Candida albicans PHO81 is required for the inhibition of hyphal ...

FEBS Letters 584 (2010) 4639–4645

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Candida albicans PHO81 is required for the inhibition of hyphal development by farnesoic acid Soon-Chun Chung a, Tae-Im Kim a, Chan-Hong Ahn a, Jongheon Shin b,⇑, Ki-Bong Oh a,⇑ a b

Department of Agricultural Biotechnology, College of Agriculture and Life Science, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, Republic of Korea Natural Products Research Institute, College of Pharmacy, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea

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Article history: Received 6 August 2010 Revised 12 October 2010 Accepted 12 October 2010 Available online 21 October 2010 Edited by Judit Ovádi Keywords: Farnesoic acid PHO81 Hyphal development Dimorphism Signaling pathway

a b s t r a c t Farnesoic acid is a signaling molecule that inhibits the transition from budding yeast to filament formation in Candida albicans, but the molecular mechanism regulated by this substance is unknown. In this study, we analyzed the function of CaPHO81, which is induced by farnesoic acid. The pho81D mutant cells existed exclusively as filaments under favorable yeast growth conditions. Furthermore, the inhibition of hyphal growth and repression of CPH1, EFG1, HWP1, and GAP1 mRNA expression in response to farnesoic acid were defective in pho81D mutant cells. These data suggest a role for CaPHO81 in the inhibition of hyphal development by farnesoic acid. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

1. Introduction Candida albicans, an opportunistic human pathogen, undergoes a reversible morphogenetic transition between a budding yeast form and a filamentous hyphal or pseudohyphal form. This dimorphism is thought to contribute to the virulence of the organism [1–3]. The morphogenesis of C. albicans is triggered by various environmental signals in vitro: serum, high temperatures, a high ratio of CO2 to O2, and a neutral pH, and nutrient-poor media induce the cells to sprout true hyphae. Conversely, low temperatures, a high ratio of O2 to CO2, an acidic pH (4–6), and enriched media promote yeast cell growth [4,5]. Many signaling pathways and regulators involved in hyphal development have been identified as a result of strong molecular conservation among C. albicans and Saccharomyces cerevisiae in many cellular processes [6]. For example, a mitogen-activated protein kinase (MAPK) pathway involving CST20, HST7, CEK1, and CPH1, and a cAMP-dependent protein kinase A (PKA) pathway involving CAP1, TPK1, TPK2, and EFG1 promote filamentous growth [7–12]. Furthermore, the small GTP-binding protein Ras1 is required for the regulation of both MAPK and cAMP-dependent PKA pathways [13]. Three other C. albicans genes, TUP1, RFG1, and NRG1, act to repress filamentous growth [14–17], but despite these data, the regulatory mechanisms of dimorphism are poorly ⇑ Corresponding authors. Fax: +82 2 762 8332 (J. Shin), +82 2 873 3112 (K.-B. Oh). E-mail addresses: [email protected] (J. Shin), [email protected] (K.-B. Oh).

understood, possibly because the upstream regulatory components are unclear [18]. The dimorphism of C. albicans is controlled by at least two morphogenic autoregulatory substances (ARSs), which accumulate in the medium as the cells proliferate. Farnesoic acid and farnesol are sesquiterpenes produced by C. albicans that act as ARSs to inhibit hyphal development [18,19]. An in vitro inhibitory activity assay revealed that farnesoic acid exerted a weaker inhibitory effect (IC50 = 4.56 lg/ml) than farnesol (IC50 = 2.82 lg/ml) [20]. However farnesoic acid had no detectable effect on yeast cell growth at concentrations below 200 lg/ml, unlike farnesol [18,20]. In C. albicans, farnesol reduces the expression of HST7 and CPH1, which are components of MAPK cascades [21], induces the cell cyclin gene PCL2, and represses HGC1 and CLN3, which are required for filamentous growth [22]. Recently, farnesol was reported to inhibit a Ras1-mediated cAMP-dependent PKA pathway [23]. Nevertheless, how these substances regulate C. albicans hyphal development remains unknown because the regulatory factor controlled by these signaling molecules has not been identified. Previously, we identified several genes that were upregulated by farnesoic acid as a first step toward elucidating the mechanism of action for farnesoic acid in C. albicans morphogenesis [24]. Here, we show that CaPHO81 induced by farnesoic acid is required for inhibition of the yeast-to-hypha transition. CaPHO81 expression was increased twofold by treatment with farnesoic acid within 40 min. The pho81D mutant cells existed exclusively as filaments under favorable yeast growth conditions and were insensitive to

