However, as shown inFigures 8A and S1, some sequence flexibility exists in this motif. 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 UK 370106 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. == Introduction == Defensins are small cysteine-rich proteins present in all plants and constitute an ancient and diverse set of natural antimicrobial proteins. These 45- to 54-residue proteins usually contain four intramolecular disulfide bonds and adopt a similar cysteine-stabilized / (CS/) motif in which one -helix is stabilized through disulfide bridging to a three-strand antiparallel -sheet [1,2]. Because plant defensins lack a distinct hydrophobic core, the C10rf4 protein fold is stabilized primarily by the disulfide bonds. Although 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]. Several plant defensins exhibit antifungal activityin vitroat low micromolar concentrations, [2-4]. These defensins also display differing antifungal properties. 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. Indeed, transgenic plants overexpressing defensins exhibit resistance to a range of fungal and oomycete pathogens [8]. In order to fully harness the potential of these proteins for bioengineering crops with robust resistance to fungal and oomycete pathogens, it UK 370106 is essential to understand their structure-activity relationships and modes of antifungal action. Nearly two decades of studies have revealed that plant defensins interact with fungal-specific cell wall and plasma membrane components and inhibit fungal growth from the extracellular or intracellular side of fungal cells [9,10]. For example,Raphanus sativusAFP2 (RsAFP2) andDahlia merckiiAMP1 (DmAMP1) bind with high affinity to UK 370106 distinct sphingolipids present in the plasma membrane or cell wall of their target fungi. Such interactions are a prerequisite for antifungal activity [11-13]. RsAFP2 interacts with glucosylceramide (GlcCer) present in the cell wall causing accumulation of apoptosis-inducing ceramides [14], whereas DmAMP1 binds specifically to mannosyl diinositolphosphoryl ceramide present in the plasma membrane [15].Medicago sativadefensin 1 (MsDef1) is also thought to interact with GlcCer since aFusarium graminearum mutant Fggcs1lacking GlcCer exhibits strong resistance to this defensin [16]. In some instances, a defensin must be internalized by fungal cells after cell wall binding to cause cell death [17,18]. For Nicotiana alata defensin 1 (NaD1), this process is mediated by the premeabilization of fungal hyphae using a cell wall-dependent process [19]. Once inside fungal cells, these defensins likely target intracellular processes critical for fungal growth. For example,Pisum sativumdefensin 1 (Psd1) is active against the model filamentous fungusNeurospora crassaand once inside the cells of this fungus it interacts with a nuclear cyclin-like protein involved in cell-cycle control and co-localizes in the nuclei [17]. The precise mechanisms of the antifungal action of defensins capable of entry into fungal cells are still not fully understood and the sequence motifs mediating fungal cell entry of these defensins remain to be identified. To date, all known antifungal plant defensins containing disulfide bonds possess a highly conserved -core motif (GXCX39C, where X is any amino acid) consisting of 2 and 3 strands with an interposed loop that carries a net positive charge and participates in one to four disulfide bonds [20]. Structure-activity studies indicate that the major determinants of antifungal activity reside in the -core motif [21,22]. MtDef4 is an evolutionarily conserved defensin fromM. truncatulaand its homologs are found in all plants examined to date [22,23]. MtDef4 carries a net positive UK 370106 charge of + 6. It inhibits the growth of several filamentous fungi includingF. graminearumat micromolar concentrations. Since it does not induce hyperbranching of fungal hyphae, it is thought to be a nonmorphogenic defensin [24]. Homology-based structure of MtDef4 predicts a -core motif GRCRGFRRRC composed of the 2 2 and 3 strands connected by the RGFRRR loop. When the -core.