Donald Danforth Plant Science Center, St Louis, Missouri, USA.
Earth and Biological Sciences Directorate, Pacific Northwest National Laboratory, Richland, Washington, USA.
Mol Plant Pathol. 2023 Aug;24(8):896-913. doi: 10.1111/mpp.13336. Epub 2023 Apr 10.
Chemical fungicides have been instrumental in protecting crops from fungal diseases. However, increasing fungal resistance to many of the single-site chemical fungicides calls for the development of new antifungal agents with novel modes of action (MoA). The sequence-divergent cysteine-rich antifungal defensins with multisite MoA are promising starting templates for design of novel peptide-based fungicides. Here, we experimentally tested such a set of 17-amino-acid peptides containing the γ-core motif of the antifungal plant defensin MtDef4. These designed peptides exhibited antifungal properties different from those of MtDef4. Focused analysis of a lead peptide, GMA4CG_V6, showed that it was a random coil in solution with little or no secondary structure elements. Additionally, it exhibited potent cation-tolerant antifungal activity against the plant fungal pathogen Botrytis cinerea, the causal agent of grey mould disease in fruits and vegetables. Its multisite MoA involved localization predominantly to the plasma membrane, permeabilization of the plasma membrane, rapid internalization into the vacuole and cytoplasm, and affinity for the bioactive phosphoinositides phosphatidylinositol 3-phosphate (PI3P), PI4P, and PI5P. The sequence motif RRRW was identified as a major determinant of the antifungal activity of this peptide. While topical spray application of GMA4CG_V6 on Nicotiana benthamiana and tomato plants provided preventive and curative suppression of grey mould disease symptoms, the peptide was not internalized into plant cells. Our findings open the possibility that truncated and modified defensin-derived peptides containing the γ-core sequence could serve as promising candidates for further development of bio-inspired fungicides.
化学杀菌剂在保护作物免受真菌病害方面发挥了重要作用。然而，许多单一作用位点的化学杀菌剂的真菌抗药性不断增加，这就需要开发具有新作用模式（MoA）的新型抗真菌剂。具有多作用位点 MoA 的序列差异的富含半胱氨酸的抗真菌防御素是设计新型肽类杀菌剂的有前途的起始模板。在这里，我们通过实验测试了一组含有抗真菌植物防御素 MtDef4 的γ-核心基序的 17 个氨基酸肽。这些设计的肽表现出与 MtDef4 不同的抗真菌特性。对一种先导肽 GMA4CG_V6 的重点分析表明，它在溶液中是无规卷曲的，几乎没有或没有二级结构元件。此外，它对植物真菌病原体 Botrytis cinerea 表现出强大的耐阳离子抗真菌活性，Botrytis cinerea 是水果和蔬菜灰霉病的病原体。其多作用位点 MoA 涉及主要定位于质膜、质膜透化、快速内化到液泡和细胞质以及与生物活性磷酸肌醇 3-磷酸（PI3P）、PI4P 和 PI5P 的亲和力。RRRW 序列基序被确定为该肽抗真菌活性的主要决定因素。虽然 GMA4CG_V6 在 Nicotiana benthamiana 和番茄植株上的局部喷雾应用提供了对灰霉病症状的预防和治疗抑制，但该肽并未被内化到植物细胞中。我们的研究结果为含有γ-核心序列的截断和修饰防御素衍生肽作为进一步开发基于生物灵感的杀菌剂的有前途的候选物提供了可能性。
