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 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.

Keywords: 12-oxo-phytodienoic acid; CBB, calmodulin binding buffer; CBP, calmodulin-binding peptide; CaMV, cauliflower mosaic virus; FDR, false discovery rate; MS, mass spectrometry; OPDA, 12-oxo-phytodienoic acid; PGA1, prostaglandin A1; PPA1, phytoprostane A1; RubisCo, ribulose-1,5-bisphosphate carboxylase; SA, salicylic acid; SAR, systemic acquired resistance; TAP, tandem affinity purification; TEV, tobacco etch virus; Y2H, yeast 2-hybrid; bZIP, basic region/leucine zipper motif; glutathione-S-transferase; lipid stress; protein complex; thale cress.

Figure 1.

TGA2 expression in transgenic A. thaliana lines. Individual transgenic lines containing a 35S::TGA2 construct with a C-terminal TAP-tag in the genetic background of the tga2,5,6 mutant are shown on the abscissa. Bars represent means of quantitative RT-PCR from 2 biological replicates. TGA2 expression was normalized to the expression of actin. Expression in wild type was arbitrarily set to 1 and expression values in all other genotypes were relative to it.

Figure 2.

Functional analysis of recombinant TGA2 in transgenic A. thaliana seedlings grown in liquid medium. (A) Immunoblot analysis of TGA2 expression using a polyclonal αTGA2-C antiserum. Molecular weight markers and genotypes are indicated on the left and on the top, respectively. Coomassie staining demonstrated that lanes were equally loaded (data not shown). The arrow indicates TAP-tagged TGA2, the white arrowhead indicates a cross-reactive band, and the other arrowheads indicate putative degradation products. The gray arrowhead indicates the predicted size of native TGA2 without the TAP-tag. (B) TGA-dependent and stimulus-induced expression of GST25. Expression relative to actin was based on quantitative RT-PCR. Means and standard errors of 3 biological replicates are shown. Asterisks indicate significant differences between treatment means (P < 0.05) based on Relative Expression Software Tool (REST).

Figure 3.

In vivo pull-down of NPR1 with TAP-tagged TGA2 from leaves using a calmodulin (CaM) affinity resin. Affinity purification of TAP-tagged TGA2 was followed by immunoblotting using αTGA2-C antiserum. Fractions are shown on the top; extracts (Extr.) from 6-week old plants treated without or with 1 mM salicylic acid (SA) for 30 h, fraction unbound to CaM affinity matrix (Unb.), wash fraction, EGTA elution steps (Elu1 and Elu2), and protein remaining on the CaM affinity matrix (Bead). Immunoblot using αNPR1 antibody shows copurification of NPR1 with TGA2. A band of ∼35 kDa cross-reacting with the NPR1 antibody is also visible. Coomassie staining of the blot, which shows approximately equal loading of the lanes containing the extracts. Reduced staining in lanes labeled Elu1, Elu2, and Bead demonstrates partial purification of TGA2 and the associated NPR1.

Figure 4.

Expression of TAP-tagged TGA2 in transgenic A. thaliana treated with 1 mM salicylic acid (SA) or not treated. In this experiment, samples were taken 26 hours after spraying of 6-week old plants. This immunoblot was stained with Ponceau S prior to antibody detection. The αTGA2-C antiserum slightly cross-reacts with the large subunit of ribulose-1,5-bisphosphate carboxylase (RubisCo). Note that the SA treatment does not alter the abundance of recombinant TGA2 in this experiment.

Figure 5.

TAP-tag purification of TGA2 from seedlings grown in liquid medium. (A) Immunoblot analysis of TGA2 using the αTGA2-C antiserum. TAP-tagged TGA2 was bound to IgG-sepharose for 90 min at 20°C. The resin was washed with Tris-saline Tween 20. Tobacco etch virus (TEV) protease was used to cleave off the IgG-binding domain. Any remaining TGA2 was eluted with 0.5 M acetic acid, pH 3.4 (see Materials and Methods for details). Molecular weight markers are indicated on the left. The black arrow indicates TAP-tagged TGA2, the white arrow indicates TEV-cleaved tagged TGA2 and black arrowheads indicate putative degradation products. (B) Coomassie staining of the immunoblot shown to the left. Note that the 27 kDa band represents TEV protease. The bands in the lane of the acid elution step likely represent large and small IgG subunits. (C) Immunoblot analysis of TGA2; second purification step using calmodulin (CaM) affinity resin. TGA2 was bound to CaM affinity resin for 90 min at 20°C, washed with CaM binding buffer (see Materials and Methods), and eluted with 5 mM EGTA. Proteins remaining on the column after this elution were released by boiling in SDS sample buffer (CaM beads). Molecular weight markers, arrows and arrowheads are as indicated above. (D) Silver staining of a gel containing fractions collected during CaM purification.

Figure 6.

TAP-tag purification of TGA2 from plants grown in soil treated or not treated with salicylic acid (SA). Proteins were cross-linked in situ prior to extraction. (A) Immunoblot analysis of TGA2 using αTGA2-C antiserum. TAP-tagged TGA2 was bound to IgG-sepharose (IgG bnd). TGA2 was cleaved off the resin with Tobacco etch virus (TEV) protease to remove the IgG-binding domain. Proteins were bound to calmodulin (CaM) affinity resin, washed, and finally eluted (CaM bnd). Molecular weight markers are indicated on the left. The largest band is similar to the predicted size of TEV-cleaved tagged TGA2. Putative degradation products are smaller. (B) SYPRO Ruby staining of putative TGA2-interacting proteins. The approximate sizes of the standard (Std) are shown to the left.