Using a simple oligo selection procedure, we have previously identified a tobacco sequence-specific DNA-binding activity, TDBA12, that increases markedly during the tobacco mosaic virus (TMV)-induced hypersensitive response (HR). Based on the binding specificity and the two cDNA clones isolated, TDBA12 is related to a novel class of DNA-binding factors containing WRKY domains. In the present study, we report that TDBA12 could be induced not only by TMV infection but also by treatment with salicylic acid (SA) or its biologically active analogs capable of inducing pathogenesis-related (PR) genes and enhanced resistance. TDBA12 was sensitive to temperature and the protein dissociating agent sodium deoxycholate, suggesting that it may be a multimeric factor in which protein–protein interaction is important for the enhanced DNA-binding activity. Pre-treatment of nuclear extracts with alkaline phosphatase abolished TDBA12, suggesting that protein phosphorylation is important for its high DNA-binding activity. TDBA12 specifically recognized the elicitor response element of the tobacco class I basic chitinase gene promoter. The increase in the levels of TDBA12 following TMV infection or SA treatment preceded the induced expression of the tobacco chitinase gene. These results strongly suggest that certain WRKY DNA-binding proteins may be activated by enhanced protein phosphorylation and regulate inducible expression of defense-related genes during pathogen- and SA-induced plant defense responses.
During the last decade, significant progress has been made in understanding plant disease resistance at the molecular level. A key difference between resistant and susceptible plants is the timely recognition of the invading pathogen and the rapid and effective activation of host defense mechanisms ( Yang et al. 1997 ). Timely recognition of the pathogen is achieved through the specific interaction between pathogen-encoded molecules called elicitors and plant host receptors (many of which may be encoded by disease resistance genes). This specific interaction probably activates a signal transduction cascade that may involve protein phosphorylation, ion fluxes and reactive oxygen species (ROS), and subsequently leads to differential regulation of a large number of plant genes associated with defense responses. The best characterized plant defense genes include those that encode β-glucanases (PR-2) and chitinases (PR-3), which have direct antimicrobial activities, by digesting the cell wall components glucan and chitin of many microbial pathogens (Key & Kosuge 1985). Despite their established biological functions in the plant defense response, the molecular mechanisms responsible for their induced expression during plant defense responses are largely unknown.
Transcriptional regulation of gene expression may be mediated by the change of the level or activity of sequence-specific DNA-binding (SSDB) factors. Because transcriptional regulation of a large number of genes is a vital and major part of plant defense responses ( Rushton & Somssich 1998), identification and characterization of SSDB factors differentially regulated during the activation of this response may provide novel insights into the molecular mechanisms by which plant defense responses are regulated. For this purpose, we have been interested in identifying differentially regulated SSDB activities associated with the interaction of tobacco with tobacco mosaic virus (TMV). Infection of tobacco with TMV results in one of two distinct responses depending upon the genetic background of the host and the viral strain ( Whitham et al. 1994 ). In a susceptible tobacco cultivar, infection with TMV (e.g. strain U1) results in the systemic spread of the virus from the original point of entry to distal parts of the host. In contrast, tobacco plants carrying the dominant N genes respond to TMV infection by rapid, localized host cell death (hypersensitive response; HR), which resembles animal programmed cell death. In addition to the localized HR, a resistant tobacco plant responds to TMV infection by activating defense responses in upper uninfected parts of the plant so that the entire plant becomes more resistant to subsequent infections by TMV or other pathogens, a phenomenon commonly referred to as systemic acquired resistance (SAR). Plant HR and SAR are common disease resistance responses found in many plant-pathogen systems ( Yang et al. 1997 ).
