Summary: Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of a fungal pathogen, Fusarium graminearum, at micromolar concentrations. However, molecular mechanisms by which they inhibit the growth of this fungus are not known. We have characterized a functional role of the fungal sphingolipid glucosylceramide in regulating sensitivity of the fungus to MsDef1 and MtDef4. A null mutation of the FgGCS1 gene encoding glucosylceramide synthase results in a mutant lacking glucosylceramide. The ΔFggcs1‐null mutant becomes resistant to MsDef1, but not to MtDef4. It shows a significant change in the conidial morphology and displays dramatic polar growth defect, and its mycelia are resistant to cell wall degrading enzymes. Contrary to its essential role in the pathogenicity of a human fungal pathogen, Cryptococcus neoformans, GCS1 is not required for the pathogenicity of F. graminearum. The ΔFggcs1 mutant successfully colonizes wheat heads and corn silk, but its ability to spread in these tissues is significantly reduced as compared with the wild‐type PH‐1 strain. In contrast, it retains full virulence on tomato fruits and Arabidopsis thaliana floral and foliar tissues. Based on our findings, we conclude that glucosylceramide is essential for MsDef1‐mediated growth inhibition of F. graminearum, but its role in fungal pathogenesis is host‐dependent.

Antifungal defensins, MsDef1 and MtDef4, from Medicago spp., inhibit the growth of Fusarium graminearum, which causes head blight disease in cereals. In order to determine the signalling cascades that are modulated by these defensins, we have isolated several insertional mutants of F. graminearum that exhibit hypersensitivity to MsDef1, but not to MtDef4. The molecular characterization of two of these mutants, designated enhanced sensitivity to defensin (esd), has revealed that the Mgv1 and Gpmk1 MAP kinase signalling cascades play a major role in regulating sensitivity of F. graminearum to MsDef1, but not to MtDef4. The Hog1 MAP kinase signalling cascade, which is responsible for adaptation of this fungus to hyperosmotic stress, does not participate in the fungal response to these defensins. Significantly, the esd mutants also exhibit hypersensitivity to other tested defensins and are highly compromised in their pathogenesis on wheat heads and tomato fruits. The studies reported here for the first time implicate two MAP kinase signalling cascades in a plant defensin-mediated alteration of fungal growth. Based on our findings, we propose that specific MAP kinase signalling cascades are essential for protection of a fungal pathogen from the antimicrobial proteins of its host plant.

The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogenFusarium graminearumand complement aPichia pastorisC-9-MT-null mutant. The ΔFgmt1mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungusP. pastoris. The ΔFgmt2mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms inArabidopsis thaliana. Surprisingly, the ΔFgmt2mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1and ΔFgmt2mutants, as well as theP. pastorisstrain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.