WRKY Transcription Factors Involved in Activation of SA Biosynthesis Genes
© van Verk et al; licensee BioMed Central Ltd. 2011
Received: 22 December 2010
Accepted: 19 May 2011
Published: 19 May 2011
Increased defense against a variety of pathogens in plants is achieved through activation of a mechanism known as systemic acquired resistance (SAR). The broad-spectrum resistance brought about by SAR is mediated through salicylic acid (SA). An important step in SA biosynthesis in Arabidopsis is the conversion of chorismate to isochorismate through the action of isochorismate synthase, encoded by the ICS1 gene. Also AVR PPHB SUSCEPTIBLE 3 (PBS3) plays an important role in SA metabolism, as pbs3 mutants accumulate drastically reduced levels of SA-glucoside, a putative storage form of SA. Bioinformatics analysis previously performed by us identified WRKY28 and WRKY46 as possible regulators of ICS1 and PBS3.
Expression studies with ICS1 promoter::β-glucuronidase (GUS) genes in Arabidopsis thaliana protoplasts cotransfected with 35S::WRKY28 showed that over expression of WRKY28 resulted in a strong increase in GUS expression. Moreover, qRT-PCR analyses indicated that the endogenous ICS1 and PBS3 genes were highly expressed in protoplasts overexpressing WRKY28 or WRKY46, respectively. Electrophoretic mobility shift assays indentified potential WRKY28 binding sites in the ICS1 promoter, positioned -445 and -460 base pairs upstream of the transcription start site. Mutation of these sites in protoplast transactivation assays showed that these binding sites are functionally important for activation of the ICS1 promoter. Chromatin immunoprecipitation assays with haemagglutinin-epitope-tagged WRKY28 showed that the region of the ICS1 promoter containing the binding sites at -445 and -460 was highly enriched in the immunoprecipitated DNA.
The results obtained here confirm results from our multiple microarray co-expression analyses indicating that WRKY28 and WRKY46 are transcriptional activators of ICS1 and PBS3, respectively, and support this in silico screening as a powerful tool for identifying new components of stress signaling pathways.
KeywordsWRKY28 WRKY46 ICS1 PBS3 salicylic acid plant defense signal transduction transcription factors
Because of their sessile nature, plants have evolved very sophisticated mechanisms to actively cope with different sorts of stresses. The various defense mechanisms are controlled by signaling molecules like salicylic acid (SA), jasmonic acid (JA), and ethylene, or by combinations of these signal compounds. SA accumulates locally in infected leaves, as well as in non-infected systemic leaves after infection with biotrophic pathogens and mediates the induced expression of defense genes, resulting in an enhanced state of defense known as systemic acquired resistance (SAR) [1–5]. SAR is a long-lasting broad-spectrum resistance against a variety of pathogenic fungi, bacteria and viruses [6, 7]. Also exogenous application of SA results in induced expression of defense related genes [8, 9]. Among the genes that are induced during SAR is a set of genes collectively known as PR (pathogenesis-related) genes, with members encoding anti-fungal β-1,3-glucanases (PR-2), chitinases (PR-3, PR-4) and PR-1, which are often used as molecular markers for SAR [7, 9–11].
Genetic studies have revealed important components of the SA signal transduction pathway, briefly outlined as follows: After perception of pathogen attack by cytoplasmic TIR-NB-LRR receptors, several genes are involved in initiation of the defense response. One of these genes is ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1), which is probably activated after elicitor perception . EDS1 heterodimerizes with PHYTOALEXIN DEFICIENT 4 (PAD4) and their nuclear localization is important for subsequent steps in the signaling pathway [13, 14]. Both EDS1 and PAD4 are induced by pathogen infection and SA application. Another enhanced disease susceptibility gene (EDS5) that is also situated upstream of SA biosynthesis is expressed at high levels upon pathogen infection in an EDS1- and PAD4-dependent manner . The eds5 mutant plants are no longer able to accumulate high levels of SA upon pathogen infection and are unable to initiate the SAR response .
