In vivo interaction between NPR1 and transcription factor TGA2 leads to salicylic acid-mediated gene activation in Arabidopsis

Abstract

The Arabidopsis NPR1 protein is a key regulator of salicylic acid (SA)-mediated gene expression in systemic acquired resistance. Based on yeast two-hybrid analysis, NPR1 has been suggested to interact with members of the TGA family of transcription factors, including TGA2 (AHBP-1b). However, genetic evidence demonstrating that the NPR1-TGA interaction occurs in planta is still lacking, and the role of this interaction in SA-mediated gene activation has yet to be determined. In this study, we expressed a truncated form of TGA2 in Arabidopsis and found that the resulting transgenic lines displayed phenotypes similar to those of npr1 mutants. This dominant-negative effect of the TGA2 mutant shows that TGA2 and NPR1 interact in planta. We also present biochemical evidence indicating that this interaction is specific and enhanced by SA treatment. Moreover, using a chimera reporter system, we found that a chimeric TGA2GAL4 transcription factor activated a UAS(GAL)::GUS reporter gene in response to SA and that this activation was abolished in the npr1 mutant. NPR1 is required for the DNA binding activity of the transcription factor. These genetic data clearly demonstrate that TGA2 is a SA-responsive and NPR1-dependent transcription activator.

Figure 1.

In Vitro Characterization of TGA2CT. (A) TGA2 and 35S::TGA2CT. The TGA2 transcription factor consists of the NT domain (amino acids 1 to 46), the bZIP domain (amino acids 47 to 94), and the CT domain (amino acids 95 to 330). TGA2CT was put under the control of the 35S CaMV, and the resulting protein was tagged with a His₆ tag at the C terminus. (B) GMSA. The recombinant proteins were purified from E. coli. The proteins (0.1 μg of TGA2 or 1 μg of TGA2CT [CT]) were incubated with ³²P-labeled as-1 element (4 × 10⁴ cpm) in the presence (+) or absence (−) of 5 ng of unlabeled probe as a competitor. The TGA2–as-1 complexes are indicated by asterisks. (C) In vitro copurification analysis. Purified recombinant TGA proteins (5 μg) were mixed with 20 μL of protein extract from an NPR1-expressing insect cell line (Zhang et al., 1999) and loaded onto Ni-NTA columns. The His-tagged TGA proteins and their interactors were eluted, and the eluates were run on an SDS-PAGE gel. As a control, the NPR1 extract alone was loaded onto an Ni-NTA column, and the eluate was loaded onto the gel. As an additional control, 1 μL of the NPR1 extract (5% input) was applied directly to the gel. The NPR1 protein that coeluted with the TGA factors was detected by protein gel blot analysis using an antibody against NPR1 (Cao et al., 1998).

Figure 2.

Overexpression of TGA2CT in Wild-Type Plants Results in an npr1-Like Phenotype. (A) Protein gel blot analysis of the TGA2CT protein in 35S::TGA2CT transformants. Protein extracts were made from 2-week-old plants. Independent 35S::TGA2CT transformants (5, 6, 17, and 18) in the wild-type background (35S::TGA2CT [WT]) were compared with untransformed wild-type plants (WT). The His₆-tagged TGA2CT was detected using an antibody against the His tag. (B) RNA gel blot analysis of PR-1 gene expression. Plants were grown for 2 weeks on MS medium with (+) or without (−) 20 μM INA. Total RNA (10 μg) was used to make the blot, which was probed for the PR-1 and UBQ5 mRNAs. (C) Disease symptoms caused by P. syringae pv maculicola ES4326. Leaves from 4-week-old wild-type, 35S::TGA2CT (WT) (line 17), and npr1-2 plants were infiltrated with a P. syringae pv maculicola ES4326 suspension of OD₆₀₀ = 0.0001. Photographs were taken of representative leaves 4 days after infection. (D) Growth of plants on MS medium containing 0.5 mM SA. The photographs were taken 7 days after germination.

Figure 3.

The Dominant-Negative Effect of TGA2CT Is Dependent on Interaction with NPR1. (A) Protein gel blot analysis of the TGA2CT protein in independent 35S::TGA2CT transformants 2 and 4 in the npr1-2 mutant background. Protein extracts were made from 4-week-old npr1-2 control and 35S::TGA2CT transgenic lines. The amount of His₆-tagged TGA2CT protein in each line was determined using an antibody against the His₆ tag. (B) RNA gel blot analysis of PR-1 gene expression in wild-type (WT), npr1-2, and 35S::TGA2CT (npr1-2) transformants. Plants were grown for 2 weeks on MS medium with (+) or without (−) 20 μM INA. Total RNA (10 μg) was used for RNA gel blot analysis using probes specific for PR-1 and UBQ5. (C) Copurification of TGA2CT and NPR1 in 35S::TGA2CT transgenic plants. Equal amounts of protein extract from the wild type and npr1-2 transformed with 35S::TGA2CT (lines 4 and 17, respectively) were loaded onto Ni-NTA columns. The input protein extracts (I) and the eluates (E) were run on a 6 to 16% gradient SDS-PAGE gel. The presence of TGA2CT and NPR1 proteins was detected by protein gel blot analysis using antibodies against the His₆ tag and NPR1, respectively. (D) Effect of SA on TGA2CT-NPR1 copurification. Protein extracts were made from 35S::TGA2CT transformant line 17 in the wild-type background with (+) or without (−) a 24-h treatment of 1 mM SA. The amounts of the TGA2CT-NPR1 complex were measured as described in (C).

