Due to rapidly emerging resistance to single‐site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation‐tolerant MtDef4 variant was generated that bound to β‐glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation‐tolerant variant as a peptide‐based fungicide.

中文翻译：

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植物防御素 MtDef4 作为肽类生物杀菌剂的作用模式和潜力

由于植物真菌病原体对单位点杀菌剂的抗性迅速出现，对安全和多位点杀菌剂的需求日益增长。具有多位点作用模式 (MoA) 的植物抗真菌肽具有作为仿生杀菌剂的潜力。蒺藜苜蓿此前有报道称，防御素 MtDef4 对真菌病原体表现出有效的抗真菌活性。其作用机制涉及质膜破坏和与细胞内靶标的结合。然而，这种防御素抑制并导致细胞死亡的具体生化过程尚未确定。在这里，我们证明 MtDef4 对灰葡萄孢。它导致这种病原体的幼苗出现严重的质膜和细胞器不规则性。它与真菌核糖体结合并在体外抑制蛋白质翻译。缺乏抗真菌活性的 MtDef4 变体表现出大大降低的蛋白质翻译抑制活性。产生了一种阳离子耐受的 MtDef4 变体，它以比 MtDef4 更高的亲和力与真菌细胞壁的 β-葡聚糖结合。当外源施用时，与 MtDef4 相比，它还可以更大程度地减少灰霉病症状本塞姆氏烟草植物、番茄果实和玫瑰花瓣。我们的研究结果揭示了蛋白质合成的抑制是 MtDef4 的一个可能目标，以及其阳离子耐受变体作为肽类杀菌剂的潜力。