Roles of arabidopsis WRKY18, WRKY40 and WRKY60 transcription factors in plant responses to abscisic acid and abiotic stress
© Chen et al; licensee BioMed Central Ltd. 2010
Received: 11 September 2009
Accepted: 19 December 2010
Published: 19 December 2010
WRKY transcription factors are involved in plant responses to both biotic and abiotic stresses. Arabidopsis WRKY18, WRKY40, and WRKY60 transcription factors interact both physically and functionally in plant defense responses. However, their role in plant abiotic stress response has not been directly analyzed.
We report that the three WRKYs are involved in plant responses to abscisic acid (ABA) and abiotic stress. Through analysis of single, double, and triple mutants and overexpression lines for the WRKY genes, we have shown that WRKY18 and WRKY60 have a positive effect on plant ABA sensitivity for inhibition of seed germination and root growth. The same two WRKY genes also enhance plant sensitivity to salt and osmotic stress. WRKY40, on the other hand, antagonizes WRKY18 and WRKY60 in the effect on plant sensitivity to ABA and abiotic stress in germination and growth assays. Both WRKY18 and WRKY40 are rapidly induced by ABA, while induction of WRKY60 by ABA is delayed. ABA-inducible expression of WRKY60 is almost completely abolished in the wrky18 and wrky40 mutants. WRKY18 and WRKY40 recognize a cluster of W-box sequences in the WRKY60 promoter and activate WRKY60 expression in protoplasts. Thus, WRKY60 might be a direct target gene of WRKY18 and WRKY40 in ABA signaling. Using a stable transgenic reporter/effector system, we have shown that both WRKY18 and WRKY60 act as weak transcriptional activators while WRKY40 is a transcriptional repressor in plant cells.
We propose that the three related WRKY transcription factors form a highly interacting regulatory network that modulates gene expression in both plant defense and stress responses by acting as either transcription activator or repressor.
Plants are constantly exposed to a variety of biotic and abiotic stresses and have evolved intricate mechanisms to sense and respond to the adverse conditions. Phytohormones such as salicylic acid (SA), ethylene (ET), jasmonic acid (JA) and abscisic acid (ABA) play important roles in the regulation of plant responses to the adverse environmental conditions. In Arabidopsis, mutants deficient in SA biosynthesis (e.g. sid2) or signalling (e.g. npr1) exhibit enhanced susceptibility to biotrophic pathogens, which parasitize on plant living tissue [1, 2]. ET- and JA-mediated signaling pathways, on the other hand, often mediate plant defense against necrotrophic pathogens that promote host cell death at early stages of infection . ABA is extensively involved in plant responses to abiotic stresses including drought, extreme temperatures and osmotic stress [4, 5]. ABA also plays a regulatory role in important plant growth and developmental processes including seed development, dormancy, germination and stomatal movement. Recent studies have reported crosstalk of signaling pathways regulated by these signal molecules that contributes to either antagonistic or synergistic interactions between abiotic and biotic interactions [6, 7].
A large body of evidence indicates that plant WRKY DNA-binding transcription factors play important role in plant defense responses. In Arabidopsis, a majority of its WRKY genes are induced by pathogen infection or SA treatment . A large number of plant defense or defense related genes including pathogenesis-related (PR) genes and the regulatory NPR1 gene contain W box sequences in their promoters that are recognized by WRKY proteins . A number of studies have shown that these W-box sequences are necessary for the inducible expression of these defense genes. Mutant analyses in Arabidopsis have revealed direct links between specific WRKY proteins and complex plant defense responses. Mutations of WRKY70 enhance plant susceptibility to both biotrophic and necrotrophic pathogens including Erwinia carotovora, Hyaloperonospora parasitica, Erysiphe cichoracearum and Botrytis cinerea [10–12]. Disruption of WRKY33 results in enhanced susceptibility to necrotrophic fungal pathogens and impaired expression of JA/ET-regulated defense genes . Mutations of other WRKY genes including WRKY7, WRKY11, WRKY17, WRKY48, WRKY38 and WRKY62, on the other hand, enhance basal plant resistance to virulent P. syringae strains, suggesting that they function as negative regulators of plant basal defense [14–17].
