The Vps21 signalling pathway regulates white-opaque switching and mating in Candida albicans

Figures & data

Figure 1. Function of Vps21 and its variants in the regulation of white-to-opaque switching. White cells of the WT control, vps21/vps21 mutant, and reconstituted strains were initially grown on Lee’s glucose medium (pH 6.8) at 25 °C for 7 days. Candida albicans cells of homogeneous white colonies were replated onto Lee’s GlcNAc medium (pH 6.8) and incubated at 25 °C for 5 days. wh, white; op, opaque; op-s, opaque-sectored (colonies containing both white and opaque cells). Scale bar for cells, 10 μm; Scale bar for colonies, 2 mm. Strains used: WT (FDZF208); vps21/vps21 (FDZF263); vps21/vps21 + VPS21 (FDZF489); vps21/vps21 + VPS21^S24N (FDZF475); and vps21/vps21 + VPS21^Q69L (FDZF473). The mating type of all strains used was MTLa/Δ. The switching frequencies are presented in Table 1.

Table 1. White-opaque switching frequencies of the WT control and mutant strains of the Vps21 signalling pathway.

Strain	Wh-to-op (%)		Op-to-wh (%)
	Lee’s GlcNAc	Lee’s glucose	Lee’s GlcNAc	Lee’s glucose
WT	14.6 ± 0.2	<0.2	0.7 ± 0.8	7.3 ± 3.4
vps21/vps21	30.9 ± 2.3	<0.4	<0.3	8.3 ± 3.7
vps21/vps21 + VPS21	10.9 ± 1.0	<0.2	<0.3	5.4 ± 2.5
vps21/vps21 + VPS21^Q69L	12.4 ± 0.6	<0.2	<0.2	8.9 ± 0.6
vps21/vps21 + VPS21^S24N	30.8 ± 3.0	<0.2	<0.2	7.6 ± 1.2
vps9/vps9	44.1 ± 3.5	<0.2	<0.3	19.8 ± 3.8
vps9/vps9 + VPS9	19.0 ± 1.2	<0.3	0.3 ± 0.6	7.8 ± 2.6
vps3/vps3	94.3 ± 3.1	<0.3	<0.2	27.6 ± 2.1
vps3/vps3 + VPS3	8.1 ± 0.6	<0.3	<0.2	6.5 ± 2.1
vac1/vac1	91.7 ± 3.4	<0.3	0.3 ± 0.6	31.4 ± 4.4
vac1/vac1 + VAC1	8.9 ± 4.2	<0.4	1.5 ± 1.2	9.8 ± 2.5
pep12/pep12	96.9 ± 1.8	<0.2	<0.3	32.4 ± 4.5
pep12/pep12 + PEP12	8.1 ± 1.7	<0.3	<0.2	15.4 ± 5.3

White and opaque cells of the WT control and mutant strains were initially grown on Lee’s glucose medium (pH 6.8) for 7 days. Candida albicans cells of homogeneous white or opaque colonies were replated onto Lee’s glucose (pH 6.8) or Lee’s GlcNAc medium (pH 6.8) and incubated at 25 °C for 5 days. Wh-to-op (%), white-to-opaque switching frequency (percentage); op-to-wh (%), opaque-to-white switching frequency (percentage); <, no colonies with the alternative phenotype were observed. Strains used: WT (FDZF208); vps21/vps21 (FDZF263); vps9/vps9 (FDZF213); vps3/vps3 (FDZF259); vac1/vac1 (FDZF531); pep12/pep12 (FDZF546); vps21/vps21 + VPS21 (FDZF489); vps21/vps21 + VPS21^S24N (FDZF475); vps21/vps21 + VPS21^Q69L (FDZF473); vps9/vps9 + VPS9 (FDZF491); vps3/vps3 + VPS3 (FDZF493); vac1/vac1 + VAC1 (FDZF548); and pep12/pep12 + PEP12 (FDZF549). The mating type of all strains used was MTLa/Δ.