In a previous study, we reported our use of a simple oligo selection procedure to identify several differentially regulated SSDB activities associated with TMV-induced HR in resistant tobacco plants ( Wang et al. 1998 ). One of these differentially regulated SSDB activities identified (denoted TDBA12) is markedly enhanced in resistant tobacco plants following TMV infection ( Wang et al. 1998 ). TDBA12 recognizes a TTGAC DNA sequence that has been previously found to be both sufficient and essential for induced expression of a number of plant defense genes. Two genes encoding proteins that bind to the same TTGAC sequence have been isolated and found to be homologous to the novel WRKY DNA-binding protein genes that recognize the elicitor responsive elements of the parsley PR-1 gene promoters ( Rushton et al. 1996 ). Thus, certain members of WRKY DNA-binding proteins may serve as common regulators of plant defense genes in different plant species. In the present study, we have further studied the activation and regulation of TDBA12 during plant defense responses. Using the electrophoretic mobility shift assay (EMSA), we demonstrate that TDBA12 is activated not only by TMV infection in resistant tobacco plant, but also by the plant defense signal molecule SA and its biologically active analogs. Experiments using the protein dissociating agent sodium deoxycholate and alkaline phosphatase have demonstrated that TDBA12 may be a multimeric protein complex that requires protein–protein interaction and protein phosphorylation for its enhanced DNA-binding activity. Furthermore, we have identified a tobacco class I basic chitinase gene as a potential target gene for TDBA12. From these results we propose that certain WRKY DNA-binding factors may serve as components in pathogen-induced signal transduction pathways in plant host cells and regulate expression of certain plant defense genes.
Activation by both TMV infection and SA treatment
Because inoculation of resistant tobacco plants with even a high titer of TMV leads to infection of only a small portion of plant cells, there is a high background of uninfected cells that can mask any differentially regulated DNA-binding activities. To reduce this background, we took advantage of the temperature sensitivity of TMV-induced HR ( Whitham et al. 1994 ). Thus, we first inoculated the resistant tobacco plants with TMV and maintained them at a higher temperature (32°C), under which the host HR is not activated and the virus replicates and spreads throughout the plant to cause systemic infection. After 3–4 days at 32°C, these infected plants were shifted to 22°C to induce the HR. Since systemic infection had occurred prior to the activation of HR, there was a low background of uninfected cells in these plants. TMV-infected susceptible tobacco, which do not exhibit TMV-induced HR under either the higher or the lower temperature, were used as controls. TDBA12 increased markedly in nuclear extracts prepared from TMV-infected resistant plants, harvested 9 h after the temperature shift from 32°C to 22°C ( Fig. 1a). In contrast, no significant increase was observed in TMV-infected susceptible tobacco ( Fig. 1a) or uninfected resistant plants (data not shown).
Since TMV-induced HR in tobacco is associated with increased levels of SA, which plays an essential role in the establishment of SAR and induced expression of a large number of defense-related genes ( Malamy et al. 1990 ; Malamy et al. 1992 ), we then examined whether the levels of TDBA12 could also be induced by SA. Indeed, spraying of tobacco leaves with SA induced TDBA12 ( Fig. 1b). Furthermore, a biologically active analog of SA (acetylSA) which induces PR genes and enhanced resistance also increased the levels of TDBA12, whereas two biologically inactive analogs (3-hydroxybenzoic acid and 4-hydroxybenzoic acid) did not ( Fig. 1b).
Expression of tWRYK1 and tWRKY2 genes
The marked increase in the level of TDBA12 during TMV-induced HR or following SA treatment could be caused by increased expression of genes encoding proteins associated with this defense-associated DNA-binding activity. To test this possibility, we examined the expression patterns of cloned tWRKY1 and tWRKY2 genes in tobacco plants after TMV infection. As shown in Fig. 2, the tWRKY1 mRNA levels started to increase in TMV-infected resistant tobacco plants between 6 and 9 h after the temperature shifted from 32°C to 22°C. There was no significant change in the tWRKY2 mRNA levels throughout the same time period after the temperature shifting ( Fig. 2). As described below, TDBA12 started to increase in TMV-infected resistant tobacco plants between 3 to 6 h after the temperature shifting. Thus, the marked increase in the levels of TDBA12 was not fully correlated with increased transcription of either the tWRKY1 or tWRKY2 genes.