There is also evidence that WRKY transcription factors are involved in plant responses to abiotic stresses. Microarray experiments have identified WRKY genes that are induced by various abiotic stresses. In Arabidopsis, for example, WRKY genes were among several families of transcription factor genes that are induced by drought, cold or high-salinity stress [18–20]. The barley Hv-WRKY38 gene is rapidly and transiently induced during exposure to low non-freezing temperature in ABA-independent manner and exhibits continuous induction during dehydration and freezing treatment . In tobacco, a WRKY transcription factor is specifically induced during a combination of drought and heat shock . Regulated expression of WRKY genes during plant stress responses provides circumstantial evidence that implicates WRKY proteins in plant responses to abiotic stress. In Creosote bush (Larrea tridentate) that thrives in vast arid areas of North American, a WRKY protein (LtWRKY21) is able to activate the promoter of an ABA-inducible gene, HVA22, in a dosage-dependent manner . A number of rice WRKY proteins regulate positively or negatively ABA signalling in aleurone cells [23, 24]. Overexpression of soybean GmWRKY13, GmWRKY21 and GmWRKY54 conferred differential tolerance to abiotic stresses in transgenic Arabidopsis plants . However, stable or transient overexpression of a gene in transgenic plants can often lead to pleiotropic phenotypes that may or may not reflect the true biological functions of the gene. Very recently, Jiang and Yu  have reported that Arabidopsis wrky2 knockout mutants are hypersensitive to ABA responses during seed germination and postgermination early growth, suggesting an important role of the stress-regulated WRKY gene in plant stress responses.
Arabidopsis WRKY18, WRKY40 and WRKY60 are pathogen-induced and encode three structurally related WRKY proteins . We have previously shown that WRKY18, WRKY40 and WRKY60 interact physically with themselves and with each other through a leucine-zipper motif at their N-terminus . Analysis with both knockout alleles and overexpresison lines indicated that the three pathogen-induced WRKY transcription factors have a partially redundant negative effect on SA-mediated defense but exerted a positive role in JA-mediated defense. . Likewise, ABA plays a complex role in plant defense response. In Arabidopsis, ABA counteracts SA-dependent defense against the hemibitrophic bacterial pathogen Pseudomonas syringae , but is a signal required for resistance to the necrotrophic pathogens Pythium irregulare and Alternaria brassicicola . In the present study, we report that Arabidopsis WRKY18, WRKY40 and WRKY60 proteins indeed function in a complex pattern in plant responses to ABA and abiotic stresses. The complex roles of the three WRKY transcription factors in plant biotic and abiotic stress responses are consistent with the complex nature of their expression, transcription-regulating activities and physical interactions.
Altered ABA Sensitivity of Mutants and Overexpression Plants
We have previously shown that structurally related WRKY18, WRKY40 and WRKY60 interact both physically and functionally in the regulation of plant basal defense . To determine possible functional interactions among the three WRKY proteins, we compared the ABA sensitivity of their double and triple knockout mutants (Figure 1D, E, G and 1F; Additional file 1). Germination rates of the wrky18 wrky60 double mutant at relatively low ABA concentrations (< 2 μM) were higher than those of wild type and were similar to those of the wrky60 single mutant (Figure 1E). At higher ABA concentrations (3 and 5 μM), however, the germination rates of the double mutant were 10-15% higher than those of the wrky60 single mutant (Figure 1E). Thus, WRKY18 and WRKY60 act additively in enhancing seed sensitivity to ABA in germination assays. The germination rates of the wrky18 wrky40 double mutant at various ABA concentrations were substantially lower than those of wild type and the wrky40 single mutant (Figure 1D). Interestingly, the germination rates of the wrky40 wrky60 double mutant were significantly higher than those of wild type. However, at certain ABA concentrations (e.g. 1.5 and 2.0 μM) the wrky40 wrky60 double mutant didn't germinate as well as the wrky60 single mutant (Figure 1). There was no significant difference between wild type and the wrky18 wrky40 wrky60 triple mutant in germination at the various ABA concentrations tested (Figure 1).
Altered tolerance of mutants and overexpression plants to abiotic stress
Induced expression by ABA and abiotic stress
We also analyzed responses of the three WRKY genes to salt and drought(PEG) treatments. Wild-type seedlings (7 days old) were transferred to a MS growth medium with or without 150 mM NaCl or 250 g/l PEG and the seedlings were harvested 24 hours later for isolation of total RNA and qRT-PCR analysis. As shown in Figure 4B, the transcript levels for WRKY18, WRKY40 and WRKY60 were elevated by the NaCl treatment 6.5, 18.7 and 4.9 fold, respectively. After PEG treatment, the three WRKY genes were also induced 4 to 7 fold (Figure 4B). These results indicated that the three WRKY genes were also responsive to abiotic stresses. Induced expression of the WRKY genes by ABA and abiotic stresses have also been observed from previously reported microarray analysis [29, 30].
