Figure 1.
Identification of the genetic network in hyphal and yeast populations.
(A) A diagram depicting the pheromone and filamentation signaling pathways and their downstream cellular processes in Cryptococcus. (B) The PCTR4-2-ZNF2 strain and the wildtype strain H99 were grown under the condition that inhibits mating activation in order to perform comparative transcriptome analyses of homogeneous yeast and hyphal populations without the complication pheromone stimulation. (C) Histogram and the Venn diagram depicting the difference in two transcriptome analyses. The current one (red) used the homogeneous morphotypes and yielded a higher number of genes with significantly differential expression between the yeast and filamentous growth. The former one (black) used wildtype population in response to mating stimulation and the population showed pronounced heterogeneity in cell morphotype, which likely obstructed the previous effort in identifying many of the potential Znf2 downstream targets. The significant overlap of the lists from this study and the previous one verified our current approach and also indicated some factors that are likely to be specific to the growth conditions (hypergeometric p-value of significant overlap <0.001). (D) The list of enriched GO terms with differentially expressed genes in different morphotypes. The DAVID gene ontology program was used for the analysis and gene-enrichment in the annotated terms was evaluated based on the EASE Score. The EASE Score is a modified Fisher Exact P-Value and the default threshold is 0.1 [29]. Asterisks indicate GO terms with values above the default threshold. These GO terms are included because of their potential biological significance based on previous studies in C. neoformans or other fungi [11], [12], [30], [47]. The extremely mating-suppressive conditions used in this study may result in the under-representation of these classes and their lower statistical significance.
Figure 2.
The Znf2 downstream target Pum1 directs filamentation.
(A) The 29 Znf2 downstream targets were placed under the control of the constitutive PGDP1 promoter to construct a Cryptococcus overexpression library. The screen for their effects on filamentation was conducted under filamentation-suppressing or filamentation-inducing conditions with a 5-day incubation period. CNAG_0‘number’ indicates the gene locus name used in Broad Institute C. neoformans H99 sequence annotation, and these locus names were used because they are previously uncharacterized genes. The gene locus number of PUM1 is CNAG_04003. (B) Pum1 positively controls filamentation. The overnight cultures of the wildtype strain XL280, the pum1Δ mutant, and the PUM1 overexpression strain of the same cell density were dropped onto the V8 agar (mating-inducing) or the YPD agar (mating-suppressing) and photographed after 5 days of growth. Scale bar: 1 mm. (C) The Pum1-mCherry shows a biased expression in the hyphal subpopulation. The images of the fluorescence labeled wildtype strain were taken at 72 hrs post mating stimulation. The localization patterns of Pum1 in the figure are representative for each given cell types (at least 40 cells for each cell type were examined). See Materials and Methods for the detailed description of the experimental condition for the sub-cellular localization of Pum1-mCherry. Scale bar: 10 µm.
Figure 3.
Pum1 controls filamentation but not early mating processes.
(A) Transcriptional dynamics of MF1α and PUM1 during mating. The RNA from the a+α mixture co-cultured on V8 agar at 22°C in the dark at indicated time points was subjected to the transcriptional analysis by real time PCR. There was a delay in the transcriptional induction of PUM1 compared to that for the pheromone gene MF1α. (B) Overexpression of PUM1did not increase the transcript level of the genes in the pheromone pathway. The expression level of the corresponding genes in the wildtype strain XL280 grown under the same condition was arbitrarily set as 1 for comparison. (C) Pum1 is modestly inhibitory for cell fusion. The α and a partners from wildtype strains or the pum1Δ mutant marked with different auxotrophic markers were co-cultured on V8 agar or YPD liquid medium for 22 hours at 22°C in the dark. The cocultures were then collected, washed, and plated onto YNB minimal medium to select for fusion products. The cell fusion efficiency of the wildtype coculture was arbitrarily set as 1 for comparison. (D) Overexpression of PUM1 and CFL1 are sufficient to trigger filamentation even under mating-suppressive conditions. The wildtype strain, the PCTR4-2-PUM1 strain, and the PCTR4-2-CFL1 strain were grown in the YPD liquid medium. Filamentation and cell aggregation (cell-cell adhesion) were observed in the CFL1oe and PUM1oe strains but not in the wildtype strain. Scale bar: 20 µm. (E) The PGPD1-PUM1 strain was significantly less virulent in a murine intranasal inhalation infection model compared with the wildtype strain XL280 and the pum1Δ mutant (P<0.01). (F) Pum1-mCherry is expressed at a relatively constant level throughout hyphal growth and basidial development (>40 hyphae for each stage were examined). Scale bar: 10 µm.
Figure 4.
Cfl1 and Pum1 represent two major circuits downstream of Znf2 in directing aerial hyphae formation.
