Expression of TGA Proteins in the Yeast Two-Hybrid System.
Immunoblot analysis of yeast cells demonstrating that noninteracting TGA–GAL4 transcriptional activation domain fusions are expressed at levels similar to those of interacting TGA–GAL4 transcriptional activation domain fusions. Yeast cells containing the NPR1–GAL4 DNA binding domain fusion together with the TGA2–GAL4 transcriptional activation domain (lane 1), the TGA1–GAL4 transcriptional activation domain (lane 2), or the TGA4–GAL4 transcriptional activation domain (lane 3) fusions were grown on synthetic dextrose medium lacking leucine and tryptophan only. Proteins were extracted, fractionated on 8% SDS–polyacrylamide gels, and immunoblotted with an anti–GAL4 transcriptional activation domain antibody.

Expression of Wild-Type and Mutant NPR1 Proteins in the Yeast Two-Hybrid System.
Immunoblot analysis of yeast cells demonstrating similar expression levels of wild-type and mutant NPR1 proteins. Yeast cells containing the NPR1–GAL4 DNA binding domain fusion (lane 1), the NPR1-1–GAL4 DNA binding domain fusion (lane 2), the NPR1-2–GAL4 DNA binding domain fusion (lane 3), the NIM1-2–GAL4 DNA binding domain fusion (lane 4), or the empty GAL4 DNA binding domain vector (lane 5), together with a full-length TGA3–GAL4 transcriptional activation domain fusion (lanes 1 to 5), were grown on synthetic dextrose medium lacking leucine and tryptophan only. Proteins were extracted, fractionated on 8% SDS–polyacrylamide gels, and immunoblotted with an anti-NPR1 antibody.

TGA2 Interacts with NPR1 but Not with the NIM1-2 Mutant Protein in Vitro.
(A) Biotinylated NPR1 (lanes 2 and 5) or NIM1-2 (lane 3) was produced in Escherichia coli and coupled to streptavidin–agarose beads. The beads were mixed and incubated for 1 hr with in vitro–translated radiolabeled TGA2 (lanes 2 and 3) or TGA4 (lane 5). After washing the beads, the bound proteins were eluted by boiling in SDS sample buffer, resolved by 8% SDS-PAGE, and visualized by autoradiography. Lanes 1 and 4 contain 20% of the input radiolabeled TGA2 and TGA4, respectively.
(B) Immunoblot analysis of NPR1 and NIM1-2 biotinylated resins, demonstrating that they contain similar amounts of coupled proteins. Biotinylated NPR1 and NIM1-2 were produced in E. coli and coupled to streptavidin–agarose beads. The NPR1 (lane 1) and the NIM1-2 (lane 2) beads were boiled in SDS sample buffer, resolved by 8% SDS-PAGE, and immunoblotted with an anti-NPR1 antibody. The position of the intact NPR1 fusion protein is marked by an arrow.

TGA Binding Activity Is Deregulated in npr1 Mutant Plants.
(A) Extracts from wild-type (Wt; lanes 3 and 4) or npr1 mutant (lanes 5 and 6) Arabidopsis plants treated for 1 hr with water (lanes 3 and 5) or with a solution of 0.5 mM SA (lanes 4 and 6) were subjected to an EMSA, using the as-1 element as a probe (lanes 1 to 6). The EMSA reaction in lane 1 contained no protein (−Prot.), whereas the one in lane 2 contained in vitro–translated TGA3.
(B) Extracts from SA-treated Arabidopsis plants were subjected to an EMSA using the as-1 element as a probe (lanes 1 to 3). A 10-fold molar excess of mutant as-1 (lane 2) or wild-type as-1 (lane 3) DNA was added as competitor. In lane 1, the dash denotes a reaction in which no competitor was added.
In (A) and (B), the brackets indicate the position of the specific complexes; asterisks indicate the positions of a nonspecific complex.

Binding of TGA2 to the LS7 SA Response Element of the Arabidopsis PR-1 Promoter Is Enhanced by NPR1 but Not by the NIM1-2 Mutant Protein.
(A) Nucleotide sequence of the PR-1 promoter surrounding the LS7 element (boxed). The numbers indicate the position of the element relative to the PR-1 RNA start site. LS7 and LS7mut indicate the sequence of the wild-type and mutant oligonucleotides, respectively, used as probes in EMSAs. Note that the LS7 oligonucleotide contains mutated LS6 and LS8 sites. Dashes indicate nucleotides identical to the top strand of the wild-type sequence.
(B) EMSA using in vitro–translated TGA2 (lanes 2 to 4), NPR1 (lane 5), NIM1-2 (lane 6), or the reticulocyte (Retic.) extract alone (lane 1), together with the wild-type as-1 probe (lane 2), the wild-type LS7 (lanes 1, 3, 5, and 6), or a mutated version of the LS7 element (lane 4) as the probe.
(C) EMSA using in vitro–translated TGA2 (lanes 2 to 4) and the LS7 DNA element as a probe (lanes 1 to 4). NPR1 (lane 3) or NIM1-2 (lane 4) was added to the TGA2–LS7 EMSA reaction. Equal amounts of NPR1 and NIM1-2 were used in these experiments (see Figure 4C). For (B) and (C), an equal amount of reticulocyte lysate extract was used in all lanes. The black arrow indicates the position of the TGA2 complex. Retic. indicates that these EMSA reactions contain only protein from the reticulocyte lysate extract.

