Our previous work pointed to cooperation between NtWRKY12 and TGA transcription factors in the activation of the PR-1a promoter. To analyze a possible protein–protein interaction between NtWRKY12 and tobacco TGA factors in vivo and in vitro, we used FRET analysis and in vitro pull-down assays, respectively.

To elaborate the cellular localization of NtWRKY12, TGA1a, TGA2.1, TGA2.2, and NtNPR1 for the FRET analyses we transfected Arabidopsis protoplasts with plasmids in which the corresponding cDNAs were cloned upstream of the YFP or CFP coding sequence. Examples of imaging of the fusion proteins in living protoplasts by confocal laser scanning microscopy are shown in Figure 1. Whereas fluorescence of unfused CFP and YFP was dispersed throughout the cytoplasm and nucleus (Figures 1A,B), NtWRKY12:CFP, TGA2.1:YFP, and TGA2.2:YFP (Figures 1C–E) fluorescence localized mainly in the nucleus. The same results were obtained when the proteins were fused to the other chromophore (data not shown). Interestingly, the signals of both NtNPR1:CFP and NtNPR1:YFP were always concentrated in small nuclear spots (Figure 1F). Furthermore, it is noteworthy that we never detected fluorescence in protoplasts transformed with constructs containing TGA1a fused to either CFP or YFP. These results show that tobacco TGAs 2.1 and 2.2 localize to the nucleus, as was previously found in tobacco and for the Arabidopsis homologs (Pontier et al., 2002; Johnson et al., 2003; Thurow et al., 2005). Due to the extreme brightness of the uneven distributed small nuclear spots of the NtNPR1 chromophore fusions, these could not be used for FRET analysis.

Fluorescence resonance energy transfer analysis is based on overlapping emission/excitation spectra of donor fluorophore CFP and acceptor fluorophore YFP. Emitted fluorescence from CFP can only excite YFP when both fluorophores are in close (less than 10 nm apart) spatial proximity (Wu and Brand, 1994). Thus, a close association of two proteins with fusions to the respective fluorophores would result in an increase of acceptor fluorescence and quenching of the donor fluorescence. As a positive control for FRET, Arabidopsis protoplasts were transfected with an expression plasmid encoding a YFP:CFP tandem fusion, while cotransfection with uncoupled CFP- and YFP-encoding plasmids was used as negative control. The protoplasts were incubated for 24 h, after which FRET measurements were performed. The result is shown in Figure 2A. For the negative control, protoplasts were selected that showed both CFP (475 nm) and YFP (527 nm) emission after excitation of the respective fluorophores to confirm transfection with both CFP and YFP plasmids. Excitation of CFP with 457 nm UV light in these protoplasts resulted in an emission spectrum with a maximum at 475 nm and a certain level of bleeding at 527 nm. CFP excitation of the YFP:CFP fusion protein in the positive control protoplasts resulted in quenched emission at 475 nm, as part of the emission energy was used to excite the YFP fluorophore of the fusion protein, which was subsequently emitted at 527 nm. Thus, the slope of the line connecting the normalized emission intensities at 475 and 527 nm is a measure of the amount of FRET. Similarly, FRET assays were performed on protoplasts cotransfected with combinations of plasmids encoding NtWRKY12 and TGA chromophore fusion proteins.

