In Vitro Binding by Nuclear TGA Factors to the LS7 Element of PR-1.
(A) Gel-shift binding assay with leaf nuclear extracts. Lanes 1 to 10, DNA–protein complexes between labeled LS7 probe of the PR-1 promoter and leaf nuclear proteins; lane 1, probe alone; lanes 2 to 4, 1, 3, and 9 μg of nuclear protein from leaves of untreated wild-type plants (wt); lanes 5 to 7, 1, 3, and 9 μg of nuclear protein from leaves of SA-treated wild-type plants (wt + SA); lanes 8 to 10, 1, 3, and 9 μg of nuclear protein from leaves of SA-treated npr1-1 mutant plants (npr1-1 + SA); lanes 11 to 14, labeled LS7 mutant probe (LS7m) alone (lane 11) or with 9 μg of nuclear protein from leaves of untreated wt (lane 12), wt + SA (lane 13), or npr1-1 + SA (lane 14) plants; lanes 15 to 18, labeled G-box probe (G-1A) alone (lane 15) or with 9 μg of nuclear protein from leaves of untreated wt (lane 16), wt + SA (lane 17), or npr1-1 + SA (lane 18) plants. Closed and open arrowheads indicate TGA factor and G-box factor complexes with their respective LS7 and G-1A probes. The asterisk indicates the presence of a lower mobility complex bound to the G-1A probe.
(B) Immunodepletion assay of TGA2 and TGA3. In each case, 9 μg of nuclear protein from SA-treated wild-type leaves (wt + SA) was incubated with protein A–Sepharose resin (Prot. A; lane 1), 1 μg of rabbit control (IgG; lane 2), anti-TGA2 antibody (α-TGA2; lane 3), or anti-TGA3 antibody (α-TGA3; lane 4) bound to resin. After a brief spin, supernatants from these samples were used in standard gel-shift assays with radiolabeled LS7 as a probe.

Trans-activation by Chimeric TGA3 Factors.
Effector and reporter constructs were used in transfection assays. Reporter constructs were as follows. 5xGAL-m35S-LUC, a synthetic promoter with five tandem GAL4 cis elements, was fused to the minimal TATA box–containing promoter (−46-bp region) of the 35S promoter of Cauliflower mosaic virus (CaMV) located upstream of the firefly luciferase (LUC) gene; 35S-GUS, a full-length CaMV 35S promoter was fused to the bacterial β-glucuronidase (GUS) gene (Jefferson et al., 1987). Effector constructs were as follows. The DNA binding domain (DBD) of the yeast GAL4 factor was used as a control or fused in frame to the cDNA of TGA3 or TGA3ΔbZIP. Both factors then were cloned downstream of the full-length CaMV 35S promoter in the pMON 999 vector. TGA3 is composed of an N-terminal domain (NT; amino acids [aa.] 2 to 100), a basic domain/Leu zipper domain (bZIP; amino acids 101 to 145), and a C-terminal domain (CT; amino acids 146 to 384). For the transient transfection assay, Arabidopsis rosette leaves were transfected by particle bombardment with reporter and effector DNA. The 35S-GUS reporter was used as an internal control for transfection efficiency in all experiments. All effector DNAs were used in saturating amounts that resulted in maximal expression of the 5xGAL-m35S-LUC reporter gene. After bombardment, leaves were treated with SA and incubated for 20 h. LUC activity was normalized to the activity of the GUS internal control (expressed as a ratio) and expressed as arbitrary units of activity. Means and standard errors for data from three independent experiments are shown.

Immunodetection of TGA2 and TGA3 in Leaf Nuclear Extracts.
(A) Specificity of anti-TGA factor antibodies. Lanes 1 to 6, input fractions of in vitro–synthesized, ³⁵S-Met–labeled Arabidopsis TGA factors (TGA1 to TGA6); lanes 7 to 18, products from immunoprecipitation reactions with normalized input fractions of the indicated TGA factors and 1 μg of affinity-purified antibodies against TGA2 (α-TGA2; lanes 7 to 12) or TGA3 (α-TGA3; lanes 13 to 18). Molecular masses (in kilodaltons) of protein markers are indicated.
(B) Immunodetection of nuclear TGA2 and TGA3 proteins. To enrich for these factors, leaf nuclear proteins (500 μg) were incubated with 1 μg of either rabbit control (IgG; lanes 1 and 5) or specific antibodies against TGA2 (α-TGA2; lanes 2 to 4) or TGA3 (α-TGA3; lanes 6 to 8). These conditions resulted in the quantitative and complete recovery of TGA factor antigens from nuclear extracts (ANE; lanes 9 and 11), because the unbound fraction after immunoprecipitation (supernatant [Supt]) lacked detectable amounts of either TGA2 (lane 10) or TGA3 (lane 12). Immunocomplexes were recovered with protein A–Sepharose, washed with RIPA buffer, and fractionated by SDS-PAGE before being examined by protein gel blot analysis with the anti-TGA factor antibodies indicated. Leaf nuclear extracts were obtained from wild-type (wt), SA-treated wild-type (wt/SA), or SA-treated npr1-1 mutant (npr1-1/SA) plants. The expected positions for TGA2 and TGA3 are indicated. IgG heavy chain (IgG_H) polypeptide from the primary antibodies is present in all immunoprecipitation reactions, as expected.
(C) Coimmunoprecipitation of TGA2 and TGA3 complexes. Immunodetection of TGA2 and TGA3 was performed as described above except that the antibodies against TGA2 (α-TGA2) and TGA3 (α-TGA3) used for the initial immunoenrichment were reversed in a subsequent detection step involving protein gel blot analysis. The leaf nuclear extracts studied were from wild-type (wt) and SA-treated wild-type (wt/SA) plants. Positions corresponding to TGA2 and TGA3 polypeptides are indicated, in addition to that of the IgG heavy chain (IgG_H) polypeptide from immunoprecipitation reactions.
(D) Coimmunoprecipitation assays of in vitro–synthesized TGA2 and TGA3. To test for heterodimer formation between TGA2 and TGA3, constructs that encode full-length versions of these factors were transcribed and translated in vitro in the presence of ³⁵S-Met to label de novo proteins. Immunoprecipitation reactions were performed as described above with antibodies against either TGA2 (α-TGA2) or TGA3 (α-TGA3), with samples containing TGA2 alone (lane 1), a post-translational mixture of TGA2 and TGA3 (lanes 2 and 5), cotranslated TGA2 and TGA3 (lanes 3 and 6), or TGA3 alone (lane 4). After immunoprecipitation, immunocomplexes were fractionated by SDS-PAGE and detected by fluorography. Full-length TGA2 and TGA3 polypeptides are as shown. The arrow indicates the presence of a truncated product of TGA3.