Biosynthesis of SA can occur via two different pathways, the pathway that synthesizes SA from phenylalanine , and the isochorismate pathway. Inhibition of the phenylalanine pathway still allows accumulation of SA [18, 19]. An important step in the isochorismate pathway is the conversion of chorismate to isochorismate (ICS). Expression of a bacterial ICS gene in plants causes accumulation of SA, constitutive expression of PR genes and constitutive SAR , whereas the sid2 mutant corresponding with a defective ICS1 gene, is compromised in accumulation of SA and unable to mount SAR [16, 21]. Expression of the ICS1 gene is rapidly induced after infection . AVRPPHB SUSCEPTIBLE 3 (PBS3), of which the pathogen-induced expression is highly correlated with expression of ICS1, is acting downstream of SA. In the pbs3 mutant, accumulation of SA-glucoside and expression of PR-1 are drastically reduced. The PBS3 gene product is a member of the auxin-responsive GH3 family of acyl-adenylate/thioester forming enzymes of which some have been shown to catalyze hormone-amino acid conjugation, like the protein encoded by the JAR1 gene that catalyzes the formation of JA-isoleucine. However, the observation that PBS3 is not active on SA, INA and chorismate leads to the hypothesis that PBS3 must be placed upstream of SA [22–24].
Although many mutants have been reported to affect SA accumulation, no direct transcriptional regulators of genes like ICS1 or PBS3 have been identified. For ICS1 the presence of many TGAC core sequences, as present in the binding sites for WRKY transcription factors, has been hypothesized to be important for transcriptional regulation of ICS1 gene expression . Here we describe two WRKY transcription factors that were previously identified in our group via a bioinformatics analysis to be closely co-expressed with ICS1 and PBS3. Co-expression analyses in protoplasts showed that WRKY28 and WRKY46 positively regulated the expression of ICS1 and PBS3, respectively. In addition, the binding sites for WRKY28 in the ICS1 promoter were identified.
Our results indicate that WRKY28 and WRKY46, which themselves are both rapidly induced by pathogen elicitors [26, 26], link pathogen-triggered defense gene expression to the accumulation of SA via induction of ICS1 and PBS3 gene expression.
WRKY28 Activates ICS1::GUSGene Expression in Arabidopsis Protoplasts
Characterization of the WRKY28 Binding Sites in the ICS1Promoter
To further analyze the requirements for binding of WRKY28, pairwise mutations of the sequence around the core at -445 were scanned in an m1 background (Figure 4B). The results are shown in Figure 4A, Lanes 9 to 24. Mutations m2.1 and m2.4 show binding to WRKY28 (Figure 4A, Lanes 10 and 16). As expected, mutations within the core sequence completely abolished binding of WRKY28 (m2.2 and m2.3, Figure 4A, Lanes 12 and 14). Since the TGAC core at -460 has TC upstream of the core and the inverted core at -445 has a CT in this position, we checked to which extend the T or C nucleotides are important for binding. Changing CT to TC resulted in a binding of WRKY28 that was as strong as to the wild type sequence (m2.5, Figure 4A, Lane 18). Changing CT to TT significantly lowered binding (m2.6, Figure 4A, lane 20), suggesting that the presence of a C at either position -1 or -2 from the core is important for binding WRKY28. We further analyzed the effect of mutations at positions -3/-4 and +3/+4 from the core. Pairwise mutation of nucleotides at -3/-4 did not alter the binding of WRKY28 (m2.8, Figure 4A, Lane 24), however no shift was observed when the nucleotides at +3/+4 were mutated, indicating that this flanking sequence is important for binding of WRKY28 (m2.7, Figure 4A, Lane 22).
Mutational Analysis of WRKY28-Mediated Activationof ICS1::GUSGene Expression in Arabidopsis Protoplasts
Chromatin Immunoprecipitation Analysis
The transactivation experiments in protoplasts and the in vitro binding studies described above support a role for WRKY28 as a transcriptional activator of ICS1. To check if WRKY28 is able to bind to the ICS1 promoter in vivo, chromatin immunoprecipitation (ChIP) assays were set up using Arabidopsis protoplasts, as described by . The WRKY28 coding sequence was fused to a haemagglutinin (HA) tag and expressed in Arabidopsis protoplasts. The resulting WRKY28-HA fusion protein was able to induce GUS expression when cotransfected with an ICS1 promoter::GUS construct, indicating that the HA tag did not interfere with WRKY28's functionality (Results not shown).