The control experiments with combinations of NtWRKY12:YFP, TGA2.1:YFP, or TGA2.2:YFP with unfused CFP did not result in increased 527 nm emission (dashed lines in Figures 2B–D, respectively), showing that neither NtWRKY12 nor the TGAs interacted with the CFP chromophore, which would preclude the use of FRET for analyzing interactions between these proteins. The angles of the solid lines in Figures 2B–D indicate the amount of FRET obtained between the various YFP and CFP fusion proteins. In addition to providing the control that the YFP chromophore does not interact with NtWRKY12, the lack of raised 527 nm emission with the combination of NtWRKY12:YFP/NtWRKY12:CFP indicates that NtWRKY12 is not able to form homodimers (Figure 2B). Similarly, although 475 nm emission in the protoplasts transfected with the NtWRKY12:CFP/TGA2.1:YFP plasmids was quenched, 527 nm emission was not significantly higher than in the control protoplasts, showing that no strong interaction occurred between TGA2.1 and NtWRKY12 (Figure 2C). On the other hand, the large amount of FRET in the protoplasts expressing the combination NtWRKY12:CFP/TGA2.2:YFP demonstrates that NtWRKY12 strongly interacted with TGA2.2 (Figure 2D). Although we could not detect the TGA1a:chromophore fusion proteins in our localization experiments (see above), we did perform a cotransfection of protoplasts with TGA1a:YFP and NtWRKY12:CFP. While it was not surprising to find no YFP signal in these protoplasts, we had not expected the reproducible total absence of protoplasts showing CFP emission.

To confirm the interaction between NtWRKY12 and TGA2.2, in vitro pull-down assays were performed with Escherichia coli-expressed GST and Strep/HIS fusion proteins purified using affinity chromatography. In addition to the interaction between NtWRKY12 and TGA2.2, also interactions with TGA1a, TGA2.1, and NtNPR1 were assayed. Figure 3 shows the results of these pull-down assays; the data obtained in Figures 3A–C are summarized in Figure 3D. Figure 3A shows the interactions between different TGA proteins and NtNPR1. GST:NtNPR1 was incubated with various Strep:TGA:HIS fusion proteins and with a Strep:NtNPR1:HIS fusion, after which the complexes were pulled down using Streptactin beads. The pulled-down proteins were analyzed on Western blots using anti-GST antibody conjugate. Strong NtNPR1–TGA2.2 and NtNPR1–NtNPR1 interactions were observed (Figure 3A, lanes 3 and 5), whereas no interactions between NtNPR1 and TGA2.1 or TGA1a were detectable (Figure 3A, lanes 2 and 4). Figure 3A, lanes 6 to 10 show the controls with single fusion proteins. The low background signal obtained with GST:NtNPR1 (Figure 3A, lane 6) was also visible in Figure 3A, lane 4. Homodimer formation as seen with the tobacco NtNPR1 has been reported for Arabidopsis NPR1, where it relies on interaction of NPR1’s BTB/POZ domain, which is independent of SA (Boyle et al., 2009).

In the experiments shown in Figures 3B,C, GST fusions of NtWRKY12 and NtNPR1 were incubated with various Strep:TGA:HIS fusions, and protein complexes were bound to Glutathione–Sepharose 4B beads. The pulled-down proteins were analyzed on Western blots using anti-HIS antibodies. Interactions of NtWRKY12 were observed with TGA2.2 (Figure 3B, lane 1), but not with TGA1a or TGA2.1 (Figure 3C, lanes 1 and 5). Moreover, the conclusion from Figure 3A that NtNPR1 interacts with TGA2.2, but not with TGA1a or TGA2.1 was confirmed in this system (Figure 3B, lane 2; Figure 3C, lanes 2 and 6).

As a first step toward the characterization of the NtWRKY12 sequence involved in the interaction with TGA2.2, two NtWRKY12 deletion mutants were made. NtWRKY12ΔC lacks the C-terminal 87 aa of the 220 aa long protein; NtWRKY12BD lacks the N-terminal 113 aa. Both mutants were found to interact with TGA2.2 (Figure 3B, lanes 6 and 7). Either the overlap between the two mutant proteins (aa 114–133) is involved in the interaction of NtWRKY12 with TGA2.2, or NtWRKY12 contains two independent binding sites for TGA2.2, possibly involved in the interaction with a TGA dimer.