In Vivo Recruitment of TGA2 and TGA3 to the PR-1 Genomic Promoter.
(A) Diagram of the PR-1 locus and flanking regions. The PR-1 locus on Arabidopsis chromosome 2 (T6B13 clone) contains the transcriptionally divergent genes PR-1 and a putative XET spaced ∼2.65 kb apart. (Note that “PR-1-like protein” is the designation for PR-1 in the TAIR database [http://www.tair.org].) In the shared intergenic region between PR-1 and XET, only two as-1–type elements (LS5 and LS7) are present (Lebel et al., 1998; our observations using the plantCARE software of Lescot et al. [2002]). Untranslated regions (black boxes), exons (white boxes), and the direction and start site of transcription (solid arrows at +1) are indicated. Also, primer pairs used in ChIP and RT-PCR analyses are those that amplify intergenic sequences present downstream of PR-1 (black arrows), the LS5- and LS7-containing intergenic region of the PR-1 promoter (white arrows), and coding sequences of the XET gene (hatched arrows).
(B) Effect of the SA and NPR1 signal pathway on PR-1 and XET expression in leaves. Transcripts of PR-1 and XET were measured by RT-PCR using total RNA from leaves of Arabidopsis wild-type (wt) and npr1-1 mutant (npr1-1) plants that had been treated with SA for 0 to 32 h. DNA products of these reactions were fractionated by agarose gel electrophoresis and stained with ethidium bromide for quantification using the Eagle Eye II still-video system. To facilitate comparisons between genes, changes in the amount of their corresponding transcripts were converted to fold induction by normalizing all values from SA-treated samples to those of the untreated (0-h) sample. Transcripts of a constitutively expressed β-tubulin gene (TUB8) also were analyzed as an internal control for the specificity of induction.
(C) ChIP assays of in vivo DNA binding by TGA2 and TGA3. In vivo DNA–protein complexes in chromatin were covalently cross-linked by formaldehyde and recovered from leaves of wild-type (Wt) and npr1-1 (npr1-1) Arabidopsis plants that had been treated with SA for 0 h (lanes 1 to 3), 2 h (lanes 4 to 6), or 16 h (lanes 7 to 9). Chromatin then was sonicated to yield a population of soluble fragments and incubated with BSA as a negative control (lanes 1, 4, and 7) or with specific antibodies against either TGA2 (lanes 2, 5, and 8) or TGA3 (lanes 3, 6, and 9). Target PR-1 promoter sequences were detected by PCR using specific primers and α-³²P-dCTP. Reaction products were fractionated by PAGE and visualized using autoradiography. Crude chromatin (inputs) corresponding to ∼1/200th of the total sample used in the other lanes was analyzed similarly by PCR (lanes 10 to 12) to show that equal amounts of chromatin template were programmed in ChIP reactions. Lane C shows a standard PCR result using cloned PR-1 promoter as a template.
(D) Relative recoveries of chromatin sequences bound in vivo by TGA2 and TGA3. ChIP assays were performed as in (C) with chromatin from wild-type leaves treated for 16 h with SA. The resulting ChIP products then were analyzed by PCR using primers that amplify the coding sequence of a control β-tubulin gene (TUB8) that lacks identifiable TGA factor binding sites, the PR-1 promoter (PR-1), the coding sequence of the flanking XET gene (XET), and the intergenic region that is 3′ to PR-1 (3′ downstream). Histogram plots of a single experiment are shown, with PCR products corresponding to ChIP control reactions in the absence of specific antibody (white bars) or with test reactions with either anti-TGA2 antibody (α-TGA2; black bars) or anti-TGA3 antibody (α-TGA3; gray bars). Values were plotted as the relative fold accumulation of ChIP products from specific immunoprecipitation versus control (no-antibody) reactions.

Model of PR-1 Transcriptional Regulation.
(A) Wild-type plants. In untreated leaves of wild-type plants, NPR1 is localized to the cytoplasm. Under these conditions, TGA2 or TGA3 in the nucleus interacts with a nuclear repressor (octagon) that inhibits its DNA binding activity, whereas an inhibitor protein (solid oval) at the LS5 element keeps basal transcription to a minimum (small arrow). After exposure to SA, the cytoplasmic form of NPR1 is mobilized to the nucleus, where it binds TGA factors and consequently displaces the repressor. The NPR1-TGA factor complex then is recruited to the target PR-1 promoter, resulting in the release of NPR1 and a net increase in the expression of this gene (large arrow).
(B) npr1-1 mutant plants. The absence of a functional NPR1 protein (open circle) results in a constitutive interaction between the repressor and its cognate TGA factor. Consequently, PR-1 expression no longer is activated by SA (small arrow).