Sensitivity to temperature and protein dissociating agent
Because of the failure to fully correlate the induction of TDBA12 with enhanced expression of the two cloned WRKY genes, we examined the possible involvement of certain post-translational events in the activation of TDBA12. Because of the established importance of activated protein kinase cascades in plant defense responses ( Yang et al. 1997 ), we first examined the possible involvement of protein phosphorylation in the regulation of TDBA12 by analyzing its sensitivity to in vitro pre-treatment of nuclear extracts with alkaline phosphatase. These studies revealed that the DNA-binding activity of TDBA12 was unstable at 37°C, the condition under which the pre-treatment with alkaline phosphatase was performed ( Fig. 3a). Interestingly, increasing the glycerol concentration from 10% to 15–40% in the buffer stabilized its DNA-binding activity at this modest temperature ( Fig. 3a). This result suggested that active TDBA12 may be a multimeric protein complex that dissociates at 37°C with low concentrations of glycerol. In support of this hypothesis, inclusion of the protein dissociating agent sodium deoxycholate in the nuclear extract at a final concentration of 0.5% completely abolished the DNA-binding activity of TDBA12 ( Fig. 3b). Interestingly, the DNA-binding activity of purified recombinant tWRKY1 and tWRKY2 proteins were also sensitive to treatment with sodium deoxycholate, but not to the incubation at 37°C (data not shown). These results suggested that the active forms of these WRKY proteins are multimeric and that in the TDBA12 complex there is a very labile or unstable component that is important for its high DNA-binding activity. Using the same protein dissociating agent at a slightly higher concentration (1%), it has been previously demonstrated that a potato DNA-binding factor (PBF-1) that binds to the elicitor responsive elements of potato PR-10a gene promoter might also exist as a multimeric protein complex ( Despres et al. 1995 ). In contrast, the DNA-binding activity that recognizes the CaMV as-1 cis-element following SA treatment increases after treatment with 0.2–1.2% sodium deoxycholate ( Jupin & Chua 1996). Thus, protein–protein interactions within a multimeric DNA-binding protein complex can either promote or inhibit the DNA-binding activity.
Sensitivity to alkaline phosphatase
After establishing that TDBA12 is stable at 37°C in the presence of higher glycerol concentrations, we used the same glycerol concentration (15%) and buffer compositions in all nuclear extracts in our in vitro studies on the sensitivity of TDBA12 to alkaline phosphatase treatment. As shown in Fig. 4(a), treatment of nuclear extracts with alkaline phosphatase resulted in almost complete loss of the DNA-binding activity of TDBA12 from TMV-infected resistant tobacco plants after temperature shifting. This sensitivity has been reproducibly detected with five independently prepared nuclear extracts. Furthermore, the enhanced DNA-binding activity of TDBA12 after SA treatment was also sensitive to the phosphatase treatment ( Fig. 4b). To test whether the decreased activity of TDBA12 after incubation with alkaline phosphatase was indeed due to the phosphatase activity, we included a phosphatase inhibitor sodium fluoride in the reaction mixture and found that this phosphatase inhibitor inhibited the decrease of the TDBA12 caused by the phosphatase treatment ( Fig. 4).
Recognition of the cis-element of a tobacco basic chitinase gene by a DNA-binding activity similar to TDBA12
Tobacco class I basic chitinase genes are induced by pathogen infection or treatment with pathogen-derived elicitors, SA and biologically active SA analogs (such as 2,6-dichloroisonicotinic acid, INA) ( Fukuda et al. 1991 ; Ward et al. 1991 ). Molecular analysis has previously shown that the DNA region between –788 and –345 from the initiation site of transcription in the promoter of the basic class I chitinase gene, CHN50, is required for a fungal elicitor-induced expression in tobacco suspension cultures ( Fukuda & Shinshi 1994; Fukuda 1997). Within this DNA region, a sequence (TGACTNNNTGACC) that contains two TGAC direct repeats spaced by four nucleotides is specifically recognized by a DNA-binding activity present in nuclear extracts prepared from elicitor-treated cultured cells but not in extracts from untreated cells ( Fukuda 1997). Deletion of this sequence abolishes elicitor-induced expression of a reporter gene, indicating that this direct-repeated sequence is an essential cis-acting element ( Fukuda 1997). Since the second type of W-boxes (TGACNNNNNNGTCA) found in the parsley PR1 gene promoter contain two TGAC convergent repeats, the two TGAC direct repeats found in the CHN50 gene promoter could represent a third type of W-boxes recognized by pathogen-induced WRKY DNA-binding factors ( Rushton & Somssich 1998). To test this possibility, we examined whether this sequence is recognized by either a purified tobacco WRKY DNA-binding protein (tWRKY1) or pathogen-induced TDBA12 in the nuclear extracts from TMV-infected resistant tobacco plants. As shown in Fig. 5(a), when the recombinant tWRKY1 protein was incubated with the CHN probe (Pchn), a retarded band was detected with a mobility similar to that detected with probe P12a, which contains the TTGACC binding sequence. In fact, tWRKY1 appeared to have a higher affinity for Pchn than for P12a based on the intensities of the retarded complexes detected by these two probes ( Fig. 5a). When incubated with nuclear extracts, Pchn also detected a TMV-induced DNA-binding activity similar to TDBA12 detected with P12a. Like the purified tWRKY1 protein, this TMV-induced DNA-binding activity recognized Pchn with a higher affinity than P12a based on the intensities of the retarded complexes. To determine whether the sequence of the two direct repeats in the Pchn probe is important for the recognition by both purified tWRKY protein and TMV-induced TDBA12, we constructed a mutant probe (mPchn) in which the direct repeat sequence was changed from TGAC to TGAA. As shown in Fig. 5, this mutant probe failed to be recognized by either the purified recombinant tWRKY1 protein or the TMV-induced TDBA12. Previously, it has been shown that this mutant oligo failed to detect the elicitor-induced DNA-binding activity in tobacco cell cultures ( Fukuda 1997).