Previously, yeast one-hybrid screening for tobacco proteins binding to the PR-1a promoter resulted in the isolation of a protein corresponding to the C-terminal 107 aa of NtWRKY12 fused to the GAL4 activation domain. This protein contained the conserved WRKY and Zn-finger domains and, apparently, a DNA-BD (van Verk et al., 2008). Moreover, it was shown that full-length (220 aa) NtWRKY12 was able to activate PR-1a::HIS gene expression in yeast, indicating that in addition to a BD, NtWRKY12 also contains an activating domain (AD). To further characterize functional domains of NtWRKY12, deletion mutants of NtWRKY12 were assayed in the one-hybrid system in three different ways. First, the mutants were fused to the GAL4 BD and assayed for their ability to activate GAL4 promoter::Ade reporter gene expression (Figure 4; results summarized in the column with the caption “BD”). Fusions, which activated the reporter gene, were concluded to contain the NtWRKY12 AD. Second, the mutants were fused to the GAL4 AD and assayed for their ability to activate PR-1a::HIS gene expression (Figure 4; results summarized in the column with the caption “AD”). Fusions which activated gene expression were concluded to contain the NtWRKY12 BD. Third, the mutants were expressed as non-fused proteins and assayed for their ability to activate PR-1a::HIS expression (Figure 4; results summarized in the column with the caption “–”). Mutants which activated gene expression were concluded to contain both the AD and BD domains of NtWRKY12.

Figure 4 (column “BD”) shows that GAL4 BD fusions lacking the C-terminal 37 aa (construct 4) or N-terminal 40 aa (construct 7) of NtWRKY12, and a protein with both these deletions (construct 15) were able to activate GAL4::Ade expression. Apparently, the NtWRKY12 AD function is contained within the aa 41–183 region of the protein. The GAL4 AD fusion of the smallest NtWRKY12 deletion mutant that was able to activate PR-1a::HIS expression was construct 18 (Figure 4, column “AD”). Thus, the NtWRKY12 BD is localized in the sequence of aa 121–201. This region encompasses both the conserved WRKY and Zn-finger domains. Apparently, aa upstream of the WRKY domain are also necessary for DNA binding, as a deletion mutant with only seven aa in front of the WRKY domain (Figure 4, construct 13, aa 132–220) was not able to activate HIS gene expression. Construct 17 (aa 41–201) combined the minimal sequences with NtWRKY12 AD and BD activity (construct 15, aa 41–183, and construct 18, aa 121–201). However, the non-fused protein encoded by construct 17 was not able to activate PR-1a::HIS expression (Figure 4, column “–“). To permit both AD and BD activity, the sequence of construct 17 had to be extended by either the N-terminal 40 aa of NtWRKY12 (Figure 4, construct 2, aa 1–201) or the C-terminal 19 aa (Figure 4, construct 7, aa 41–220). Possibly, the lack of activity of the protein encoded by construct 17 (aa 41–201) was due to instability or misfolding of the polypeptide.

In our previous paper we showed that cotransfection of Arabidopsis protoplasts with 35S::NtWRKY12 and PR-1a::GUS constructs resulted in a strong increase in GUS expression (van Verk et al., 2008). NtWRKY12 activates PR-1a::GUS expression in Arabidopsis protoplasts by binding to the WK-box (TTTTCCAC) at position −564 in the PR-1a promoter (van Verk et al., 2008). The as-1-like element (CGTCA[N]₆TGACG) at position −592 is the likely binding site for TGA transcription factors. This raises the possibility that NtWRKY12 and TGA2.2 bind to the PR-1a promoter in close proximity, and binding of NtWRKY12 and TGA2.2 might be stabilized by interactions between the two factors that were observed in vivo and in vitro (Figures 2 and 3). To further investigate the role of NtWRKY12, TGA and NPR1 on activation of PR-1a driven expression, additional transactivation assays were set up in protoplasts isolated from leaves of Arabidopsis seedlings grown on MS medium. To avoid interfering effects of NtWRKY12 binding to the far upstream WK binding site (−859), this WK site in the PR-1a::GUS reporter gene used in these experiments was mutated (TTTTCCAC into TCCCTTGC). Figure 5A shows the effects of overexpression of NtWRKY12, TGA2.1, TGA2.2, and NPR1 on PR-1a::GUS expression in wild-type Arabidopsis protoplasts. Obviously, NtWRKY12 greatly enhanced beta-glucuronidase expression from the PR-1a promoter (sixfold over background level). Overexpression of TGA2.1, TGA2.2 or NtNPR1, or combinations of the TGAs with NtNPR1 did not result in enhanced GUS expression. Neither did TGA2.2, alone or in combination with NtNPR1, affect the level of NtWRKY12 enhanced PR-1a::GUS expression, whereas overexpression of TGA2.1, alone or together with NtNPR1, notably reduced NtWRKY12 activated GUS expression.

