A tandem affinity purification tag of TGA2 for isolation of interacting proteins in Arabidopsis thaliana

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 TAPtag 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 copurified 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.

Primary root response to combined drought and heat stress is regulated via salicylic acid metabolism in maize

“…In addition, TGA transcription factors are involved in the regulation of plant root growth, stress response, flowering regulation and other physiological metabolic processes. Stotz et al [48] showed that TGA2 transcription factors participated in the oxidative stress pathway and the REDOX regulation pathway of ROS induced by environmental stress in Arabidopsis thaliana, restoring plant growth and development to avoid the impact of external adverse conditions on plants. In tobacco, glyoxalase (GLO) promoted the interaction between TGA and NPR1 and recognized the specific sequence TGA CGT to activate the expression of PR1 and to respond to the ROS pathway in plants [49].…”

Prediction and Investigation of Novel Proteins in DNA Double Stranded Break Repair

“…This TF is linked to the promoter of reporter genes such as LacZ which produces b-galactosidase, a quantifiable colored product, or HIS, URA3 and ADE2 auxotrophic selection markers (Suter B et al 2008 (Puig O et al 2001). Over the past two decades, this technique has been applied to plants (Stotz HU et al 2014), mammalian (Knuesel M et al 2003) and bacterial cell (Hu P et al 2009) lines to investigate PPIs in vivo. In this method, the protein of interest is fused to a double tag at its C-or N-terminus and isolated in two rounds of affinity purification (Jessulat M et al 2010).…”