SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis.

The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)(2)C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)(2)C-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 beta-1,6-glucan biosynthesis. We demonstrate that a Deltaskn1 mutant lacks M(IP)(2)C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)(2)C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae.

Neutral glycolipids of the filamentous fungus Neurospora crassa: altered expression in plant defensin-resistant mutants.

To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705-1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as beta-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta3-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-beta-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.

Defensins from insects and plants interact with fungal glucosylceramides.

Growth of the yeast species Candida albicans and Pichia pastoris is inhibited by RsAFP2, a plant defensin isolated from radish seed (Raphanus sativus), at micromolar concentrations. In contrast, gcs-deletion mutants of both yeast species are resistant toward RsAFP2. GCS genes encode UDP-glucose:ceramide glucosyltransferases, which catalyze the final step in the biosynthesis of the membrane lipid glucosylceramide. In an enzyme-linked immunosorbent assay-based binding assay, RsAFP2 was found to interact with glucosylceramides isolated from P. pastoris but not with soybean nor human glucosylceramides. Furthermore, the P. pastoris parental strain is sensitive toward RsAFP2-induced membrane permeabilization, whereas the corresponding gcs-deletion mutant is highly resistant to RsAFP2-mediated membrane permeabilization. A model for the mode of action of RsAFP2 is presented in which all of these findings are linked. Similarly to RsAFP2, heliomicin, a defensin-like peptide from the insect Heliothis virescens, is active on C. albicans and P. pastoris parental strains but displays no activity on the gcs-deletion mutants of both yeast species. Furthermore, heliomicin interacts with glucosylceramides isolated from P. pastoris and soybean but not with human glucosylceramides. These data indicate that structurally homologous anti-fungal peptides present in species from different eukaryotic kingdoms interact with the same target in the fungal plasma membrane, namely glucosylceramides, and as such support the hypothesis that defensins from plants and insects have evolved from a single precursor.

Isolation and characterization of Neurospora crassa mutants resistant to antifungal plant defensins.

Twenty-five Neurospora crassa mutants obtained by chemical mutagenesis were screened for increased resistance to various antifungal plant defensins. Plant defensin-resistant N. crassa mutants were further tested for their cross-resistance towards other families of structurally different antimicrobial peptides. Two N. crassa mutants, termed MUT16 and MUT24, displaying resistance towards all plant defensins tested but not to structurally different antimicrobial peptides were selected for further characterization. MUT16 and MUT24 were more resistant towards plant defensin-induced membrane permeabilization as compared to the N. crassa wild-type. Based on the previously demonstrated key role of fungal sphingolipids in the mechanism of growth inhibition by plant defensins, membrane sphingolipids of MUT16 and MUT24 were analysed. Membranes of these mutants contained structurally different glucosylceramides, novel glycosylinositolphosphorylceramides, and an altered level of steryl glucosides. Evidence is provided to link these clear differences in sphingolipid profiles of N. crassa mutants with their resistance towards different plant defensins.

DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae.

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.

Interactions of antifungal plant defensins with fungal membrane components.

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.