Antifungal symbiotic peptide NCR044.1 exhibits unique structure and multi-faceted mechanisms of action that confer plant protection

NCR044.1 is a 36-amino acid nodule-specific cysteine-rich antimicrobial peptide expressed in the developing nodules of . Here, we determined its unique NMR structure to be largely disordered, one four-residue α-helix and one three-residue anti-parallel β-sheet stabilized by two disulfide bonds, suggesting it is highly dynamic. NCR044.1 exhibited potent fungicidal activity against multiple plant fungal pathogens. It breached the fungal plasma membrane, bound to multiple phosphoinositides, and induced reactive oxygen species. Time-lapse confocal and super-resolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, and subsequent accumulation in the cytoplasm with elevated levels in nucleoli. Nucleolar localization of NCR044.1 was unique amongst plant antifungal peptides, suggesting its potential interaction with ribosomes and inhibition of translation. Spray-applied NCR044.1 significantly reduced gray mold disease symptoms caused by the fungal pathogen in tomato plants and post-harvest products demonstrating its potential as a spray-on peptide-based biofungicide.