0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.10.026

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Table 1 Strains and plasmids used in this study. Strains or plasmids Strains SC5314 CAI4 CAG CAG4 CAG47 CAG4711 Plasmids pMB7 pMB7-1 pMB7-2 pMB7-3 pMB7-4 YPB-ADHpt pADH-CaPHO81 pADH-Ras1 pADH-Ras1G13V

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Prototrophic clinical isolate Dura3::imm434/Dura3::imm434 CAI4 Dpho81::hisG(I-SceI)-URA3-hisG(ISceI)/PHO81 CAI4 Dpho81::hisG(I-SceI)/PHO81 CAI4 Dpho81::hisG(I-SceI)-URA3-hisG(ISceI)/Dpho81::hisG(I-SceI) CAI4 Dpho81::hisG(I-SceI)/Dpho81::hisG(ISceI)

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(I-SceI) hisG-URA3-hisG (I-SceI) in pUC18 1–383 bp of PHO81 open reading frame in BglII site of pMB7 3516–3993 bp of PHO81 open reading frame in SalI site of pMB7-1 1–383 bp of PHO81 open reading frame in SalI site of pMB7 3014–3458 bp of PHO81 open reading frame in BglII site of pMB7-3 Promoter and terminator regions of ADH1 gene in YPB1 CaPHO81 in YPB-ADHpt RAS1 in YPB-ADHpt RAS1G13V in YPB-ADHpt

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manufacturer’s instructions. Cells were grown in 6 ml of highphosphate glucose minimal medium (10 mM phosphate), washed in low-phosphate (10 lM) minimal medium, and then resuspended in 1 ml of low-phosphate medium. An aliquot was then transferred to 20 ml of fresh high- or low-phosphate medium, grown to an optical density at 600 nm of 0.3–0.5, washed in citrate buffer (90 mM, pH 4.8), and resuspended in 100 ll of citrate buffer. A total of 10 ll from each cell suspension was used in the acid phosphatase assay. 2.4. Morphological characterization of the pho81D mutant

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The wild-type cells (CAI4), pho81D mutant cells (CAG4711) containing empty YPB-ADHpt vector which carries the ADH1 promoter, terminator sequence, and C. albicans URA3 (Table 1) were streaked onto SD plates without uridine and incubated at 28 °C for 3–4 days. A single colony was inoculated in liquid media and cultured at 28 °C to the early stationary phase. To induce hyphal growth, cells (1 107 cells/ml) were incubated in liquid media or 25 mM Tris–HCl buffer (pH 7.0) for 6 h at 37 °C. For the farnesoic acid inhibition assay, cells (1 107 cells/ml) were inoculated in glucose-salts (GS) medium (5 g of glucose/0.26 g of Na2HPO412H2O/0.66 g of KH2PO4/0.88 g of MgSO47H2O/0.33 g of NH4Cl/16 lg of biotin per liter) [18] and incubated for 6 h without shaking at 37 °C, with or without 20 lg/ml farnesoic acid. 2.5. Northern hybridization

