. The expression of MAT2 (B) and CRK1 (C) during bisexual mating was examined by real-time qRT-PCR analysis. Triplicate reactions for each sample were conducted. Error bar represents the standard deviation from the mean of three replicates. The results were normalized to C. neoformans GPD1 expression. (** indicates p < 0.005).

Figure 5

Overexpression of MAT2 increased bisexual filamentation and upregulated CRK1 expression. (A) Crosses involved C. neoformans MATa and MATα wild-type and MATa wild-type and MATα P_GPD1::MAT2 strains were conducted on V8 agar plates incubated at 26 °C in the dark. Photos were taken 24 h post-incubation at 100× magnification. The same crosses were also subjected to gene expression studies and samples were collected at 0 and 24 h post-incubation. The expression of MAT2 (B), SXI1α (C), and CRK1 (D) during mating was determined by real-time qRT-PCR analysis. Triplicate reactions for each sample were conducted. Error bar represents the standard deviation from the mean of three replicates. The results were normalized to C. neoformans GPD1 expression. (** indicates p < 0.005; * indicates p < 0.05).

Figure 6

Dikaryotic filamentation and aerial hyphae formation were enhanced in the bilateral gat1 mutant and GAT1^T1164A mutant crosses. (A) C. neoformans MATa and MATα gat1 mutant and overexpression strains were crossed as indicated and compared to the wild type cross. (B) C. neoformans MATa and MATα GAT1^T1164A bilateral mutant cross was performed and compared to the crosses as indicated. Small photos reveal the density of aerial hyphae. Yellow arrow indicates aerial hyphae. Mating was conducted on V8 agar plates and incubated at 26 °C in the dark. Photos were taken 24 h post-incubation at 100× magnification.

Figure 7

The expression of mating- and hyphal extension-related genes was increased in the bilateral gat1 mutant cross. Bilateral crosses involved the MATa and MATα wild-type and gat1 mutants were conducted on V8 agar plates and incubated at 26 °C in the dark. Samples were collected at 0, 12, 14, 16, 18, 20, 22 and 24 h post-incubation. The expression of GAT1 (A), MFα (B), MAT2 (C), and PUM1 (D) was examined by real-time qRT-PCR analysis. Error bar represents the standard deviation from the mean of three replicates.Triplicate reactions for each sample were conducted. The results were normalized to C. neoformans GPD1 expression. (** indicates p < 0.005; * indicates p < 0.05).

Figure 8

The crk1 and crk1gat1 mutants were phenotypically identical in bisexual mating. C. neoformans MATa and MATα gat1, crk1, and crk1gat1 mutant strains were crossed on V8 agar plates at 26 °C in the dark and compared to the wild type strains. The edges of mating mixtures were photographed 16 h and 24 h post-incubation at 100× magnification. Photos of mating filaments were also recorded at 400× magnification. Small photos illustrate the tip of dikaryotic filament and yellow arrow indicates basidium.

Figure 9

The GAT1^T1164D phospho-mimetic active allele dramatically repressed dikaryotic filamentation in the wild-type cross but showed slight effects on mating under crk1 mutant background. (A) C. neoformans MATa and MATα strains were crossed as indicated. Mating was conducted on V8 agar plates and incubated at 26 °C in the dark. Photos were taken 16 h and 24 h post-incubation at 100× magnification. (B) Small photos demonstrate the basidia in the crk1 and crk1GAT1^T1164D bilateral mutant crosses respectively. Yellow arrow indicates basidium. Photos were taken 24 h post-incubation at 400× magnification.

Figure 10

The expression of mating-related genes was down-regulated by the GAT1 phospho-mimetic allele. C. neoformans MATa and MATα strains were crossed as indicated. Mating was conducted on V8 agar plates and incubated at 26 °C in the dark. Samples were collected at 0, 18, and 24 h post-incubation. The expression of GAT1 (A), MFα (B), MAT2 (C), CSA1 (D), and DMC1 (E) was examined by real-time qRT-PCR analysis. Triplicate reactions for each sample were conducted. Error bar represents the standard deviation from the mean of three replicates. The results were normalized to C. neoformans GPD1 expression. (** indicates p < 0.005; * indicates p < 0.05).

Figure 11

C. neoformans Crk1 and Gat1 negatively regulated the expression of Mat2 to coordinately modulate sexual differentiation. Mat2 is a major transcription factor downstream the Cpk1-MAPK signaling pathway to regulate pheromone production and mating responses in C. neoformans. The protein kinase Crk1 negatively regulated MAT2 expression via phosphorylating Gat1 or other target(s) to reduce the MAT2 transcript level and repress pheromone production and other mating responses. The expression of CRK1 was possibly also regulated by Mat2. Crk1-Gat1 may form a regulatory circuit to properly regulate MAT2/Mat2 levels to control the progression of sexual filamentation and transition to basidium stage. We propose this model how Crk1 regulates pheromone production, elongation of dikaryotic filaments, and basidium formation during bisexual mating.