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UME6 is a crucial downstream target of other transcriptional regulators of true hyphal development in Candida albicans

Ute Zeidler, Thomas Lettner, Caroline Lassnig, Mathias Müller, Robert Lajko, Helmut Hintner, Michael Breitenbach, Arnold Bito
DOI: http://dx.doi.org/10.1111/j.1567-1364.2008.00459.x 126-142 First published online: 1 February 2009

Abstract

True hyphal growth of Candida albicans can be induced by several environmental conditions and contributes significantly to the high virulence of this pathogenic fungus. The transcriptional network that governs hyphal morphogenesis is complex, depends on several regulators and is not completely understood. Recently, CaUME6, a homolog of the Saccharomyces cerevisiae UME6 gene, has been shown to be required for hyphal elongation. In the present study, the C. albicans ume6Δ strain showed a complete defect in hyphae formation under all the growth conditions tested. UME6 was repressed by the Nrg1-Tup1 repressor in yeast-form cells but NRG1 was not repressed by Ume6p under hyphal growth conditions. Wild-type UME6 expression depended on each hyphal regulator tested, and ectopic UME6 expression in efg1Δ, cph1Δ and ras1Δ cells rescued the hyphal defects of these mutants under some hyphal growth conditions. Thus, UME6 is a common downstream target of regulators promoting hyphal development. Ectopic UME6 expression promoted both germ tube formation and hyphal elongation. The expression of all hyphae-specific genes investigated depended on UME6 expression. A model for transcriptional regulation of hyphal development and the role of Ume6p is proposed.

Keywords
  • UME6
  • hyphal development
  • Candida albicans
  • germ tube
  • virulence
  • NRG1

Introduction

Candida albicans is a polymorphic fungus that, although present as a harmless commensal in most healthy people colonizing several mucosal tissues of the human host, may become a pathogen under distinct host conditions (Kullberg & Filler, 2002; Ruhnke, 2002). Depending on different environmental growth conditions but also influenced by reversible epigenetic switching, C. albicans is able to grow in distinct yeast-like cell forms (Soll, 2003), pseudohyphal and true hyphal filaments (Sudbery, 2004). Several studies have shown that each of these forms contributes to the virulence of C. albicans in the mammalian host (reviewed by Kumamoto & Vinces, 2005). Most seriously, it can cause systemic infections in patients with a compromised immune system caused either by some disease (e.g. AIDS) or by immune suppressive treatments. Thus, hyphal development of C. albicans has been studied extensively and a number of components involved in the regulation of true hyphal development have been identified (reviewed recently by Biswas, 2007; Whiteway & Bachewich, 2007). Because several different environmental conditions, for example growth at 37 °C, presence of serum, neutral to alkaline pH, nutrient starvation and N-acetylglucosamine, are able to induce hyphal development and because some regulators act specifically under only one particular condition, the overall model that emerged (Biswas, 2007) proposes that entry into hyphal development is controlled by a regulatory network comprising parallel pathways that cross talk with each other.

Hyphae formation is the result of polarized growth that depends on the polarisome and the Spitzenkörper, a hyphae-specific organelle (Crampin, 2005). The essential role of members of the Ras G protein superfamily, Cdc42 and its GDP–GTP exchange factor Cdc24 and Rac1, in hyphal development has been identified (Ushinsky, 2002; Bassilana, 2005; Bassilana & Arkowitz, 2006). Among a number of other factors, several cyclins play a prominent role in hyphal development (Loeb, 1999; Bachewich & Whiteway, 2005; Bensen, 2005; Chapa y Lazo, 2005). Most importantly, Hgc1p, which is exclusively expressed during hyphal development and present only in the apical cell of the hyphal filament (Zheng, 2004; Wang, 2007), is, in complex with the cyclin-dependent kinase Cdc28p, required for polarized hyphal growth (Sinha, 2007; Zheng, 2007).

However, a transcriptional regulator that is required for hyphal development under each of the alternative hyphae-inducing environmental conditions and that may be the common downstream target of all regulatory pathways has not been identified. Another unanswered question is whether germ tube formation and subsequent formation of true hyphae (hyphal elongation) are two different steps of hyphal development that are controlled by distinct regulatory proteins and mechanisms.

In a systematic phenotypic study of C. albicans homologs of several yeast meiotic genes, we have found that the null mutant strain of CaUME6 is unable to form hyphal filaments. We further showed that UME6 functions in promoting both germ tube formation and hyphal elongation and it is expressed only in hyphal cells in the wild-type strain. Using strains allowing strictly controlled ectopic UME6 expression in strains mutant for several other hyphal regulators, and by UME6 expression analysis, we were able to identify UME6 as a downstream target of each regulatory pathway of hyphal development investigated. We also found that the expression of hyphae-specific genes (HSGs) in wild-type cells depends on UME6 expression. Based on these results we propose a model for the transcriptional regulation of hyphal development. Finally, we have shown that UME6 contributes to the virulence of C. albicans in a murine model of systemic infection.

Recently, while performing the final experiments of this study and preparing the manuscript, a study of C. albicans UME6 was published by Banerjee (2008). The results and the conclusions made therein overlap to some extent with the present study. However, using different approaches for investigating the role of UME6 in hyphal development, we obtained additional results that allowed conclusions that differ from that made by Banerjee & colleagues.

Materials and methods

Strains, media and growth conditions

The strains used in this study are listed in Table 1. For growing C. albicans strains in the yeast form, cells were cultured at 30 °C either in YPD (2% yeast extract, 1% Bacto peptone and 2% dextrose) or in SD medium (synthetic dextrose: 0.17% yeast nitrogen base, 0.5% ammonium sulfate and 2% dextrose) supplemented with the appropriate amino acids for auxotrophic strains. For hyphal growth in liquid media, cells were incubated at 37 °C in either of the following media: YPD+10% bovine serum (PAA Laboratories GmbH, Austria) with or without buffering with 20 mM potassium phosphate to pH 7.5, 2.5 mM N-acetylglucosamine in 0.335% yeast nitrogen base with ammonium sulfate, RPMI-1640 (GIBCO-BRL), Spider medium (Liu, 1994), Lee's medium, pH 6.8 (Lee, 1975) and for alkaline growth conditions SD medium buffered with 150 mM HEPES at pH 8.0. Agar plates (2% agar) for hyphal growth were prepared using either Spider medium or Lee's medium. For hyphal growth under embedded conditions at 25 °C YPS agar (2% yeast extract, 1% Bacto peptone, 2% sucrose and 1% agar) was prepared and the cells embedded as described (Brown, 1999). Pseudohyphal growth of C. albicans strains at 30 °C was promoted in liquid synthetic high-phosphate medium (Hornby, 2004) for several hours. Where indicated, doxycycline was added to media to a final concentration of 50 μg mL−1.

