title: FPLC and liquid-chromatography mass spectrometry identify candidate necrosis-inducing proteins from culture filtrates of the fungal wheat pathogen Zymoseptoria tritici.
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.
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands; Laboratory of Plant Molecular Physiology, Center of Biotechnology of Borj Cedria, BP 901, Hammam-Lif 2050, Tunisia.
authors.forename: Sarrah
authors.initials: S
authors.lastname: Ben M'Barek
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands; Center for BioSystems and Genomics, 6700AA Wageningen, The Netherlands; Netherlands Proteomics Center, Utrecht, The Netherlands.
authors.forename: Jan H G
authors.initials: JH
authors.lastname: Cordewener
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands; Safiabad Agricultural and Natural Resource Research Center, P.O. Box 333, Dezfoul, Iran.
authors.forename: Seyed M
authors.initials: SM
authors.lastname: Tabib Ghaffary
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands.
authors.forename: Theo A J
authors.initials: TA
authors.lastname: van der Lee
authors.affiliation: Department of Plant Pathology, North Dakota State University, Fargo, ND 58108, USA.
authors.forename: Zhaohui
authors.initials: Z
authors.lastname: Liu
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands; Department of Plant Protection, College of Agriculture, University of Tehran, Plant Pathology Building, Karaj, Iran.
authors.forename: Amir
authors.initials: A
authors.lastname: Mirzadi Gohari
authors.affiliation: Cereal Research Department, Seed and Plant Improvement Institute, Karaj, Iran.
authors.forename: Rahim
authors.initials: R
authors.lastname: Mehrabi
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands; Center for BioSystems and Genomics, 6700AA Wageningen, The Netherlands; Netherlands Proteomics Center, Utrecht, The Netherlands.
authors.forename: Antoine H P
authors.initials: AH
authors.lastname: America
authors.affiliation: Florimond Desprez, BP41, 59242 Cappelle-en-Pévèle, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Robert
authors.affiliation: USDA-ARS, Northern Crop Science Laboratory, Fargo, ND 58102, USA.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Friesen
authors.affiliation: Laboratory of Genetics, National Agronomic Institute of Tunisia, 43 Avenue Charles Nicolle, 1082 Tunis, Tunisia.
authors.forename: Sonia
authors.initials: S
authors.lastname: Hamza
authors.affiliation: Department of Plant Pathology, University of California Davis, 578 Hutchison Hall, One Shields Avenue, Davis, CA 95616-8751, USA.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Stergiopoulos
authors.affiliation: Laboratory of Phytopathology, Wageningen University, 6708 PB Wageningen, The Netherlands.
authors.forename: Pierre J G M
authors.initials: PJ
authors.lastname: de Wit
authors.affiliation: Wageningen University & Research Center, Plant Research International, 6708 PB Wageningen, The Netherlands. Electronic address: gert.kema@wur.nl.
authors.forename: Gerrit H J
authors.initials: GH
authors.lastname: Kema
mesh_terms: D001203:Ascomycota / Q000737:chemistry*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000032:analysis* / Q000737:chemistry; D008027:Light; D013058:Mass Spectrometry; D009336:Necrosis / Q000382:microbiology*; D010935:Plant Diseases / Q000382:microbiology*; D055550:Protein Stability; D013696:Temperature; D014908:Triticum / Q000382:microbiology*; D037521:Virulence Factors / Q000032:analysis* / Q000737:chemistry
chemical_list: D005656:Fungal Proteins; D037521:Virulence Factors
keywords: Culture filtrates; Fast protein liquid chromatography; Liquid chromatography mass spectrometry; Necrosis-inducing proteins; Zymoseptoria tritici