Snoj Tina, Lukan Tjaša, Gruden Kristina, Anderluh Gregor
Department of Molecular Biology and Nanobiotechnology, National Institute of Chemistry, Hajdrihova 19, 1000, Ljubljana, Slovenia.
Graduate School of Biosciences, Biotehnical Faculty, University of Ljubljana, 1000, Ljubljana, Slovenia.
J Membr Biol. 2024 Dec 18. doi: 10.1007/s00232-024-00330-3.
Plants are attacked by various pathogens that secrete a variety of effectors to damage host cells and facilitate infection. One of the largest and so far understudied microbial protein families of effectors is necrosis- and ethylene-inducing peptide-1-like proteins (NLPs), which are involved in important plant diseases. Many NLPs act as cytolytic toxins that cause cell death and tissue necrosis by disrupting the plant's plasma membrane. Their mechanism of action is unique and leads to the formation of small, transient membrane ruptures. Here, we capture the interaction of the cytotoxic model NLP from the oomycete Pythium aphanidermatum, NLP, with plant cell-mimicking membranes of giant unilamellar vesicles (GUVs) and tobacco protoplasts using confocal fluorescence microscopy. We show that the permeabilization of GUVs by NLP is concentration- and time-dependent, confirm the small size of the pores by observing the inability of NLP monomers to pass through them, image the morphological changes of GUVs at higher concentrations of NLP and confirm its oligomerization on the membrane of GUVs. In addition, NLP bound to plasma membranes of protoplasts, which showed varying responses. Our results provide new insights into the interaction of NLP with model lipid membranes containing plant-derived sphingolipids.
植物受到各种病原体的攻击，这些病原体分泌多种效应子来破坏宿主细胞并促进感染。坏死和乙烯诱导肽-1样蛋白（NLPs）是效应子中最大且迄今研究较少的微生物蛋白家族之一，它们与重要的植物病害有关。许多NLPs作为溶细胞毒素，通过破坏植物的质膜导致细胞死亡和组织坏死。它们的作用机制独特，会导致形成小的、短暂的膜破裂。在这里，我们使用共聚焦荧光显微镜捕捉了卵菌瓜果腐霉的细胞毒性模型NLP与巨型单层囊泡（GUVs）和烟草原生质体的植物细胞模拟膜之间的相互作用。我们表明，NLP对GUVs的通透作用是浓度和时间依赖性的，通过观察NLP单体无法穿过它们来确认孔的小尺寸，在较高浓度的NLP下对GUVs的形态变化进行成像，并确认其在GUVs膜上的寡聚化。此外，NLP与原生质体的质膜结合，原生质体表现出不同的反应。我们的结果为NLP与含有植物来源鞘脂的模型脂质膜之间的相互作用提供了新的见解。