WRKY28 and WRKY46 Activate Expression of ICS1 and PBS3, Respectively
Our in silico co-expression analysis of Arabidopsis transcription factor genes and genes involved in stress signaling suggested many putative new components of the signal transduction pathways . Among the genes resulting from this screening were two encoding WRKY transcription factors linked to genes involved in SA metabolism. The gene encoding the WRKY type II member WRKY28 was found to be closely co-regulated with the ICS1 gene involved in SA biosynthesis, whereas the type III WRKY46 gene linked to PBS3. Based on this finding we decided to investigate the effects of these WRKYs on transcriptional activation of ICS1 and PBS3. Indeed, overexpression of WRKY28 in Arabidopsis protoplasts led to enhanced GUS activity from a co-expressed GUS reporter gene under control of a 1 kb ICS1 promoter, and also expression of the endogenous ICS1 gene was increased (Figures 1 and 2). Likewise, overexpression of WRKY46 resulted in increased accumulation of PBS3 mRNA, supporting the notion that WRKY46 is a transcriptional activator of PBS3 (Figure 2). GUS activity was not enhanced from a co-expressed 1 kb PBS3 promoter::GUS gene. This suggests that WRKY46 may activate the PBS3 gene by binding at a position in the promoter further upstream than 1 kb. However, we cannot exclude the possibility that the 1 kb promoter used for the construction of the reporter construct and which was derived from curated genome sequence data by The Arabidopsis Information Resource (TAIR), is not the actual PBS3 promoter. A detailed analysis of the region upstream of the coding sequence in the Arabidopsis genome shows that the intron of almost 1 kb suggested to be present in the 5'-UTR of PBS3 contains several putative binding sites for transcription factors like WRKYs and TGAs. It will be interesting to investigate if the suggested "intron" is the actual PBS3 promoter.
Functional analysis that would further support the important role of WRKY28 in ICS1 gene expression were hampered by the lack of WRKY28 knock-out mutants or T-DNA insertion lines, while our efforts to achieve silencing of WRKY28 through Agrobacterium-mediated transformation with pHANNIBAL constructs via flower dip only resulted in seedlings that died shortly after germination. These findings suggest that WRKY28 also plays an essential role during early plant development.
DNA Binding Site of WRKY28
Several studies on DNA binding characteristics of WRKY transcription factors have led to the generally accepted consensus binding sequence TTGAC[C/T], commonly referred to as the W-box [25, 30, 34–39]. Recently, we identified a variant binding site for the tobacco NtWRKY12 transcription factor . NtWRKY12 binds to a WK-box (TTTTCCAC), which deviates significantly from the W-box consensus sequence.
In this study we have characterized two sites in the ICS1 promoter that have a high affinity for WRKY28. The consensus WRKY28 binding site that emerged from this analysis has some characteristics that differ from the W-box consensus (Figure 5C). We found that, unlike the consensus W-box, a C may be present at position -1 in front of the TGAC core, and although a T is also allowed at -1, a C is then required at -2. Similarly, for the sequence after the core, in one of the binding sites an A is present at +1, which in the W-box is usually either a C or a T.
To disable binding of WRKY28 to the 30-bp EMSA probe harboring the binding sites at -460 and -445, mutation of both these sites was necessary. With only one site intact, binding was still possible (Figure 4A, Lanes 4 and 6). Nevertheless, with the 1 kb promoter, mutation of only one of the sites had a severe effect on reporter gene expression and expression was not further reduced when both sites were mutated. Apparently, for transcriptional activation both sites are required. Possibly, activation requires that WRKY28 binds as a dimer, similar to WRKYs 18, 40 and 60, which were found to form functionally relevant homo- and heterodimers .
The transactivation experiments also showed that mutation of the sites at -460 (m1) and -445 (m2) did not completely knock out reporter gene expression. In comparison to the GUS activity obtained with the wild type construct, approximately 20% remained. Furthermore, the reduction in basal expression levels seen with the mutant ICS1 promoters in the absence of overexpressed WRKY28 indicates that also endogenous factors binding to the sites at -460 and -445 contribute to the expression level. qRT-PCR has shown that the WRKY28 gene is much higher expressed in protoplasts than in suspension cells from which the protoplasts were made (Results not shown), suggesting that possibly these factors include endogenous WRKY28. Besides the direct activation of ICS1 gene expression, WRKY28 might also indirectly effect the ICS1 gene via transcriptional activation of genes encoding other transcription factors acting on the ICS1 promoter. Moreover, the residual GUS expression remaining with the m1, m2 and m1+2 mutant promoters could indicate that other sites in the ICS1 promoter are still able to bind WRKY28, although the existence of such sites was not supported by the results of the ChIP analysis.
