• Plant defensin;
  • Sphingolipid;
  • Antifungal;
  • Mode of action;
  • Glycosylphosphatidylinositol-anchored protein;
  • Saccharomyces cerevisiae


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

DmAMP1, an antifungal plant defensin from Dahlia merckii, was shown previously to require the presence of sphingolipids for fungicidal action against Saccharomyces cerevisiae. Sphingolipids may stabilize glycosylphosphatidylinositol (GPI)-anchored proteins, which interact with DmAMP1, or they may directly serve as DmAMP1 binding sites. In the present study, we demonstrate that S. cerevisiae disruptants in GPI-anchored proteins showed small or no increased resistance towards DmAMP1 indicating no involvement of these proteins in DmAMP1 action. Further, studies using an enzyme-linked immunosorbent assay (ELISA)-based binding assay revealed that DmAMP1 interacts directly with sphingolipids isolated from S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol. Therefore, DmAMP1 antifungal action involving membrane interaction with sphingolipids and ergosterol is proposed.






  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Plant defensins are small (45–54 amino acid residues), highly basic, cysteine-rich peptides that possess antifungal and/or antibacterial activity at micromolar concentrations (reviewed in [1,2]). They are active against a broad range of phytopathogenic fungi and human pathogens (such as Candida albicans) [3]. Plant defensins appear not to be toxic to either mammalian or plant cells. Experimental evidence points towards a role of the plant defensins in defending the host plant from fungal attack (reviewed in [1,4]). Up to date, more than 80 different defensins have been identified in 38 different plant species belonging to various plant families [1]. The global fold of plant defensins comprises a cysteine-stabilized αβ motif (CSαβ motif) consisting of an α-helix and a triple-stranded β-sheet, organized in a βαββ architecture and stabilized by four disulfide bridges. Regarding amino acid composition, the plant defensin family is quite diverse; sequence conservation is restricted to eight structurally important cysteines.

DmAMP1, a plant defensin isolated from seeds of dahlia (Dahlia merckii), induces an array of relatively rapid responses in fungal membranes, including increased K+ efflux, increased Ca2+ uptake, increased uptake of fluorescent dyes, and membrane potential changes [5,6]. Furthermore, the existence of high-affinity binding sites for DmAMP1 on fungal cells and plasma membrane fractions was demonstrated [7]. Via a genetic complementation approach, IPT1 was identified as a gene determining sensitivity towards DmAMP1 in Saccharomyces cerevisiae[8]. IPT1 encodes an enzyme involved in the last step of the synthesis of the sphingolipid mannosyldiinositolphosphorylceramide (M(IP)2C) [9]. M(IP)2C, mannosylinositolphosphorylceramide (MIPC) and inositolphosphorylceramide (IPC) are the three major classes of sphingolipids in S. cerevisiae. S. cerevisiae strains with a non-functional IPT1 allele lacked M(IP)2C in their membranes, bound significantly less DmAMP1 compared to parental S. cerevisiae strain, and were highly resistant to DmAMP1-induced membrane permeabilization [8]. Recently, we have shown that DmAMP1 sensitivity is not linked with the presence of a functional IPT1-encoding protein (Ipt1p) but with the presence of M(IP)2C in the fungal plasma membrane [8,10]. Possibly, membrane patches containing sphingolipids act as binding sites for DmAMP1, or alternatively, are required to anchor membrane or cell wall-associated proteins that interact with DmAMP1. Subsequently, this interaction could lead to insertion of DmAMP1 into the membrane resulting in membrane destabilization.

