Structural and Functional Studies of a Phosphatidic Acid-Binding Antifungal Plant Defensin MtDef4: Identification of an RGFRRR Motif Governing Fungal Cell Entry

Abstract

MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.

The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

“…Besides phospholipid-binding antibodies, innate defense molecules can also target the altered cell surface phospholipid profile of tumor cells. Cationic antimicrobial peptides (CAPs) are a class of small (o5 kDa) positively charged innate defense molecules that act primarily to destabilize or penetrate the membrane of microbial cells, in some cases via interactions with specific phospholipids 47,48 (functioning as phospholipid detectors). Interestingly, CAPs originated from different species including plants, insects, amphibians, mammals have been shown to possess antiproliferative activity towards mammalian tumor cells both in vitro and in vivo (reviewed in detail by Riedl et al 49 ).…”

“…The antifungal activity of MtDef4 was proposed to be mediated through an interaction between a cationic loop region of the defensin and PA on the fungal hyphal membrane. 48 Likewise, the aforementioned ornamental tobacco defensin, NaD1, which binds PI(4,5)P 2 as well as a range of other phosphorylated phosphatidylinositol species, also exhibits antifungal activity against filamentous fungi, in part mediated by phospholipid binding. 69 NaD1 was solved by X-ray crystallography in complex with PI(4,5)P 2 , revealing the formation of a dimeric 'cationic grip' PI(4,5)P 2 binding pocket, repeated to form an extended oligomeric arch.…”

The Tomato Defensin TPP3 Binds Phosphatidylinositol (4,5)-Bisphosphate via a Conserved Dimeric Cationic Grip Conformation To Mediate Cell Lysis

“…Similarly, membrane permeabilization of Saccharomyces cerevisiae by the dahlia defensin DmAMP1 depends on expression of mannosyldiinositolphosphorylceramide (10). More recently, binding to phosphatidic acid (PA) has been implicated in the membrane-permeabilizing activity of MtDef4 on Fusarium graminearum (11). Despite the identification of these lipid ligands for defensins, the molecular basis of the interactions remains poorly defined.…”

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).…”