View this table:
1

Candida albicans strains used in this study

StrainParentRelevant genotype (otherwise see parent genotype)Source
SC5314Wild-type clinical isolateGillum (1984)
SN87leu2Δ/leu2Δhis1Δ/his1ΔURA3/ura3ΔNoble & Johnson (2005)
IRO1/iro1Δ
UZ24SN87ume6Δ∷CmLEU2/UME6 his1Δ/his1ΔThis study
UZ43UZ24ume6Δ∷CdHIS1/ume6Δ∷CmLEU2This study
UZ73UZ43ume6Δ∷CdHIS1/ume6Δ∷CmLEU2UME6This study
(PACT1-CaSAT1)
UZ121UZ43ADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)This study
ume6Δ∷CdHIS1/ume6Δ∷CmLEU2
UZ149SC5314ADH1/adh1∷ Ptet-UME6 (PACT1-CaSAT1)This study
UME6/UME6
HLC52efg1hisG/efg1hisG-URA3-hisGLo (1997)
JKC19ura3Δ/ura3ΔLiu (1994)
cph1hisG/cph1hisG-URA3-hisG
HLC54ura3Δ/ura3Δcph1hisG/cph1hisGLo (1997)
efg1hisG/efg1hisG-URA3-hisG
Can52ura3Δ/ura3Δras1Δ∷hisG/ras1Δ∷hphFeng (1999)
BCa2-9tup1Δ∷hisG/tup1Δ∷hisG ura3Δ/ura3ΔBraun & Johnson (1997)
BCa5tup1Δ∷hisG/tup1Δ∷hisG
mal3p455- MAL-TUP1-URA3 ura3Δ/ura3ΔBraun & Johnson (1997)
DK129rfg1Δ/rfg1Δ∷URA3 ura3Δ∷λimm434/ura3Δ∷λimm434Kadosh & Johnson (2001)
iro1Δ∷λimm434/iro1Δ∷λimm434
MMC3ura3Δ/ura3Δnrg1hisG-URA3-hisG/nrg1hisGMurad (2001a, b)
AS15ura3/ura3 tec1Δ∷hisG/tec1Δ∷hisG (pVEC)Schweizer (2000)
YJB4899ura3Δ/ura3Δhis1hisG/his1hisGBensen (2002)
arg4hisG/arg4hisG fkh2HIS1/fkh2ARG4
CCF3ura3Δ/ura3Δflo8hisG/flo8hisG-URA3-hisGCao (2006)
HLY1927cph2ARG4/cph2HIS1 ura3Δ/ura3ΔLane (2001b)
his1hisG/his1hisG arg4hisG/arg4hisG
CKY157czf1hisG/czf1hisG ura3Δ/ura3ΔBrown (1999)
UZ207JKC19cph1Δ/cph1ΔADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)This study
UZ200HLC52efg1Δ/efg1ΔADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)This study
UZ209HLC54cph1Δ/cph1Δefg1Δ/efg1ΔADH1/adh1∷Ptet-UME6This study
(PACT1-CaSAT1)
UZ205Can52ras1Δ/ras1ΔADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)This study
UZ203DK129rfg1Δ/rfg1ΔADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)This study
UZ202BCa5tup1Δ∷hisG/tup1Δ∷hisG, mal3p455- MAL-TUP1This study
ADH1/adh1∷Ptet-UME6 (PACT1-CaSAT1)

Escherichia coli strains used for plasmid constructions and isolation were cultured in L broth or on L broth agar (Bertani, 1951) containing ampicillin (100 mg L−1).

UME6 deletion and reintegration

The heterozygous and homozygous ume6 deletion strains, UZ24 and UZ43 (Table 1), were generated as described by Noble & Johnson (2005). Briefly, the regions (around 300 bp) immediately upstream and downstream of the UME6 ORF, and the marker gene cassettes CdHIS1 and CmLEU2 were amplified either from the Candida genome or from the plasmids pSN52 and pSN40 (Noble & Johnson, 2005), respectively. The PCR primers used are listed in Supporting Information, Table S1. The gene deletion cassette was generated by fusion PCR and integrated into the genome of strain SN87.

The reconstituted strain UZ73 was created by integration of plasmid pCaUme6-Sat2 (Table S2), which had been linearized before by EcoRV digestion, at the native UME6 promoter of the ume6Δ strain, thereby reconstituting one wild-type UME6 gene copy. Construction of pCaUme6-Sat2 was carried out as follows. The UME6 gene including 110 bp upstream of the ORF was PCR amplified with oligonucleotides CA0423-1 and CA0423-2. The PCR product was digested with XbaI and SacI and inserted into YCplac33 linearized by the same enzymes. Into the resulting plasmid, pCaUme6-1, a gene cassette coding for nourseothricin resistance and carrying the SAT1 ORF (Reuss, 2004) downstream of the ACT1 promoter was inserted between the NarI and AatII sites resulting in the plasmid pCaUme6-Sat1. To do so, the SAT1 cassette was PCR amplified from plasmid pSDS4 (constructed by us; not published; and carrying modified fragments from pNIM1; Reuss, 2004) using the oligonucleotides CaAct1-Spe-Nar and CaAdh1-Aat and digested with NarI and AatII. Thereafter, pCaUme6-Sat2 was constructed by enlargement of the UME6 promoter region (to 450 bp) in pCaUme6-Sat1 by integration of a PCR product, amplified from genomic DNA using primers CaUme6-14 and CaUme6-d3, between the SacI and EcoRV sites. For both the heterozygous and the homozygous ume6 deletion strains and the reconstituted strain, the expected structural genomic alterations were confirmed by both PCR and Southern blots (Fig. S1).

Ectopic UME6 expression by the Tet promoter

In order to create a gene cassette for ectopic UME6 expression, the following plasmids were constructed. pCaTet-MCS2 was constructed by insertion of a double-stranded oligonucleotide comprising a multiple cloning site into the BglII site of plasmid pNIM1. The latter plasmid carries the SAT1 marker gene and a recombinant Tet promoter system, which allows tightly controlled expression of the gene of interest upon adding doxycycline to the growth medium (Park & Morschhäuser, 2005). Thereafter, the UME6 ORF was PCR amplified from genomic C. albicans DNA using oligonucleotide primers CaUme6-11 and CaUme6-12, and inserted into the BamHI site of pCaTet-MCS2. The resulting plasmid pCaUme6-3 (Table S2) was digested with ApaI and PmlI and inserted at the ADH1 locus of several different strains, namely SC5314, UZ43, JKC19, HLC52, HLC54, Can52, DK129 and BCa5. Correct genomic integrations of the gene cassette were confirmed by both PCR and Southern blots. Ectopic UME6 expression was induced by growing the strains in medium containing 50 μg doxycycline mL−1.

RNA isolation and Northern analysis

Gene expression was studied by Northern blot analysis. Total RNA was isolated from cells using the RNeasy Mini and RNeasy Midi kits from Qiagen (Düsseldorf, Germany). After electrophoretic separation of 2.5 μg RNA on 1.2% denaturing agarose gels, the RNA was transferred onto Amersham Hybond-N+nylon membranes (purchased from GE Healthcare, Munich, Germany) via semi-dry electroblotting using the PerfectBlue ‘Semi-Dry’ Elektroblotter from Peqlab (Erlangen, Germany). Hybridization with [α-32P]-labeled probes and blot washing was performed as described (Sambrook, 1989).

For the synthesis of hybridization probes, PCR products were amplified using gene-specific oligonucleotide primers (Table S1). After verification of the PCR products by gene-specific restriction analysis, the antisense strand was labeled by incorporation of [α-32P]-dATP (Hanse Analytik GmbH, Bremen, Germany) in a single-stranded PCR reaction. After hybridization, transcripts were made visible by phosphoimaging (Fujifilm BAS-1800 II phosphoimager). As a control for RNA quantity and transfer efficiency, each blot was rehybridized with an ACT1 gene-specific probe.

Virulence study

For challenging mice, yeast cells of different C. albicans strains were grown with continuous agitation for 4 h at 30 °C in fresh YPD medium. Harvested cells were washed once with phosphate-buffered saline (PBS, pH 7.2), counted with a hemacytometer and diluted in PBS to a final concentration of 2 × 107 or 6 × 107 yeast cells mL−1. Groups of 8–10-week-old BALB/c mice (n=10) were infected intraperitoneally with 0.5 mL C. albicans suspensions or with PBS for control. The mice were monitored at least twice daily and sacrificed when moribund (defined as hunched posture, minimal motor activity and weight loss of >15% of starting body weight). All procedures involving mice were approved by the Institutional Animal Care and Use Committee, in accordance with the National Institutes of Health guidelines for housing and care.

