Tetorya, M. | bioRxiv Science

bioRxiv2024

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. HighlightsThe 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.

pathology↗

bioRxiv2024

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 2 or 3 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 +9 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression in Pichia pastoris resulted 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 translation in vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused by Botrytis 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 structural framework for design of novel, potent antifungal peptides for agricultural use. Author SummaryFungal pathogens cause significant pre-harvest and post-harvest losses of crop yield, making them a serious biological threat to global food security. Chemical fungicides are effective in controlling fungal diseases across various crops. However, rapid evolution of fungal pathogen resistance to single-site chemical fungicides in agriculture has created an urgent need for development of safe, sustainable, and cost-effective multi-site fungicides. Nodule-specific cysteine-rich (NCR) peptides expressed in the inverted-repeat lacking clade legume plants exhibit potent antifungal activity; however, their modes of action (MoA) are poorly understood. Particularly, the specific contribution of disulfide pairing to the potency and spectrum of antifungal activity against fungal plant pathogens and MoA of these peptides remains to be identified. Chickpea NCR13 expressed in P. pastoris generates two peptide variants that differ in their disulfide cross-linking pattern. These variants exhibit striking differences in their three-dimensional structures and potency of antifungal activity against multiple fungal pathogens and MoA. They also differ in their ability to confer resistance to gray mold in pepper and tomato plants. Our study highlights the major impact a specific pattern of disulfide pairing can have on the in vitro and in planta antifungal activity of an NCR peptide.

plant biology↗

bioRxiv2023

Small cationic cysteine-rich defensin-derived antifungal peptide controls white mold in soybean

White mold disease caused by a necrotrophic ascomycete pathogen Sclerotinia sclerotiorum results in serious economic losses of soybean yield in the USA. Lack of effective genetic resistance to this disease in soybean germplasm and increasing pathogen resistance to fungicides makes white mold difficult to manage. Small cysteine-rich antifungal peptides with multi-faceted modes of action hold potential for development as sustainable spray-on bio-fungicides. We have previously reported that GMA4CG_V6 peptide, a 17-amino acid variant of the MtDef4 defensin-derived peptide GMA4CG containing the active {gamma}-core motif, exhibits potent antifungal activity against the gray mold fungal pathogen Botrytis cinerea in vitro and in planta. This peptide exhibited antifungal activity against an aggressive field isolate of S. sclerotiorum 555 in vitro. It markedly reduced white mold disease symptoms when applied to detached soybean leaves, pods, and stems. Spray-application on soybean plants provided robust control of the white mold disease. GMA4CG_V6 at sub-lethal concentrations reduced sclerotia production. It was also non-phytotoxic to soybean plants. Our results demonstrate that GMA4CG_V6 peptide has high potential for development as a bio-fungicide for white mold control in soybean.

plant biology↗