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SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis
FEBS 29392
FEBS Letters 579 (2005) 1973–1977
SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis Karin Thevissena,*, Jola Idkowiak-Baldysb, Yang-Ju Imb, Jon Takemotob, Isabelle E.J.A. Franc¸oisa, Kathelijne K.A. Ferketa, An M. Aertsa, Els M.K. Meerta, Joris Winderickxc, Johnny Roosenc, Bruno P.A. Cammuea a
Centre for Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium b Department of Biology, Utah State University, Logan, UT 84322-5305, USA c Laboratory for Functional Biology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Heverlee, Belgium Received 8 November 2004; revised 4 February 2005; accepted 15 February 2005 Available online 2 March 2005 Edited by Gerrit van Meer
Abstract The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)2C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)2C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in b-1,6-glucan biosynthesis. We demonstrate that a Dskn1 mutant lacks M(IP)2C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)2C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Sphingolipid; Antifungal; Plant defensin; Syringomycin E; SKN1; Saccharomyces cerevisiae
1. Introduction Plant defensins are small, basic and cysteine-rich peptides that possess antimicrobial activities at micromolar concentrations [1]. They are active against a broad range of phytopathogenic fungi and human pathogens (such as Candida albicans). Plant defensins are non-toxic to either mammalian or plant cells and are produced by plants for defense against fungal attack [1,2]. Most cationic antimicrobial peptides (AMPs) induce membrane permeabilization after initial electrostatic binding to negatively charged phospholipids on the target cell surface. In contrast, plant defensins induce membrane permeabilization through a specific interaction with high-affinity binding sites on fungal cells [3–6]. These plant defensin binding sites have been identified as complex sphingolipids. Yeast mutants affected in the biosynthesis of such sphingolipids are resistant
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Corresponding author. Fax: +32 16 32 19 66. E-mail address: [email protected] (K. Thevissen). Abbreviations: AMP, antimicrobial peptide; M(IP)2C, mannosyl diinositolphosphoryl ceramide
to certain plant defensins: a Saccharomyces cerevisiae ipt1deletion mutant is at least 20-fold more resistant to DmAMP1, a plant defensin isolated from Dahlia merckii, and a C. albicans gcs-deletion mutant is 20-fold more resistant to RsAFP2, a plant defensin isolated from Raphanus sativus, as compared to the corresponding wild-type yeast strains [7,8]. IPT1 encodes an enzyme involved in the last step of the synthesis of the acidic sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)2C) [9], whereas GCS encodes an enzyme involved in the synthesis of the neutral sphingolipid glucosylceramide [10]. Fungal sphingolipids associate with sterols in the plasma membrane to form patches or rafts [11]. A model for the mode of action of plant defensins was presented in which plant defensins interact with membrane patches consisting of sphingolipids and induce membrane permeabilization in susceptible fungi [7,8]. Whether fungal membrane permeabilization results from direct insertion of the plant defensins in the fungal membrane or from activation of an endogenous sphingolipid-mediated signaling pathway is currently not clear. In the present study, we aimed at identifying additional plant defensin-sensitivity genes in the yeast S. cerevisiae in order to further unravel their mode of action. To this end, we screened a S. cerevisiae deletion mutant library for enhanced resistance against the plant defensin DmAMP1. Besides IPT1, we identified a novel DmAMP1-sensitivity gene in S. cerevisiae, namely SKN1. SKN1 was previously identified as a multicopy suppressor for the Dkre6 null phenotype, restoring defects in cell wall b-1,6-glucan synthesis and anchorage of cell wall proteins in Dkre6 disruptants [12]. Remarkably, in this study, we further demonstrate that SKN1 plays an important role in the biosynthesis of the yeast sphingolipid M(IP)2C, similarly to the previously defined IPT1 gene. This finding increases the importance of this sphingolipid as a primary target for DmAMP1 antifungal activity.
2. Materials and methods 2.1. Materials and microorganisms DmAMP1 was isolated as described previously [13]. Yeast strains used are S. cerevisiae strain BY4741, the BY4741-derived deletion mutant library (Invitrogen, Carlsbad, CA) and a BY4741-derived Dskn1 deletion mutant from the Euroscarf collection (www.unifrankfurt.de/fb15/mikro/euroscarf/index.html).
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.02.043
1974 2.2. Screening of a yeast deletion library for DmAMP1 resistance Microtiter plates were filled with 100 ll yeast minimal medium (YMM; 0.8 g/l CSM, complete amino acid supplement mixture, Bio 101 Systems; 6.5 g/l YNB, yeast nitrogen base; 20 g/l glucose) supplemented with 8 lM DmAMP1, inoculated with mutant strains of the library and incubated at 30 C. The liquid cultures in the microtiter plates were microscopically scored for growth after 24 h. Strains resistant to 8 lM DmAMP1 were retested in an antifungal activity assay. 2.3. Antifungal activity assay Antifungal activity of DmAMP1 against yeast strains was assayed as described previously [4]. Briefly, 100 ll of an overnight yeast culture grown in YMM or YMM-HIS (YMM without histidine) was inoculated in 10 ml YMM or YMM-HIS. Eighty micoliters aliquots of these cultures were added to 20 ll of twofold dilution series of DmAMP1 in microtiter plates. Growth of the yeast cultures at 30 C was scored microscopically after 24 h. 2.4. Construction of Dipt1Dskn1 double deletion mutant Gene disruption of SKN1 in Dipt1 deletion mutant was accomplished by PCR-mediated gene disruption as described by Brachmann [14]. Following primers were used: 5 0 -ATGTCTGTGCGAAACCTCACCAATAATCGTCATTCCAACAGCGAAAATAGAGATTGTACTGAGAGTGCAC-3 0 and 5 0 -TCAGGATAATGAGAATTTTGAACTACTACAACCTCCTGTTAGAATACTGTGCGGTATTTCACACCG-3 0 , comprising the universal sequences of 20 nucleotides (bold) for amplifying the auxotrophic marker gene LEU on pRS425, and the first 50 bp and last 46 bp of the coding region of SKN1, respectively. Verification of the deletion was performed by PCR analysis using primer 5 0 -CATTACTAGCTAGTGTTACA-3 0 annealing 36 bp upstream of SKN1 and primer 5 0 -TGAACTACTACAACCTCCTG-3 0 annealing at the last 55 bp of the coding region of SKN1. 2.5. Complementation of Dskn1 or Dipt1 deletion mutant with SKN1 or IPT1 A Ycp50 plasmid bearing a functional SKN1 gene [12], was digested with SalI and EcoRI. The resulting 3.7 kb fragment with the SKN1 coding sequence was ligated into the yeast multicopy shuttle vector pRS423 [15], yielding the plasmid pRS423(SKN1). pRS423(SKN1) as well as pRS423(IPT1) bearing a functional IPT1 gene [7], and the empty plasmid pRS423 were transformed into BY4741 strain, Dipt1 or Dskn1 deletion mutant (Invitrogen). Transformants were selected on YMM-HIS. 2.6. Sequencing of IPT1 alleles in Dskn1 mutant The IPT1 gene of Dskn1 mutant (Invitrogen) was amplified by PCR with PfuTurbo polymerase (Stratagene, La Jolla, CA) on genomic DNA, using primers 5 0 -ATGAATGTCATATTTTCTTTGGC-3 0 and 5 0 -CTCTTATCAAACCGGCAGCAAAC-3 0 for the amplification of the IPT1 coding region from 1 to 811 bp and primers 5 0 -GACACCGAACATGTTAATTACACC-3 0 and 5 0 -CTATGCAAGCGGATCAAAAAACCA-3 0 for the amplification of the IPT1 coding region from 757 to 1584 bp. All PCRs were done in triplicate to check for errors generated by PfuTurbo polymerase itself. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA), purified PCR products were used for sequencing on an ABI-3100Avant apparatus (Applied BioSystems). 2.7. Sphingolipid analyses Yeast strains were grown and radiolabeled in YPD medium and sphingolipids were analyzed by two-dimensional thin-layer chromatography (2D-TLC) as described previously [16]. 2.8. RNA isolation and Northern blot analysis Isolation and quantification of total RNA from yeast strains were performed as described previously [17]. Northern blots were prepared by electrophoretic separation of total RNA (18 lg total RNA/lane) in gels containing 1% agarose in 50 mM boric acid, 1 mM sodium citrate, 5 mM NaOH, pH 7.5, and 1% formaldehyde. RNA was transferred by capillary blotting to a Hybond-N membrane (Amersham Biosciences) using 10· SSC buffer (1.5 M NaCl, 0.15 M Na-citrate, pH 7.0). These blots were hybridized with [a32P]dCTP-labeled probe of the coding re-
K. Thevissen et al. / FEBS Letters 579 (2005) 1973–1977 gion of IPT1, constructed using the High Prime kit from Boehringer Mannheim/Roche Diagnostics. The blots were analyzed using the Fuji BAS-1000 Phosphorimager and PCBAS 2.0 and TINA 9.0 software (Fujifilm Medical Systems, Stanford, CT, USA).
3. Results 3.1. Screening of a yeast deletion library for DmAMP1 resistance To address the question whether genes other than IPT1 are involved in DmAMP1-sensitivity, a S. cerevisiae deletion mutant library was screened for resistance towards DmAMP1. This deletion mutant library consists of single gene knock-outs in S. cerevisiae BY4741 parental strain, and covers all 4385 open reading frames (ORFs) encoding non-essential proteins out of 6218 known ORFs in the fully sequenced S. cerevisiae genome. Besides IPT1, one other gene was identified that might play a role in DmAMP1-sensitivity, namely SKN1. Indeed, deletion of SKN1 resulted in at least 8-fold increased DmAMP1-resistance as compared to BY4741 (Table 1). An independently BY4741-derived S. cerevisiae Dskn1 deletion mutant, obtained from the Euroscarf project, was also found to be resistant against DmAMP1 (results not shown). In addition, we constructed a Dipt1Dskn1 double mutant, which was also found to be resistant against DmAMP1 (Table 1). 3.2. Complementation of Dskn1 and Dipt1 deletion mutants with SKN1 and IPT1 genes To analyze whether Dskn1 deletion mutant can be complemented with a functional SKN1, we cloned the SKN1 gene in pRS423 resulting in pRS423(SKN1). The plasmids pRS423(SKN1) and empty pRS423 were subsequently transformed to both BY4741 and the Dskn1 deletion mutant and DmAMP1-sensitivity of the resulting transformants was tested (Table 1). Reintroduction of SKN1 in the Dskn1 deletion mutant via pRS423(SKN1) indeed restored its DmAMP1-sensitivity to the same level as DmAMP1-sensitivity of BY4741 transformed with pRS423(SKN1) or empty pRS423. Hence,
Table 1 Antifungal activity of DmAMP1 towards various S. cerevisiae strains and presence/absence of M(IP)2C in their membranes Yeast strain
MIC (lM)a DmAMP1
Presence (+) or absence () of M(IP)2C in the membrane
Parental strain BY4741 BY4741 + pRS423 BY4741 + pRS423(SKN1) BY4741 + pRS423(IPT1) Dipt1 mutant Dipt1 + pRS423 Dipt1 + pRS423(SKN1) Dskn1 mutant Dskn1 + pRS423 Dskn1 + pRS423(SKN1) Dskn1 + pRS423(IPT1) Dipt1Dskn1 double mutant
3.0 8.0b 7.5b 7.0b >20 >20b >20b >20 >20b 7.0b 8.0b >20
+ + NDc ND + +
a MIC values are the minimal concentrations (lM) of the peptides required to inhibit the growth of a yeast strain by 100%. Data are means of duplicate measurements. Standard errors were typically below 6.5%. b Growth medium was selective medium YMM-HIS. c Not determined.
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SKN1 can be regarded as a DmAMP1-sensitivity gene in S. cerevisiae. Interestingly, transformation of Dskn1 deletion mutant with pRs423(IPT1) bearing a functional IPT1 [7], also fully restored its DmAMP1-sensitivity (Table 1). To investigate the possibility that Dskn1 deletion mutant is affected in its IPT1 gene, the IPT1 gene was sequenced. However, no mutations in the IPT1 gene of Dskn1 mutant could be identified (results not shown). Moreover, to check the transcription of IPT1 in Dskn1 mutant, we performed Northern blot analysis on total RNA from BY4741 and Dskn1 mutant. No significant differences in hybridization signal intensities for IPT1 on both strains were observed, indicating that expression of IPT1 is not affected in Dskn1 mutant as compared to BY4741. Additionally, to figure out whether overexpression of SKN1 in Dipt1 mutant is also sufficient to induce DmAMP1-sensitivity, we transformed Dipt1 deletion mutant with pRS423(SKN1) or with empty pRS423. However, Dipt1 transformed with pRS423(SKN1) remained as resistant to DmAMP1 as Dipt1 transformed with empty pRS423, indicating that Skn1p cannot functionally complement for Ipt1p in defined minimal medium (Table 1). 3.3. Role of Skn1p in sphingolipid biosynthesis Since the sphingolipid biosynthesis gene IPT1 can restore DmAMP1-sensitivity of Dskn1 yeast mutant, we performed sphingolipid analyses using 2D-TLC on BY4741, Dskn1, Dipt1 and Dipt1Dskn1. Also the sphingolipid profile of the Dskn1 deletion mutant transformed with pRS423(IPT1) and pRS423(SKN1) was examined. As depicted in Fig. 1, the presence of inositolphosphoryl ceramide (IPC) and mannosyl inositolphosphoryl ceramide (MIPC) in all strains is clear; double spots represent the C18- and C20-sphingoid base forms of IPC and MIPC. In contrast to the parental strain BY4741, mem-
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branes of deletion mutants Dipt, Dskn1 and Dipt1Dskn1 completely lack M(IP)2C (Fig. 1). Transformation of Dskn1 with pRS423(IPT1) or pRS423(SKN1), however, restored M(IP)2C production (Fig. 1; Table 1) and DmAMP1-sensitivity (see Section 3.2). Transformation of Dipt1 with pRS423(SKN1), however, could not restore M(IP)2C production (Table 1) nor DmAMP1-sensitivity (see Section 3.2). These data indicate that Ipt1p can functionally substitute for Skn1p but not vice versa when grown in defined minimal medium. Interestingly, we previously demonstrated that M(IP)2C reappears in Dipt1 mutant grown under nutrient limitation, i.e., in 1/2 PDB [16]. Therefore, we analyzed sphingolipid profiles of BY4741, Dskn1, Dipt1 and Dipt1Dskn1 grown in 1/2 PDB. Both, Dipt1 and Dskn1 mutants showed reappearance of M(IP)2C in their membranes when grown in 1/2 PDB, whereas M(IP)2C was completely absent in membranes of Dipt1Dskn1 grown in 1/2 PDB (results not shown). Consistently, we found that Dipt1Dskn1 mutant remains at least 4fold more resistant to DmAMP1 in 1/2 PDB, whereas Dipt1 and Dskn1 are as sensitive toward DmAMP1 in 1/2 PDB as compared to BY4741 (results not shown). Hence, reappearance of M(IP)2C in Dipt1 mutant grown under nutrient limitation is indeed dependent on SKN1. These data point towards a functional substitution between IPT1 and SKN1 in yeast, only under nutrient limitation.