Results

Candida albicans UME6 is required for hyphal development under each hyphae-promoting condition tested

The ORF of CaUME6 codes for an 843-amino acid-long protein. Compared with its yeast homolog the protein sequence of CaUme6p has the highest sequence identity (31%) in the predicted DNA-binding domain located at the C-termini of both proteins. In order to study the function of the gene in C. albicans, we made heterozygous and homozygous strains, UZ24 and UZ43 (Table 1), respectively, that lack one or both alleles of UME6. The UME6 ORF was replaced by the marker genes CdHIS1 and CmLEU2 (see Materials and methods). In addition, as a control for phenotypic analyses, a reconstituted strain, UZ73, was created. In this strain, one copy of the wild-type UME6 gene had been reintegrated into the ume6Δ strain at the native genomic locus.

The phenotype of ume6Δ, the heterozygous and the reintegrated strains and the wild-type strain SC5314 grown under conditions that promote either the yeast form or the formation of true hyphae (Ernst, 2000), respectively, was investigated. This was done both in liquid and on agar media. The strains were also tested for filamentation after embedding cells in YPS agar and growth at 25 °C. Yeast-form ume6Δ cells grew as fast as wild-type cells and without any obvious difference in size and shape (not shown). In contrast to the wild-type, heterozygous and reconstituted strains, the ume6 null mutant strain did not form long hyphal filaments under any of these liquid and agar conditions or when the cells were embedded in agar (Fig. 1a, b and d). Although rough colony morphology of the ume6Δ strain grown on Lee's agar (pH 6.8) indicated the formation of true hyphae, microscopic inspection of resuspended colonies showed that they are composed of yeast cells and pseudohyphae and we did not find any true hyphae. In all liquid media tested, ume6Δ cells were able to form germ tubes after 1 h. However, subsequent growth resulted in the formation of only short filaments. The filaments grown in different media (Fig. 1d, 4 h) showed distinct appearance, either more true hyphae (YPD+serum, GlcNAc) or pseudohyphae (Spider, RPMI). During prolonged growth (8 and 16 h; not shown), the amount of short pseudohyphal elements and single elongated cells increased, and germ tubes and hyphae-like forms disappeared completely. Calcofluor staining of cells grown in YPD+serum for 8 h showed (Fig. 1c) a mixture of short filaments (with a maximum of four cells), composed of hyphal cells without constrictions at the septae, or of cells of more pseudohyphal shape with constrictions and cells with elongated daughter cells. Sometimes a filament consisted of the blastospore, followed by a hyphal cell, a pseudohyphal cell and at the end a yeast cell that is just budding off the pseudohyphal cell. Because all these different cell and short filament forms were not easy to distinguish from each other, it was not possible to determine the corresponding fractions reliably. Overall, this indicated that these chains of cells did not represent dead-end forms, but were able to form yeast-like cells by a budding mechanism. In conclusion, under hyphal conditions, ume6Δ cells are unable to continue hyphal growth after germ tube formation but switch to pseudohyphal or yeast growth.

1

Growth of ume6Δ cells under several distinct conditions promoting either hyphal or pseudohyphal growth. The wild-type, the ume6Δ and the reconstituted strains were grown on/in several distinct media promoting hyphal development: (a) on agar, (b) embedded in YPS agar or (c, d) in liquid. Colonies were photographed or inspected under the microscope. Cells grown in liquid were observed under the microscope. Because the colony morphology of the ume6Δ strain indicated hyphal growth on Lee's agar (a), the colony edges were inspected under the microscope. In addition, colonies were resuspended and examined under the microscope. Whereas true hyphal filaments were found in wild-type and reconstituted strain colonies, only yeast cells and pseudohyphae were found in colonies of the mutant strain. When embedded in YPS agar (b), the ume6Δ strain did not show formation of hyphal filaments when grown up to 10 days but a few sections of some colonies showed strong pseudohyphal growth. Grown in liquid media (d), the ume6Δ strain formed germ tubes after 1 h and short filaments after 4 h. Staining of ume6Δ cells grown in YPD+serum at 37°C for 8 h with Calcofluor (c) showed a mixture of short cell chains (with a maximum of four cells), composed of hyphal cells without constrictions at the septae or of cells of more pseudohyphal shape with constrictions and cell pairs with an elongated daughter cell.

Because pseudohyphae formation was observed under conditions promoting true hyphal growth and was not affected in the mutant upon growth at 30 °C in synthetic high-phosphate medium (not shown), a condition known to promote pseudohyphae formation of C. albicans (Hornby, 2004), UME6 appears to have no role in this mode of filamentous growth.

UME6 is not expressed in C. albicans yeast cells but is induced upon hyphal induction

By semi-quantitative reverse-transcription PCR (RT-PCR) we could detect UME6 transcripts in cells induced for hyphal development but not at all in cells grown in the early logarithmic phase (not shown). For further confirmation, UME6 mRNA expression under several distinct growth conditions and at different time points was determined by Northern blot analysis. The growth media and conditions used were as follows: (1) YPD at either 30 or 37 °C (early exponential to stationary phase); (2) YPD, shifting the culture that had been pregrown at 30 °C up to an OD600 nm of 0.2 to 37 °C and continued growth for 6 h; and (3) hyphal growth in YPD+serum at 37 °C. The results confirmed the RT-PCR result and showed that UME6 transcripts were not detectable in C. albicans cells grown in the yeast form but were present in cells harvested from hyphal cultures (Fig. 2a). UME6 expression was highest early after hyphal induction and constantly decreased in the course of subsequent hyphal growth. After 6 h, only barely detectable amounts of UME6 transcripts were seen. We have also detected UME6 transcripts in wild-type cells induced for hyphal growth in liquid Spider medium, although to a lesser extent compared with induction by serum-containing medium at 37 °C (not shown).

2

(a) Wild-type and (b) ectopic UME6 expression under conditions promoting yeast or hyphal growth. (a) For growth in the yeast form, YPD medium was inoculated with wild-type cells (UME6/UME6) and cells were grown at either 30°C (two cultures) or 37°C (one culture) for 12 h. At this time, the cultures had reached an OD600 nm of 0.2, which represents the start point of the experiment. Growth of each culture was continued at 30 or 37°C, and one culture was shifted from 30 to 37°C. Cells were harvested either after the cultures had reached a certain OD (to investigate the dependence of UME6 expression from the growth phase of the yeast-cell culture) or at certain time points. The OD600 nm of stationary phase cultures (stat) was 25. After isolation of total RNA, UME6 expression was determined by Northern analysis. Only stationary phase cells show very low expression with sometimes strong degradation of UME6 transcripts. Hyphal growth was started by inoculating prewarmed YPD+10% serum with a certain fraction of a separate stationary phase culture to give an OD600 nm of 0.2. Cells were harvested up to 6 h of hyphal growth. UME6 expression was highest after 30 min (first hyphal sample taken) and decreased constantly and was barely detectable after 6 h. An extensive smear indicates a high degradation rate of the transcript. As expected, there are no UME6 transcripts detectable in ume6Δ cells. The UME6 transcripts marked by arrowheads have a calculated length of 8–10 kb (dominating upper band) and 3 kb (one lower band; that has to be expected from the UME6 ORF size). (b) Strain UZ121 (ume6Δ/ume6ΔADH1/adh1∷Ptet-UME6) was grown under similar conditions as in (a). There is no (yeast cells) or barely detectable (hyphal conditions) UME6 expression under the noninducing growth condition (medium without doxycycline). In the presence of DOX (50 μg mL−1), UME6 is weakly expressed at 30°C compared with growth at 37°C. In contrast to UME6 expression from the native promoter in the wild type (a), expression from the recombinant Tet promoter remains at a high level up to 6 h. Note that strain UZ121 is only able to express a UME6 transcript from the Tet promoter that has a size of about 3 kb. For comparison of the UME6 expression patterns of UZ121 and the wild-type strain, see Fig. 5.

