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

Authors: BEN M'BAREK, SARRAH - Wageningen University And Research Center; CORDEWENER, JAN H. - Wageningen University And Research Center; TABIB GHAFFARY, SEYED - Wageningen University And Research Center; VAN DER LEE, THEO A. - Wageningen University And Research Center; LIU, ZHAOHUI - North Dakota State University; GOHARI, AMIR - Wageningen University And Research Center; MEHRABI, RAHIM - Seed And Plant Improvement Institute; AMERICA, ANTOINE H. - Wageningen University And Research Center; Friesen, Timothy; HAMZA, SONIA - Institut National Agronomique De Tunisie (INAT); STERGIOPOULOS, IOANNIS - University Of California; DE WIT, PIERRE J.G. - Wageningen University; KEMA, GERRIT H. - Wageningen University And Research Center

Submitted to: Fungal Genetics and Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/27/2015
Publication Date: 3/27/2015
Publication URL: http://handle.nal.usda.gov/10113/61008
Citation: Ben M'Barek, S., Cordewener, J.H.G., Tabib Ghaffary, S.M., Van Der Lee, T.A.J., Liu, Z., Mirzadi Gohari, A., Mehrabi, R., America, A.H.P., Robert, O., Friesen, T.L., Hamza, S., Stergiopoulos, I., De Wit, P.J.G.M., Kema, G.H.J. 2015. FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici. Fungal Genetics and Biology. 79:54-62.

Interpretive Summary: Zymoseptoria tritici is a pathogen of wheat that causes yield and quality losses by inducing necrosis on wheat leaves. Z. tritici cultures were grown in liquid media and culture filtrates (CFs) were infiltrated into leaves of various wheat cultivars. The necrosis-inducing factors were partially purified and were shown to be proteinaceous, heat stable and the interactions were temperature and light dependent. Protease experiments suggest that the necrosis inducing proteins (NIPs) were either 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 30 min and cannot be reversed even after PK treatment. Further proteomic analysis revealed that most of the proteins identified had not been described before. The two most prominent ZtNIP encoding candidates (ZtNIP1 and ZtNIP2) were expressed in yeast and infiltrated on a range of wheat cultivars. These two proteins showed differential necrosis on these lines indicating their potential as virulence factors.

Technical 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 30 min 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 30 min. 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.