4. Discussion Previous studies on the mode of action of DmAMP1 identified the M(IP)2C-biosynthesis gene IPT1 as a DmAMP1sensitivity gene [7]. In this study, a search for additional
Fig. 1. Glycosphingolipid compositions of yeast strains. 2D-TLC (using CHCl3:CH3OH:4.2NNH4OH (9:7:2 v/v) and CHCl3:CH3OH:CH3COOH:H2O (15:6:4:1.6 v/v) as developing solvents) was performed on deacylated total lipid extracts of [32P]phosphate-labeled cells of strains BY4741 (panel A), Dskn1 transformed with pRS423(IPT1) (panel B), Dipt1 (panel C), Dskn1 (panel D) and Dipt1Dskn1 (panel E) and the chromatograms were subjected to autoradiography. The location where the samples were spotted on the TLC plate is indicated by \.
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DmAMP1-sensitivity genes was undertaken by screening a S. cerevisiae deletion mutant library for increased DmAMP1resistance. We identified SKN1 as a second putative DmAMP1-sensitivity gene, based on an observed 8-fold increased DmAMP1-resistance for a Dskn1 mutant compared to the parental S. cerevisiae strain. Introduction of a functional SKN1 gene in the Dskn1 mutant restored DmAMP1-sensitivity, confirming SKN1 as a DmAMP1-sensitivity gene. SKN1 was previously identified as a multicopy suppressor for the Dkre6 null phenotype, restoring defects in cell wall b1,6-glucan synthesis and anchorage of cell wall proteins in Dkre6 disruptants [12]. Using hydrophobic cluster analysis, it was found that Kre6p and Skn1p show significant similarities to a family of 16 glycoside hydrolases, indicating that they are glucosyl hydrolases or transglucosylases [18]. Despite the high similarity between Skn1p and Kre6p, Dkre6 disruptants were as sensitive to DmAMP1 as wild-type yeast, as well as yeast disruptants in other genes encoding proteins involved in b1,6-glucan biosynthesis, such as ROT2, KRE5, CNE1, KRE11, KRE9, KNH1, and KRE1 (results not shown). These findings indicate that b-1,6-glucan content of yeast strains and their DmAMP1-sensitivity are not correlated. Surprisingly, DmAMP1-sensitivity of Dskn1 mutant could also be restored via complementation with the IPT1 gene, pointing towards a possible non-functional Ipt1p in the Dskn1 mutant. Sequence analysis and Northern blot analysis of the IPT1 gene in the Dskn1 mutant, however, revealed that its IPT1 gene was intact and its transcription not affected. Further biochemical characterization of the Dskn1 mutant showed a lack of M(IP)2C in its membranes, as in case of the Dipt1 mutant. These data point to an important but yet unidentified direct role of Skn1p in sphingolipid biosynthesis. However, introduction of a functional SKN1 gene in Dipt1 mutant could not restore M(IP)2C synthesis nor DmAMP1-sensitivity in defined minimal medium. These data indicate that IPT1 can functionally complement for SKN1 in defined minimal medium but not vice versa. Recently, microarray analysis of the Dlcb4Dlcb5 yeast mutant, which is deficient in sphingolipid biosynthesis, confirmed the involvement of SKN1 in sphingolipid biosynthesis: the expression of SKN1 was found to be about 4-fold upregulated, whereas expression of KRE6 was not altered in this Dlcb4Dlcb5 mutant (Dr. Cowart, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, US, personal communication). Furthermore, the expression of SKN1 was found to be significantly upregulated (>1.8-fold) in yeast strains upon entry into stationary phase [19] or the addition of compounds mimicking nutrient starvation conditions such as methyl methanesulfonate, 3aminotriazole [20] and rapamycin (Roosen, unpublished results). Interestingly, it was previously demonstrated that M(IP)2C reappears in membranes of a Dipt1 yeast mutant grown under nutrient limitation, and hence, a Dipt1 deletion mutant was found to be as sensitive towards DmAMP1 as the corresponding wild-type yeast strain in these conditions [16]. Thus, the observed upregulation of SKN1 under nutrient limitation could be responsible for M(IP)2C production, resulting in DmAMP1-sensitivity of Dipt1 deletion mutant under nutrient limitation. This hypothesis is further confirmed in this study by demonstrating that membranes of Dipt1Dskn1 double mutant grown under nutrient limitation do not contain M(IP)2C and this mutant concomitantly remains resistant
K. Thevissen et al. / FEBS Letters 579 (2005) 1973–1977
to DmAMP1 in 1/2 PDB. In conclusion, since reappearance of M(IP)2C in Dipt1 mutant strain under nutrient limitation is dependent on SKN1, it seems that SKN1 and IPT1 are functional homologous genes in yeast under nutrient limitation. This is in contrast to growth in defined minimal medium in which Ipt1p can functionally complement for Skn1p but not vice versa. Another antifungal peptide that interacts with the fungal sphingolipid M(IP)2C is syringomycin E (SRE), a cyclic lipodepsinonapeptide produced by Pseudomonas syringae [21]. SRE affects fungal membrane permeability by processes that are likely related to channel formation [22,23]. Several sphingolipid biosynthetic genes, one of which is IPT1, were found to be involved in SRE-susceptibility of S. cerevisiae [23]. Interestingly, the Dskn1 mutant was also found to be at least 4-fold resistant towards SRE as compared to BY4741 (results not shown). As a result of this study primarily aiming at the identification of DmAMP1-sensitivity genes in S. cerevisiae, it can be concluded that only two genes (IPT1 and SKN1) could be determined from a genome-wide screening of non-essential yeast genes implicated in DmAMP1-sensitivity. Interestingly, both appear to be involved in the biosynthesis of the sphingolipid M(IP)2C, pointing towards a crucial role of this sphingolipid in the antifungal activity of DmAMP1. Moreover, for one of these genes, SKN1, we identified a novel function, namely its involvement in the biosynthesis of complex sphingolipids. Hitherto, based on sequence comparison, Skn1p was only thought to be involved in b-1-6-glucan biosynthesis. To get more insight in the mechanism of action of this gene in sphingolipid biosynthesis, it would be interesting to investigate its effect on M(IP)2C synthase activity. This could be done directly at membrane level, e.g., using an assay as described by Dickson et al. [9]. Moreover, cellular localization studies of Skn1p and Ipt1p in wild-type yeast strain and the different deletion mutants would be invaluable to indicate a possible physical interaction between these proteins. It is obvious that future research is needed to specifically address the precise functions of these proteins in yeast sphingolipid biosynthesis and their possible physical and functional interactions. Acknowledgments: This work was supported by grants from FWO-Vlaanderen (to B.P.A.C. and J.W.) and the Utah Agricultural Experiment Station (project 607, to J.T.). I.E.J.A.F. acknowledges the receipt of a postdoctoral grant from the IWT-Vlaanderen. Doctoral fellowship from the IWT-Vlaanderen to A.M.A. and J.R. are gratefully acknowledged. We thank Dr. A. Cowart for informing us on microarray analysis of Dlcb4Dlcb5 yeast mutant, and Professor D. De Vos (COK, KU Leuven, Belgium) for confirmation experiments in sphingolipid analysis.