There were two characteristics in all blots of RNA isolated from wild-type cells and hybridized with UME6 probes during this study: (1) there was >1 transcript detectable, with the largest one (calculated size 8–10 kb) being quantitatively predominant, and (2) a smear below these bands was always visible, which indicates degradation of the transcripts. RNA isolation from strains with distinct genotype (but UME6 wild-type) with different isolation protocols and hybridization with different UME6-specific probes always resulted in similar hybridization patterns. Moreover, the same RNA preparations were used for Northern analysis of other genes as well, and for nearly all of them one single transcript (or one strongly dominating) was detected that did not show significant degradation. Thus, it is very unlikely that the multiple transcripts and the degradation products detected by the UME6 probes were the result of technical artifacts. Most likely, the UME6 transcript(s) have an intrinsically high degradation rate in C. albicans cells. Each of the multiple transcripts resembles the sense strand, because for probe synthesis, only the antisense strand of the UME6 ORF was labeled by single-stranded PCR. It is not clear as to which of the detected transcripts is translated into the functional Ume6 protein. These transcripts were found in all wild-type samples investigated and a nearly constant quantitative ratio among the transcripts was observed (Fig. 5).

5

UME6 expression in deletion mutants of other regulators of hyphal development. RNA for Northern analysis of UME6 expression was isolated from cells of wild-type and mutant strains that were grown in parallel in YPD at 30°C up to an OD600 nm of 0.2 (yeast conditions) and in YPD+serum at 37°C (hyphae-inducing conditions). The following strains were used: HLC52 (efg1Δ), JKC19 (cph1Δ), HLC54 (efg1Δcph1Δ), Can52 (ras1Δ), BCa2-9 (tup1Δ), DK129 (rfg1Δ), MMC3 (nrg1Δ), AS15 (tec1Δ), YJB4899 (fkh2Δ), CCF3 (flo8Δ), HLY1927 (cph2Δ) and CKY157 (czf1Δ). UZ43 (ume6Δ) and UZ121 (doxycycline-dependent UME6 expression) were used as controls. Under yeast growth conditions (upper panel), UME6 is derepressed in nrg1Δ and tup1Δ cells, but also to a lesser extent and accompanied with abundant degradation products in the efg1Δcph1Δ double mutant. The amount of UME6 transcript(s) varies among the other mutant strains when grown under hyphal conditions (lower panel) and does not reach the wild-type level in these strains. As control, hybridization of an ACT1 probe on the same blots is shown (lower part of each panel). The predominant UME6 transcript (calculated size 8–10 kb) of strains with the UME6 wild-type background is marked by a black arrowhead; the transcript expressed from the Tet promoter is marked by a white arrowhead (size around 3 kb).

Ectopic UME6 expression promotes hyphal elongation and germ tube formation

The results described above indicate that the Ume6 protein is a general regulator of hyphal elongation that is essential under each condition promoting hyphal development. Therefore, we were interested in investigating whether ectopic expression under growth conditions not promoting formation of true hyphae could force C. albicans cells into hyphal development. A gene cassette was constructed, which carries the UME6 ORF cloned behind the recombinant Tet promoter (Park & Morschhäuser, 2005) and integrated into one ADH1 allele of both the wild-type and the ume6Δ strains (see Materials and methods). As expected, the strain UZ121, which lacks both wild-type UME6 alleles but carries the Ptet-UME6 cassette, expressed UME6 only in the presence of doxycycline, under both yeast- and hyphae-promoting conditions (Fig. 2b). The expression was much higher in yeast cells grown at 37 °C than at 30 °C and remained at a high level for hours. Remarkably, whereas we detected maximal expression at 37 °C 2 h after adding doxycycline, in the presence of serum the expression was at its highest level after 30 min as found for UME6 expression in wild-type cells. This indicates that the transcript is stabilized under this strongly hyphae-inducing condition.

Phenotypic analysis showed that in the absence of doxycycline, strain UZ121 behaved like the ume6Δ strain under any condition tested and was not capable of hyphal development. Hyphal growth could be restored by adding doxycycline to the media (Fig. 3a). Compared with the wild-type strain, ectopic UME6 expression led to the formation of somewhat longer and thinner filaments in liquid YPD+serum and more extensive filamentation when hyphal growth was induced on agar plates or under embedded conditions.

3

Phenotype of ectopic UME6 expression in an otherwise wild-type background. The strain UZ121 (ume6Δ/ume6ΔADH1/adh1∷Ptet-UME6) was grown under conditions (as indicated) either promoting (a) or not promoting (b) hyphal development. Colonies were photographed or inspected under the microscope. Cells grown in liquid were observed under the microscope. Ectopic UME6 expression induced by the addition of doxycycline (50 μg mL−1) to the medium resulted in the formation of germ tubes under conditions not promoting germ tube formation in the wild type (b) and strong filamentation under promoting conditions (a). Germ tubes formed at 24 and 30°C have constrictions at the neck (black arrowheads).

As expected, ectopic UME6 expression improved the efficiency of filament elongation at 37 °C (without serum). However, and unexpectedly, ectopic UME6 expression forced the cells that were grown under conditions that do not promote germ tube formation of the wild-type strain, such as YPD without serum at 30 °C but even at 24 °C, into germ tube formation (Fig. 3b). Most germ tubes showed constrictions at the neck like most germ tubes formed at 37 °C that give rise to pseudohyphae (Sudbery, 2001). However, subsequent hyphal elongation did not occur under these growth conditions. Alternatively, these structures may represent hyperpolarized buds as described previously (Bachewich, 2003; Whiteway & Bachewich, 2007). However, in comparison with the structures shown there, the ‘germ tubes’ of ume6Δ cells were much thinner and thus, probably, represent true germ tubes. These observations were made when UME6 was ectopically expressed in the strain with both wild-type alleles (not shown) and in the strain lacking both wild-type alleles. However, in three independent experiments, the fraction of cells forming germ tubes at 30 °C was only around 5% (19/392, 22/431 and 23/481), possibly representing the fraction of cells that are in a certain cell cycle stage. Under the same growth conditions, the wild-type strain SC5314 did not form germ tubes either in the presence or in the absence of doxycycline.

In conclusion, Ume6p promotes hyphal elongation and germ tube formation.

