Fig 1.
Deletion of genes encoding C. albicans Flo8-Mfg1-Mss11 complex members has distinct impacts on filamentation induced by diverse cues.
A) C. albicans flo8Δ/flo8Δ and mfg1Δ/mfg1Δ mutants are largely blocked in filamentation in response to most cues, except in response to Hsp90 inhibition by treatment with geldanamycin (GdA). In contrast, a C. albicans mss11Δ/mss11Δ mutant was able to filament in response to all cues tested. Cells were grown in the conditions indicated, and were imaged after 3.5 hours, except for those grown in the presence of GdA, which were imaged after 24 hours. Scale bar is 20 μm. B) FLO8 is required for filamentation induced by deletion of the negative regulators of filamentation, NRG1 or LRG1, while MFG1 is not required. Cells were grown in YPD for 5 hours at 30°C. Scale bar is 20 μm.
Fig 2.
Overexpression of FLO8 or MSS11 results in filamentous growth in the absence of an inducing cue, but overexpression of MFG1 does not.
A) FLO8, MSS11 or MFG1 were overexpressed by replacing the native promoter of one allele with a tetracycline-repressible promoter, tetO. Cells were grown in YPD at 30°C for 6 hours. Scale bar is 20 μm. B) Individual overexpression of genes encoding each complex member was achieved by replacing the native promoter of one allele with a tetracycline-repressible promoter, tetO, which in the absence of tetracycline drives constitutive expression of the target gene. Cells were grown in YPD at 30°C for 3 hours. Transcript levels were normalized to ACT1 and TEF1 and error bars represent standard error of technical triplicates. Assays were performed in biological duplicate. Asterisks indicate P< 0.0001 (***) relative to the wild type (two tailed unpaired t-test). C) Filamentation was quantified by monitoring expression of the filament-specific transcript HWP1 using qRT-PCR, and normalizing to ACT1 and TEF1. Cells were grown for 4 hours in YPD at 30°C. Error bars represent standard error of technical triplicates as is representative of two biological replicates. Asterisks indicate P < 0.0001 (***) relative to parental strain (one-way ANOVA, Bonferroni Multiple Comparison Test). D) Filamentation induced by overexpression of FLO8 is largely independent of Mfg1, whereas filamentation induced by overexpression of MSS11 is completely dependent on Mfg1. Cells were grown in YPD at 30°C for 6 hours. Scale bar is 20 μm.
Fig 3.
C. albicans Mfg1 partially complements the activity of its S. cerevisiae ortholog.
A) C. albicans FLO8 or MFG1 were expressed in diploid cells of the filamentation-competent S. cerevisiae Σ1278b strain (Sc Wild type) and in the S. cerevisiae strains lacking the relevant orthologs to assess the ability of these complex members to complement the block in S. cerevisiae diploid pseudohyphal growth. Cells were grown on SLAD agar plates and incubated at 30°C for 11 days. Scale bar is 1.0 mm. B) C. albicans FLO8 or MFG1 were expressed in S. cerevisiae Σ1278b haploid cells (Sc Wild type) to assess their abilities to complement lack of FLO8 or MFG1 in S. cerevisiae haploid invasive growth. Cells were spotted onto YPD agar plates and incubated at 30°C for 4 days at which point they were washed with water and images of the plate were taken with a Canon Power Shot A610. Scale bar is 1.0 cm.
Fig 4.
C. albicans Mfg1 acts downstream of the PKA complex, as does Flo8.
A) Quantification of TPK2 expression by qRT-PCR. Overexpression of TPK2 was achieved by replacing the native promoter of one allele with a tetracycline-repressible promoter, tetO. Cells were grown in YPD for either 4 hours (30°C) or 3.5 hours (34°C). TPK2 transcript levels were monitored using qRT-PCR and normalized to ACT1 and TEF1. Error bars represent standard error of technical triplicates. Assays were performed in biological duplicate. Asterisks indicate P < 0.05 (*) and P < 0.001 (**), relative to parental strain (two-way ANOVA, Bonferroni Multiple Comparison Test). B) TPK2 was overexpressed by replacing the native promoter of one TPK2 allele with the tetracycline-repressible promoter, tetO, in either wild-type, mfg1Δ/mfg1Δ or flo8Δ/flo8Δ strains. Cells were grown in YPD for 6 hours at either 30°C or 34°C. Scale bar is 20 μm.
Fig 5.
ChIP-chip and transcriptional analyses reveal targets of Mfg1 and Flo8, and AP-MS reveals continued physical interaction under filament-inducing conditions.