References [1] Thomma, B.P.H.J., Cammue, B.P.A. and Thevissen, K. (2002) Plant defensins. Planta 216, 193–202. [2] Thevissen, K., Ferket, K.K.A., Franc¸ois, I.E.J.A. and Cammue, B.P.A. (2003) Interactions of antifungal plant defensins with components of the fungal plasma membrane. Peptides 24, 1705– 1712. [3] Thevissen, K., Osborn, R.W., Acland, D.P. and Broekaert, W.F. (1997) Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. J. Biol. Chem. 272, 32176–32181.
K. Thevissen et al. / FEBS Letters 579 (2005) 1973–1977 [4] Thevissen, K., Terras, F.R. and Broekaert, W.F. (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Environ. Microbiol. 65, 5451–5458. [5] Thevissen, K., Franc¸ois, I.E.J.A., Takemoto, J., Ferket, K.K.A., Meert, E.M.K. and Cammue, B.P.A. (2003) DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae. FEMS Microbiol. Lett. 226, 169–173. [6] Thevissen, K., Osborn, R.W., Acland, D.P. and Broekaert, W.F. (2000) Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity. Mol. Plant Microbe. Interact. 13, 54–61. [7] Thevissen, K., Cammue, B.P.A., Lemaire, K., Winderickx, J., Dickson, R.C., Lester, R.L., Ferket, K.K.A., Even, F., Parret, A.H. and Broekaert, W.F. (2000) Van A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc. Natl. Acad. Sci. USA 97, 9531– 9536. [8] Thevissen, K., Warnecke, D.C., Francois, I.E.J.A., Leipelt, M., Heinz, E., Ott, C., Thomma, B.P.H.J., Ferket, K.K.A. and Cammue, B.P.A. (2004) Defensins from insects and plants interact with fungal glucosylceramides. J. Biol. Chem. 279, 3900–3905. [9] Dickson, R.C., Nagiec, E.E., Wells, G.B., Nagiec, M.M. and Lester, R.L. (1997) Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene. J. Biol. Chem. 272, 29620–29625. [10] Leipelt, M., Warnecke, D., Za¨hringer, U., Ott, C., Mu¨ller, F., Hube, B. and Heinz, E. (2001) Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276, 33621– 33629. [11] Bagnat, M., Keranen, S., Shevchenko, A. and Simons, K. (2000) Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc. Natl. Acad. Sci. USA 97, 3254–3259. [12] Roemer, T., Delaney, S. and Bussey, H. (1993) SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13, 4039–4048. [13] Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B. and Broekaert, W.F. (1995) Isolation and characterisation of plant
1977
[14]
[15] [16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368, 257–262. Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P. and Boeke, J.D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H. and Hieter, P. (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122. Im, Y.J., Idkowiak-Baldys, J., Thevissen, K., Cammue, B.P.A. and Takemoto, J.Y. (2003) IPT1-independent sphingolipid biosynthesis and yeast inhibition by syringomycin E and plant defensin DmAMP1. FEMS Microbiol. Lett. 223, 199–203. Sherman, F. (1991) Getting started with yeast. Meth. Enzymol. 194, 3–21. Montijn, R.C., Vink, E., Muller, W.H., Verkleij, A.J., Van Den Ende, H., Henrissat, B. and Klis, F.M. (1999) Localization of synthesis of beta-1,6-glucan in Saccharomyces cerevisiae. J. Bacteriol. 181, 7414–7420. Gasch, A.P., Spellman, P.T., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G., Botstein, D. and Brown, P.O. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell. 11, 4241–4257. Natarajan, K., Meyer, M.R., Jackson, B.M., Slade, D., Roberts, C., Hinnebusch, A.G. and Marton, M.J. (2001) Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol. Cell. Biol. 21, 4347–4368. Takemoto, J.Y. (1992) Bacterial phytotoxin syringomycin and its interaction with host membranes in: Molecular Signals in Plantmicrobe Communications (Verma, D.P.S., Ed.), pp. 247–260, CRC Press, Boca Raton, FL. Feigin, A.M., Schagina, L.V., Takemoto, J.Y., Teeter, J.H. and Brand, J.G. (1997) The effect of sterols on the sensitivity of membranes to the channel-forming antifungal antibiotic, syringomycin E. Biochim. Biophys. Acta 1324, 102–110. Stock, S.D., Hama, H., Radding, J.A., Young, D.A. and Takemoto, J.Y. (2000) Syringomycin E inhibition of Saccharomyces cerevisiae: Requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups. Antimicrob. Agents Chemother. 44, 1174–1180.