To examine further the sequence specificity of the DNA–protein interactions, we performed competition assays for the binding of Pchn and P12a probes by purified tWRKY1 proteins or TMV-induced TDBA12. As shown in Fig. 6, unlabeled Pchn was very effective for competing with labeled P12a probes for being bound by either the purified tWRKY1 proteins or TMV-induced TDBA12. In contrast, the mPchn probe, which contains mutated TGAA repeats, failed to compete with labeled P12a for being bound by the purified WRKY proteins or pathogen-induced TDBA12. These results further indicated that the P12a probe with the TTGACC binding sequence and the Pchn probes with the two TGAC direct repeats were recognized by WRKY DNA-binding proteins with a high degree of sequence specificity.
Activation of TDBA12 preceded expression of the CHN50 gene
The specific recognition of the elicitor response element of tobacco CHN50 gene by TDBA12 suggests that this defense-related gene serves as a potential target gene of this pathogen-induced WRKY DNA-binding activity. To provide further evidence for this model, we determined the induction kinetics of the CHN50 gene expression in comparison with the induction of TDBA12 in tobacco plants after TMV infection and SA treatment. In TMV-infected resistant tobacco plants, the levels of TDBA12 started to increase between 3 and 6 h after the temperature shift from 32°C to 22°C ( Fig. 7a), whereas the mRNA levels of the CHN50 gene started to accumulate between 6 and 9 h after the temperature shift ( Fig. 7b). For plants sprayed with SA, TDBA12 reached its maximum levels by 4 h after the treatment ( Fig. 8a), whereas substantial accumulation of the CHN50 gene appeared to occur between 4 and 8 h after the treatment ( Fig. 8b). In a previous study with the expression of the tobacco basic chitinase gene, induction of the gene was also detected between 4 and 8 h after spraying the plants with 50 m m SA ( Ward et al. 1991 ). Thus, for both TMV infection and SA treatment, activation of TDBA12 preceded induced expression of the CHN50 gene.