Ectopic UME6 expression restores the hyphal growth defects of strains lacking EFG1, CPH1 or RAS1 but not that of hyphal repressors RFG1 and TUP1

The results shown above indicate that C. albicans UME6 might be a central transcriptional regulator of hyphal development. Moreover, it could be either the single common target gene of transcriptional regulators acting upstream of UME6 or at least one of a set of a few common target genes whose products concertedly govern hyphal development under any hyphae-promoting environmental condition. Therefore, we asked whether ectopic UME6 expression might be able to override the hyphal defects of mutant strains that lack genes that are required for hyphal development under several environmental conditions. Genes that are either known to promote hyphae formation, namely the transcriptional regulators EFG1 (Stoldt, 1997; Doedt, 2004) and CPH1 (Liu, 1994; Lo, 1997), and RAS1 (Feng, 1999), or involved in the repression of filamentation, TUP1 (Braun & Johnson, 1997, 2000) and RFG1 (Kadosh & Johnson, 2001; Khalaf & Zitomer, 2001), were chosen. Each mutant strain had either a complete or a partial defect in formation of true hyphae under several hyphae-promoting conditions, and was unable to form true hyphae in rich medium containing serum at 37 °C or had a low efficiency in doing so. After integration of the gene cassette allowing doxycycline-dependent UME6 expression into these strains, we investigated hyphal growth in YPD+10% bovine serum at 37 °C. The results are shown in Fig. 4a.

4

Phenotype of ectopic UME6 expression in mutant strains defective for hyphal growth. (a) The wild-type (WT) strain SC5314 and the mutant strains HLC52 (efg1Δ), JKC19 (cph1Δ), HLC54 (efg1Δcph1Δ), Can52 (ras1Δ) and the corresponding strains UZ200, UZ207, UZ209 and UZ205 with the inserted gene cassette carrying the Tet-promoter-UME6 construct were grown in YPD+10% bovine serum at 37°C for 8 h. The cells were inspected under the microscope for hyphae formation. Ectopic UME6 expression in the medium supplemented with doxycycline (50 μg mL−1) allowed the mutant cells to form true hyphae. Note that the ras1Δ strain forms short pseudohyphal structures when UME6 is not expressed from the Tet promoter. (b) Calcofluor staining of hyphal filaments. Hyphal development of strains UZ200, UZ207 and UZ205 was induced by growth in YPD+serum at 37°C for 8 h in the presence of doxycycline for ectopic UME6 expression. Hyphal septae are marked by white arrowheads.

The recombinant strains derived from the efg1Δ, cph1Δ, efg1Δcph1Δ and ras1Δ strains, respectively, showed the same hyphal growth defects as the corresponding parent strains when grown in the absence of doxycycline (ras1Δ cells formed short pseudohyphae). However, in the presence of doxycycline, the yeast cells of each strain were able to start germ tube formation at distinct time points after hyphal induction (thus confirming the germ-tube-promoting activity of Ume6p) and to develop long filaments after 6 h at the latest. The filaments formed consisted of true hyphal cells that were separated by unconstricted septae, as shown by Calcofluor staining (Fig. 4b). The mutant strains lacking the cassette for ectopic UME6 expression formed neither filaments nor germ tubes regardless of the presence or absence of doxycycline. These results strongly indicate that UME6 is a downstream target of these genes and the corresponding regulatory pathways of hyphal development.

Next, we studied the hyphal growth of these strains under three other environmental conditions (Spider, RPMI-1640 and GlcNAc-containing medium) when UME6 was expressed ectopically. The ability to form germ tubes and whether or not filaments (hyphal or pseudohyphal) were formed were different both under different hyphae-inducing conditions and strain backgrounds (Fig. S2). Only in Spider medium did ectopic UME6 expression restore true hyphal growth of the mutant strains. In the efg1Δcph1Δ background, UME6-dependent hyphae formation also occurred in RPMI medium. Neither strain was able to form true hyphae in GlcNAc medium. The efg1Δ, cph1Δ and ras1Δ strains did not form filaments in RPMI medium, although the latter two were able to form germ tube-like structures in the presence of GlcNAc. For the strain with efg1Δ background (other backgrounds were not investigated) doxycycline-dependent UME6 expression was at nearly the same level under each of these hyphae-promoting conditions (not shown). We conclude that although UME6 expression is required, it is not sufficient for induction and maintenance of the regulatory hyphal growth program under each environmental condition.

On the other hand, doxycycline-dependent ectopic UME6 expression in mutants that lack or do not express repressors of HSGs, namely RFG1 and TUP1, did not restore the defects of these strains in forming true hyphae in YPD+serum (not shown; other conditions were not investigated). Instead, strain UZ202, which expresses TUP1 only in maltose-containing medium, remained locked in its pseudohyphal morphology when doxycycline, but not maltose, was added. Moreover, ectopic UME6 expression did not improve the low efficiency of the rfg1Δ strain to form hyphal filaments. In conclusion, together with the results described above, this indicates that the absence of some repressors of true hyphal development is epistatic over both UME6 expression and the expression of other genes that promote the formation of true hyphae.

UME6 expression depends on several regulators of hyphal development but not vice versa

The results described above for the efg1Δ, cph1Δ, efg1Δcph1Δ, and the ras1Δ strains indicate that the UME6 gene might be expressed in the mutant strains either not at all or below a level required for hyphae formation. This was investigated by Northern analysis of RNA isolated from the corresponding mutant strains grown to early exponential phase in YPD at 30 °C. The mutants of the following genes were investigated: EFG1, CPH1, RAS1, TUP1, RFG1 (references see above), NRG1 (Braun, 2001; Murad, 2001a, b), CZF1 (Brown, 1999), TEC1 (Schweizer, 2000), FLO8 (Cao, 2006), CPH2 (Lane, 2001a,b) and FKH2 (Bensen, 2002). Under these conditions, most strains grew in the yeast form but some formed filaments of pseudohyphal morphology (tup1Δ, nrg1Δ and fkh2Δ). Alternatively, the strains were grown in the presence of serum at 37 °C to induce hyphal development. With the exception of cph2Δ and czf1Δ strains, for each of the mutant strains a complete or at least a partial defect in the development of true hyphae under this condition has been reported. Hyphal growth was monitored for 4 h and samples were taken for RNA isolation 2 and 4 h after hyphal induction. However, the results are only shown for the 2-h time point (Fig. 5, lower part), because the UME6 expression pattern after 4 h was very similar to that seen after 2 h.

The results show that in each mutant the level of UME6 expression was altered compared with the wild type. Most notably, UME6 was highly expressed in the nrg1Δ and tup1Δ cells grown in YPD at 30 °C (Fig. 5, upper part), suggesting that the Nrg1p/Tup1p pair is the responsible component that represses UME6 in yeast cells, whereas Rfg1p, another DNA-binding supposed partner of Tup1p for repressing another set of genes, has no role in UME6 repression. The efg1Δ strain and to a greater extent the efg1Δcph1Δ strain also showed derepression of UME6 in yeast cells. In cultures induced for hyphal growth, each of the mutant strains, with the exceptions of the nrg1Δ and tup1Δ strains, expressed UME6 at a lower level compared with the wild type (Fig. 5, lower part). UME6 expression was barely detectable in the cph1Δ, flo8Δ and tec1Δ strains, and the fkh2Δ strain did not express the gene at all. Interestingly, in efg1Δcph1Δ cells grown under the hyphae-inducing conditions, the largest UME6 transcript was completely absent, presumably as a result of degradation. Because the ACT1 transcript and rRNA (the latter is not shown) did not show a higher level of degradation compared with the RNA isolated from other strains, it is unlikely that the RNA from this strain was of lesser quality. In conclusion, these results and those described above from ectopic expression in several mutants indicate that (1) UME6 is the downstream target of several or even all regulatory pathways of hyphal growth and the regulatory proteins involved and (2) a decreased level or even absence of UME6 expression is the most critical parameter that causes defective hyphal growth of mutants of other regulators, but (3) at least under some growth conditions it is not the single decisive factor.