FEBS Letters 579 (2005) 1973–1977
SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis Karin Thevissena,*, Jola Idkowiak-Baldysb, Yang-Ju Imb, Jon Takemotob, Isabelle E.J.A. Franc¸oisa, Kathelijne K.A. Ferketa, An M. Aertsa, Els M.K. Meerta, Joris Winderickxc, Johnny Roosenc, Bruno P.A. Cammuea a
Centre for Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001 Heverlee, Belgium b Department of Biology, Utah State University, Logan, UT 84322-5305, USA c Laboratory for Functional Biology, Katholieke Universiteit Leuven, Kasteelpark Arenberg 31, B-3001 Heverlee, Belgium Received 8 November 2004; revised 4 February 2005; accepted 15 February 2005 Available online 2 March 2005 Edited by Gerrit van Meer
Abstract The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)2C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)2C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in b-1,6-glucan biosynthesis. We demonstrate that a Dskn1 mutant lacks M(IP)2C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)2C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae. 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Sphingolipid; Antifungal; Plant defensin; Syringomycin E; SKN1; Saccharomyces cerevisiae
1. Introduction Plant defensins are small, basic and cysteine-rich peptides that possess antimicrobial activities at micromolar concentrations [1]. They are active against a broad range of phytopathogenic fungi and human pathogens (such as Candida albicans). Plant defensins are non-toxic to either mammalian or plant cells and are produced by plants for defense against fungal attack [1,2]. Most cationic antimicrobial peptides (AMPs) induce membrane permeabilization after initial electrostatic binding to negatively charged phospholipids on the target cell surface. In contrast, plant defensins induce membrane permeabilization through a specific interaction with high-affinity binding sites on fungal cells [3–6]. These plant defensin binding sites have been identified as complex sphingolipids. Yeast mutants affected in the biosynthesis of such sphingolipids are resistant
*
Corresponding author. Fax: +32 16 32 19 66. E-mail address: [email protected] (K. Thevissen). Abbreviations: AMP, antimicrobial peptide; M(IP)2C, mannosyl diinositolphosphoryl ceramide
to certain plant defensins: a Saccharomyces cerevisiae ipt1deletion mutant is at least 20-fold more resistant to DmAMP1, a plant defensin isolated from Dahlia merckii, and a C. albicans gcs-deletion mutant is 20-fold more resistant to RsAFP2, a plant defensin isolated from Raphanus sativus, as compared to the corresponding wild-type yeast strains [7,8]. IPT1 encodes an enzyme involved in the last step of the synthesis of the acidic sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)2C) [9], whereas GCS encodes an enzyme involved in the synthesis of the neutral sphingolipid glucosylceramide [10]. Fungal sphingolipids associate with sterols in the plasma membrane to form patches or rafts [11]. A model for the mode of action of plant defensins was presented in which plant defensins interact with membrane patches consisting of sphingolipids and induce membrane permeabilization in susceptible fungi [7,8]. Whether fungal membrane permeabilization results from direct insertion of the plant defensins in the fungal membrane or from activation of an endogenous sphingolipid-mediated signaling pathway is currently not clear. In the present study, we aimed at identifying additional plant defensin-sensitivity genes in the yeast S. cerevisiae in order to further unravel their mode of action. To this end, we screened a S. cerevisiae deletion mutant library for enhanced resistance against the plant defensin DmAMP1. Besides IPT1, we identified a novel DmAMP1-sensitivity gene in S. cerevisiae, namely SKN1. SKN1 was previously identified as a multicopy suppressor for the Dkre6 null phenotype, restoring defects in cell wall b-1,6-glucan synthesis and anchorage of cell wall proteins in Dkre6 disruptants [12]. Remarkably, in this study, we further demonstrate that SKN1 plays an important role in the biosynthesis of the yeast sphingolipid M(IP)2C, similarly to the previously defined IPT1 gene. This finding increases the importance of this sphingolipid as a primary target for DmAMP1 antifungal activity.
2. Materials and methods 2.1. Materials and microorganisms DmAMP1 was isolated as described previously [13]. Yeast strains used are S. cerevisiae strain BY4741, the BY4741-derived deletion mutant library (Invitrogen, Carlsbad, CA) and a BY4741-derived Dskn1 deletion mutant from the Euroscarf collection (www.unifrankfurt.de/fb15/mikro/euroscarf/index.html).
0014-5793/$30.00 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2005.02.043
1974 2.2. Screening of a yeast deletion library for DmAMP1 resistance Microtiter plates were filled with 100 ll yeast minimal medium (YMM; 0.8 g/l CSM, complete amino acid supplement mixture, Bio 101 Systems; 6.5 g/l YNB, yeast nitrogen base; 20 g/l glucose) supplemented with 8 lM DmAMP1, inoculated with mutant strains of the library and incubated at 30 C. The liquid cultures in the microtiter plates were microscopically scored for growth after 24 h. Strains resistant to 8 lM DmAMP1 were retested in an antifungal activity assay. 2.3. Antifungal activity assay Antifungal activity of DmAMP1 against yeast strains was assayed as described previously [4]. Briefly, 100 ll of an overnight yeast culture grown in YMM or YMM-HIS (YMM without histidine) was inoculated in 10 ml YMM or YMM-HIS. Eighty micoliters aliquots of these cultures were added to 20 ll of twofold dilution series of DmAMP1 in microtiter plates. Growth of the yeast cultures at 30 C was scored microscopically after 24 h. 2.4. Construction of Dipt1Dskn1 double deletion mutant Gene disruption of SKN1 in Dipt1 deletion mutant was accomplished by PCR-mediated gene disruption as described by Brachmann [14]. Following primers were used: 5 0 -ATGTCTGTGCGAAACCTCACCAATAATCGTCATTCCAACAGCGAAAATAGAGATTGTACTGAGAGTGCAC-3 0 and 5 0 -TCAGGATAATGAGAATTTTGAACTACTACAACCTCCTGTTAGAATACTGTGCGGTATTTCACACCG-3 0 , comprising the universal sequences of 20 nucleotides (bold) for amplifying the auxotrophic marker gene LEU on pRS425, and the first 50 bp and last 46 bp of the coding region of SKN1, respectively. Verification of the deletion was performed by PCR analysis using primer 5 0 -CATTACTAGCTAGTGTTACA-3 0 annealing 36 bp upstream of SKN1 and primer 5 0 -TGAACTACTACAACCTCCTG-3 0 annealing at the last 55 bp of the coding region of SKN1. 2.5. Complementation of Dskn1 or Dipt1 deletion mutant with SKN1 or IPT1 A Ycp50 plasmid bearing a functional SKN1 gene [12], was digested with SalI and EcoRI. The resulting 3.7 kb fragment with the SKN1 coding sequence was ligated into the yeast multicopy shuttle vector pRS423 [15], yielding the plasmid pRS423(SKN1). pRS423(SKN1) as well as pRS423(IPT1) bearing a functional IPT1 gene [7], and the empty plasmid pRS423 were transformed into BY4741 strain, Dipt1 or Dskn1 deletion mutant (Invitrogen). Transformants were selected on YMM-HIS. 2.6. Sequencing of IPT1 alleles in Dskn1 mutant The IPT1 gene of Dskn1 mutant (Invitrogen) was amplified by PCR with PfuTurbo polymerase (Stratagene, La Jolla, CA) on genomic DNA, using primers 5 0 -ATGAATGTCATATTTTCTTTGGC-3 0 and 5 0 -CTCTTATCAAACCGGCAGCAAAC-3 0 for the amplification of the IPT1 coding region from 1 to 811 bp and primers 5 0 -GACACCGAACATGTTAATTACACC-3 0 and 5 0 -CTATGCAAGCGGATCAAAAAACCA-3 0 for the amplification of the IPT1 coding region from 757 to 1584 bp. All PCRs were done in triplicate to check for errors generated by PfuTurbo polymerase itself. Using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied BioSystems, Foster City, CA), purified PCR products were used for sequencing on an ABI-3100Avant apparatus (Applied BioSystems). 2.7. Sphingolipid analyses Yeast strains were grown and radiolabeled in YPD medium and sphingolipids were analyzed by two-dimensional thin-layer chromatography (2D-TLC) as described previously [16]. 2.8. RNA isolation and Northern blot analysis Isolation and quantification of total RNA from yeast strains were performed as described previously [17]. Northern blots were prepared by electrophoretic separation of total RNA (18 lg total RNA/lane) in gels containing 1% agarose in 50 mM boric acid, 1 mM sodium citrate, 5 mM NaOH, pH 7.5, and 1% formaldehyde. RNA was transferred by capillary blotting to a Hybond-N membrane (Amersham Biosciences) using 10· SSC buffer (1.5 M NaCl, 0.15 M Na-citrate, pH 7.0). These blots were hybridized with [a32P]dCTP-labeled probe of the coding re-
K. Thevissen et al. / FEBS Letters 579 (2005) 1973–1977 gion of IPT1, constructed using the High Prime kit from Boehringer Mannheim/Roche Diagnostics. The blots were analyzed using the Fuji BAS-1000 Phosphorimager and PCBAS 2.0 and TINA 9.0 software (Fujifilm Medical Systems, Stanford, CT, USA).
