In the indeterminate nodules of a model legume Medicago truncatula, ∼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel β-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea. In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.

在豆科植物蒺藜苜蓿的不确定根瘤中，表达了约 700 个具有保守半胱氨酸特征的根瘤特异性富含半胱氨酸 (NCR) 肽。 NCR 肽的序列高度多样化，其中一些阳离子肽在体外和体内表现出抗菌活性。然而，人们对其结构、抗真菌活性和对抗植物真菌病原体的作用方式缺乏了解。在此，解析了 36 个氨基酸 NCR044 肽的三维 NMR 结构。这种独特的结构很大程度上无序且高度动态，具有由两个二硫键稳定的一个四残基 α-螺旋和一个三残基反向平行 β-折叠。 NCR044 肽还对多种植物真菌病原体表现出有效的杀菌活性，包括灰葡萄孢和三种镰刀菌属。它抑制灰霉病菌静止孢子的萌发。在幼体中，它突破了真菌质膜并诱导产生活性氧。它在体外与多种生物活性磷酸肌醇结合。延时共焦和超分辨率显微镜显示真菌细胞壁强烈结合，在离散病灶处穿透细胞膜，随后膨胀逐渐丧失，随后在细胞质中积累，并且幼苗核仁中的水平升高。喷雾施用的 NCR044 显着减少了番茄和烟草植物以及采后产品中由真菌病原体灰霉病菌引起的灰霉病症状。我们的工作说明了结构独特的 NCR 肽对植物真菌病原体的抗真菌活性，并为未来开发此类肽作为喷雾抑真菌剂/杀菌剂铺平了道路。