title: Transferring an optimized TAP-toolbox for the isolation of protein complexes to a portfolio of rice tissues.
abstract: Proteins are the cell's functional entities. Rather than operating independently, they interact with other proteins. Capturing in vivo protein complexes is therefore crucial to gain understanding of the function of a protein in a cellular context. Affinity purification coupled to mass spectrometry has proven to yield a wealth of information about protein complex constitutions for a broad range of organisms. For Oryza sativa, the technique has been initiated in callus and shoots, but has not been optimized ever since. We translated an optimized tandem affinity purification (TAP) approach from Arabidopsis thaliana toward Oryza sativa, and demonstrate its applicability in a variety of rice tissues. A list of non-specific and false positive interactors is presented, based on re-occurrence over more than 170 independent experiments, to filter bona fide interactors. We demonstrate the sensitivity of our approach by isolating the complexes for the rice ANAPHASE PROMOTING COMPLEX SUBUNIT 10 (APC10) and CYCLIN-DEPENDENT KINASE D (CDKD) proteins from the proliferation zone of the emerging fourth leaf. Next to APC10 and CDKD, we tested several additional baits in the different rice tissues and reproducibly retrieved at least one interactor for 81.4 % of the baits screened for in callus tissue and T1 seedlings. By transferring an optimized TAP tag combined with state-of-the-art mass spectrometry, our TAP protocol enables the discovery of interactors for low abundance proteins in rice and opens the possibility to capture complex dynamics by comparing tissues at different stages of a developing rice organ.
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium. maarten.dedecker@cropdesign.com.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium. maarten.dedecker@cropdesign.com.|CropDesign N.V., Technologiepark 21, 9052, Ghent, Belgium. maarten.dedecker@cropdesign.com.
authors.forename: Maarten
authors.initials: M
authors.lastname: Dedecker
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Jelle
authors.initials: J
authors.lastname: Van Leene
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Nancy
authors.initials: N
authors.lastname: De Winne
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Dominique
authors.initials: D
authors.lastname: Eeckhout
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Geert
authors.initials: G
authors.lastname: Persiau
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Eveline
authors.initials: E
authors.lastname: Van De Slijke
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Bernard
authors.initials: B
authors.lastname: Cannoot
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium.
authors.forename: Leen
authors.initials: L
authors.lastname: Vercruysse
authors.affiliation: CropDesign N.V., Technologiepark 21, 9052, Ghent, Belgium.
authors.forename: Lies
authors.initials: L
authors.lastname: Dumoulin
authors.affiliation: CropDesign N.V., Technologiepark 21, 9052, Ghent, Belgium.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Wojsznis
authors.affiliation: Department of Medical Protein Research and Biochemistry, VIB, Albert Baertsoenkaai 3, 9000, Ghent, Belgium.|Department of Biochemistry, Ghent University, Albert Baertsoenkaai 3, 9000, Ghent, Belgium.
authors.forename: Kris
authors.initials: K
authors.lastname: Gevaert
authors.affiliation: CropDesign N.V., Technologiepark 21, 9052, Ghent, Belgium.
authors.forename: Steven
authors.initials: S
authors.lastname: Vandenabeele
authors.affiliation: Department of Plant Systems Biology, VIB, Technologiepark 927, 9052, Ghent, Belgium. geert.dejaeger@psb.vib-ugent.be.|Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark 927, 9052, Ghent, Belgium. geert.dejaeger@psb.vib-ugent.be.
authors.forename: Geert
authors.initials: G
authors.lastname: De Jaeger
mesh_terms: D064173:Anaphase-Promoting Complex-Cyclosome / Q000302:isolation & purification / Q000502:physiology; D003001:Cloning, Molecular; D018844:Cyclin-Dependent Kinases / Q000302:isolation & purification / Q000502:physiology; D013058:Mass Spectrometry; D012275:Oryza / Q000378:metabolism / Q000502:physiology*; D018515:Plant Leaves / Q000378:metabolism / Q000502:physiology; D010940:Plant Proteins / Q000302:isolation & purification* / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D036226:Seedlings / Q000378:metabolism / Q000502:physiology
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D064173:Anaphase-Promoting Complex-Cyclosome; D018844:Cyclin-Dependent Kinases
keywords: Anaphase promoting complex; CYCLIN-DEPENDENT KINASE D; Oryza sativa (rice); Protein–protein interactions; Tandem affinity purification coupled to mass spectrometry (TAP–MS)