Blastomyces dermatitidis septins CDC3, CDC10, and CDC12 impact the morphology of yeast and hyphae, but are not required for the phase transition

Abstract

Blastomyces dermatitidis, the etiologic agent of blastomycosis, belongs to a group of thermally dimorphic fungi that change between mold (22°C) and yeast (37°C) in response to temperature. The contribution of structural proteins such as septins to this phase transition in these fungi remains poorly understood. Septins are GTPases that serve as a scaffold for proteins involved with cytokinesis, cell polarity, and cell morphology. In this study, we use a GFP sentinel RNA interference system to investigate the impact of CDC3, CDC10, CDC12, and ASPE on the morphology and phase transition of B. dermatitidis. Targeting CDC3, CDC10, and CDC12 by RNA interference resulted in yeast with aberrant morphology at 37°C with defects in cytokinesis. Downshifting the temperature to 22°C promoted the conversion to the mold phase, but did not abrogate the morphologic defects. CDC3, CDC10, and CDC12 knockdown strains grew as mold with curved, thickened hyphae. Knocking down ASPE transcript did not alter morphology of yeast at 37°C or mold at 22°C. Following an increase in temperature from 22°C to 37°C, all septin knockdown strains were able to revert to yeast. In conclusion, CDC3, CDC10, and CDC12 septin- encoding genes are required for proper morphology of yeast and hyphae, but are dispensable for the phase transition.

Fig. 1

Septin regions targeted for RNA interference. Black bars below CDC3, CDC10, CDC12, and ASPE correspond to the regions targeted for RNA interference. In addition, conserved motifs (G1, G3, G4, S1-S4), GTPase domain, polybasic region, septin unique element, and coiled-coil domains are shown for all septins. The polybasic region is shaded gray. Diagonal lines represent the GTPase domain. The septin unique element is black. Horizontal lines indicate the coiled-coil domain.

Fig. 2

Phenotype of CDC3, CDC10, CDC12, & ASPE knockdown strains at 37°C and 22°C. (A) T53-19 GFP (reporter) and non-RNAi vector (NRV) control strains grew as broad-based budding yeast with intense green fluorescence. GFP RNAi only (GFPi) control displayed normal yeast morphology, but reduced GFP intensity. CDC3, CDC10, and CDC12 knockdown strains grew as large, misshapen yeast with reduced GFP intensity. ASPE-GFP knockdown strains had reduced GPF intensity and similar morphology to the control strains. Scale bar is 10 μm. Cells were grown on 3M agar at 37°C. (B) Following a downshift in temperature from 37°C to 22°C, control strains grew as thin, smooth, branched hyphae. In contrast, CDC3, CDC10 and CDC12 knockdown strains grew as curved mold with a knobby appearance. ASPE RNAi strains have a similar morphology as the control strains. Corresponding fluorescent images are not shown for hyphal cells because GFP, which functions as a reporter, is under control of the BAD-1 promoter and is not expressed at 22°C. The H2AB promoter, which drives transcription of the hairpin responsible for RNA interference is active at both 37°C and 22°C. Scale bar is 10 μm. Cells were grown on 3M agar at 22°C.

Fig. 3

Quantification of abnormal yeast morphology in septin knockdown strains. The majority of yeast cells for B. dermatitidis strains knocked down for CDC3, CDC10, and CDC12 had abnormal morphology (55–85%) when compared to control strains (<15%). This difference was statistically significant (adjusted P value <0.05; represented by an asterisk). ASPE knockdown strains grew as normal shaped yeast similar to controls. At least 400 cells were analyzed per strain. Numbers below the histogram bars represent the different transformants that were analyzed. 5′ and 3′ refer to the gene region targeted for RNA interference. GFP is the GFP reporter strain (T53-19 GFP). GFPi refers to the control strain knocked down for GFP only and NRV refers to the non-RNAi vector control.

Fig. 4

Quantitative real-time PCR analysis. qRT-PCR demonstrated a reduction in transcript abundance for CDC3, CDC10, CDC12, and ASPE for their respective knockdown strains when compared to GFP reporter and non-RNAi vector (NRV) control strains. NRV control had similar transcript levels to the reporter strain. Quantitative RT-PCR data was analyzed using the 2 ^−ΔΔCt method and values are expressed as fold change in gene transcript relative to the GFP reporter strain. Ct values were normalized to α-tubulin with ΔCt = (Ct of gene of interest) − (Ct of α-tubulin). ΔΔCt = (ΔCt of NRV or knockdown strain) − (ΔCt of GFP reporter). ANOVA was used to analyze transcript abundance between septin knockdown strains and controls. Transcript reduction in knockdown strains was not statistically significant when compared to controls; however, the sharp differences in morphology between knockdown and controls indicated that septin transcript in knockdown strains fell below a threshold required for normal cellular function.

Fig. 5

Growth curve for septin knockdown strains. At 37°C, yeast strains knocked down for CDC3, CDC10, and CDC12 grew substantially slower than T53-19 GFP reporter and non-RNAi vector control strains. ASPE knockdown yeast grew faster than other septin RNAi strains, but slower than control strains. By day 2 in liquid culture, all septin knockdown strains demonstrated a statistically significant reduction in growth when compared to the reporter and non-RNAi controls (adjusted P value < 0.05). Yeast cultures were grown in liquid HMM medium over a period of 6 days and growth was measured daily by absorbance at 600 nm. Numbers next to CDC3, CDC10, CDC12, and ASPE represent the strains analyzed. A representative experiment is shown.