Figure 4.

Construction of the TGA2GAL4 Chimeric Transcription Factor and the UAS^GAL::GUS Reporter. The bZIP domain of TGA2 (residues 47 to 94) was replaced by the DNA binding domain of the yeast GAL4 transcription factor (GAL4-DBD), and the resulting chimeric transcription factor was put under the control of the 35S promoter to generate 35S::TGA2GAL4. The GAL4-responsive reporter UAS^GAL::GUS was created by inserting the coding region of β-glucuronidase into plasmid pTA7001 (Aoyama and Chua, 1997) under the control of the synthetic promoter UAS^GAL, which contains the minimal 35S promoter sequence and six GAL4 binding sites. The plasmid (pGVG-GUS) carrying the UAS^GAL::GUS reporter also contains the chimeric transcription factor GVG, which consists of the DNA binding domain of GAL4 (GAL4-DBD), the activation domain of VP16 (VP16-AD), and the hormone binding domain of glucocorticoid receptor (GR-HBD).

Figure 5.

Characterization of TGA2GAL4 Transcriptional Activity in SAR Induction. (A) Quantitative GUS assay of plants transformed with the chimera reporter system. Plants were grown on MS medium with and without 50 μM INA for 2 weeks, and GUS activities were measured using 4-methylumbelliferyl β-d-glucuronide as a substrate. The fold induction by INA was calculated for each genotype by comparing the GUS activity of INA-treated plants with that of untreated plants. The values represent averages of three replicates ±se. A transgenic line containing the INA-responsive BGL2::GUS reporter (Bowling et al., 1994; Cao et al., 1994) was used as a positive control. Three independent 35S::TGA2GAL4/UAS^GAL::GUS transformants in the wild-type background (WT; lines 28, 36, and 65) and three in the npr1-2 background (lines 40, 42, and 49) were examined. (B) Protein gel blot analysis of the TGA2GAL4 protein in plants transformed with the chimera reporter system. Protein extracts were made from wild-type plants and wild-type and npr1-2 plants transformed with the chimera reporter system (lines 65 and 49, respectively). TGA2GAL4 and GVG were immunoprecipitated using a rabbit polyclonal antibody against GAL4-DBD (amino acids 1 to 147). The TGA2GAL4 protein then was detected using a mouse monoclonal antibody against residues 94 to 147 of GAL4-DBD. The GVG protein, which contains only amino acids 1 to 74 of GAL4-DBD, was not detected on this blot. (C) Histochemical staining of GUS activities in plants transformed with the chimera reporter system. Plants were grown on MS medium (MS) or MS medium with 50 μM INA (INA) for 2 weeks. As controls, some plants for each genotype were taken from the MS plates, treated with 1 μM DEX in liquid MS medium for 24 h (DEX), and then stained for GUS. (D) GMSA. Protein extracts were made from 2-week-old plants grown in MS medium with (+) or without (−) 50 μM INA or treated with DEX for 24 h (DEX). 35S::TGA2GAL4/UAS^GAL::GUS transgenic line 65 in the wild-type background and line 49 in the npr1-2 background were used. Each extract (20 μg) was incubated with a ³²P-labeled probe (17-mer) containing UAS^GAL (4 × 10⁴ cpm), and the mixtures were run on a 4% native PAGE gel. The protein-DNA complexes were detected by autoradiography. The arrow points to the TGA2GAL4-UAS^GAL complex, the diamond marks the GVG-UAS^GAL complex, and the asterisks indicate nonspecific complexes. FP, free probe.

Figure 6.

Proposed Model for the Regulatory Mechanism of TGA2 in SAR-Related Gene Expression. (A) In wild-type plants, NPR1 forms complexes with TGA2 and other TGA factors (TGA?). Under noninducing conditions, these complexes are unable to bind to DNA. Upon SAR induction (+SA), more NPR1 becomes available to form the NPR1-TGA2 complex. Through an unknown mechanism, NPR1 enhances the DNA binding activity of TGA2, leading to PR gene induction. (B) In an npr1 mutant, the NPR1–TGA2 interaction is disrupted, so TGA2 cannot bind to DNA. Some TGA factors (TGA?) may be able to bind to DNA in the absence of NPR1, but they are not active in inducing PR gene expression.