3. Results 3.1. Screening of a yeast deletion library for DmAMP1 resistance To address the question whether genes other than IPT1 are involved in DmAMP1-sensitivity, a S. cerevisiae deletion mutant library was screened for resistance towards DmAMP1. This deletion mutant library consists of single gene knock-outs in S. cerevisiae BY4741 parental strain, and covers all 4385 open reading frames (ORFs) encoding non-essential proteins out of 6218 known ORFs in the fully sequenced S. cerevisiae genome. Besides IPT1, one other gene was identified that might play a role in DmAMP1-sensitivity, namely SKN1. Indeed, deletion of SKN1 resulted in at least 8-fold increased DmAMP1-resistance as compared to BY4741 (Table 1). An independently BY4741-derived S. cerevisiae Dskn1 deletion mutant, obtained from the Euroscarf project, was also found to be resistant against DmAMP1 (results not shown). In addition, we constructed a Dipt1Dskn1 double mutant, which was also found to be resistant against DmAMP1 (Table 1). 3.2. Complementation of Dskn1 and Dipt1 deletion mutants with SKN1 and IPT1 genes To analyze whether Dskn1 deletion mutant can be complemented with a functional SKN1, we cloned the SKN1 gene in pRS423 resulting in pRS423(SKN1). The plasmids pRS423(SKN1) and empty pRS423 were subsequently transformed to both BY4741 and the Dskn1 deletion mutant and DmAMP1-sensitivity of the resulting transformants was tested (Table 1). Reintroduction of SKN1 in the Dskn1 deletion mutant via pRS423(SKN1) indeed restored its DmAMP1-sensitivity to the same level as DmAMP1-sensitivity of BY4741 transformed with pRS423(SKN1) or empty pRS423. Hence,
Table 1 Antifungal activity of DmAMP1 towards various S. cerevisiae strains and presence/absence of M(IP)2C in their membranes Yeast strain
MIC (lM)a DmAMP1
Presence (+) or absence () of M(IP)2C in the membrane
Parental strain BY4741 BY4741 + pRS423 BY4741 + pRS423(SKN1) BY4741 + pRS423(IPT1) Dipt1 mutant Dipt1 + pRS423 Dipt1 + pRS423(SKN1) Dskn1 mutant Dskn1 + pRS423 Dskn1 + pRS423(SKN1) Dskn1 + pRS423(IPT1) Dipt1Dskn1 double mutant
3.0 8.0b 7.5b 7.0b >20 >20b >20b >20 >20b 7.0b 8.0b >20
+ + NDc ND + +
a MIC values are the minimal concentrations (lM) of the peptides required to inhibit the growth of a yeast strain by 100%. Data are means of duplicate measurements. Standard errors were typically below 6.5%. b Growth medium was selective medium YMM-HIS. c Not determined.
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SKN1 can be regarded as a DmAMP1-sensitivity gene in S. cerevisiae. Interestingly, transformation of Dskn1 deletion mutant with pRs423(IPT1) bearing a functional IPT1 [7], also fully restored its DmAMP1-sensitivity (Table 1). To investigate the possibility that Dskn1 deletion mutant is affected in its IPT1 gene, the IPT1 gene was sequenced. However, no mutations in the IPT1 gene of Dskn1 mutant could be identified (results not shown). Moreover, to check the transcription of IPT1 in Dskn1 mutant, we performed Northern blot analysis on total RNA from BY4741 and Dskn1 mutant. No significant differences in hybridization signal intensities for IPT1 on both strains were observed, indicating that expression of IPT1 is not affected in Dskn1 mutant as compared to BY4741. Additionally, to figure out whether overexpression of SKN1 in Dipt1 mutant is also sufficient to induce DmAMP1-sensitivity, we transformed Dipt1 deletion mutant with pRS423(SKN1) or with empty pRS423. However, Dipt1 transformed with pRS423(SKN1) remained as resistant to DmAMP1 as Dipt1 transformed with empty pRS423, indicating that Skn1p cannot functionally complement for Ipt1p in defined minimal medium (Table 1). 3.3. Role of Skn1p in sphingolipid biosynthesis Since the sphingolipid biosynthesis gene IPT1 can restore DmAMP1-sensitivity of Dskn1 yeast mutant, we performed sphingolipid analyses using 2D-TLC on BY4741, Dskn1, Dipt1 and Dipt1Dskn1. Also the sphingolipid profile of the Dskn1 deletion mutant transformed with pRS423(IPT1) and pRS423(SKN1) was examined. As depicted in Fig. 1, the presence of inositolphosphoryl ceramide (IPC) and mannosyl inositolphosphoryl ceramide (MIPC) in all strains is clear; double spots represent the C18- and C20-sphingoid base forms of IPC and MIPC. In contrast to the parental strain BY4741, mem-
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branes of deletion mutants Dipt, Dskn1 and Dipt1Dskn1 completely lack M(IP)2C (Fig. 1). Transformation of Dskn1 with pRS423(IPT1) or pRS423(SKN1), however, restored M(IP)2C production (Fig. 1; Table 1) and DmAMP1-sensitivity (see Section 3.2). Transformation of Dipt1 with pRS423(SKN1), however, could not restore M(IP)2C production (Table 1) nor DmAMP1-sensitivity (see Section 3.2). These data indicate that Ipt1p can functionally substitute for Skn1p but not vice versa when grown in defined minimal medium. Interestingly, we previously demonstrated that M(IP)2C reappears in Dipt1 mutant grown under nutrient limitation, i.e., in 1/2 PDB [16]. Therefore, we analyzed sphingolipid profiles of BY4741, Dskn1, Dipt1 and Dipt1Dskn1 grown in 1/2 PDB. Both, Dipt1 and Dskn1 mutants showed reappearance of M(IP)2C in their membranes when grown in 1/2 PDB, whereas M(IP)2C was completely absent in membranes of Dipt1Dskn1 grown in 1/2 PDB (results not shown). Consistently, we found that Dipt1Dskn1 mutant remains at least 4fold more resistant to DmAMP1 in 1/2 PDB, whereas Dipt1 and Dskn1 are as sensitive toward DmAMP1 in 1/2 PDB as compared to BY4741 (results not shown). Hence, reappearance of M(IP)2C in Dipt1 mutant grown under nutrient limitation is indeed dependent on SKN1. These data point towards a functional substitution between IPT1 and SKN1 in yeast, only under nutrient limitation.