In Arabidopsis, PR gene expression is dependent on NPR1 and there is accumulating evidence that NPR1 orthologs similarly effect expression of PR genes in other plant species (Rayapuram and Baldwin, 2007; Anand et al., 2008; Le Henanff et al., 2009). We wondered whether the lack of effects of overexpressed NtNPR1 on PR-1a::GUS expression in the cotransfection experiments could be due to the presence of saturating levels of functionally equivalent Arabidopsis NPR1. However, the results of transactivation assays in protoplasts from npr1-1 mutant plants were virtually identical to those of the wild-type protoplasts (compare Figures 5A,B). This implies that PR-1a gene expression mediated by NtWRKY12 in Arabidopsis protoplasts is independent of NPR1.

On the basis of sequence homology, tobacco TGA2.2 belongs to the group II TGA proteins together with Arabidopsis TGAs 2, 5, and 6 (Xiang et al., 1997). To exclude the possibility that the absence of effects of overexpressed tobacco TGA on PR-1a::GUS expression in the Arabidopsis protoplasts was caused by functionally similar Arabidopsis TGAs, cotransfection experiments were performed in Arabidopsis protoplasts derived from tga2-1 tga5-1 tga6-1 (tga256) and tga2-1 tga3-1 tga5-1 tga6-1 (tga2356) triple and quadruple mutant plants (Figure 6). Also in these mutant backgrounds, overexpression of NtWRKY12 led to activation of PR-1a::GUS expression (ninefold over background level in the triple mutant (Figure 6A), although the enhancement in the quadruple mutant was greatly reduced (twofold, Figure 6B). Likely, this reduced GUS expression is the result of reduced production of NtWRKY12 from the transfected 35S::NtWRKY12 gene, similar to the reduced GUS activity from the 35S::GUS gene in tga2356 protoplasts compared to wild-type or tga256 protoplasts (Figure 6C).

Together, the results of the cotransfection assays in Arabidopsis leaf protoplasts suggest that NtWRKY12 is an important transcriptional activator of PR-1a::GUS expression and that TGA2.1, TGA2.2, or NtNPR1, alone or in combination, do not positively affect activation.

The open reading frames of NtWRKY12 and NtNPR1, and mutants encoding the 133 amino acids (aa) of the N-terminal half (NtWRKY12ΔC) or 107 aa of the C-terminal half (NtWRKY12BD) were cloned in frame behind the GST open reading frame of expression vector pGEX-KG (Guan and Dixon, 1991), expressed and purified according to van Verk et al. (2008).

The full-length coding sequence of N. tabacum TGA1a, TGA2.1, TGA2.2, and NPR1 were cloned in frame of expression vector pASK-IBA45 plus harboring a Strep and HIS tag (IBA). These plasmids were transformed into E. coli XL1. For induction of protein expression, cultures were grown to mid-log phase at 37°C, after which tetracycline was added to a final concentration of 0.2 μg mL⁻¹ and incubation continued for 3.5 h at 29°C. The cells were harvested by centrifugation, resuspended in 1/25th volume lysis buffer [1× PBS containing 1% (v/v) NP40, 2 mM DTT, and 1/50th volume Complete (Roche) protease inhibitors] and lysed by sonication (Vibracell, Sonics en Materials Inc., USA). Soluble protein fraction was collected by centrifugation, and expressed fusion proteins were analyzed using 12% (w/v) SDS-PAGE.

