Modes of Action and In Planta Antifungal Activity of <i>Olea europaea</i> Defensin OefDef1.1-Derived Peptide Variant

Peptide-based biopesticides represent a promising strategy for sustainable disease control in agriculture. Synthetic antifungal peptides incorporating the γ-core motif of plant defensins offer multiple modes of action (MoA) and potential as biofungicides. We investigated a synthetic short-chain variant of the olive defensin OefDef1.1 for antifungal activity, structure-function relationships, and MoA against <i>Botrytis cinerea</i>, a necrotrophic pathogen causing gray mold disease in fruits and vegetables. A disulfide-bridged peptide, GMAOe1C_V1*, derived from OefDef1.1 (G32-Y53) modified with hydrophobic amino acid substitutions inhibited <i>B. cinerea</i> growth in vitro and reduced lesion formation in detached leaves. Foliar application of GMAOe1C_V1* suppressed disease symptoms in pepper plants. Mechanistically, GMAOe1C_V1* rapidly permeabilized fungal plasma membrane and accumulated in vacuole, triggering vacuolar expansion and cell death. It also inhibited protein synthesis in vitro and in vivo, suggesting a role as a translation inhibitor. Alanine scanning mutagenesis of the non-disulfide bridged variant identified the ⁷RHSKH¹¹ motif as essential for antifungal activity. Circular dichroism revealed an unstructured conformation with minimal secondary structure. Transcriptomic analysis of GMAOe1C_V1* treated <i>B. cinerea</i> showed downregulation of genes involved in mitochondrial function and amino acid biosynthesis. These findings demonstrate the potential of an olive defensin-derived peptide as a bio-inspired antifungal agent with multi-faceted MoA for crop protection.