Induction of WRKY60 by ABA was relatively slow when compared to that of WRKY18 and WRKY40 (Figure 4A). In wild type, no significant induction of WRKY60 transcripts was observed during the first five hours after ABA treatment. However, WRKY60 transcripts increased about 10 fold by 12 hours after the treatment and then declined gradually during the remaining period of the experiments (Figure 4A). In the wrky18 mutant, the induction of WRKY60 was drastically reduced, with only a small increase observed after 24 hours of treatment (Figure 5C). In the wrky40 single mutant and wrky18 wrky40 double mutant, ABA induction of WRKY60 was completely abolished (Figure 5C). Thus both WRKY18 and WRKY40 are necessary for ABA-induced WRKY60 expression.
Recognition of WRKY60 promoter by WRKY18 and WRKY40
To determine whether the W boxes from the WRKY60 gene promoter are recognized by WRKY18 and WRKY40 proteins, we generated and labelled a double-stranded DNA probe containing these three W boxes (PW60) (Figure 6B). When incubated with recombinant WRKY18 or WRKY40 proteins, the probe produced a retarded band in electrophoretic mobility shift assays (Figure 6C). A similar retarded band was also produced when the probe was incubated with a mixture of WRKY18 and WRKY40 recombinant proteins (Figure 6C). To determine whether the W-boxes in the PW60 probe were important for the recognition, we also tested a mutant probe (mPW60) in which the TTGAC sequence of each W-box was changed to TTGAA (Figure 6B). As shown in Figure 6C, this mutant probe failed to detect retarded bands when incubated with WRKY18 or WRKY40 proteins. Thus, WRKY18 and WRKY40 proteins recognize the W-box sequences in the WRKY60 gene promoter.
Activation of the WRKY60 Promoter by WRKY18 and WRKY40 in Protoplasts
To determine whether WRKY18 and WRKY40 can activate the WRKY60 promoter in protoplasts, we generated the WRKY18 and WRKY40 effector constructs under control of the constitutive CaMV 35S promoter. As shown in Figure 7B, coexpression of WRKY18 or WRKY40 led to only a very small increase in the reporter gene expression from the W60:GUS construct in the wrky18/wrky40 mutant protoplasts (Figure 7B). On the other hand, coexpression of both WRKY18 and WRKY40 activated the the reporter gene expression the W60:GUS construct by almost 5-fold in the wrky18/wrky40 mutant protoplasts (Figure 7B). This activation of the WRKY60 promoter by coexpression of WRKY18 and WRKY40 was not observed from the mW60:GUS construct (Figure 7B). Thus, WRKY18 and WRKY40 cooperate in the activation of the WRKY60 gene expression mostly likely through recognition of the W box sequence in the WRKY60 gene promoter.
Transcription-regulating activity of WRKY18, WRKY40 and WRKY60
To generate the WRKY18, WRKY40 and WRKY60 effectors, we fused their coding sequences with that of the DNA-binding domain (DBD) of LexA (Figure 8A). The fusion constructs were subcloned behind the steroid-inducible Gal4 promoter in pTA7002  and transformed into transgenic plants that already contain the GUS reporter construct. Unfused WRKY and LexA DBD genes were also subcloned into pTA7002 and transformed into transgenic GUS reporter plants as controls (Figure 8A). For comparison, we also include WRKY48, a strong transcription activator , and WRKY7, a transcription repressor , in the assays. Transgenic plants containing both the reporter and an effector construct were identified through antibiotic resistance screens. To determine the effect of the effectors on GUS reporter gene expression, we determined the changes of GUS activities in the transgenic plants after induction of the effector gene expression by spraying 20 μM dexamethasone (DEX), a steroid. In the transgenic plants that expressed unfused WRKY18, WRKY40, WRKY60 or LexA DBD effector, there were little changes in the GUS activities after 18-hour DEX treatment (Additional file 3). In the transgenic plants harboring the LexA DBD-WRKY18 effector gene, induction of the fusion effector after DEX treatment resulted in 1.4 - fold increase in GUS activity (Additional file 3). A slightly higher 1.6-fold increase in GUS activity was observed in the transgenic plants harboring the LexA DBD-WRKY60 effector gene after DEX treatment (Additional file 3). By comparison, as previously reported , transgenic plants harboring the LexA DBD-WRKY48 effector gene, DEX treatment resulted in ~24-fold increase in GUS activity. These results indicate that both WRKY18 and WRKY60 are weak transcriptional activators. By contrast, in the transgenic plants harboring the LexA DBD-WRKY40 effector gene, induction of the fusion effector after DEX treatment resulted in a 2-fold reduction in GUS activity (Additional file 3). In transgenic plants harboring the LexA DBD-WRKY7 effector gene, DEX treatment resulted in ~5-fold reduction in GUS activity. Thus, WRKY40 is a relatively weak transcriptional repressor.
