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.
Highlights
Defensins are small cysteine-rich proteins present in all plants and constitute an ancient and diverse set of natural antimicrobial proteins
We sought to determine if MtDef4 is internalized by F. graminearum cells using immunogold labeling and transmission electron microscopy (TEM) of freeze substituted material
TEM immunogold localization experiments as well as confocal microscopy of fungal cells treated with fluorescently-labeled MtDef4 have revealed that MtDef4 is internalized by F. graminearum cells and dispersed in the cytoplasm
Summary
Defensins are small cysteine-rich proteins present in all plants and constitute an ancient and diverse set of natural antimicrobial proteins. Structural features of plant defensins are highly conserved, a comparison of their primary amino acid sequences reveals a rich diversity of variants [2,3]. This sequence diversity is responsible for the functional diversity observed in defensins [4,5]. Morphogenic defensins inhibit hyphal elongation with a concomitant increase in hyphal branching, whereas nonmorphogenic defensins reduce hyphal elongation without causing significant morphological changes [6,7] Because of their potent antifungal activity, defensins have been widely exploited in agrobiotechnological applications to generate disease resistant crops. In order to fully harness the potential of these proteins for bioengineering crops with robust resistance to fungal and oomycete pathogens, it is essential to understand their structure-activity relationships and modes of antifungal action
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