inhibition of the yeast-to-hypha transition by farnesoic acid. Furthermore, the cells showed defective repression of CHP1, EFG1, GAP1, and HWP1 expression in response to farnesoic acid. These results indicate that CaPHO81 is a novel regulatory factor that functions to repress filamentous growth, suggesting that it is required for the inhibition of hyphal development in response to farnesoic acid. 2. Materials and methods 2.1. Candida albicans strains and culture conditions The C. albicans strains used in this study are indicated in Table 1. Cells were grown in yeast extract/peptone/dextrose (YPD) medium (1% yeast extract, 2% peptone, and 2% glucose), while strains carrying plasmids or introduced gene disruption cassettes were grown in SD medium (0.67% yeast nitrogen base without amino acids, 0.192% yeast synthetic dropout medium supplement without uracil, and 2% glucose). 2.2. Gene disruption To disrupt the first chromosomal CaPHO81 allele, pMB7-2 was digested with SalI and SacI, and 5 lg of the linearized pho81D::hisG-URA3-hisG gene disruption cassette was introduced into strain CAI4 using the lithium acetate method [25]. To disrupt the second chromosomal CaPHO81 allele, C. albicans PHO81/pho81D::hisG cells (CAG4) were again transformed with the linearized pho81D::hisGURA3-hisG gene disruption cassette (i.e., pMB7-4 digested with SalI and SacI). Spontaneous uridine auxotrophs were selected on media containing 1 mg/ml 5-fluoroorotic acid (5-FOA). The genotypes of the mutant strains were confirmed by PCR and Southern hybridization (Supplementary Fig. 1). 2.3. Acid phosphatase assays Acid phosphatase assays were performed using an Acid Phosphatase Assay Kit (Sigma, St. Louis, MO, USA) according to the

Aliquots (10 lg) of total RNA were denatured and subjected to 1.2% agarose electrophoresis in the presence of 1 M formaldehyde. The size-fractionated RNAs were then transferred to a nylon membrane and fixed by UV irradiation. CPH1-, EFG1-, TUP1-, HWP1-, and GAP1-specific probes were generated using a Random Primer DNA Labeling Kit (Takara Shuzo, Kyoto, Japan) with [a-32P]dCTP (Amersham, Piscataway, NJ, USA). Radioactivity was detected using a BioImaging Analyzer System (BAS 2500; FujiFilm, Tokyo, Japan). 2.6. Detection of GTP-Ras1 in vivo GTP-Ras1 was detected using an Active Ras Pull-Down and Detection Kit (Thermo Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. A total of 1 mg of total protein was incubated for 1 h at 4 °C with GST-Raf1-RBD in the presence of GST-agarose beads, washed, and eluted with 2 SDS sample buffer (125 mM Tris–HCl, pH 6.8, 2% glycerol, 4% SDS, and 0.05% bromophenol blue). The eluted proteins and 25 lg of total protein were separated by SDS–PAGE, blotted onto PVDF membranes, and detected with anti-Ras antibodies (1:500 dilution; Thermo Scientific). Goat anti-mouse IgG-horseradish peroxidase (HRP) conjugate (Thermo Scientific) was used as the secondary antibody in this procedure; protein bands were visualized with the ECL system using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The ratio of GTP-Ras1/total Ras1 was determined by densitometric analysis (Scion-Image Software, Frederick, MD, USA). For the control reaction, total protein (1 mg) was incubated for 1 h at 4 °C, either with 0.1 mM GTPcS or 1 mM GDP, and incubated with GST-Raf1-RBD in the presence of GST-agarose beads (Fig. 6A). 3. Results 3.1. Identification and characterization of CaPHO81 Previously, we identified several genes that are upregulated by farnesoic acid [24]. Of these, CA11 expression was increased twofold by treatment with farnesoic acid for 40 min. CA11 shares


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Fig. 1. Sequence of C. albicans Pho81. The SPX domain (A) and ankyrin repeats (B) in Pho81 from C. albicans and S. cerevisiae, as conceptually translated from their DNA sequence, are compared. The alignment was performed using ClustalW; identities are highlighted. (C) The PHO81 gene products and relative arrangement of their N-terminal conserved domains (black) and ankyrin repeat motifs (gray) are shown.