(A) Transcriptional analysis by qPCR indicates that alterations of Cfl1 or Pum1 do not affect the transcript level of each other. The expression level of genes in the wildtype strain XL280 was arbitrarily set as 1 for comparison. (B) The production or the release of Cfl1 proteins was not drastically affected in the pum1Δ mutant. The expression of Cfl1 proteins was abolished in the znf2Δ mutant and enhanced when the compatible mating partner was present. The mCherry-labeled Cfl1 was used to measure the Cfl1 protein level by fluorescence microscopy and released Cfl1 proteins was detected by colony immunoblot. (C) Pum1 is dispensable for the expression or the processing of Cfl1. By comparison, the Cfl1 protein expression was below the detectable level in the znf2Δ mutant. This served as a negative control. The asterisk indicates the non-specific band, which is also shown in control strain XL280 without mCherry. (D) Double deletion of CFL1 and PUM1 resulted in a much more severe defect in aerial filamentation during bisexual mating compared to either of the single deletion. (E) Pum1 and Cfl1 represent two major downstream regulatory branches of Znf2 in initiating filamentation. (F) Pum1 is also involved in hyphal extension. The deletion of PUM1 resulted in shorter filaments during bisexual mating. Such a phenotype was not observed in the cfl1Δ mutant. Scale bar: 20 µm.
Figure 5.
Pum1 directs hyphal initiation and extension partially through a novel hypha-specific extracellular protein Fas1.
(A) FAS1, FAD1, and DHA1 are the genes encoding extracellular proteins overlapped in the regulons of Znf2 and Pum1. (B) Fas1 recapitulates the role of Pum1 in unisexual hyphal production. The fas1Δ mutant, like the pum1Δ mutant, showed reduced abundance of aerial hyphae. (C) The indicated strains were grown on V8 agar plate containing 200 µM BCS in the dark at 22°C. Images of the colonies were photographed after 5 days of incubation. Released Fas1 proteins were detected by colony immune-blot. Only overexpression of Fas1 with intact signal peptide led to a more robust filamentation and longer hyphae. Thus, the secretion of Fas1 is crucial for its biological function. (D) Fas1-mCherry is observed on hyphal surface and in vesicles at 72 hrs post unisexual mating stimulation. (E) The FAS1 expression level was positively regulated by Pum1 based on the transcriptional analysis of its transcript level in the pum1Δ mutant and the PGPD1-PUM1 strain by qPCR. The deletion or the overexpression of CFL1 did not significantly affect the FAS1 expression level. (F and G) Pum1 increased the intensity of Fas1 expression and reduces its stochasticity. The average intensity of Fas1-mCherry fluorescence was dramatically reduced in the absence of Pum1, but not Cfl1. The deletion of PUM1 led to a much higher variability in the Fas1-mCherry level among hyphal cells during unisexual filamentation (Inset of Figure? 5G). The frequencies of Fas1-mCherry's mean intensity in the hyphal population were plotted for the wildtype strain, the cfl1Δ mutant, and the pum1Δ mutant (software: ZEN 2011).
Figure 6.
Unisexual meiotic progression is only observed in the Pum1-controlled aerial hyphal population.
(A) Pum1, but not Cfl1, affects the expression of DMC1 during unisexual reproduction. (B) Dmc1 expression is associated with a portion of hyphal cells but not yeast cells. Scale bar: 20 µm. (C) Fas1 is exclusively expressed in the hyphal population and it is used as a molecular indicator to visualize the hyphal population in a mating colony. A side-view of a single-cell derived colony (3 days old) shows the topology of Fas1-expressing hyphal population in the community. (D) Details of the morphology and Dmc1 expression for aerial hyphae and root hyphae (invasive hyphae) from the unisexual mating community are shown. Fungal cell wall was stained blue with calcofluor white. Dmc1 expression was exclusively observed in aerial hyphae but not in invasive hyphae (more than 200 invasive hyphae were examined). Scale bar: 10 µm. (E) The dynamics of Dmc1-mCherry monitored during unisexual development. Scale bar: 10 µm. The localization patterns of Dmc1 in the figure are representative for each given stage of development (>40 hyphae for each stage were examined).
Figure 7.
Pum1 coordinates filamentation and sexual reproduction.
(A and B) The deletion of PUM1 impairs the expression of Dmc1 in basidia in a stochastic manner. Some but not all differentiating basidial heads lost the expression of Dmc1-mCherry without Pum1. Those that did expressed Dmc1at lower levels. Scale bars: 20 µm. (C) The unisexual sporulation was perturbed in the absence of the meiosis-specific protein Dmc1 and the global regulator Pum1. The wildtype strain XL280, the dmc1Δ mutant, and the pum1Δ mutant were diluted on V8 agar to form single colonies. Typical mature basidia observed from these strains were shown. Scale bar: 10 µm. (D and E) The deletion of PUM1 and DMC1 also dramatically reduced the sporulation efficiency during bisexual mating. (F) The effect of the deletion of ZNF2, PUM1, or DMC1 on aerial hyphal morphogenesis at the colony level during both bisexual and unisexual development. Scale bar: 1 mm.
Figure 8.
Filamentation pathway coordinates sexual reproduction and morphogenesis in Cryptococcus neoformans.
Activation of Znf2 promotes the filamentous growth and modestly inhibits the early mating behaviors that initiate the transition from the yeast form to the hypha form [7], [12]. Cfl1 and Pum1 represent two parallel major branches downstream of Znf2 in the orchestration of this morphological switch. Pum1 plays additional important roles in sustaining hyphal growth and in bridging hyphal development and sexual reproduction partially through its control over the expression of DMC1 and FAS1. The question mark indicates unidentified factors controlled by Pum1 that regulate meiosis and sporulation.