Binding of TGA2 to the LS5 Negative Regulatory Element of the Arabidopsis PR-1 Promoter Is Enhanced by NPR1 but Not by the NIM1-2 Mutant Protein.
(A) Nucleotide sequence of the PR-1 promoter surrounding the LS5 element (boxed). The numbers indicate the position of the element relative to the PR-1 RNA start site. LS5 indicates the sequence of the wild-type oligonucleotide used as a probe in EMSA. The sequences of mutant oligonucleotide LS3mut, LS4mut, LS5mut, and LS6mut used as competitors in EMSAs are also shown. Dashes indicate nucleotides identical to the top strand of wild-type LS5.
(B) EMSA using in vitro–translated TGA2 (lanes 3 to 10), NPR1 (lane 1), or NIM1-2 (lane 2), together with the wild-type as-1 probe (lane 3) or the wild-type LS5 probe (lanes 1, 2, and 4 to 10). The as-1 (lane 5), LS5 (lane 6), LS3mut (lane 7), LS4mut (lane 8), LS5mut (lane 9), and LS6mut (lane 10) denote double-stranded oligonucleotides added in the EMSA reactions as competitors. The black arrow indicates the position of the TGA2 complex. An equal amount of reticulocyte lysate extract was used in all lanes.
(C) EMSA using in vitro–translated TGA2 (lanes 1 to 3) and the LS5 DNA element as a probe (lanes 1 to 3). NPR1 (lane 2) or NIM1-2 (lane 3) was added to the TGA2–LS5 EMSA reaction. Equal amounts of NPR1 and NIM1-2 were used in these experiments (see Figure 4C). The black arrow indicates the position of the TGA2 complex; the asterisk indicates the position of the slower migrating band detected in reactions containing NPR1 proteins. An equal amount of reticulocyte lysate extract was used in all lanes.

Binding of TGA2 to the as-1 Element Is Enhanced by NPR1 but Not by the NIM1-2 Mutant Protein.
(A) EMSA using in vitro–translated TGA2 (lanes 2 and 3), NPR1 (lane 4), NIM1-2 (lane 5), or the reticulocyte (Retic.) extract alone (lane 1), together with the wild type (lanes 1, 2, 4, and 5) or a mutated version of the as-1 element (lane 3) as the probe. An equal amount of reticulocyte lysate extract was used in all lanes.
(B) EMSA using in vitro–translated TGA2 (lanes 2 to 4) or TGA4 (lanes 5 and 6) and the as-1 DNA element as a probe (lanes 1 to 6). NPR1 (lane 3) or NIM1-2 (lane 4) was added to the TGA2–as-1 EMSA reaction. NPR1 (lane 6) was added to the TGA4–as-1 EMSA reaction. The open arrow indicates the position of the TGA2 complex, the black arrow indicates the position of the TGA4 complex, the asterisk indicates the position of the slower migrating band detected in reactions containing NPR1 proteins, and the bracket indicates the position of the free probe. An equal amount of reticulocyte lysate extract was used in all lanes.
(C) Autoradiography of 4 μL of reticulocyte lysates after separation on 8% SDS–polyacrylamide gels, demonstrating the presence of similar amounts of NPR1 and NIM1-2 used in the EMSAs. The arrow marked 66 kD denotes the apparent molecular mass of NPR1 and NIM1-2.

Immunoblot Analysis of NPR1 Protein in Wild-Type Unstimulated Arabidopsis.
(A) Specificity of the NPR1 antibody. Aliquots (5 μg) of total E. coli protein extracts expressed from an empty pBluescript SK+ vector (Stratagene) (lane 1), a Pinpoint–NPR1 fusion (lanes 2 and 3), or a pRSETB–ICK2 fusion (lane 4) were fractionated on 8% SDS–polyacrylamide gels and immunoblotted with an anti-NPR1 antibody. Lane 2 contains one-tenth (0.5 μg) the amount of proteins loaded in the other lanes.
(B) Specificity of the anti-NPR1 antibody. Arabidopsis proteins from the cytoplasmic (240 μg) and the nuclear (20 μg) fractions were pooled and then fractionated on 8% SDS–polyacrylamide gels (lanes 1 and 2). Proteins in lane 1 were immunoblotted with the anti-NPR1 antibody that had been depleted by treatment with streptavidin–agarose beads bound by the protein moiety produced from the Pinpoint vector. Proteins in lane 2 were immunoblotted with the anti-NPR1 antibody that was depleted on streptavidin–agarose beads bound by the NPR1 fusion protein produced from the Pinpoint–NPR1 vector. Exposure of the films was 1 min for lane 1 and 10 min for lane 2.
(C) Localization of NPR1 in Arabidopsis extracts. Tissues were ground in a nuclei-stabilizing buffer. Nuclei were pelleted, and the supernatant was kept as the cytoplasmic fraction (lane 1). Nuclei were further purified on a Percoll gradient and lysed, and the recovered proteins were kept as the nuclear fraction (lane 2). The proteins were immunoblotted with an anti-NPR1 antibody.
Numbers in (B) and (C) refer to the molecular mass standards expressed in kilodaltons. The cytoplasmic (240 μg) and the nuclear (20 μg) fractions were separated on 3- and 1.5-mm-thick SDS–polyacrylamide gels, respectively. The running conditions were otherwise identical. The position of the NPR1 bands relative to one another was determined by superimposing the molecular mass standards run with each gel. In (A) to (C), arrows denote the position of the NPR1 protein.