1. FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.
- Author
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Ben M'Barek S, Cordewener JH, Tabib Ghaffary SM, van der Lee TA, Liu Z, Mirzadi Gohari A, Mehrabi R, America AH, Robert O, Friesen TL, Hamza S, Stergiopoulos I, de Wit PJ, and Kema GH
- Subjects
- Chromatography, Liquid, Electrophoresis, Polyacrylamide Gel, Fungal Proteins chemistry, Light, Mass Spectrometry, Protein Stability, Temperature, Virulence Factors chemistry, Ascomycota chemistry, Fungal Proteins analysis, Necrosis microbiology, Plant Diseases microbiology, Triticum microbiology, Virulence Factors analysis
- Abstract
Culture filtrates (CFs) of the fungal wheat pathogen Zymoseptoria tritici were assayed for necrosis-inducing activity after infiltration in leaves of various wheat cultivars. Active fractions were partially purified and characterized. The necrosis-inducing factors in CFs are proteinaceous, heat stable and their necrosis-inducing activity is temperature and light dependent. The in planta activity of CFs was tested by a time series of proteinase K (PK) co-infiltrations, which was unable to affect activity 30min after CF infiltrations. This suggests that the necrosis inducing proteins (NIPs) are either absent from the apoplast and likely actively transported into mesophyll cells or protected from the protease by association with a receptor. Alternatively, plant cell death signaling pathways might be fully engaged during the first 30min and cannot be reversed even after PK treatment. Further fractionation of the CFs with the highest necrosis-inducing activity involved fast performance liquid chromatography, SDS-PAGE and mass spectrometry. This revealed that most of the proteins present in the fractions have not been described before. The two most prominent ZtNIP encoding candidates were heterologously expressed in Pichia pastoris and subsequent infiltration assays showed their differential activity in a range of wheat cultivars., (Copyright © 2015 Elsevier Inc. All rights reserved.)
- Published
- 2015
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