Modes of action and bio-fungicide potential of peptides derived from the bi-domain plant defensin MtDef5.

The Medicago truncatula bi-domain defensin MtDef5 exhibits activity at sub-micromolar concentrations against some fungal pathogens. It is comprised of two single-domain defensins, MtDef5A and MtDef5B, connected with the linker peptide APKKVEP. MtDef5B exhibits greater antifungal potency compared to MtDef5A. We identified amino acid residues important for antifungal activity of MtDef5B and elucidated its modes of action (MoA). MtDef5B inhibited spore germination of Botrytis cinerea (Bc) at low micromolar concentrations. However, it did not inhibit spore germination of Colletotrichum gloeosporioides (Cg). MtDef5B permeabilized the plasma membrane, induced the production of reactive oxygen species, and localized to the nucleoli in Bc germlings. The carboxy-terminal MtDef5A-derived GMA5AC peptide was selected for mutagenesis because of its lower cationic charge than the corresponding MtDef5B-derived peptide. GMA5AC inhibited spore germination of Bc, but not of Cg. However, GMA5AC_V2, a variant of GMA5AC, inhibited spore germination of Cg and exhibited a multi-faceted MoA. Spray-application of GMA5AC_V2 on the leaves of pepper plants resulted in pre- and post-inoculation control of the gray mold disease. Furthermore, when applied topically on tomato fruits pre-inoculated with the pathogen Cg, this peptide reduced anthracnose disease symptoms. This study highlights the potential of short chain defensin-derived peptides for management of fungal diseases.