Abstract
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.
Highlights
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
The sensitivity of S. cerevisiae to DmAMP1, a defensin isolated from the seeds of Dahlia merckii [14], was shown to be dependent on IPT1 [15], a gene required for the final step in the biosynthesis of the complex yeast sphingolipid mannose-(inositol-phosphate)2-ceramide [16]
We isolated and characterized two neutral glycolipid components from N. crassa not previously identified in this species; more significantly, we detected changes in their expression patterns in mutant strains selected for resistance to a radish defensin, RsAFP2 [11]
Summary
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. 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 -glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Neutral glycolipids of the filamentous fungus Neurospora crassa: altered expression in plant defensin-resistant mutants. Plants possess an impressive arsenal of antimicrobial compounds that are either constitutively arrayed within certain tissues or synthesized in direct response to attack by pathogens Among these compounds are defensins, a class of evolutionarily and structurally related small, highly basic, cysteine-rich peptides, many of which display antifungal activity (reviewed in 1–3). The structural characterization of these neutral glycolipid components, isolated from wild-type and defensin-resistant mutant N. crassa strains, is described in this report
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