For example, RsAFP2 from Raphanus sativus and DmAMP1 from Dahlia merckii bind to distinct sphingolipids in membranes of fungi and this AG-013736 VEGFR/PDGFR inhibitor interaction with sphingolipids is required for their antifungal activity. Other plant defensins like MsDef1 and ZmES4 likely act on ion channels. MsDef1, a 45-amino acid protein from the seed of (+)-JQ1 Medicago sativa, inhibits the growth of a filamentous fungus, Fusarium graminearum, at micromolar concentrations. MtDef4 is a 47-amino acid protein that is expressed constitutively and in response to biotic and abiotic stress in many tissues of a model legume, M. truncatula. Based on their effects on the morphology of fungal hyphae, antifungal plant defensins are divided into two different subgroups, referred to as morphogenic and nonmorphogenic. Morphogenic defensins inhibit hyphal growth with a concomitant increase in hyphal branching, whereas nonmorphogenic defensins inhibit hyphal growth without causing marked morphological alterations. MsDef1 is a morphogenic defensin that induces extensive hyperbranching of fungal hyphae, whereas MtDef4 is a nonmorphogenic defensin that does not induce hyperbranching. MtDef4 is more potent against F. graminearum than MsDef1. Two lines of evidence indicate that MsDef1 and MtDef4 have different modes of antifungal action. First, insertional mutants of F. graminearum that were isolated as being hypersensitive to MsDef1 exhibit no change in their sensitivity to MtDef4. The analysis of these mutants revealed two mitogen-activated protein kinase signaling cascades that were required for the protection of the fungus from the toxic effects of MsDef1. Second, a mutant depleted in the plasma membrane sphingolipid glucosylceramide, designated DFggcs1, was found to be highly resistant to MsDef1, but retained the wild-type sensitivity to MtDef4. Little is known about the structural determinants of the in vitro antifungal activity of MsDef1 and MtDef4. Since all plant defensins whose 3-D structures have been determined have a similar backbone, any differences in their antifungal activities and specificities are likely to arise primarily from differences in the amino acid composition and charge distribution of solventexposed loops. The calculated net positive charge of +6 for MtDef4 is significantly higher than the calculated net positive charge of +3 for MsDef1. Also, the predicted solvent exposed ccore of MtDef4 has significantly higher positive charge than that of MsDef1. We previously reported that the carboxyterminal amino acid sequence was a major determinant of the in vitro antifungal activity of MsDef1 and that R38Q mutation significantly reduced its antifungal activity. This sequence spans the b2 and b3 strands and the interposed loop on the homology-based 3-D structure of MsDef1, previously referred to as AlfAFP.