Sphingolipids associate with sterols in the plasma membrane to form patches (also referred to as rafts) that are highly enriched in glycosylphosphatidylinositol (GPI)-anchored membrane proteins [11]. In this study, we investigated whether such GPI-anchored proteins are involved in constituting the binding site for DmAMP1 or whether DmAMP1 interacts directly with the S. cerevisiae sphingolipids. Finally, we investigated a possible effect of ergosterol, the main fungal sterol, on the interaction between DmAMP1 and yeast sphingolipids.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

2.1Materials and microorganisms

DmAMP1 was isolated as described previously [12]. Anti-DmAMP1 serum from rabbit was purchased from Eurogentec (Seraing, Belgium). Phosphatase-coupled goat anti-rabbit immunoglobulins, bovine serum albumin (BSA) and ergosterol were purchased from Sigma (St. Louis, MO, USA). Yeast strains used in this study were S. cerevisiae parental strain BY4741 (Invitrogen, Carlsbad, CA, USA) and S. cerevisiae BY4741 disruption mutants (Euroscarf, Frankfurt, Germany) in the following genes encoding non-essential GPI-anchored proteins: YBR067c, YER011w, YIL011w, YJR150c (CCW13), YKL096w (CWP1), YKL096w-a (CWP2), YLR040c, YOR009w, YOR010c, YAR050w, YKR102w, YIR019c, YDR077w, YER150w, YEL040w, YGR189c, YJR004c, YNR044w, YCR089w, YHR126c, YJL171c, YJL078c, YKL046c (DCW1), YLR042c, YLR110c (CCW12), YLR194c, YLR391w-a (CCW14), YNL300w, YNL327w, YOL155c, YOR214c, YOR382w, YOR383c, YPL130w, YMR307w, YLR343w, YMR215w, YOL132w, YOL030w, YDR144c, YDR349c, YLR120c, YLR121c, YBR078w, YCL048w, YDR055w, YDR522c, YMR006c, YMR008c, YDR261c, YMR200w, YNL190w, YNL322c, YPL261c. S. cerevisiae parental strain SEY6210 and corresponding double and triple yeast disruption mutants Δccw12Δccw13 and Δccw12Δccw13Δccw14 were kindly provided by Prof. Widmar Tanner (Universität Regensburg, Regensburg, Germany).

2.2Antifungal activity assay

Antifungal activity of DmAMP1 against yeast strains was assayed by microscopic analysis of liquid cultures grown in microtiter plates as described previously [7].

2.3Isolation of S. cerevisiae sphingolipids

S. cerevisiae sphingolipids were prepared from commercial freeze-dried yeast (Saf-instant, SAFMEX S.A. DE C.V., Mexico) using the procedures of Smith and Lester [13]. After hydration (24 g dry weight of cells per 100 ml water), treatment with 5% trichloroacetic acid (15 min, room temperature), washing the precipitate (by centrifugation with 0.5% KH2PO4), and heating (95°C, 7 min), the lipids were extracted with 95% ethanol:diethylether:pyridine (15:5:1 vol per vol) and deacylated (glacial acetic acid, pH 5.5). A mixture of sphingolipids composed of IPC, MIPC, and M(IP)2C was purified from the extract of non-deacylated lipids using the alternative method of Smith and Lester [13] by methanol precipitation for several days followed by Chelex 100 (Sigma Chem. Co, St. Louis, MO, USA) chromatography. Purity and composition were analyzed by silica gel thin-layer chromatography as described by Im et al. [10], and typical preparations showed approximately equal proportions of IPC, MIPC, and M(IP)2C as judged by orcinol-H2SO4 staining of thin-layer chromatographic plates [13].

2.4Construction of S. cerevisiae double deletion mutants in GPI-anchored proteins

Gene disruption of CWP2 and YHR126 in both Δcwp1 and Δykl046c S. cerevisiae deletion mutants was accomplished by replacing the coding region of CWP2 and YHR126 with the URA auxotrophic marker gene as described by Brachmann et al. [27], resulting in four double deletion mutants, namely Δcwp1Δcwp2, Δykl046cΔcwp2, Δcwp1Δyhr126c and Δykl046cΔyhr126c. Correct gene transplacement was verified by polymerase chain reaction (PCR).

