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

Medicago truncatula bi-domain defensin MtDef5 exhibits antifungal activity at sub-micromolar concentrations against some fungal pathogens. It comprises two single-domain defensins, MtDef5A and MtDef5B, connected with a linker APKKVEP. MtDef5B is a more potent antifungal defensin than 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 reactive oxygen species and traveled to the nucleoli in germlings of Bc . Furthermore, a carboxy-terminal MtDef5A-derived GMA5AC peptide was selected for mutagenesis because of its lower cationicity 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 multi-faceted MoA. Spray-application of GMA5AC_V2 on the leaves of pepper plants demonstrated preventive and curative 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. Highlights The bi-domain MtDef5-derived antifungal peptides exhibit multiple modes of action and confer resistance against the gray mold and anthracnose diseases in pepper plants and tomato fruits, respectively.