Open AccessCo‐immunoprecipitation‐based identification of putative BAX INHIBITOR ‐1‐interacting proteins involved in cell death regulation and plant–powdery mildew interactions
Author(s) -
Weis Corina,
Pfeilmeier Sebastian,
Glawischnig Erich,
Isono Erika,
Pachl Fiona,
Hahne Hannes,
Kuster Bernhard,
Eichmann Ruth,
Hückelhoven Ralph
Publication year - 2013
Publication title -
molecular plant pathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.945
H-Index - 103
eISSN - 1364-3703
pISSN - 1464-6722
DOI - 10.1111/mpp.12050
Subject(s) - powdery mildew , biology , immunoprecipitation , microbiology and biotechnology , mutant , programmed cell death , bimolecular fluorescence complementation , unfolded protein response , endoplasmic reticulum , apoptosis , biochemistry , botany , gene
Summary 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 A rabidopsis 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 ( RPN 2) family protein, VACUOLAR ATP SYNTHASE SUBUNIT A ( VHA ‐ A ), cytochrome P 450 83 A 1 ( CYP 83 A 1), H + ‐ ATPASE 1 ( AHA 1) and PROHIBITIN 2 ( PHB 2), were further investigated with regard to their role in BI ‐1‐associated processes. To this end, we analysed a set of A rabidopsis mutants in the interaction with the adapted powdery mildew fungus E rysiphe 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 A lternaria 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 CYP 83 A 1 at the ER .