Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

Abstract

Cationic nodule-specific cysteine-rich (NCR) peptides of nitrogen-fixing legume plants exhibit antifungal activity and can be repurposed for development as biofungicides. Chickpea NCR13 is a highly cationic peptide with six cysteines forming three disulfide bonds. Expression of NCR13 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. PFV1 and PFV2 differed profoundly in the potency of antifungal activity and their multi-faceted modes of action (MoA). PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities and greater stability once inside the fungal cells. PFV1 was more effective in binding to fungal ribosomal RNA and inhibiting protein translation in vitro. When sprayed on pepper and tomato plants, PFV1 was more effective in controlling the gray mold disease caused by Botrytis cinerea. 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.