We have previously identified a sequence-specific DNA-binding activity denoted TDBA12 that is markedly activated during TMV-induced HR in resistant tobacco plants ( Wang et al. 1998 ). In the present study, we have further investigated the regulation of TDBA12 during the activation of plant defense responses. We have shown that TDBA12 is induced in tobacco plants not only by TMV infection ( Fig. 1a), but also by SA and its biologically active analogs ( Fig. 1b). DNA-binding activities that are activated by SA have been identified before. The most characterized SA-responsive DNA-binding activity is ASF-1, which binds the as-1 cis-element from CaMV 35S promoter and the as-1-like ocs and nos sequences from Agrobacterium octopine and nopaline synthase promoters, respectively ( Bouchez et al. 1989 ; Fromm et al. 1989 ; Fromm et al. 1991 ; Lam et al. 1990 ). Although TDBA12 and ASF share a number of characteristics such as responsiveness to SA and sensitivity to phosphatase treatment, they also differ in a number of aspects. First, promoters containing as-1-like elements have been previously found to be activated not only by SA but also by inactive SA analogs incapable of inducing PR genes and enhanced disease resistance ( Ulmasov et al. 1994 ). In fact, these cis-elements are also activated by both active and inactive auxin analogs ( Ulmasov et al. 1994 ). These results suggest that activation of ASF-1 may represent a general stress response. In contrast, although SA and its biologically active analog acetylSA effectively induced TDBA12, biologically inactive analogs did not ( Fig. 1b). This observation indicated that induction of TDBA12 may be part of a more specific defense response than ASF-1. Second, while ASF-1 belongs to the basic leucine zipper (bZiP) family of transcription factors, which have been found in other types of organisms, TDBA12 belongs to WRKY proteins that appear to be unique to plants ( Rushton & Somssich 1998). Third, ASF-1 and TDBA12 recognize different cis-elements. ASF-1 recognized ACGT elements ( Foster et al. 1994 ), whereas TDBA12 recognize several types of W-boxes with a TGAC core sequence. Finally, activity of ASF-1 is enhanced by treatment with protein dissociation agents, suggesting that protein–protein interaction suppresses its DNA-binding activity ( Jupin & Chua 1996). By contrast, TDBA12 was sensitive to both a modest temperature and a protein dissociation agent ( Fig. 3), suggesting that protein–protein interaction is important for its enhanced DNA-binding activity.
Chitinases are believed to play an important role in plant defense against pathogen infection because of their antimicrobial activities ( Brunner et al. 1998 ). In tobacco, there are three identified classes of chitinases that accumulate in response to infection by pathogens or treatment with SA ( Lawton et al. 1992 ). In previously published studies, Fukedsa and Shinshi have identified a cis-acting elicitor responsive element that is essential for the induced expression of the CHN50 gene in response to treatment with a fungal elicitor ( Fukuda & Shinshi 1994; Fukuda 1997). This cis-acting element contains a direct repeat of TGAC spaced by four nucleotides and binds to a DNA-binding activity that is preferentially present in the nuclear extracts from elicitor-treated tobacco cell suspension cultures ( Fukuda 1997). In the present study, we have shown that this elicitor-induced DNA-binding activity was very similar and possibly identical to TDBA12 based on binding specificity and pathogen inducibility ( Fig. 5 and 6). Thus, the elicitor response element of the tobacco CHN50 gene promoter, which contains a direct repeat of TGAC, may represent a third type of W-boxes recognized by pathogen-induced WRKY DNA-binding proteins. Previously, WRKY proteins have been shown to specifically bind DNA molecules with a TTGAC sequence or a convergent repeat of TGAC spaced by six nucleotides ( Rushton et al. 1996 ; Wang et al. 1998 ). The ability of WRKY proteins to bind to the TGAC repeats of different directions may be related to the fact that many of these WRKY DNA-binding proteins contain two DNA-binding domains and can oligomerize to form multimeric protein complexes.
In support of the involvement of TDBA12 in the expression of CHN50, we have further demonstrated that induction of TDBA12 by TMV infection or SA treatment preceded the increase in mRNA level for the tobacco chitinase gene ( Fig. 7 and 8). These results suggest that the tobacco basic chitinase gene may be one of the target genes for pathogen-induced WRKY DNA-binding proteins. In addition to the tobacco class I basic chitinase gene, pathogen-induced WRKY DNA-binding factors recognize the elicitor-response elements of the parsley PR-1 genes ( Rushton et al. 1996 ), suggesting that members of this group of DNA-binding factors may also regulate the expression of other PR genes. Moreover, as reviewed by Rushton & Somssich (1998), DNA sequences similar to the W boxes have been found in the maize PR-1 class gene Prms ( Raventos et al. 1995 ), the potato glutathione S-transferase gst1 (prp1) ( Hahn & Strittmatter 1994), the PR10 gene from asparagus (AoPR10) ( Warner et al. 1993 ), the potato PR-10a gene ( Despres et al. 1995 ), and the stilbene synthase gene (Vst1) from grapevine encoding an enzyme of phytoalexin biosynthesis ( Schubert et al. 1997 ). Thus, the pathogen-induced WRKY DNA-binding proteins may serve as common transcriptional activators that regulate the expression of a large set of pathogen-responsive genes throughout the plant kingdom.