For the in vitro pull-down assay, GST-fusion proteins were mixed with Strep-fusion-HIS proteins in binding buffer [1× PBS, 1% (w/v) NP40, 2 mM DTT] and incubated on an orbital shaker for 1 h at room temperature. To this mixture Glutathione–Sepharose 4B beads (GE Healthcare) or Strep–Tactin Sepharose beads (IBA) in buffer W (100 mM Tris pH 8.0, 150 mM NaCl, 1 mM EDTA) were added, and incubation was continued for an additional hour. The beads were washed five times with PBS [with 1% (w/v) NP40 for Glutathione beads] after which the beads were collected, resuspended in Laemmli buffer, and heated at 95°C for 2 min.

The proteins bound to the beads were separated by SDS-PAGE and transferred onto Hybond P membrane (GE Healthcare). Membranes were incubated with the anti-GST antibody (GE Healthcare), or anti-HIS antibody (5 Prime) according to manufacturers instructions and exposed to X-ray film.

A tetramer fragment of the tobacco PR-1a promoter corresponding to the region −605 to −513 relative to the transcription start site cloned in front of the HIS3 gene and integrated in the Saccharomyces cerevisiae genome of strain Y187 (van Verk et al., 2008) was used to screen for the DNA-BD of NtWRKY12 and presence of both an activation (AD) and BD. Deletion mutants of NtWRKY12 were cloned in pACT2 to screen for the presence of an BD or p415GPD-HA to screen for both the BD and AD. Mutants were screened for HIS-independent growth with addition of 3AT up to 20 mM. To locate the AD, deletion mutants were cloned into pAS2-1 and transformed in yeast strain PJ69-4A containing the Gal4 binding site in front of the Ade gene. Mutants were screened for adenine independent growth.

For microscopy protoplasts were prepared from Arabidopsis thaliana ecotype Col-0 cell suspensions according to van Verk et al. (2008).

The leaves from approximately 50 four-week-old seedlings (Col-0, npr1-1, tga256, tga2356) grown on sterile medium were cut in small pieces and protoplasts were prepared according to He et al. (2007). In total 1 × 10⁵ protoplasts were transformed per transfection using polyethylene glycol [40% (w/v) PEG 4000, 0.2 M mannitol, 0.1 M CaCl₂].

Protoplasts were cotransfected with 2 μg of plasmid carrying PR-1a promoter::GUS construct and 6 μg of 35S::effector plasmid pRT101 (Töpfer et al., 1987). As a control, cotransformation of PR-1a::GUS construct with the empty expression vector pRT101 was carried out. The protoplasts were harvested 16 hrs after transformation and GUS activity was determined. GUS activities from triplicate experiments were normalized against total protein level.

Protoplasts were cotransfected with 10 μg of plasmid carrying protein::CFP and 10 μg of protein::YFP constructs. As controls 2.5 μg of plasmid containing unfused CFP/YFP or 10 μg YFP:CFP fusion was used. Protoplasts expressing the fusion proteins were analyzed with a Leica DM IRBE confocal laser scanning microscope with a 63× water objective, digital zoom, and 51% laser intensity. The fluorescence was visualized with an Argon laser for excitation at 457 nm with 471–481 nm emission filter for CFP and 514 nm excitation with a 522- to 532-nm filter for YFP. A transmitted light picture was used as reference. For FRET analysis Lambda scanning was performed by excitation at 457 nm and by measuring emission from 468 to 587 nm in a total of 30, 5 nm wide intervals using a RSP465 filter. Of every interval the intensity of the whole cell was quantified using ImageJ. The intensity of five protoplasts were averaged and normalized. The slopes between the 475- and 527-nm point were compared for differences in quenched donor emission and increased acceptor emission in comparison to the controls. Similar results were obtained for three independent transfections.