If it is true that UME6 is the downstream transcriptional target of other hyphal growth regulators, the expression of the corresponding genes should be mostly independent of UME6 expression. Therefore, we investigated the expression of several regulatory genes, namely EFG1, CPH1, CZF1, TEC1, RFG1, NRG1 and TUP1 in ume6Δ cells and cells of the strain allowing doxycycline-dependent ectopic UME6 expression. This was done by Northern analysis of total RNA isolated from cells grown either in YPD at 30 °C or in YPD+serum at 37 °C. The analysis of the expression patterns (Fig. S3) revealed that the expression level of none of the genes studied appears to be markedly influenced by the expression of UME6. For a reliable confirmation of this finding, quantitative RT-PCR should be performed.

Expression of HSGs is under the control of UME6

From its homology to the Saccharomyces cerevisiae Ume6 protein, in particular the very high similarity of the predicted DNA-binding domains, it appears very probable that CaUme6p is a transcriptional regulator. We concentrated on two groups of genes that might be under transcriptional control by Ume6p. One group included HSGs, namely HWP1 (Staab & Sundstrom, 1998; Staab, 1999), ALS3 (Hoyer, 1998), ECE1 (Birse, 1993) and HYR1 (Bailey, 1996). A second group of genes codes for cyclins, in particular HGC1 (Zheng, 2004), CLB2 and CLB4 (Bensen, 2005), CCN1 (Loeb, 1999), CLN3 (Bachewich & Whiteway, 2005) and the orthologs of S. cerevisiae PCL1 and PCL2 (Enjalbert & Whiteway, 2005). For several of the cyclins, a role in the yeast-to-hyphae transition is inferred from the mutant phenotypes (reviewed by Berman, 2006). With the exception of HGC1 (which is also an HSG), these genes are also expressed in yeast-form wild-type cells. Expression of the genes was determined by Northern analysis of total RNA isolated from cells of the wild-type strain, the ume6Δ strain and the strain allowing doxycycline-dependent ectopic UME6 expression. The cells were grown under both yeast- and hyphae-promoting conditions as used in the previous part of the paper.

Among the HSGs, two groups could be clearly distinguished. Under hyphal growth conditions, expression of HWP1, ALS3 and ECE1 partially depended on UME6 expression (Fig. 6b). These genes were expressed weakly in the absence of UME6 expression but were strongly upregulated in the presence of doxycycline. This indicates that apart from Ume6p the wild-type expression level of these HSGs depends on at least one other transcriptional regulator. Remarkably, ectopic UME6 expression resulted in much weaker or even no expression of HSGs under yeast growth conditions compared with hyphal growth conditions (see also Discussion). However, the expression level of HYR1 in cells grown under hyphal growth conditions appeared to depend strictly on UME6 expression (Fig. 6a).

6

Differential expression of HSGs in the presence or absence of UME6 expression. Northern analysis of (a) HGC1 and HYR1 and (b) ALS3, ECE1 and HWP1 expression in cells grown under conditions that result in UME6 expression or do not (compare Fig. 2). The wild-type strain SC5314, strain UZ43 (ume6Δ) and strain UZ121, which lacks both native UME6 alleles but carries the gene cassette for ectopic UME6 expression, were compared. For quantitative controls, hybridization of an ACT1 probe on the same blots (a) or ethidiumbromide-stained rRNA (b) are shown.

One of the cyclin-encoding genes, HGC1, which has been shown to be expressed specifically in the tip cell of a hyphal filament (Wang, 2007), showed a partial dependency on UME6 expression as well (Fig. 6a) and was expressed weakly in the strain allowing ectopic UME6 expression also under yeast growth conditions in the presence of doxycycline. Expression of the other cyclins in yeast and hyphal cells was observed at the same level in wild-type and ume6Δ cells and was not influenced by the presence or absence of UME6 expression (not shown). Thus, these genes are not transcriptionally controlled by the Ume6 protein.

UME6 is required for full virulence of C. albicans

The role of UME6 in the virulence of C. albicans was analyzed using a mouse model for intraperitoneal systemic infection. The results clearly show (Fig. S4) that the ume6Δ strain had a significantly lower virulence, requiring a threefold higher dose to cause a similar mortality rate compared with both the wild-type and the reconstituted strain.

Discussion

Candida albicans has two homologs of the S. cerevisiae UME6 gene, whose product is the most important transcriptional regulator of genes expressed in the early stages of yeast meiosis (reviewed by Kassir, 2003). In C. albicans, meiosis and ascospore development have never been observed, and from analysis of the genome sequence of C. albicans (Arnaud, 2007; van Het Hoog, 2007) it appears unlikely that this organism is able to undergo meiosis, because homologs of many genes that are essential for yeast meiosis are lacking.

In the present study, we show that in C. albicans one ScUME6 homolog, CaUME6, functions as a regulator of hyphal development. The deletion of CaUME7, the second homolog of ScUME6, did not result in any phenotype and the ume7Δ strain appears to be as efficient as the wild-type strain in forming hyphae under any environmental condition tested (not shown).

Very recently, Banerjee (2008) published a study on C. albicans UME6 as well. The experimental approaches used, the results obtained and the conclusions made overlap with that in this study. However, there are some major differences. Banerjee & colleagues performed epistasis analysis in order to study the dependencies and genetic interactions of UME6 with genes encoding repressors of HSGs. We have chosen to construct strains with strictly controllable doxycycline-dependent ectopic UME6 expression in several distinct mutant backgrounds lacking genes that code for either transcriptional activators or repressors of hyphal morphogenesis. Ectopic UME6 expression in a strain lacking both wild-type alleles allowed us to study its contribution to hyphal development under several environmental conditions and also the expression of HSGs and genes involved in the regulation of hyphal morphogenesis. Moreover, we have studied the growth of the ume6Δ strain under more environmental conditions promoting hyphal growth.

Like Banerjee (2008), we found that UME6 contributes significantly to the virulence of C. albicans. This agrees with several studies (reviewed by Kumamoto & Vinces, 2005) showing that C. albicans' ability to form filaments contributes considerably to the virulence and pathogenicity of this fungal organism. In contrast to Banerjee & colleagues, who used the intravenous route of infection, we chose the intraperitoneal route. A comparison of the infection routes revealed that the mutant strain was less virulent in the intraperitoneal model. This result is not unexpected due to the fact that the invading pathogen has to overcome one additional barrier before dissemination into the body. Based on former studies, it is commonly acknowledged that its ability to form hyphal filaments helps C. albicans cells to penetrate the host tissues and to escape from phagocytic cells.

UME6 is a common downstream target of several hyphal regulators

We present proof that UME6 is a common downstream target of several regulatory pathways that act under distinct hyphae-promoting environmental growth conditions. First, the ume6 null mutant strain has a defect in hyphae formation under any environmental condition tested, regardless of whether the cells were grown in liquid, on agar or under embedded conditions. Secondly, the wild-type expression level of UME6 depends on each of the major known hyphal regulators that promote hyphal development. Because mutant cells lacking any of these genes showed a significant reduction in UME6 expression, their inability to form true hyphae may be caused mainly by insufficient or otherwise inappropriate UME6 expression. On the other hand, none of the hyphae-regulatory genes tested in this study appeared to depend on UME6 transcription for its regulation. Thirdly, we showed that artificial ectopic UME6 expression was able to overrule the absence of every regulatory gene tested in the corresponding mutant and thereby enabled mutant cells to enter and maintain hyphal development. However, ectopic UME6 expression did not restore hyphal development of the efg1Δ, cph1Δ, efg1Δcph1Δ and ras1Δ strains under all environmental conditions (Fig. S2). This indicates that Efg1p, Cph1p and Ras1p regulate genes that are required essentially in addition to Ume6p for entry into and maintenance of hyphal development under certain growth conditions (further discussed below).