Fig. 2. Acid phosphatase assays of the wild-type (CAI4) and pho81D mutant (CAG4711) cells. Cells grown under high (10 mM) or low (10 lM) phosphate conditions were assayed for inducible acid phosphate activity using p-nitrophenol phosphate as the substrate.

open reading frame (ORF) consisting of 1328 amino acids with 32% identity to Pho81 from S. cerevisiae over its entire length. The SPX domain (residues 1–188) and six ankyrin repeats (residues 457–657) were conserved, and the primary sequence showed 52% and 38% homology with ScPho81 (Fig. 1). We therefore renamed the gene C. albicans PHO81 (GenBank Accession Number DQ074694). In S. cerevisiae, Pho81 inhibits the kinase activity of the Pho80Pho85 cyclin–CDK complex, resulting in the transcription of phosphate-responsive genes, including PHO5, an acid phosphatase gene, under phosphate starvation conditions [26,27]. Therefore, to assess the functional similarity between C. albicans PHO81 and S. cerevisiae PHO81, we compared the acid phosphatase activity [28] in the wild-type and pho81D mutant cells. Under low-phosphate conditions, acid phosphatase activity in the pho81D mutant cells was found to be 26%, compared to 100% in the wild-type cells (Fig. 2). The results indicate that the CaPho81p is a phosphatases, and therefore likely homolog of the yeast protein, ScPho81p, which is also a CDK inhibitor. Whether Pho81p inhibits CDK remains to be determined. 3.2. Deletion of CaPHO81 results in filamentous growth

98% homology with orf19.7475 (108–1843 bp) from the Candida Genome Database (http://candidagenome.org), which is thought to be a homolog of S. cerevisiae PHO81, a cyclin-dependent protein kinase (CDK) inhibitor. To confirm the full-length cDNA sequence of CA11, we performed 30 and 50 rapid amplification of the cDNA ends (RACE). Sequencing and conceptual translation revealed an

To investigate the role of CaPHO81 in C. albicans morphogenesis, morphological differences between the wild-type and pho81D mutant cells were compared under the microscope. Both strains were grown under conditions known to favor the yeast form (YPD). Under these conditions, the wild-type cells exhibited the expected

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Fig. 3. Morphological characterization of the pho81D mutant. (A) Cells were incubated in YPD to the early stationary phase at 28 °C or for 6 h at 37 °C. (B) Cells were placed on cornmeal agar plus Tween 80 (0.33%) plates under a coverslip and grown for 20 h at 25 °C. Photographs were taken at a magnification of 40 with phase optics. Scale bar = 50 lm.

yeast form, whereas the homozygous pho81D mutant cells existed mainly in the filamentous form, creating more extended filaments than wild-type cells at high temperatures (Fig. 3A). The pho81D mutant cells expressing CaPHO81 under control of the ADH1 promoter exhibited a wild-type phenotype. Similar results were obtained when the cells were incubated in GS [18], Spider, SLAD, or SHAD media (Supplementary Fig. 2). Heterozygous PHO81/pho81 cells showed an intermediate phenotype between the wild-type and homozygous cells on nutrient-poor media such as cornmeal agar by micro-aerobic growth under coverslips (Fig. 3B). The heterozygous cells were morphologically similar to the wild-type cells on most media. These results suggest that CaPHO81 has a role in suppressing filamentous growth.

ted cells with those in control cells. Northern blot analysis showed that CPH1 and EFG1 mRNA expression was repressed in the farnesoic acid-treated wild-type cells, but that TUP1 mRNA expression was not (Fig. 4B). Furthermore, GAP1 and HWP1 mRNA, which represent Cph1- and Efg1-dependent genes specific for hyphal induction [29,30], respectively, decreased in farnesoic acid-treated C. albicans. The repression of these genes was defective in farnesoic acid-treated pho81D mutant cells (Fig. 4C). These data suggest that CaPHO81 acts downstream of the signaling pathways that regulate the inhibition of hyphal growth by farnesoic acid, but upstream of both MAPK and cAMP-dependent PKA pathways.

3.3. The pho81D mutant cells do not respond to farnesoic acid

Because Ras1 is required for the regulation of both MAPK and cAMP-dependent PKA pathways [13], we next assessed whether CaPho81 contributes to regulation of the Ras1 signaling. CAI4Ras1 containing the wild-type RAS1 gene and CAI4-Ras1G13V carrying a dominant-active RAS1 gene were incubated in 25 mM Tris–Cl (pH 7.0) buffer at 37 °C with no added inducer. The CAI4-Ras1G13V strain formed hypha under these conditions, whereas the CAI4Ras1 strain did not (Fig. 5A). Thus, hyperactivation of the Ras1 signaling pathway due to the presence of a dominant-active Ras1 variant (Ras1G13V) resulted in hyphal growth under these conditions. Following incubation with 20 lg/ml farnesoic acid under the same conditions, hyphal formation was inhibited in the CAI4-Ras1G13V strain, but not in the pho81D-Ras1G13V strain (Fig. 5B). These results were shown that CaPHO81 is required for inhibition of Ras1 signaling by farnesoic acid. Ras proteins are members of the small GTPase superfamily, which cycle between an inactive GDP-bound and an active GTPbound form [31]. The active form of Ras stimulates downstream signaling pathways through direct interactions with effectors such