Using the protein dissociation agent sodium deoxycholate and alkaline phosphatase phosphatase, we have demonstrated that the enhanced DNA-binding activity of tobacco WRKY-related TDBA12 requires both protein–protein interaction and protein phosphorylation ( Fig. 3 and 4). A potato elicitor-induced DNA-binding factor, PBF-1, that recognizes the elicitor response element of the potato PR-10a gene promoter is also sensitive to both sodium deoxycholate and alkaline phosphatase ( Despres et al. 1995 ). PBF-1 was detected with the DNA sequence between –135 and –105 upstream of the transcription start of the potato PR-10a gene. Within this DNA region, there are a number of distinct DNA motifs, including two AT-rich elements (ATAAAAT and AAAAAT), an AP-1 binding site (TGACACA) and an E-box (CAAATG) ( Despres et al. 1995 ). These authors have previously suggested that PBF-1 binds to the AP-1 site or the E-box because both AP-1 and E box-recognizing helix-loop-helix factors interact with DNA as multimers ( Despres et al. 1995 ). Interestingly, the AP-1 binding site in the same DNA molecule can also form a TGAC-N6-GTCA W box with adjacent sequences. Therefore, potato PBF-1 could be an elicitor-induced WRKY factor that requires both protein–protein interaction and protein phosphorylation for enhanced DNA-binding activity as demonstrated in the present study. The possible involvement of protein phosphorylation in the activation of these pathogen-induced WRKY DNA-binding factors is consistent with the in vivo study with protein kinase or phosphatase inhibitors. In potato, the protein kinase inhibitor staurosporine blocks both the induction of the PR-10a gene and the activation of PBF-1 following elicitor treatment. The protein phosphatase inhibitor okadaic acid, on the other hand, induces PR-10a gene expression and activation of PBF-1 in the absence of elicitor treatment ( Despres et al. 1995 ). More recently, evidence has been provided that a functional homolog of mammalian protein kinase c participates in the elicitor-induced activation of PBF-1 and expression of potato PR-10a gene ( Subramaniam et al. 1997 ). In tobacco, protein kinase inhibitor staurosporine also blocks the induction of CHN50 gene expression and the activation of the elicitor-induced DNA-binding activity that recognizes its W box cis-acting element ( Fukuda 1997). These results collectively suggest that protein phosphorylation plays an important role in the regulation of pathogen-induced WRKY DNA-binding proteins and consequently the expression of their potential target genes.
On the other hand, other studies have indicated that the regulation of pathogen-induced WRKY DNA-binding proteins may also involve de novo protein synthesis. In parsley, three WRKY genes have been isolated, two of which are rapidly induced in cell cultures following elicitor treatment ( Rushton et al. 1996 ). Interestingly, this rapid induction of parsley WRKY genes was not accompanied by enhanced DNA-binding activity as detected by EMSA in the nuclear extracts prepared from elicitor-treated cell cultures ( Rushton et al. 1996 ). In contrast, we found that during the first 6 h after temperature shifting TDBA12 markedly increases in TMV-infected resistant tobacco without concomitant induction of two isolated tobacco WRKY genes ( Fig. 1 and 2). These contrasting results may suggest that while pathogen-induced WRKY proteins play a general role in regulating plant defense gene expression, the mechanisms for their induction or activation may vary among different plant species. Alternatively, since WRKY proteins are encoded by a family of genes, the two isolated tobacco WRKY genes may not be the members of this gene family that respond to pathogen infection or elicitor treatment. In support of this possibility, Fukuda (1997) has shown that activation of the TDBA12-equivalent DNA-binding activity from elicitor-treated tobacco cell cultures can be blocked by protein synthesis inhibitor cycloheximide. These result suggest that regulation of pathogen-induced WRKY proteins may require both de novo synthesis and post-transcriptional modification of WRKY proteins. Understanding these complex mechanisms should contribute to our understanding of the signal transduction pathways that are activated upon pathogen infection.
[32P]dATP (> 3000 Ci mmol–1) was obtained from New England Nuclear; other common chemicals were purchased from Sigma. Tobacco (Nicotiana tabacum cv. Xanthi nc) plants were grown at 22°C in a 14 h light cycle. The U1 strain of tobacco mosaic virus (TMV) was used throughout the experiments. SA and its analogues were dissolved in water as 100 m m stock solutions and adjusted to pH 6.5 with KOH
Isolation of nuclear extracts
Preparation of tobacco nuclear extracts was performed as described previously ( Green et al. 1987 ).
