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FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici

Sarrah Ben M’Barek, Jan H.G. Cordewener, Seyed M. Tabib Ghaffary, Theo A.J. van der Lee, Zhaohui Liu, Amir Mirzadi Gohari, Rahim Mehrabi, Antoine H.P. America, Olivier Robert, Timothy L. Friesen, Sonia Hamza, Ioannis Stergiopoulos, Pierre J.G.M. de Wit, Gerrit H.J. Kema
Fungal genetics and biology 2015 v.79 pp. 54-62
Mycosphaerella graminicola, Komagataella pastoris, necrosis, polyacrylamide gel electrophoresis, signal transduction, wheat, mass spectrometry, liquid chromatography, Triticum aestivum, mesophyll, pathogens, leaves, cultivars, apoplast, temperature, peptidase K, fractionation, heat stability, time series analysis, fungal proteins, culture filtrates, fungi
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.