2.5Microtiter plate binding assay (enzyme-linked immunosorbent assay (ELISA))

Interaction of DmAMP1 with sphingolipids was evaluated by using an ELISA-based assay as described previously [3,14–16]. Stock solutions of sphingolipids and ergosterol were prepared in methanol:chloroform:water (16:16:5; vol:vol:vol) at a concentration of 1 mM. Lipids were applied in 75-μl aliquots to the wells of microtiter plates and allowed to dry overnight at room temperature. All subsequent handling steps were performed at 37°C. Blocking buffer was 3% (w/v) BSA in phosphate-buffered saline (PBS) and washing buffer was 10% blocking buffer. Anti-DmAMP1 rabbit antiserum and phosphatase-coupled goat anti-rabbit immunoglobulin were 1000-fold diluted in washing buffer. Plotted values are means of triplicates adjusted for the plate background. Plate background values are the absorbance readings of methanol:chloroform:water (16:16:5; vol:vol:vol)-coated wells incubated with DmAMP1 and antisera.


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

3.1Involvement of GPI-anchored proteins in constituting the DmAMP1 interaction site

In a first instance, we investigated the possibility that a member of the group of so-called GPI-anchored proteins might constitute the DmAMP1 binding site, or at least might be involved in DmAMP1 activity. Use of the Von Heijne algorithm and homology searches allowed the identification of 58 open reading frames (ORFs) in the genome of S. cerevisiae that encode proteins with a putative GPI attachment signal in their signal sequences [26]. S. cerevisiae disruptants in the 54 non-essential plasma membrane and cell wall GPI-anchored proteins were tested for increased resistance towards DmAMP1. S. cerevisiae disruptants in GPI-anchored cell wall proteins (GPI-CWPs) Δyhr126c and Δykl046c showed a 2-fold increase in DmAMP1 resistance (data not shown). Besides Δyhr126c and Δykl046c, none of these yeast deletion mutants showed an increased level of resistance against DmAMP1 as compared to S. cerevisiae parental strain BY4741 (data not shown). It therefore seems unlikely that a specific GPI-anchored protein acts as a docking site for DmAMP1, facilitating its insertion into the plasma membrane. However, a remaining possibility is that DmAMP1 interacts with a common constituent, present on several GPI-anchored proteins. To test this hypothesis, various double deletion mutants in non-essential GPI-CWPs were constructed. Since (i) CWP1 and CWP2 are the most abundant GPI-CWPs in S. cerevisiae[17] and (ii) based on the above-mentioned results on a putative involvement of YHR126c and YKL046c in DmAMP1 sensitivity, four S. cerevisiae double deletion mutants were generated, namely Δcwp1Δcwp2, Δykl046cΔcwp2, Δcwp1Δyhr126c and Δykl046cΔyhr126c, and tested for increased DmAMP1 resistance. In addition, DmAMP1 resistance of a double (Δccw12Δccw13) and triple (Δccw12Δccw13Δccw14) S. cerevisiae deletion mutant in mannosylated GPI-CWPs [18] was assessed (Table 1). However, none of these S. cerevisiae deletion mutants showed more than 2-fold increased DmAMP1 resistance as compared to the corresponding S. cerevisiae parental strains. These data indicate that none of the tested GPI-anchored proteins plays a principal role in the fungal growth inhibiting process by DmAMP1.

Table 1.  DmAMP1 sensitivity of double and triple yeast deletion mutants in GPI-CWPs
  1. IC50 values are means of triplicate measurements. Standard errors were typically below 6.5%. Parental strains are in bold.

  2. aConcentration of DmAMP1 required to inhibit 50% yeast growth.

Yeast strain/mutantIC50 (μM)aReference/origin
S. cerevisiae BY47412.0Invitrogen
Δcwp1Δcwp22.0this study
Δykl046cΔcwp23.5this study
Δcwp1Δyhr126c3.5this study
Δykl046cΔyhr126c4.0this study
S. cerevisiae SEY62102.0[25]

3.2Interaction of DmAMP1 with sphingolipids isolated from S. cerevisiae

To get more insight in the involvement of fungal sphingolipids in DmAMP1-mediated growth inhibition, the interaction between DmAMP1 and sphingolipids isolated from S. cerevisiae was assessed. To this end, we developed an ELISA-based binding assay in which sphingolipids are coated to the wells of microtiter plates and interacting peptides are detected immunologically [3,14–16].

