Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle
Antifungal Pisum sativum defensin 1 interacts with Neurospora crassa cyclin F related to the cell cycle
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DOI：
10.1021/bi061441j
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发表时间：
2007-01-30
期刊：
影响因子：
2.9
通讯作者：
Kurtenbach, Eleonora
中科院分区：
文献类型：
--
作者：
Lobo, Denise S.;Pereira, Iuri B.;Kurtenbach, Eleonora
植物防御素是植物先天免疫系统的组成部分，是一种富含阳离子半胱氨酸的抗真菌肽。文献[Thevissen， K., et al. (2003) Peptides 24, 1705-1712]的证据表明，含有甘露糖基二肌醇磷酸化神经酰胺和葡萄糖神经酰胺的真菌膜斑块分别是从大丽花（Dahlia merckii）和莴苣（Raphanus sativus）中分离的植物防御素的选择性结合位点。植物防御素是否直接或间接地与真菌细胞内靶标相互作用尚不清楚。以抗真菌植物肽Pisum sativum defense in 1 （Psd1）为诱饵，建立了以gal4为基础的酵母双杂交体系，以鉴定蛋白质与蛋白质之间的物理相互作用。目的蛋白在粗神经孢子虫cDNA文库中筛选。11个双杂交候选物中有9个是核蛋白。其中一个克隆在每次筛选中检测到的频率很高，其序列与周期蛋白样蛋白相似，具有F-box和WD-repeat结构域，与细胞周期控制有关。GST下拉实验证实了这种双杂交相互作用。荧光显微镜下对fitc标记的Psd1和dapi染色的真菌细胞核进行分析，发现植物肽Psd1与细胞核在体内共定位。流式细胞术分析粗裂稻分生孢子DNA含量表明，Psd1介导细胞周期损伤，导致分生孢子发生内复制。以新生大鼠发育中的视网膜为模型，观察在Psd1存在下，有组织组织从细胞周期S期向M期增殖过程中的核相互动力学迁移。结果表明，植物防御素Psd1调控视网膜成神经细胞的核迁移。
Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.