title: Co-immunoprecipitation-based identification of putative BAX INHIBITOR-1-interacting proteins involved in cell death regulation and plant-powdery mildew interactions.
abstract: The endoplasmic reticulum (ER)-resident BAX INHIBITOR-1 (BI-1) protein is one of a few cell death suppressors known to be conserved in animals and plants. The function of BI-1 proteins in response to various biotic and abiotic stress factors is well established. However, little is known about the underlying mechanisms. We conducted co-immunoprecipitation (co-IP) experiments to identify Arabidopsis thaliana BI-1-interacting proteins to obtain a potentially better understanding of how BI-1 functions during plant-pathogen interactions and as a suppressor of cell death. Liquid chromatography and tandem mass spectrometry (LC-MS/MS) identified 95 proteins co-immunoprecipitated with green fluorescing protein (GFP)-tagged BI-1. Five selected candidate proteins, a RIBOPHORIN II (RPN2) family protein, VACUOLAR ATP SYNTHASE SUBUNIT A (VHA-A), cytochrome P450 83A1 (CYP83A1), H(+) -ATPASE 1 (AHA1) and PROHIBITIN 2 (PHB2), were further investigated with regard to their role in BI-1-associated processes. To this end, we analysed a set of Arabidopsis mutants in the interaction with the adapted powdery mildew fungus Erysiphe cruciferarum and on cell death-inducing treatments. Two independent rpn2 knock-down mutants tended to better support powdery mildew, and a phb2 mutant showed altered responses to cell death-inducing Alternaria alternata f.sp. lycopersici (AAL) toxin treatment. Two independent cyp83a1 mutants showed a strong powdery mildew resistance phenotype and enhanced sensitivity to AAL toxin. Moreover, co-localization studies and fluorescence resonance energy transfer (FRET) experiments suggested a direct interaction of BI-1 with CYP83A1 at the ER.
authors.affiliation: Lehrstuhl für Phytopathologie, Technische Universität München, 85354 Freising, Germany.
authors.forename: Corina
authors.initials: C
authors.lastname: Weis
authors.forename: Sebastian
authors.initials: S
authors.lastname: Pfeilmeier
authors.forename: Erich
authors.initials: E
authors.lastname: Glawischnig
authors.forename: Erika
authors.initials: E
authors.lastname: Isono
authors.forename: Fiona
authors.initials: F
authors.lastname: Pachl
authors.forename: Hannes
authors.initials: H
authors.lastname: Hahne
authors.forename: Bernhard
authors.initials: B
authors.lastname: Kuster
authors.forename: Ruth
authors.initials: R
authors.lastname: Eichmann
authors.forename: Ralph
authors.initials: R
authors.lastname: Hückelhoven
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism / Q000382:microbiology*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001203:Ascomycota / Q000502:physiology*; D016923:Cell Death; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D054884:Host-Pathogen Interactions; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: C491862:ATBI-1 protein, Arabidopsis; D029681:Arabidopsis Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003577:Cytochrome P-450 Enzyme System; C094565:CYP83A1 protein, Arabidopsis