Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing 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 multisite 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 that 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, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the 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 preventive and curative suppression of grey mould 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 of bio‐inspired fungicides.

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 multiple MoA are promising starting templates for design of novel peptide based fungicides. Here, we designed a set of 17-amino acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide GMA4CG_V6 showed it had a random coil in solution with potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea. Its multi-site MoA involved plasma membrane permeabilization, internalization into the vacuole and cytoplasm, and binding to phosphoinositides. Topical spray-application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventative and curative suppression of gray mold disease symptoms. Our findings open the possibility that truncated and modified defensin-derived peptides could serve as promising candidates for further development as bioinspired fungicides.

Peroxisomes are single membrane-bound organelles that play a pivotal role in various developmental processes in all eukaryotic cells. This study targeted the PEX6 gene, which encodes for peroxisomal biogenesis factor 6, by RNA interference (RNAi) in Fusarium oxysporum f. sp. lycopersici. Fusarium oxysporum is a soilborne filamentous, hemibiotrophic fungus that invades tomato roots and colonizes the xylem vessels, thereby causing complete wilting of infected tomato plants. The expression of FoPEX6 in F. oxysporum was found to be higher during early stages of growth and development. The FoPEX6 gene was isolated and a hairpin RNAi construct was prepared and introduced into F. oxysporum 4471 through glass-bead transformation. The fungal transformation status, i.e. integration, expression and presence of the intended small interfering RNAs (siRNAs), was confirmed by PCR, qPCR and stem-loop PCR, respectively. The silenced fungal transformants exhibited reduced pigmentation and a significant reduction in sporulation as compared to the wild type. They also showed dramatic reduction in pathogenicity (virulence) on tomato, based on root infection and fruit invasion assays. These results suggest that PEX6 has a central role in pigmentation, sporulation and pathogenicity in F. oxysporum.

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