Figure 2. Deletion of VPS9, VPS3, VAC1, and PEP12 promotes white-to-opaque switching. (a) Schematic diagram of the Vps21 signaling pathway. Vps9, a guanine nucleotide exchange factor (GEF) of Vps21; Vps3, a CORVET tethering complex component; Vac1, a vesicle transport protein; and Pep12, a t-SNARE involved in prevacuolar trafficking. (b) White-to-opaque switching assays. White cells of the WT control, vps9/vps9, vps3/vps3, vac1/vac1, and pep12/pep12 mutant, and corresponding reconstituted strains were initially grown on Lee’s glucose medium (pH 6.8) for 7 days. Candida albicans cells of homogeneous white colonies were replated onto Lee’s GlcNAc medium (pH 6.8) and incubated at 25 °C for 5 days. Wh, white; op, opaque. Scale bar for cells, 10 μm; Scale bar for colonies, 2 mm. Strains used: WT (FDZF208); vps9/vps9 (FDZF213); vps3/vps3 (FDZF259); vac1/vac1 (FDZF531); pep12/pep12 (FDZF546); vps9/vps9 + VPS9 (FDZF491); vps3/vps3 + VPS3 (FDZF493); vac1/vac1 + VAC1 (FDZF548); and pep12/pep12 + PEP12 (FDZF549). The mating type of all strains used was MTLa/Δ. The switching frequencies are presented in Table 1.

Table 2. Mating efficiencies of the WT control and mutant strains of the Vps21 signalling pathway.

Cross (a x α)	Mating efficiency
	Lee’s glucose	Lee’s GlcNAc
WT a x WT α	(4.7 ± 1.9) × 10^−2	(5.6 ± 1.9) × 10^−3
vps21/vps21 a x vps21/vps21 α	(5.7 ± 0.6) × 10^−3	(2.9 ± 0.6) × 10^−5
vps9/vps9 a x vps9/vps9 α	(4.2 ± 3.0) × 10^−3	(5.1 ± 1.6) × 10^−5
vps3/vps3 a x vps3/vps3 α	(9.0 ± 0.8) × 10^−5	(2.2 ± 3.7) × 10^−8
vac1/vac1 a x vac1/vac1 α	(8.2 ± 3.1) × 10^−5	< (4.2 ± 1.6) × 10^−8
pep12/pep12 a x pep12/pep12 α	(7.8 ± 3.0) × 10^−5	(8.0 ± 2.9) × 10^−8

Opaque MTLa/Δ and MTLΔ/α cells were mixed and incubated on Lee’s glucose (pH 6.8) or Lee’s GlcNAc (pH 6.8) medium at 25 °C for 3 days. The mixtures were then plated onto selective media for mating efficiency assays. <, no colonies with the alternative phenotype were observed. Strains used: WTa (FDZF208); WTα, (FDZF171); vps21/vps21a (FDZF263); vps21/vps21α (FDZF266); vps9/vps9a (FDZF213); vps9/vps9α (FDZF212); vps3/vps3a (FDZF259); vps3/vps3α (FDZF250); vac1/vac1a (FDZF531); vac1/vac1α (FDZF533); pep12/pep12a (FDZF546); and pep12/pep12α (FDZF534).

Figure 3. Relative transcriptional expression of mating associated genes in the WT control and mutant strains of the Vps21 signalling pathway. Opaque cells of the WT control, vps21/vps21, vps9/vps9, vps3/vps3, vac1/vac1, and pep12/pep12 mutant strains were incubated in liquid Lee’s glucose medium (pH 6.8) for 24 hours at 25 °C. Total RNA was extracted for quantitative RT-PCR assays. The expression level of ACT1 was used for normalisation. The average value of the WT control strain for each gene was set as “1”. “*” indicates significant difference between the WT control strain and mutant strain (p < 0.05, two-tailed Student’s t-test). Strains used: WT (FDZF171); vps21/vps21 (FDZF266); vps9/vps9 (FDZF212); vps3/vps3 (FDZF250); vac1/vac1 (FDZF533); and pep12/pep12 (FDZF534). The mating type of all strains used was MTLΔ/α.

Figure 4. Relative transcriptional expression of MFA1 and STE2 in the absence and presence of α-pheromone in the WT control and mutant strains of the Vps21 signalling pathway. Opaque cells of the WT control, vps21/vps21, vps9/vps9, vps3/vps3, vac1/vac1, and pep12/pep12 mutant strains were incubated in liquid Lee’s glucose medium (pH 6.8) and treated with or without synthetic α-pheromone (50 μg/mL) for 12 hours at 25 °C. Total RNA was extracted for quantitative RT-PCR assays. The expression level of ACT1 was used for normalisation. The average value of the WT control strain for each gene without pheromone treatment was set as “1”. “*” indicates significant difference between the WT control strain and mutant strain (p < 0.05, two-tailed Student’s t-test). Strains used: WT (FDZF208); vps21/vps21 (FDZF263); vps9/vps9 (FDZF213); vps3/vps3 (FDZF259); vac1/vac1 (FDZF531); and pep12/pep12 (FDZF546). The mating type of all strains used was MTLa/Δ.