Using the ELISA-based binding assay, DmAMP1 was found to interact in a dose-dependent manner with purified sphingolipids from S. cerevisiae. Saturability of the interaction of DmAMP1 with 15 pmol of coated sphingolipids occurred at a DmAMP1 concentration of 1 μM (which corresponds to 75 pmol DmAMP1 per well, a 5-fold molar DmAMP1 excess) (Fig. 1A). The interaction of 1 μM DmAMP1 with different amounts of coated sphingolipids is presented in Fig. 1B. Optimal interaction of 1 μM DmAMP1 with fungal sphingolipids was observed at 15 pmol of sphingolipids coated per well.


Figure 1. Interaction of DmAMP1 with S. cerevisiae sphingolipids. Dose–response curves are presented for the interaction of A: DmAMP1 with 15 pmol of coated sphingolipids and B: 1 μM DmAMP1 with different concentrations of sphingolipids. Data are means±S.E.M. of triplicates.

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Since sphingolipids are associated with sterols in the plasma membrane to form patches, we further investigated the effect of ergosterol, the main fungal sterol, on the interaction of DmAMP1 with fungal sphingolipids. In a first instance, interaction of 1 μM DmAMP1 with different concentrations of ergosterol (ranging from 1 pmol to 1 nmol coated per well) was assessed. In these conditions, no interaction of DmAMP1 with ergosterol could be observed (data not shown). Furthermore, interaction of 1 μM DmAMP1 with lipid mixtures consisting of 15 pmol sphingolipids and different amounts of ergosterol was assessed. As can be seen in Fig. 2, addition of ergosterol at concentrations ranging from 7.5 to 150 pmol (corresponding to 25 to 90 mol% ergosterol) increased the interaction of DmAMP1 with sphingolipids with 30–50%, with a maximal effect at equimolar concentrations of sphingolipids and ergosterol.


Figure 2. Effect of ergosterol on the interaction of DmAMP1 with S. cerevisiae sphingolipids. A dose–response curve is presented for the interaction of 1 μM DmAMP1 with 15 pmol of coated sphingolipids, in the presence of different concentrations of ergosterol. Data are means±S.E.M. of triplicates.

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References

Our studies show that DmAMP1 interacts in a dose-dependent manner with sphingolipids isolated from the plasma membrane of S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol, the main fungal sterol. These findings point towards a direct role of sphingolipids in the fungal growth inhibiting process by DmAMP1. Sphingolipids are, together with sterols and phospholipids, one of the three major classes of eukaryotic membrane components. The composition of the plasma membrane of fungal cells is asymmetric, which is typical of eukaryotic cells, with phosphatidylserine mainly in the inner leaflet, and sterols and sphingolipids in the outer leaflet [19]. The ergosterol to sphingolipid ratio of the yeast plasma membrane can be estimated at 1.4 [19]. We have demonstrated optimal DmAMP1 interaction with an equimolar mixture of ergosterol and yeast sphingolipids, reflecting the in vivo yeast plasma membrane composition. It has been shown that sphingolipids and sterols are enriched in specific domains in the outer plasma membrane, the so-called membrane rafts [11,20]. Possibly, DmAMP1 interacts with fungal sphingolipids, which are concentrated in such specific rafts. Interaction of DmAMP1 with these rafts could result in high local concentrations of these membrane-bound defensins. Whether a threshold concentration of membrane-bound DmAMP1 is a prerequisite for membrane disruption, as hypothesized for the structurally related human β-defensins [21], remains unclear. After interaction with the rafts, DmAMP1 is supposed to insert into the plasma membrane, resulting in membrane permeabilization. Whether fungal growth arrest is a direct consequence of increased membrane permeability or results from interaction of DmAMP1 with an intracellular target is currently under investigation.