A) Mfg1 and Flo8 bind and regulate overlapping and distinct targets, which are condition specific. Venn diagrams indicate number of genes whose promoters were bound by Mfg1 or Flo8 in filament-inducing conditions (either 1 hour or 3 hours), and for which expression was altered in the respective null mutants at the respective time point. Select targets in serum-treated conditions that are related to filamentation are indicated below the relevant category. B) AP-MS was performed with Mfg1-GFP cells grown in untreated conditions, or in the presence of serum at 37°C for 1 hour or 3 hours. Shown are prey proteins with a minimum of 25 detected peptides averaged between two biological replicates, in at least one of the three growth conditions. Mfg1, Flo8, and Mss11 are indicated in red. The separate Nop5 entries correspond to unique BioGrid IDs. Inside circle colour indicates average spectral counts, size of the circle indicates relative protein abundance, and the rim of the circle indicates the Bayesian false discovery rate (BFDR).
Fig 6.
Mfg1 and Flo8 bind to the promoter of TEC1 to regulate its expression, and overexpression of FLO8 drives TEC1 expression, enabling filamentation in the absence of Mfg1.
A) Overexpression of TEC1 results in filamentous growth in the absence of MFG1 or FLO8. Cells were grown in YPD at 30°C or in YPD with 10% serum at 37°C for 5 hours. Scale bar is 20 μm. B) Binding of Flo8-TAP and Mfg1-TAP to the promoter of TEC1 was assessed using ChIP-qPCR. Shown is the fold-enrichment over the untagged parental strain, which is set at 1. Asterisks indicate P < 0.01 (**) or P < 0.05 (*), relative to the wild-type parent in the respective condition (two-tailed unpaired t-test). Error bars represent the standard deviation of technical triplicates. Assays were performed in biological duplicate. C) TEC1 expression is reduced in the absence of Flo8 or Mfg1. Cells were grown in YPD at 30°C for 3.5 hours, pelleted and washed, and then transferred to YPD at 30°C or to YPD at 37°C with 10% serum for 1 hour. Transcript levels were monitored using qRT-PCR and normalized to PMA1 and RIP1. Error bars represent standard error of technical triplicates. Assays were performed in biological duplicate. Asterisks indicate P < 0.001 (***), relative to the wild-type strain in each respective condition (two-way ANOVA, Bonferroni Multiple Comparison Test). D) TEC1 expression is increased in strains overexpressing FLO8. Cells were grown in YPD at 30°C for 4.5 hours. Transcript levels were monitored using qRT-PCR and normalized to PMA1 and RIP1. Error bars represent standard error of technical triplicates. Assays were performed in biological duplicate. Asterisks indicate P < 0.0001 (***), relative to the respective parental strain, or as indicated (one-way ANOVA, Bonferroni Multiple Comparison Test).
Fig 7.
Amplification of chromosome 6 restores the ability of an mfg1Δ/mfg1Δ mutant to filament.
A) Independent colonies from an mfg1Δ/mfg1Δ mutant expressing the NAT (nourseothricin) resistance marker from the HWP1 promoter were plated on solid YPD containing 10% serum and 250 μg/mL NAT and grown at 37°C for 2 days. Shown are three independently evolved mutants that were able to filament in liquid conditions in response to serum (Evo #A1, Evo #A2, and Evo #A3). Cells were grown in YPD at 30°C or in YPD with 10% serum at 37°C for 6 hours. Scale bar is 20 μm. B) The genomes of three independently evolved filamentous mutants (Evo #A1, Evo #A2 and Evo #A3) in the mfg1Δ/mfg1Δ background were sequenced and profiled for copy number variants using YMAP. C) Deletion of one allele of FLO8 from the evolved mutant Evo #A2 impairs its ability to filament. One allele of FLO8 was replaced with CdARG4, and cells were grown as in A). Scale bar is 20 μm.
Fig 8.
Evolution of filament-capable mfg1Δ/mfg1Δ mutants with FLO8 at a non-native locus is not accompanied by chromosome 6 triplication.
A) An mfg1Δ/mfg1Δ mutant with FLO8 located on chromosome 5 (at the HIS1 locus) and expressing the NAT (nourseothricin) resistance marker under the control of the HWP1 promoter evolved the ability to filament upon selection on NAT and serum. Mutants Evo #C1-1 and Evo #C1-2 arose from the same overnight culture but showed distinct phenotypes in their ability to filament at later time points. Mutants Evo #C1, C2, C3, and C4 are all independently generated. Cells were grown in YPD at 30°C or in YPD with 10% serum at 37°C for 6 hours. Scale bar is 20 μm. B) The genomes of five evolved filamentous mutants in the mfg1Δ/mfg1Δ background were sequenced and profiled for copy number variants using YMAP.