University of Hertfordshire

Documents

  • Henrik Stotz
  • Simone Findling
  • Ella Nukarinen
  • Wolfram Weckwerth
  • Martin J. Mueller
  • Susanne Berger
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Original languageEnglish
Article numbere972794
JournalPlant Signaling and Behavior
Journal publication date22 Dec 2014
Volume9
Issue10
Early online date31 Oct 2014
DOIs
Publication statusPublished - 22 Dec 2014

Abstract

Tandem affinity purification (TAP) tagging provides a powerful tool for isolating interacting proteins in vivo. TAP-tag purification offers particular advantages for the identification of stimulus-induced protein interactions. Type II bZIP transcription factors (TGA2, TGA5 and TGA6) play key roles in pathways that control salicylic acid, ethylene, xenobiotic and reactive oxylipin signaling. Although proteins interacting with these transcription factors have been identified through genetic and yeast 2-hybrid screening, others are still elusive. We have therefore generated a C-terminal TAP-tag of TGA2 to isolate additional proteins that interact with this transcription factor. Three lines most highly expressing TAP-tagged TGA2 were functional in that they partially complemented reactive oxylipin-responsive gene expression in a tga2 tga5 tga6 triple mutant. TAP-tagged TGA2 in the most strongly overexpressing line was proteolytically less stable than in the other 2 lines. Only this overexpressing line could be used in a 2-step purification process, resulting in isolation of co-purifying bands of larger molecular weight than TGA2. TAP-tagged TGA2 was used to pull down NPR1, a protein known to interact with this transcription factor. Mass spectrometry was used to identify peptides that co-purified with TAP-tagged TGA2. Having generated this TGA2 TAP-tag line will therefore be an asset to researchers interested in stimulus-induced signal transduction processes.

Notes

This is an Accepted Manuscript of an article published by Taylor & Francis Group in Plant Signaling & Behavior, on 31 October 2014, available online: https://doi.org/10.4161/15592316.2014.972794.

ID: 8442320