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Abstract 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 pathogenBotrytis 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 onNicotiana benthamianaand 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.
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This content will become publicly available on December 2, 2025
Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action
Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression inPichia pastorisresulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translationin vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused byBotrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use.
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- Award ID(s):
- 2037981
- PAR ID:
- 10564832
- Editor(s):
- Dinesh-Kumar, Savithramma P
- Publisher / Repository:
- PLOS PATHOGENS
- Date Published:
- Journal Name:
- PLOS Pathogens
- Volume:
- 20
- Issue:
- 12
- ISSN:
- 1553-7374
- Page Range / eLocation ID:
- e1012745
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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