The genomic region upstream of the UME6 ORF is very large (20.7 kb) compared with other genes of C. albicans and does not contain an ORF that could be translated into a polypeptide of more than 110 aa. For already known, or predicted (from the S. cerevisiae homolog), recognition motif sequences, we found several to many potential binding sites for the transcriptional regulators Nrg1p, Rfg1p, Cph1p, Efg1p, Cph2p and Flo8p. However, it should be noted that due to the small size of the motifs, the chance of random occurrence of one or more copies in such a large DNA region is high. Nevertheless, the potential Nrg1p binding motif (CCCCCT) was found much more frequently than expected (17 copies).

UME6-specific probes detected at least three sense transcripts of UME6 of different lengths, among which the largest one (calculated size 8–10 kb) predominates in quantity. One transcript is similar in size to the UME6 ORF expressed from the Tet promoter (3 kb) and one has a size of 2 kb. Whether these smaller transcripts are products of splicing, or represent intermediate degradation products resulting from endonucleolytic activity, or result from transcription from distinct start sites remains to be determined. We also observed a higher degradation rate of UME6 transcripts compared with other genes whose expression was determined. Both a large promoter region and a high transcript instability are typical of genes encoding crucial regulators of eukaryotic cell differentiation.

The present study revealed that, like many other genes (Murad, 2001a, b; Kadosh & Johnson, 2005), UME6 is repressed in yeast cells by the Nrg1p–Tup1p transcriptional repressor pair, although, in order to confirm direct repression, the predicted binding of Nrg1p to the UME6 promoter has yet to be shown. This was also found by Banerjee (2008), who proposed a model for the transcriptional regulation of hyphae formation. In this model, Nrg1p and Ume6p act in a negative feedback loop to control gene expression during hyphal elongation. Based on the observation of reverse abundance of the NRG1 and UME6 transcripts in the course of hyphal growth from its entry to 5 h later, it was suggested that Ume6p represses NRG1. However, we have shown (Fig. S3) that the NRG1 expression level does not depend on the presence of UME6 at all, either under yeast- or under hyphae-promoting growth conditions, thus contradicting the model. Because the NRG1 transcript level appears to be strongly reduced already before UME6 is expressed upon hyphal induction (Banerjee, 2008; fig. 6 therein), there must be another gene product acting on NRG1 repression or transcript stability. Consistent with this, germ tubes that are formed when UME6 is artificially expressed under yeast-promoting conditions are unable to form true hyphal filaments (Fig. 3b). This may be the consequence of repression of some HSGs whose expression is essential for hyphal development by Nrg1p.

In contrast to Banerjee (2008), who showed weak derepression of UME6 in the rfg1Δ strain under yeast growth conditions, we found that UME6 was not repressed by Rfg1p. This is in agreement with microarray data presented by Kadosh & Johnson (2005). Because we used the same rfg1Δ strain as Banerjee & colleagues, confirmed its rfg1Δ genotype by PCR and found the same growth behavior as formerly described (Kadosh & Johnson, 2001), we do not know the reason for the conflicting results. However, even if Rfg1p might function as a transcriptional repressor of UME6, it seems probable that the gene would stay in the repressed state in the rfg1Δ strain due to the presence of Nrg1p.

The overall picture of transcriptional regulation of UME6 that emerges from our results is shown in Fig. 7a.

7

(a) Dependence of UME6 expression on upstream regulatory proteins and (b) model for transcriptional regulation of HSGs. (a) The model shows the activating and repressing activities of upstream regulatory proteins of hyphal development on UME6 expression as indicated from the expression analysis shown in Fig. 5. In order to depict the interaction of regulatory proteins and the corresponding pathways among each other, we followed the current model of the hyphal regulatory network as summarized by Biswas (2007). (b) The model for transcriptional regulation of HSGs as suggested by the results of this study is described and discussed in the main text.

UME6 functions in both germ tube formation and hyphal elongation

The ability of ume6Δ cells to form germ tubes after hyphal induction but to fail in forming long hyphal filaments indicates that the UME6 product might be a regulator of hyphal elongation only, as has been concluded by Banerjee & colleagues from their study. However, we have shown by ectopic expression that Ume6p promotes germ tube formation as well. This was observed (1) in mutant strains defective in germ tube formation under hyphae-promoting conditions and (2) in the wild-type strain and the strain lacking both native UME6 alleles under growth conditions that do not promote hyphal growth of the wild-type strain. A role of UME6 in germ tube formation in wild-type cells as well is conceivable when looking at its expression and the timescale of morphological changes after hyphal induction. Banerjee (2008) have shown that the UME6 expression level is highest 15 min after hyphal induction (in contrast, we took the first samples 30 min after hyphal induction). At this time, germ tubes are not visible in the wild-type strain but emerge 15–20 min later at the earliest. Moreover, we observed in several experiments that the fraction of ume6Δ cells forming germ tubes was always significantly lower (50–70%) compared with wild-type cells (>90% in YPD+serum).

Target genes and the role of Ume6p in hyphal development

Banerjee (2008) discuss a model of hyphal development, which suggests that hyphae formation is composed of two independent developmental stages: germ tube formation and subsequent hyphal elongation, each requiring a somewhat different gene expression pattern. In this model, the germ tube would be formed independently of UME6 after hyphal induction and UME6 expression would commit the germ tube for hyphal elongation, and if the gene is not expressed or is absent, the germ tube is not able to form an extended true hyphal filament.

In contrast, based on our results we propose that germ tube formation and hyphal elongation, although morphologically distinct steps, are not regulated by distinct sets of regulatory genes. Hyphal development of fungi is an example of eukaryotic cell differentiation. The apical cell of the filament resembles a cell with proliferative potential (comparable to a ‘stem cell’) and undergoes asymmetric cell division. The progeny are (1) a differentiated cell, the subapical, highly vacuolated hyphal cell that remains arrested in the G1 phase of the cell cycle (Hazan, 2002; Barelle, 2003) and (2) the new apical cell, which is able to undergo further cell division cycles. In this view, the yeast cell induced for hyphal development would be the first stem cell that after cell division becomes the first subapical cell which is arrested in G1 (until hyphal branching) as the other subapical cells formed later as well.

It should be emphasized that gene expression in hyphal cultures is a mixture of at least two specific patterns: one in the differentiated hyphal cells and another in hyphal tip cells (e.g. HGC1 is expressed exclusively in the hyphal tip cell). Under continued hyphae-promoting growth conditions, the population of arrested hyphal cells increases. As a consequence, there should be a relative decrease in gene transcripts expressed by hyphal tip cells in the overall culture. This may complicate the detection of genes required for hyphal development and may be one major reason why neither the present study nor the study of Banerjee (2008) clearly identified genes that encode proteins that are essentially required for hyphal morphogenesis and whose expression is strictly UME6 dependent. We identified one gene, HYR1, whose activation strictly depends on Ume6p because we could not detect transcripts in strains that do not express UME6 under hyphae-promoting conditions. This is consistent with the strong HYR1 expression in the nrg1Δ strain (Banerjee, 2008) because UME6 is also derepressed in this strain (Fig. 5 this study and Banerjee, 2008). However, HYR1 is not required for hyphal morphogenesis and its product is a predicted hyphal wall protein of yet unknown function (Bailey, 1996). Several other HSGs (HWP1, ECE1, ALS3) appear to be under the partial control of Ume6p. However, it cannot be excluded that their much higher expression level in the presence of UME6 expression is an indirect consequence and could have more to do with ongoing hyphal development and the sustained activity of its regulatory program that is promoted by Ume6p.