To determine whether CaPHO81 contributes to the inhibition of hyphal development in C. albicans by farnesoic acid, we tested farnesoic acid inhibition assay using previously described methods [18]. The wild-type and pho81D mutant cells were incubated in GS medium with or without farnesoic acid (20 lg/ml) at 37 °C for 6 h. As expected, farnesoic acid inhibited hyphal development in wildtype cells. The pho81D mutant cells grew exclusively as filaments under farnesoic acid treated condition (Fig. 4A), although the filaments of the farnesoic acid-treated pho81D mutant cells were shorter and thicker than those of the untreated cells. This observation indicates that the pho81D mutant cells exhibited filamentous growth in the presence of farnesoic acid. Hyphal development is regulated by MAPK and cAMP-dependent PKA pathways, as well as the transcriptional repressor Tup1 [6]. To determine whether farnesoic acid contributes to one of them in inhibition of the yeast-to-hypha transition, we compared the transcript levels of CPH1, EFG1, and TUP1 in farnesoic acid-trea-

3.4. CaPHO81 contributes to regulation of Ras1 signaling


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Fig. 4. Response of the pho81D mutant cells to farnesoic acid. (A) The wild-type (CAI4) and pho81D mutant (CAG4711) cells containing YPB-ADHpt were incubated in GS medium with 1% dimethyl sulfoxide (FA) or 20 lg/ml farnesoic acid (+FA) for 6 h at 37 °C. Photographs were taken at a magnification of 40 with phase optics. Scale bar = 50 lm. (B) The wild-type (CAI4) and (C) pho81D mutant (CAG4711) cells containing YPB-ADHpt were incubated in GS medium with (+FA) or without (FA) 20 lg/ml farnesoic acid for the indicated times at 37 °C.

as Raf protein kinase [32]. We also measured the amount of GTPbound Ras1 in vivo in the wild-type and pho81D mutant cells using an active Ras pull-down assay, which exploits the specific interaction between GTP-Ras and the Ras-binding domain (RBD) of Raf-1 [33–35]. We first found that RBD-Raf1 specifically interacted with GTP-Ras1 (Fig. 6A). Our active Ras pull-down assay results using total protein extracts from the wild-type and pho81D mutant cells indicate that the GTP-Ras1 levels in the pho81D mutant cells were twofold higher than those in the wild-type cells (Fig. 6B and C). These observations imply that CaPHO81 contributes to regulation of the Ras1 signaling in inhibition of hyphal development by farnesoic acid.

4. Discussion Farnesoic acid is a morphogenetic autoregulatory substance that accumulates in the medium as cells proliferate [18]. This molecule does not affect yeast cell growth, but it does inhibit hyphal development. However, the molecular mechanism underlying farnesoic acid’s role in C. albicans morphogenesis is unclear. In this study, we found that CaPHO81 is required for the inhibition of hyphal development by farnesoic acid. CaPHO81 expression decreased in hyphal cells relative to yeast cells (Supplementary Fig. 3); specifically, it increased twofold following treatment with farnesoic acid within 40 min [24]. We also found that the inhibi-

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Fig. 5. CaPHO81 was required for inhibition of the hyperactive Ras1 signaling by farnesoic acid. (A) The wild-type (CAI4) cells containing pADH-Ras1 or pADHRas1G13V were incubated in 25 mM Tris–HCl buffer (pH 7.0) for 6 h at 37 °C. (B) The wild-type (CAI4) and pho81D (CAG4711) cells containing pADH-Ras1G13V were incubated in 25 mM Tris–HCl buffer (pH 7.0) for 6 h at 37 °C with (+FA) or without (FA) 20 lg/ml farnesoic acid.