4. Discussion Previous studies on the mode of action of DmAMP1 identified the M(IP)2C-biosynthesis gene IPT1 as a DmAMP1sensitivity gene [7]. In this study, a search for additional
Fig. 1. Glycosphingolipid compositions of yeast strains. 2D-TLC (using CHCl3:CH3OH:4.2NNH4OH (9:7:2 v/v) and CHCl3:CH3OH:CH3COOH:H2O (15:6:4:1.6 v/v) as developing solvents) was performed on deacylated total lipid extracts of [32P]phosphate-labeled cells of strains BY4741 (panel A), Dskn1 transformed with pRS423(IPT1) (panel B), Dipt1 (panel C), Dskn1 (panel D) and Dipt1Dskn1 (panel E) and the chromatograms were subjected to autoradiography. The location where the samples were spotted on the TLC plate is indicated by \.
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DmAMP1-sensitivity genes was undertaken by screening a S. cerevisiae deletion mutant library for increased DmAMP1resistance. We identified SKN1 as a second putative DmAMP1-sensitivity gene, based on an observed 8-fold increased DmAMP1-resistance for a Dskn1 mutant compared to the parental S. cerevisiae strain. Introduction of a functional SKN1 gene in the Dskn1 mutant restored DmAMP1-sensitivity, confirming SKN1 as a DmAMP1-sensitivity gene. SKN1 was previously identified as a multicopy suppressor for the Dkre6 null phenotype, restoring defects in cell wall b1,6-glucan synthesis and anchorage of cell wall proteins in Dkre6 disruptants [12]. Using hydrophobic cluster analysis, it was found that Kre6p and Skn1p show significant similarities to a family of 16 glycoside hydrolases, indicating that they are glucosyl hydrolases or transglucosylases [18]. Despite the high similarity between Skn1p and Kre6p, Dkre6 disruptants were as sensitive to DmAMP1 as wild-type yeast, as well as yeast disruptants in other genes encoding proteins involved in b1,6-glucan biosynthesis, such as ROT2, KRE5, CNE1, KRE11, KRE9, KNH1, and KRE1 (results not shown). These findings indicate that b-1,6-glucan content of yeast strains and their DmAMP1-sensitivity are not correlated. Surprisingly, DmAMP1-sensitivity of Dskn1 mutant could also be restored via complementation with the IPT1 gene, pointing towards a possible non-functional Ipt1p in the Dskn1 mutant. Sequence analysis and Northern blot analysis of the IPT1 gene in the Dskn1 mutant, however, revealed that its IPT1 gene was intact and its transcription not affected. Further biochemical characterization of the Dskn1 mutant showed a lack of M(IP)2C in its membranes, as in case of the Dipt1 mutant. These data point to an important but yet unidentified direct role of Skn1p in sphingolipid biosynthesis. However, introduction of a functional SKN1 gene in Dipt1 mutant could not restore M(IP)2C synthesis nor DmAMP1-sensitivity in defined minimal medium. These data indicate that IPT1 can functionally complement for SKN1 in defined minimal medium but not vice versa. Recently, microarray analysis of the Dlcb4Dlcb5 yeast mutant, which is deficient in sphingolipid biosynthesis, confirmed the involvement of SKN1 in sphingolipid biosynthesis: the expression of SKN1 was found to be about 4-fold upregulated, whereas expression of KRE6 was not altered in this Dlcb4Dlcb5 mutant (Dr. Cowart, Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, US, personal communication). Furthermore, the expression of SKN1 was found to be significantly upregulated (>1.8-fold) in yeast strains upon entry into stationary phase [19] or the addition of compounds mimicking nutrient starvation conditions such as methyl methanesulfonate, 3aminotriazole [20] and rapamycin (Roosen, unpublished results). Interestingly, it was previously demonstrated that M(IP)2C reappears in membranes of a Dipt1 yeast mutant grown under nutrient limitation, and hence, a Dipt1 deletion mutant was found to be as sensitive towards DmAMP1 as the corresponding wild-type yeast strain in these conditions [16]. Thus, the observed upregulation of SKN1 under nutrient limitation could be responsible for M(IP)2C production, resulting in DmAMP1-sensitivity of Dipt1 deletion mutant under nutrient limitation. This hypothesis is further confirmed in this study by demonstrating that membranes of Dipt1Dskn1 double mutant grown under nutrient limitation do not contain M(IP)2C and this mutant concomitantly remains resistant
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to DmAMP1 in 1/2 PDB. In conclusion, since reappearance of M(IP)2C in Dipt1 mutant strain under nutrient limitation is dependent on SKN1, it seems that SKN1 and IPT1 are functional homologous genes in yeast under nutrient limitation. This is in contrast to growth in defined minimal medium in which Ipt1p can functionally complement for Skn1p but not vice versa. Another antifungal peptide that interacts with the fungal sphingolipid M(IP)2C is syringomycin E (SRE), a cyclic lipodepsinonapeptide produced by Pseudomonas syringae [21]. SRE affects fungal membrane permeability by processes that are likely related to channel formation [22,23]. Several sphingolipid biosynthetic genes, one of which is IPT1, were found to be involved in SRE-susceptibility of S. cerevisiae [23]. Interestingly, the Dskn1 mutant was also found to be at least 4-fold resistant towards SRE as compared to BY4741 (results not shown). As a result of this study primarily aiming at the identification of DmAMP1-sensitivity genes in S. cerevisiae, it can be concluded that only two genes (IPT1 and SKN1) could be determined from a genome-wide screening of non-essential yeast genes implicated in DmAMP1-sensitivity. Interestingly, both appear to be involved in the biosynthesis of the sphingolipid M(IP)2C, pointing towards a crucial role of this sphingolipid in the antifungal activity of DmAMP1. Moreover, for one of these genes, SKN1, we identified a novel function, namely its involvement in the biosynthesis of complex sphingolipids. Hitherto, based on sequence comparison, Skn1p was only thought to be involved in b-1-6-glucan biosynthesis. To get more insight in the mechanism of action of this gene in sphingolipid biosynthesis, it would be interesting to investigate its effect on M(IP)2C synthase activity. This could be done directly at membrane level, e.g., using an assay as described by Dickson et al. [9]. Moreover, cellular localization studies of Skn1p and Ipt1p in wild-type yeast strain and the different deletion mutants would be invaluable to indicate a possible physical interaction between these proteins. It is obvious that future research is needed to specifically address the precise functions of these proteins in yeast sphingolipid biosynthesis and their possible physical and functional interactions. Acknowledgments: This work was supported by grants from FWO-Vlaanderen (to B.P.A.C. and J.W.) and the Utah Agricultural Experiment Station (project 607, to J.T.). I.E.J.A.F. acknowledges the receipt of a postdoctoral grant from the IWT-Vlaanderen. Doctoral fellowship from the IWT-Vlaanderen to A.M.A. and J.R. are gratefully acknowledged. We thank Dr. A. Cowart for informing us on microarray analysis of Dlcb4Dlcb5 yeast mutant, and Professor D. De Vos (COK, KU Leuven, Belgium) for confirmation experiments in sphingolipid analysis.
