title: 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.
authors: Donald Danforth Plant Science Center, Saint Louis, Missouri, United States of America.
authors: Uma Shankar
authors: US
authors: Sagaram
authors: Kaoutar
authors: K
authors: El-Mounadi
authors: Garry W
authors: GW
authors: Buchko
authors: Howard R
authors: HR
authors: Berg
authors: Jagdeep
authors: J
authors: Kaur
authors: Raghu S
authors: RS
authors: Pandurangi
authors: Thomas J
authors: TJ
authors: Smith
authors: Dilip M
authors: DM
authors: Shah
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000935:Antifungal Agents / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D023082:Defensins / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D005670:Fusarium / Q000187:drug effects / Q000502:physiology / Q000648:ultrastructure; D046913:Medicago truncatula / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010712:Phosphatidic Acids / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D016415:Sequence Alignment; D055672:Static Electricity
chemical_list: D000596:Amino Acids; D000935:Antifungal Agents; D023082:Defensins; D010712:Phosphatidic Acids; D010940:Plant Proteins