Similarly to DmAMP1, a plant defensin from radish (RsAFP2) has been shown to directly interact with fungal glucosylceramides [3]. Besides sphingolipids, such as IPC, MIPC and M(IP)2C, glucosylceramides are another class of sphingolipids that are present in membranes of most fungi, except in S. cerevisiae. There is growing evidence that fungi maintain two separate pools of ceramides to be used for the synthesis of different sphingolipids [22]. Ceramide backbones with very long chain C24 and C26 fatty acids bound to the sphingobase 4-hydroxysphinganine are directed to the synthesis of the inositolphosphoryl-containing sphingolipids, whereas ceramide backbones with C16 or C18 fatty acids linked to the sphingobase 9-methyl-4,8-sphingadienine are exclusively used as precursors for biosynthesis of glucosylceramide. As such, RsAFP2 and DmAMP1 represent two groups of plant defensins with regard to target specificity on the fungal membrane.

Interestingly, it has recently been shown that sphingolipids are important pathogenicity determinants. A Cryptococcus neoformans mutant with lowered levels of sphingolipids showed reduced pathogenicity in a rabbit model [23]. The finding that DmAMP1 is active against the human pathogen C. albicans[3,24] and that it may target potential pathogenicity factors present in most fungi, opens interesting perspectives for the development of novel antimycotics [24].