References [1] Thomma, B.P.H.J., Cammue, B.P.A. and Thevissen, K. (2002) Plant defensins. Planta 216, 193–202. [2] Thevissen, K., Ferket, K.K.A., Franc¸ois, I.E.J.A. and Cammue, B.P.A. (2003) Interactions of antifungal plant defensins with components of the fungal plasma membrane. Peptides 24, 1705– 1712. [3] Thevissen, K., Osborn, R.W., Acland, D.P. and Broekaert, W.F. (1997) Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes. J. Biol. Chem. 272, 32176–32181.
K. Thevissen et al. / FEBS Letters 579 (2005) 1973–1977 [4] Thevissen, K., Terras, F.R. and Broekaert, W.F. (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Environ. Microbiol. 65, 5451–5458. [5] Thevissen, K., Franc¸ois, I.E.J.A., Takemoto, J., Ferket, K.K.A., Meert, E.M.K. and Cammue, B.P.A. (2003) DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae. FEMS Microbiol. Lett. 226, 169–173. [6] Thevissen, K., Osborn, R.W., Acland, D.P. and Broekaert, W.F. (2000) Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity. Mol. Plant Microbe. Interact. 13, 54–61. [7] Thevissen, K., Cammue, B.P.A., Lemaire, K., Winderickx, J., Dickson, R.C., Lester, R.L., Ferket, K.K.A., Even, F., Parret, A.H. and Broekaert, W.F. (2000) Van A gene encoding a sphingolipid biosynthesis enzyme determines the sensitivity of Saccharomyces cerevisiae to an antifungal plant defensin from dahlia (Dahlia merckii). Proc. Natl. Acad. Sci. USA 97, 9531– 9536. [8] Thevissen, K., Warnecke, D.C., Francois, I.E.J.A., Leipelt, M., Heinz, E., Ott, C., Thomma, B.P.H.J., Ferket, K.K.A. and Cammue, B.P.A. (2004) Defensins from insects and plants interact with fungal glucosylceramides. J. Biol. Chem. 279, 3900–3905. [9] Dickson, R.C., Nagiec, E.E., Wells, G.B., Nagiec, M.M. and Lester, R.L. (1997) Synthesis of mannose-(inositol-P)2-ceramide, the major sphingolipid in Saccharomyces cerevisiae, requires the IPT1 (YDR072c) gene. J. Biol. Chem. 272, 29620–29625. [10] Leipelt, M., Warnecke, D., Za¨hringer, U., Ott, C., Mu¨ller, F., Hube, B. and Heinz, E. (2001) Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276, 33621– 33629. [11] Bagnat, M., Keranen, S., Shevchenko, A. and Simons, K. (2000) Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc. Natl. Acad. Sci. USA 97, 3254–3259. [12] Roemer, T., Delaney, S. and Bussey, H. (1993) SKN1 and KRE6 define a pair of functional homologs encoding putative membrane proteins involved in beta-glucan synthesis. Mol. Cell. Biol. 13, 4039–4048. [13] Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B. and Broekaert, W.F. (1995) Isolation and characterisation of plant
1977
[14]
[15] [16]
[17] [18]
[19]
[20]
[21]
[22]
[23]
defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368, 257–262. Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P. and Boeke, J.D. (1998) Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132. Christianson, T.W., Sikorski, R.S., Dante, M., Shero, J.H. and Hieter, P. (1992) Multifunctional yeast high-copy-number shuttle vectors. Gene 110, 119–122. Im, Y.J., Idkowiak-Baldys, J., Thevissen, K., Cammue, B.P.A. and Takemoto, J.Y. (2003) IPT1-independent sphingolipid biosynthesis and yeast inhibition by syringomycin E and plant defensin DmAMP1. FEMS Microbiol. Lett. 223, 199–203. Sherman, F. (1991) Getting started with yeast. Meth. Enzymol. 194, 3–21. Montijn, R.C., Vink, E., Muller, W.H., Verkleij, A.J., Van Den Ende, H., Henrissat, B. and Klis, F.M. (1999) Localization of synthesis of beta-1,6-glucan in Saccharomyces cerevisiae. J. Bacteriol. 181, 7414–7420. Gasch, A.P., Spellman, P.T., Kao, C.M., Carmel-Harel, O., Eisen, M.B., Storz, G., Botstein, D. and Brown, P.O. (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol. Biol. Cell. 11, 4241–4257. Natarajan, K., Meyer, M.R., Jackson, B.M., Slade, D., Roberts, C., Hinnebusch, A.G. and Marton, M.J. (2001) Transcriptional profiling shows that Gcn4p is a master regulator of gene expression during amino acid starvation in yeast. Mol. Cell. Biol. 21, 4347–4368. Takemoto, J.Y. (1992) Bacterial phytotoxin syringomycin and its interaction with host membranes in: Molecular Signals in Plantmicrobe Communications (Verma, D.P.S., Ed.), pp. 247–260, CRC Press, Boca Raton, FL. Feigin, A.M., Schagina, L.V., Takemoto, J.Y., Teeter, J.H. and Brand, J.G. (1997) The effect of sterols on the sensitivity of membranes to the channel-forming antifungal antibiotic, syringomycin E. Biochim. Biophys. Acta 1324, 102–110. Stock, S.D., Hama, H., Radding, J.A., Young, D.A. and Takemoto, J.Y. (2000) Syringomycin E inhibition of Saccharomyces cerevisiae: Requirement for biosynthesis of sphingolipids with very-long-chain fatty acids and mannose- and phosphoinositol-containing head groups. Antimicrob. Agents Chemother. 44, 1174–1180.