tory effect of farnesoic acid on hyphal growth in the pho81D mutant cells was defective (Fig. 4A). Previously, a study reported that farnesoic acid inhibited hyphal growth, resulting in the conversion of hyphal tips into yeast cells [18]. Under our conditions, no such conversion was observed in farnesoic acid-treated pho81D mutant cells (data not shown). Furthermore, the repression of CPH1, EFG1, HWP1, and GAP1 in response to farnesoic acid was defective in the pho81D mutant cells (Fig. 4C). These data suggest that CaPHO81 is required for the inhibition of hyphal development by farnesoic acid. CaPho81 shows 32% identity with S. cerevisiae Pho81, which is a CDK inhibitor that regulates the phosphate-responsive signal transduction pathway (i.e., PHO pathway). The NH2-terminus of CaPho81 contains an SPX domain involved in G-protein-associated signal transduction [36], and ankyrin repeats with homology to the p16INK4 class of mammalian CDK inhibitors implicated in protein– protein interactions [37,38]. The SPX and ankyrin repeat domain in the two proteins show a high degree of primary sequence conservation (Fig. 1). Our data indicate that the deletion of CaPHO81 results in defective regulation of the PHO pathway (Fig. 2). Moreover, the pho81D mutant cells formed filaments under favorable yeast growth conditions, although the filamentous growth of the pho81D mutant was not the result of defects in the PHO pathway because the medium used contained high levels of phosphate. These data support the role of CaPHO81 in the inhibition of hyphal development by farnesoic acid. Hyphal development in C. albicans is regulated by the MAPK, cAMP-dependent PKA, and Ras1-mediated signaling pathways [6]. A study reported that the transcript levels of CPH1 and HST7 were reduced by treatment with farnesol, whereas the transcription of HWP1, ALS1, and HYR1, which is strongly induced by Efg1, did not change [21]. Other studies have shown that HWP1 expression is decreased by treatment with farnesol [39]. These reports indicate that ARSs, including farnesoic acid and farnesol, affect many signaling pathways and regulators of hyphal development. Recently, farnesol was reported to inhibit the Ras1-mediated cAMP-dependent PKA pathway [23]. The treatment of C. albicans with farnesol represses the hyperfilamentation phenotype and res-

Fig. 6. The C. albicans pho81D cells show increased GTP-bound Ras1 levels. (A) For the control reaction, 1 mg of total protein extracted from the wild-type cells containing YPB-ADHpt was bound to GTPcS or GDP (Exposure time to ECL system: 1 min). (B) Total proteins (1 mg) extracted from the wild-type (CAI4) and pho81D mutant (CAG4711) cells were incubated with GST-Raf1-RBD in the presence of GST-agarose beads. The bound proteins were eluted with 2 SDS sample buffer. The 25 lg of total proteins (total Ras1) and 15 ll of eluted proteins were separated by SDS–PAGE. Ras1 was detected by immunoblotting with anti-Ras antibodies (Exposure time to ECL system: 20 min). (C) The GTP-Ras1/total Ras1 ratio was determined by densitometric analysis.


cues the heat shock sensitivity induced by dominant-active Ras1. We identified that CaPHO81 acts upstream of both MAPK and cAMP-dependent PKA pathways (Fig. 4) and required for inhibition of the hyphal development induced by hyperactive Ras1 signaling in response to farnesoic acid (Fig. 5). Furthermore, the pho81D mutant cells showed increased GTP-bound Ras1 levels relative to wild-type cells (Fig. 6), in addition to increased heat shock sensitivity and invasive growth (data not shown). These observations suggest that CaPho81 impacts Ras1 signaling in the inhibition of hyphal development by farnesoic acid. Although how CaPho81 regulates Ras1 signaling in response to farnesoic acid is unclear, these findings have important implications for our understanding of the mechanism of hyphal development and virulence in pathogenic fungi. Acknowledgement This work was supported by the Korea Research Foundation Grant funded by the Korea Government (MOEHRD) (KRF-2007313-F00019).

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