NRG1, a repressor of filamentous growth in C.albicans, is down-regulated during filament induction

Abstract

In response to a variety of external signals, the fungal pathogen Candida albicans undergoes a transition between ellipsoidal single cells (blastospores) and filaments composed of elongated cells attached end-to-end. Here we identify a DNA-binding protein, Nrg1, that represses filamentous growth in Candida probably by acting through the co-repressor Tup1. nrg1 mutant cells are predominantly filamentous under non-filament-inducing conditions and their colony morphology resembles that of tup1 mutants. We also identify two filament-specific genes, ECE1 and HWP1, whose transcription is repressed by Nrg1 under non-inducing conditions. These genes constitute a subset of those under Tup1 control, providing further evidence that Nrg1 acts by recruiting Tup1 to target genes. We show that growth in serum at 37 degrees C, a potent inducer of filamentous growth, causes a reduction of NRG1 mRNA, suggesting that filamentous growth is induced by the down-regulation of NRG1. Consistent with this idea, expression of NRG1 from a non-regulated promoter partially blocks the induction of filamentous growth.

Fig. 1. Alignment of C.albicans Nrg1 (nrgc) with S.cerevisiae Nrg1 (nrg1s) and Nrg2 (nrg2s) (Park et al., 1999). Regions of identity are boxed. The zinc finger DNA-binding motif is shown in brackets and extends from amino acids 225 to 284 (of C.albicans Nrg1). Alignment was performed with Pileup in GCG 10 and prepared for display with SeqVu (Garvan Institute).

Fig. 2. Cell and colony morphology of nrg1, rfg1 and tup1 C.albicans mutants. Strains deleted for both copies of the indicated genes were streaked on YPD and SD plates, incubated for 4 days (SD) or 3 days (YPD) at 30°C and the resulting colonies were photographed at approximately ×3 magnification (left panels). Cells corresponding to these colonies were then visualized by Nomarski optics and photographed under ×100 magnification (right panels).

Fig. 3. Comparison of filament-specific gene expression in nrg1- and tup1-deleted cells. RNA was prepared from a wild-type strain and strains bearing homozygous deletions of tup1, nrg1 and tup1 nrg1 grown under the indicated conditions for 2 h (serum corresponds to 10% serum in YPD). Northern blots containing 5 µg of each RNA sample were then probed for expression of the indicated hyphal transcripts. An ACT1 control as well as an ethidium-stained gel showing the large and small rRNA subunits of each sample are shown to confirm equal loading of RNA in all lanes.

Fig. 4. Regulation of NRG1 mRNA abundance. Wild-type or homozygous mutant cells (as marked) were grown in the media indicated and probed for NRG1 mRNA via northern blot. Cells were grown in liquid for 1 h for YPD and YPD plus serum, and for 2 h for Spider. The only significant variation in mRNA abundance was seen after growth in 10% serum in YPD at 37°C. A time course investigating this decrease in expression is shown in the lower panel, for which cells were transferred from YPD at 30°C to YPD plus 10% FCS at 37°C and incubated with vigorous shaking for the time indicated. An ACT1 control as well as an ethidium-stained gel showing the large and small rRNA subunits of each sample are shown to confirm equal loading of RNA in all lanes. Numbers in the lower panel indicate the ratio of NRG1 to ACT1 mRNA.

Fig. 5. Unregulated expression of Nrg1 in wild-type and Tup1 deletion strains. A construct expressing Nrg1 constitutively from a C.albicans ACT1 promoter was integrated at the ACT1 locus in wild-type and Δtup1/Δtup1 strains. Strains bearing the vector only (ACT1) or the Nrg1 expression construct (ACT1::NRG1) were streaked on YPD plates and grown at 30°C for 3 days or on YPD + 10% FCS (serum) plates and grown at 37°C for 3 days (colony photographs at approximately ×3 magnification, left panel). Cells from these colonies were visualized at ×100 magnification by Nomarski optics (right panel).

Fig. 6. A model for induction of hyphal-specific genes by Nrg1 in the presence of serum at 37°C. In the absence of serum and at 37°C, Nrg1 is expressed and binds to the promoters of a subset of hyphal-specific genes. Nrg1 functions as a major transcriptional repressor of these genes via recruitment of the Tup1 co-repressor complex. In the presence of serum and at a temperature of 37°C, the levels of Nrg1 transcript are reduced (by a mechanism that is not yet determined). As a consequence, Nrg1 protein levels fall and hyphal transcripts are derepressed. This model does not exclude the possibility that other serum- and temperature-dependent transcriptional regulators may regulate identical or overlapping sets of hyphal-specific target genes.