Modes of action and bio-fungicide potential of peptides derived from the <i>Medicago truncatula</i> bi-domain defensin MtDef5

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