Antifungal Pisum sativum Defensin 1 Interacts with Neurospora crassa Cyclin F Related to the Cell Cycle
| Autor: | Rafael Linden, Maria Bellio, Reinaldo Calixto de Campos, Eleonora Kurtenbach, Jane Faria, Luciano Neves de Medeiros, Luiz M Cabral, Lucianne Fragel-Madeira, Iuri B Pereira, Denise S. Lobo |
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| Rok vydání: | 2007 |
| Předmět: |
Interkinetic nuclear migration
Antifungal Agents DNA Plant Plant defensin Biochemistry Retina Neurospora crassa Defensins Fungal Proteins Cyclins Two-Hybrid System Techniques Animals Endoreduplication Nuclear protein DNA Fungal Plant Proteins Genetics Binding Sites Base Sequence biology cDNA library Cell Cycle fungi Peas Fungal genetics food and beverages Cell cycle biology.organism_classification Recombinant Proteins Rats Cell biology Animals Newborn Protein Binding |
| Zdroj: | Biochemistry. 46:987-996 |
| ISSN: | 1520-4995 0006-2960 |
| DOI: | 10.1021/bi061441j |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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