  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. References
  • [1]
    Thomma, B.P.H.J., Cammue, B.P.A., Thevissen, K. (2002) Plant defensins. Planta 216, 193202.
  • [2]
    Broekaert, W.F., Cammue, B.P.A., De Bolle, M.F.C., Thevissen, K., De Samblanx, G.W., Osborn, R.W. (1997) Antimicrobial peptides from plants. Crit. Rev. Plant Sci. 16, 297323.
  • [3]
    Thevissen, K., Warnecke, D., 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. (2003) Fungal glucosylceramides constitute the binding site for plant and insect defensins. (submitted).
  • [4]
    Thevissen, K., Ferket, K.K.A., Francois, I.E.J.A. and Cammue, B.P.A. (2003) Interactions of antifungal plant peptides with fungal membrane components. Peptides (in press).
  • [5]
    Thevissen, K., Ghazi, A., De Samblanx, G.W., Brownlee, C., Osborn, R.W., Broekaert, W.F. (1996) Fungal membrane responses induced by plant defensins and thionins. J. Biol. Chem. 271, 1501815025.
  • [6]
    Thevissen, K., Terras, F.R., Broekaert, W.F. (1999) Permeabilization of fungal membranes by plant defensins inhibits fungal growth. Appl. Environ. Microbiol. 65, 54515458.
  • [7]
    Thevissen, K., Osborn, R.W., Acland, D.P., 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, 5461.
  • [8]
    Thevissen, K., Cammue, B.P., Lemaire, K., Winderickx, J., Dickson, R.C., Lester, R.L., Ferket, K.K., Van Even, F., Parret, A.H., Broekaert, W.F. (2000) 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, 95319536.
  • [9]
    Dickson, R.C., Nagiec, E.E., Wells, G.B., Nagiec, M.M., 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, 2962029625.
  • [10]
    Im, Y.J., Idkowiak-Baldys, J., Thevissen, K., Cammue, B.P.A., Takemoto, J.Y. (2003) IPT1-Independent sphingolipid biosynthesis and yeast inhibition by syringomycin E and plant defensin DmAMP1. FEMS Microbiol. Lett. 223, 199203.
  • [11]
    Bagnat, M., Keranen, S., Shevchenko, A., Shevchenko, A., Simons, K. (2000) Lipid rafts function in biosynthetic delivery of proteins to the cell surface in yeast. Proc. Natl. Acad. Sci. USA 97, 32543259.
  • [12]
    Osborn, R.W., De Samblanx, G.W., Thevissen, K., Goderis, I., Torrekens, S., Van Leuven, F., Attenborough, S., Rees, S.B., Broekaert, W.F. (1995) Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae. FEBS Lett. 368, 257262.
  • [13]
    Smith, S.W., Lester, R.L. (1974) Inositol phosphorylceramide, a novel substance and the chief member of a major group of yeast sphingolipids containing a single inositol phosphate. J. Biol. Chem. 249, 33953405.
  • [14]
    Mamelak, D., Lingwood, C. (2001) The ATPase domain of hsp70 possesses a unique binding specificity for 3′-sulfogalactolipids. J. Biol. Chem. 276, 449456.
  • [15]
    Mylvaganam, M., Binnington, B., Hansen, H.C., Magnusson, G., Nyholm, P.G., Lingwood, C.A. (2002) Interaction of verotoxin 1 B subunit with soluble aminodeoxy analogs of globotriaosyl ceramides. Biochem. J. 368, 769776.
  • [16]
    Mamelak, D., Mylvaganam, M., Tanahashi, E., Ito, H., Ishida, H., Kiso, M., Lingwood, C. (2001) The aglycone of sulfogalactolipids can alter the sulfate ester substitution position required for hsc70 recognition. Carbohydr. Res. 335, 91100.
  • [17]
    van der Vaart, J.M., Caro, L.H., Chapman, J.W., Klis, F.M., Verrips, C.T. (1995) Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J. Bacteriol. 177, 31043110.
  • [18]
    Mrsa, V., Ecker, M., Strahl-Bolsinger, S., Nimtz, M., Lehle, L., Tanner, W. (1999) Deletion of new covalently linked cell wall glycoproteins alters the electrophoretic mobility of phosphorylated wall components of Saccharomyces cerevisiae. J. Bacteriol. 181, 30763086.
  • [19]
    Schneiter, R., Brugger, B., Sandhoff, R., Zellnig, G., Leber, A., Lampl, M., Athenstaedt, K., Hrastnik, C., Eder, S., Daum, G., Paltauf, F., Wieland, F.T., Kohlwein, S.D. (1999) Electrospray ionization tandem mass spectrometry (ESI-MS/MS) analysis of the lipid molecular species composition of yeast subcellular membranes reveals acyl chain-based sorting/remodeling of distinct molecular species en route to the plasma membrane. J. Cell Biol. 146, 741754.
  • [20]
    Xu, X., Bittman, R., Duportail, G., Heissler, D., Vilcheze, C., London, E. (2001) Effect of the structure of natural sterols and sphingolipids on the formation of ordered sphingolipid/sterol domains (rafts). Comparison of cholesterol to plant, fungal, and disease-associated sterols and comparison of sphingomyelin, cerebrosides, and ceramide. J. Biol. Chem. 276, 3354033546.
  • [21]
    Hoover, D.M., Rajashankar, K.R., Blumenthal, R., Puri, A., Oppenheim, J.J., Chertov, O., Lubkowski, J. (2000) The structure of human beta-defensin-2 shows evidence of higher order oligomerization. J. Biol. Chem. 275, 3291132918.
  • [22]
    Leipelt, M., Warnecke, D., Zahringer, U., Ott, C., Muller, F., Hube, B., Heinz, E. (2001) Glucosylceramide synthases, a gene family responsible for the biosynthesis of glucosphingolipids in animals, plants, and fungi. J. Biol. Chem. 276, 3362133629.
  • [23]
    Luberto, C., Toffaletti, D.L., Wills, E.A., Tucker, S.C., Casadevall, A., Perfect, J.R., Hannun, Y.A., Del Poeta, M.M. (2001) Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev. 15, 201212.
  • [24]
    Thomma, B.P.H.J., Cammue, B.P.A., Thevissen, K. (2003) Mode of action of plant defensins suggests therapeutic potential. Curr. Drug Targ. Infect. Disord. 3, 18.
  • [25]
    Robinson, J.S., Klionsky, D.J., Banta, L.M., Emr, S.D. (1988) Protein sorting in Saccharomyces cerevisiae: isolation of mutants defective in the delivery and processing of multiple vacuolar hydrolases. Mol. Cell. Biol. 8, 49364948.
  • [26]
    Caro, L.H., Tettelin, H., Vossen, J.H., Ram, A.F., van den Ende, H., Klis, F.M. (1997) In silico identification of glycosyl-phosphatidylinositol-anchored plasma-membrane and cell wall proteins of Saccharomyces cerevisiae. Yeast 13, 14771489.
  • [27]
    Brachmann, C.B., Davies, A., Cost, G.J., Caputo, E., Li, J., Hieter, P., 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, 115132.