Plant defensin MtDef4-derived antifungal peptide with multiple modes of action and potential as a bioinspired fungicide

Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, mounting fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multi-site MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, causal agent of gray mold disease in fruits and vegetables. Its multi-site MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray-application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventative and curative suppression of gray mold disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development as bioinspired fungicides. Significance The rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has raised an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Short peptides containing the functionally active γ-core motif of antifungal defensins hold strong potential for development as efficacious, environmentally friendly biofungicides replacing or complementing chemical fungicides. A 17-amino acid pseudo-cyclic MtDef4 plant defensin-derived peptide exhibits potent antifungal activity and deploys multi-target modes of action against the gray mold fungal pathogen B. cinerea. Spray-application of this peptide confers strong preventative and curative antifungal activity against this pathogen in Nicotiana benthamiana and tomato plants. This work provides evidence for the enormous potential of defensin-derived peptides as bioinspired fungicides in agriculture.