Most interestingly, HGC1 is under partial transcriptional control by the UME6 product under hyphae-promoting conditions and is also weakly expressed upon ectopic UME6 expression under conditions favouring yeast growth. This gene codes for the single hyphae-specific cyclin, is essential for hyphal growth under all conditions (Zheng, 2004) and is expressed only in the apical cell of a hyphal filament (Wang, 2007). Recently its essential roles for phosphorylation of Rga2p, a Cdc42 GAP, and Cdc11 septin have been found (Sinha, 2007; Zheng, 2007). Because the UME6 and the HGC1 transcripts show a very similar and constant decrease of expression in the course of hyphal growth (compare Figs 6a and 2a, wild-type strain), we speculate that UME6 might be expressed exclusively in the hyphal tip cell as well. There it could be responsible for regulating genes that are required for ongoing hyphal development and elongation. If this were true, it could also explain that the expression of many genes, whose products are present in the subapical cells as well (e.g. HWP1), may depend only indirectly on Ume6p. A study aimed to confirm this is under way. Alternatively, as discussed for other HSGs whose expression partially depends on UME6 expression, HGC1 might not be a direct target of Ume6p but its expression may be promoted indirectly by Ume6p via another transcriptional regulator.

Remarkably, ectopic UME6 expression results in much weaker or even no expression of HSGs under yeast growth conditions compared with hyphal growth conditions. As mentioned above, we believe that this is caused by the repression of these genes by Nrg1p. As mentioned, NRG1 is not repressed by Ume6p either under yeast or hyphal growth conditions. Thus, only NRG1 downregulation or its complete absence combined with UME6 upregulation (the former is the case in wild-type cells only under hyphal growth conditions, the latter in nrg1Δ cells under all conditions) results in strong upregulation of HSGs.

Therefore, one major task for the future will be to identify genes that are required for hyphal morphogenesis, for example components of the Spitzenkörper, the polarisome, Cdc42 and other genes known to have essential functions in polarized growth in hyphal development (Berman, 2006; Whiteway & Bachewich, 2007) that may be under strict or partial control of Ume6p. That one or more regulators of hyphal development are controlled by Ume6p is evident from the fact that hyphal development of mutants lacking upstream regulatory genes of filamentation is restored by ectopic UME6 expression.

The model for transcriptional regulation of HSGs and the role of UME6 in hyphal development that emerges from this study combined with results from Banerjee (2008) is shown in Fig. 7b. Most, if not all, HSGs including UME6 are repressed by Nrg1-Tup1 and other transcriptional repressors [e.g. Rfg1p and Rbf1p (Ishii, 1997)] under yeast growth conditions. Upon induction of hyphal development, these repressors are inactivated transcriptionally and/or posttranscriptionally. Thereby HSGs and UME6 are derepressed. Several transcriptional activators of hyphal development (symbolized as A, B, C and D; for names and interactions among each other, compare Fig. 7a), some of which are transcriptionally upregulated early after hyphal induction (e.g. TEC1 and CPH1; Fig. S3), are posttranslationally activated (reviewed by Biswas, 2007) and are required for upregulation of UME6. Both these transcriptional activators and Ume6p upregulate derepressed HSGs. Three groups of HSGs may be distinguished by their dependence on Ume6p for expression: (1) genes (e.g. HYR1) whose expression only depends on Ume6p and perhaps also on Nrg1p, (2) genes (e.g. ALS3, ECE1, HWP1 and HGC1) whose wild-type expression depends on Ume6p and other transcriptional activators and (3) genes that are not controlled by Ume6p but by other transcriptional regulators and are required for hyphal development only under some environmental conditions. We did not identify genes of the latter group in this study but the results shown in Fig. S2 (ectopic UME6 expression does not compensate the hyphal growth deficiency of efg1Δ, cph1Δ and ras1Δ cells under each environmental condition) strongly indicate their existence.

Supporting Information

Fig. S1. Southern blot of genomic UME6 fragments from heterozygous and homozygous ume6 mutant and reintegrated strains. Deletion and reintegration of the UME6 gene from the genome of the corresponding wild-type strain SN87 were confirmed by digesting genomic DNA isolated from strains UZ24 (UME6/ume6Δ), UZ43(ume6Δ/ume6Δ) and UZ73 (ume6Δ/ume6Δ ADH1/adh1::UME6) with HindIII and BglII and hybridization with an [α-P32]-dATP-labeled probe. The UME6 probe was created by PCR (see Material and Methods) using oligonucleotide primers CaUme6-d1 and CaUme6-d3 (Suppl. Table 1). The length of the expected fragments are: 3720 bp for the wild-type, either 2200 bp or 3100 bp for the distinct alleles after ORF deletion and 5300 bp for the reintegrated UME6 copy.

Fig. S2. Ability for hyphal development of efg1Δ, cph1Δ and ras1Δ strains upon ectopic UME6 expression under different conditions. Hyphae formation was monitored under the microscope after incubating cells of the wild-type strain SC5314, the mutant strains HLC52 (efg1Δ), JKC19 (cph1Δ), HLC54 (efg1Δ cph1Δ), Can52 (ras1Δ) and the corresponding strains UZ200, UZ207, UZ209 and UZ205 with the inserted gene cassette carrying the Tet-promoter-UME6 construct in distinct liquid media each promoting hyphal development at 37°C for 8 hours. The strains showed distinct growth response (formation of germ tubes, or hyphal filaments or formation of neither) upon ectopic UME6 expression caused by the presence of DOX (50 μg/ml). The results are discussed in the text.

Fig. S3. Expression of hyphae-regulatory genes in the presence or absence of UME6 expression. The wild-type strain SC5314 and the strain UZ121, which lacks both native UME6 alleles but carries the gene cassette for ectopic UME6 expression, were grown under distinct conditions as indicated. The expression of TUP1, NRG1, RFG1, EFG1, TEC1, CZF1 and CPH1 was determined by Northern analysis of RNA isolated from the cells. As control, hybridization of an ACT1 probe on the same blots is shown. The transcript level of none of these genes appears to be influenced markedly by the presence or absence of UME6 expression.

Fig. S4. Virulence study in a mouse model of systemic infection. Ten female Balb/c mice were intraperitoneally injected with either 1×107 (A) or 3×107 (B) colony-forming units (cfu) of the wild-type strain SC5314, the ume6Δ strain UZ43 or the reconstituted strain UZ73. Survival of the infected animals was monitored twice daily for four weeks post challenge.

Table S1. Oligonucleotides used in this study.

Table S2. Plasmids used in this study.

Acknowledgements

We are grateful to Joachim Morschhäuser (University of Würzburg, Germany), Suzanne Noble and Alexander Johnson (University of California-San Francisco, San Francisco) for providing strains and plasmids, and for helpful discussions. We thank Alistair J.P. Brown (University of Aberdeen, UK), Joachim Ernst (University of Düsseldorf, Germany), Judith Berman (University of Minnesota, Minneapolis) and Jiangye Chen (Chinese Academy of Sciences, Shanghai, China) for providing strains and plasmids. We thank the reviewers for critical reading and reasonable suggestions. The work was supported (to A.B.) by the Forschungsförderungsfonds der Paracelsus Medizinische Privatuniversität (PMU) Salzburg (grant no. 05/01/001). M.M. is supported by Austrian Science Fund Grants SFB-F28, by the Austrian Ministry of Education, Science and Culture (BM:BWK OEZBT GZ200.074/1-VI/1a/2002) and by the Viennese Foundation for Science and Technology (WWTF Grant LS133).

Footnotes

  • Editor: Ian Dawes

References

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