Specific domains of plant defensins differentially disrupt colony initiation, cell fusion and calcium homeostasis in Neurospora crassa

Abstract

SummaryMsDef1 and MtDef4 from Medicago spp. are small cysteine-rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ-core motif (GXCX3-9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ-core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ-core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane/cell wall. Using N. crassa expressing the Ca 2+ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca 2+ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca 2+were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ-core motifs play important roles in their different modes of antifungal action.

D19S Mutation of the Cationic, Cysteine-Rich Protein PAF: Novel Insights into Its Structural Dynamics, Thermal Unfolding and Antifungal Function

“…However, PAF D19S acted fungicidal in a time dependent manner when applied at its MEC (32 μM) suggesting that fungal killing is mediated by other motifs that regulate mechanisms independently from the elevation of [Ca 2+ ] c . Our observation parallels with recent studies on plant defensins: by using defensin fragments amino acid sequences were identified that exhibit distinct antifungal features of their parental proteins [28]. …”

“…The specificity of the interference with divalent cations is further corroborated by the observation that monovalent cations like Na + had no/little effect on the antifungal activity of PAF and PAF D19S . Whereas the role of Mg 2+ ions in the activity of antifungal proteins awaits further investigations, the relevance of Ca 2+ ions in the mechanistic function of PAF and other related antifungal proteins and peptides was studied in detail [23,28,31,38] and let assume that their role goes beyond the disruption of electrostatic interactions. We assume that Ca 2+ ions may influence the PAF-response of the sensitive fungus in one or more of the following ways: (i) competition with PAF for binding to a Ca 2+ -sensitive interaction molecule, e.g.…”

“…After adding the antifungal proteins, a multimode plate reader (TriStar LB 941, Berthold Technologies) was used to measure bioluminescence at 25°C, over a time course of 60 min, taking measurements of the relative light units (RLUs) per well for 95 cycles (a cycle being the time it takes to measure all wells in the experiment). To convert relative light units (RLUs) into dynamic measurements of micromolar [Ca 2+ ] c in populations of conidial germlings during an experiment, an empirically derived equation was used as previously described [28,29]. …”

Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules

“…The different mechanisms of action of defensins can be explained by the variable primary sequences; the major determinants of the antifungal and morphogen activities of defensins reside in their g-core motifs [18,30]. MsDef1 (defensin 1.3 of M. sativa) and MtDef4 each contain a conserved g-core structural motif (GXCX 3-9 C, where X is any amino acid), which is a hallmark signature of disulfide-stabilized antimicrobial peptides [31].…”

The Plant Peptidome: An Expanding Repertoire of Structural Features and Biological Functions

“…Heterologous overexpression of various PDFs leads to increased resistance of both model plants and crops against different fungi and bacteria (Carvalho Ade and Gomes, 2011;De Coninck et al, 2013b;Gaspar et al, 2014). The modes of action of several PDFs, including radish (Raphanus sativus) AFP2, Nicotiana alata D1, alfalfa (Medicago sativa) Def1, M. truncatula Def4, and pea (Pisum sativum) d1, have been well studied and point toward specific interactions with various fungal sphingolipids and phospholipids (Thevissen et al, 2004(Thevissen et al, , 2012Aerts et al, 2007;Lobo et al, 2007;Ramamoorthy et al, 2007;van der Weerden et al, 2008van der Weerden et al, , 2010Sagaram et al, 2011Sagaram et al, , 2013Muñoz et al, 2014). Upon interaction, PDFs are either internalized by the fungal cell and interact with intracellular targets, or they stay outside the cell and induce cell death through induction of a signaling cascade (Vriens et al, 2014).…”