TMV infection and chemical treatments
Tobacco plants (5–6-weeks-old) were inoculated with TMV (0.5 μg ml–1 in 5 m m sodium phosphate, pH 7.0) by rubbing with carborundum. The inoculated plants were maintained in a growth chamber at 32°C in a 14 h light cycle to allow systemic infection. After 3–4 days at the higher temperature, the plants were shifted to a lower temperature for activation of HR. Chemical treatments were performed by spraying the plants with 2 m m SA or SA analogs. Leaves were harvested at various timepoints after the temperature shifting or chemical treatment for preparation of nuclear extracts.
Probe labeling and EMSA
Double-stranded synthetic oligonucleotides were labeled to specific activities of approximately 105 cpm ng–1 using the Klenow fragment of DNA polymerase I. Sequence-specific DNA binding was assayed with EMSA essentially as described previously ( Wang et al. 1998 ). Binding reactions contained 12 μl nuclear extraction buffer (25 m m HEPES/KOH, pH 7.5, 40 m m KCl, 0.1 m m EDTA, 10% glycerol, 1 m m DTT, 5 mg ml–1 antipain, 5 mg ml–1 leupeptin, 5 μg poly(dIdC), 5 μl proteins (10–30 μg for nuclear extracts or 0.2–1 μg for purified recombinant tWRKY1 proteins) and 1–2 ng of labeled double-stranded oligo DNA. DNA–protein complexes were allowed to form at room temperature for 20 min and resolved on a 10% polyacrylamide gel in 0.5× TBE at 4°C. Competition experiments were performed using labeled P12a as probe and unlabeled Pchn or mPchn as competitors in a 50-fold molar excess. Expression of recombinant tWRKY1 in E. coli and purification by affinity chromatography have been described previously ( Wang et al. 1998 ).
Treatment with dissociating agent or alkaline phosphatase
Sodium deoxycholate (0.5% final concentration) was added to the binding reaction and incubated for 30 min at room temperature before being loading onto non-denaturing polyacrylamide gels. For dephosphorylation experiments, alkaline phosphatase (10 units) (Gibco) was added to approximately 15 μg of proteins and incubated at 37°C for 30 min. The final glycerol concentration in the incubation mixtures with or without alkaline phosphatase was adjusted to 15%. Alkaline phosphatase was inhibited by adding NaF to the incubation mixtures (50 m m final concentration). Following the incubation, the DNA-binding activity was determined by EMSA using labeled P12a as probes.
Cloning of tobacco CHN50 cDNA
To isolate the tobacco CHN50 gene probe used for RNA gel blot analysis, two primers (5′-ATGAGGCTTAGAGAATTCACAGC-3′ and (5′-ATAGTCGCGATCCCATAGATCA-3′) were used for PCR amplification using cDNA synthesized from total RNA prepared from SA-treated tobacco leaves as a template. The amplified samples were size-fractionated on an agarose gel and fragments of the expected size were cloned into pCR2.1 (Invitrogen). The insert was verified to be tobacco CHN50 cDNA by partial DNA sequencing.
RNA isolation and Northern blotting
Total RNA from tobacco leaves was prepared using a procedure previously described by Logemann et al. (1987) . For Northern blot analysis, total RNA (12 μg) was separated on agarose–formaldehyde gels and blotted to nylon membranes following standard procedures. Blots were hybridized with (α-32P) dATP-labeled gene-specific probes. Hybridization was performed in 1 m NaCl, 50 m m Tris/Cl. pH 7.5, 1% SDS, 5 m m K3PO4, 100 μg ml–1 denatured salmon sperm DNA, 10% dextran sulfate, 0.2% BSA, 0.2% Ficoll 400 and 0.2% polyvinylpyrolidone 400 for 16 h at 42°C. The membrane was then washed for 10 min twice with 2× SSC and 1% SDS at 65°C and 10 min with 0.1× SSC and 1% SDS at room temperature.
We are grateful for Dr Allan Caplan for critically reading the manuscript. We thank Dr Imre Somssich for sharing information with us. This work was supported by US Department of Agriculture grant 96–36301–3316 to Z.C.