Integrated Model for Regulation of SA Biosynthesis by WRKY28 and WRKY46
The combined results of the work described here, lead us to propose the following model for the induction of SA biosynthesis upon pathogen attack. Induction of the basal defense response starts with the detection of a pathogen-associated molecular pattern (PAMP), like in the case of flagellin, which is perceived by the FLS receptor. The activated FLS receptor triggers a MAP kinase cascade (MAPKKK/MEKK1?, MKK4/5, MPK3/6), which leads to transcriptional activation of the WRKY28 gene . Transcription factor WRKY28 subsequently activates directly, and likely also indirectly via yet unknown transcription factors, expression of the ICS1 gene, through binding the promoter at the two binding sites at -460 and -445 and possibly at other sites, resulting in synthesis of ICS that catalyzes SA production. How the activated MAP kinase induces WRKY28 gene expression remains a matter of speculation. The activated MAPK could activate an as of yet unknown transcription factor on standby or release one from a repressor complex, or it may function itself as activator of WRKY28 expression.
Protoplast Preparation, Transfection and Analysis
For transactivation and qRT-PCR experiments, protoplasts were prepared from cell suspensions of Arabidopsis thaliana ecotype Col-0, according to van Verk et al. .
For transactivation experiments protoplasts were co-transfected with 2 μg of plasmids carrying reporter gene constructs ICS1 promoter::GUS (promoter refers to bp -1 to -960, relative to the transcriptional start site), or PBS3 promoter::GUS (promoter refers to bp -1 to -1000, relative to the transcriptional start site) and 6 μg of effector constructs 35S::WRKY28or 35S::WRKY46 in expression vector pRT101. As a control, cotransfection of promoter::GUS constructs 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.
Oligonucleotides used for qRT-PCR and ChIPqPCR analysis
Electrophorectic Shift Assays
Protein for EMSAs was purified from E. coli transformed with pGEX-KG constructs containing the open reading frame of WRKY28 cloned in frame behind the GST open reading frame, according to van Verk et al. .
EMSAs were performed essentially as described by Green et al. . DNA probes for the EMSA assays were obtained by slowly cooling down mixtures of equimolar amounts of complementary oligonucleotides with a 5'-GGG overhangs from 95°C to room temperature. Annealed oligonucleotides were subsequently end-filled using Klenow fragment and [α-32P]-dCTP, after which unincorporated label was removed by Autoseq G-50 column chromatography (Amersham-Pharmacia Biotech). EMSA reaction mixtures contained 0.5 μg purified protein, 3 μL 5× gel shift binding buffer [20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mMNaCl, 50 mMTris-HCl, pH 7.5, 0.25 mg mL-1poly(dI-dC) x poly(dIdC) (Promega)] in a total volume of 14 μL. After 10-min incubation at room temperature, 1 μL containing 30,000 cpm of labeled probe, representing approximately 0.01 pmol, was added and incubation was continued for 20 min at room temperature. Fifty- and 250-fold molar excess of unlabelled annealed oligonucleotides were added insome reactions as competitor. The total mixtures were loaded onto a 5% polyacrylamide gel in Tris-borate buffer and electrophoresed. After electrophoresis, the gel was dried, autoradiographed, and analyzed using X-ray film.
For ChIP assays, protoplasts were prepared as described above and transfected with 6 μg of 35S::WRKY28-HA or 35S::HA constructs in plasmid pRT101. After 24 h, protoplasts were harvested and ChIP assays were conducted as described by , with minor modifications. After formaldehyde fixation, the chromatin of the protoplasts was isolated and extensively sheared by sonication to obtain fragment sizes between 300-400 bp. Rat anti-HA monoclonal antibodies (clone 3F10, Roche) and Dynabeads Protein G magnetic beads (Invitrogen) were used to immunoprecipitate the genomic fragments. qPCRs were performed on the immunoprecipitated DNA using primer sets corresponding to six overlapping regions of the ICS1 promoter as shown in Figure 8A, and were corrected for their individual PCR amplification efficiencies. qPCRs with primers specific for the coding region of the PR1 gene and the promoter of PDF1.2 gene of Arabidopsis were used as controls. The primers used for the ChIP assays are listed in Table 1.
Acknowledgements and Funding
We gratefully acknowledge the group of dr. Ji Hoon Ahn for providing a detailed protocol for ChIP analysis in Arabidopsis protoplasts. Rob van Eck and Roy Baas are acknowledged for their help with some of the experiments and we are grateful to Ward de Winter for technical assistance. The work was performed without external funding.
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