We have previously shown that WRKY18, WRKY40 and WRKY60 physically interact with themselves and with each other to form both homo- and hetero-complexes . In addition, the three WRKY genes are induced by pathogen infection, SA and ABA treatment  (Figure 5). Thus, the transcription-regulating activity of the three WRKY proteins may change upon interaction with each other or with other induced proteins. To test this possibility, we examined the effects of SA and ABA treatment on the changes of GUS activities in the progeny of the transgenic effector/reporter lines after 24-hour DEX induction of the effector genes. Extension of DEX treatment from 18 to 24 hours increased significantly the expression levels the effector genes (unpublished data). In the transgenic plants that expressed unfused WRKY18, WRKY40, WRKY60 or LexA DBD effector, there were little changes in the GUS activities after DEX treatment with or without ABA or SA treatment (Figure 8B). In the transgenic plants harboring the LexA DBD-WRKY18 effector gene, induction of the fusion effector after DEX treatment resulted in 2.2 -fold increase in GUS activity (Figure 8B). ABA treatment had little effect on DEX-induced change of GUS activity, suggesting that ABA did not significantly affect the transcription-activating activity of WRKY18. On the other hand, in SA-treated transgenic plants harboring the LexA DBD-WRKY18 effector gene, there was almost no increase in GUS activity following induction of the fusion effector after DEX treatment. Thus, SA treatment almost completely abolished the transcription-activating activity of WRKY18. In the absence of ABA or SA treatment, a 2.5-fold increase in GUS activity was observed in the transgenic plants harboring the LexA DBD-WRKY60 effector gene after 24-hour DEX treatment (Figure 8B). Again ABA treatment had little effect on DEX-induced change of GUS activity while SA treatment resulted in more than 50% reduction in the increase of GUS activity following 24-hour DEX induction of the fused LexA DBD-WRKY60 effector gene (Figure 8B). In the transgenic plants harboring the LexA DBD-WRKY40 effector gene, induction of the fusion effector after DEX treatment resulted in a 2.5-fold reduction in GUS activity (Figure 8B). Neither ABA nor SA treatment had significant effect on the change of GUS activities in the transgenic plants harboring the LexA DBD-WRKY40 effector gene (Figure 8B). Thus, the transcription-regulating activity of both WRKY18 and WRKY60, but not WRKY40, was substantially altered by SA treatment.
Expression of ABA related genes
Differential roles of WRKY18, WRKY40 and WRKY60 in ABA and abiotic stress responses
Over the last several years, there has been growing evidence that plant WRKY transcription factors are involved in plant ABA signaling and abiotic stress responses. In rice and barley, ABA induces expression of a number of WRKY genes in aleurone cells [23, 24, 33, 34]. When transiently overexpressed in aleurone cells, some of these ABA-inducible WRKY genes activate or repress ABA-inducible reporter genes. A number of studies have also shown that WRKY genes are induced by a variety of abiotic stress conditions and overexpression of some WRKY genes altered plant stress tolerance. In the present study, we have determined the role of three Arabidopsis WRKY genes in plant ABA signaling by analyzing the effects of ABA on germination, root growth of their knockout mutants and overexpression lines. We have demonstrated that while disruption of WRKY18 and WRKY60 caused reduced sensitivity to ABA, disruption of WRKY40 increased ABA sensitivity for inhibition of germination and root growth (Figures 1 and 2). Likewise, we have demonstrated that the wrky18 and wrky60 mutants but not the wrky40 mutant are more tolerant to salt and osmotic stress (Figure 3). The differential roles of the three structurally related WRKY proteins in plant ABA and abiotic stress responses were also demonstrated from the analysis of the double and triple knockout mutants and overexpression lines (Figure 1, 2 and 3).
The roles of WRKY18, WRKY40 and WRKY60 in ABA signaling are consistent with the ABA-inducible expression of the three genes (Figure 4). Interestingly, the three WRKY genes display distinct expression patterns upon ABA treatment. WRKY18 and WRKY40 are rapidly induced upon ABA treatment and are required for ABA-induced WRKY60 expression (Figure 4). On the other hand, ABA-induced expression of WRKY60 is delayed but also prolonged (Figure 4). In addition, WRKY60 and WRKY40 act partially redundantly in repressing WRKY18 expression (Figure 5). This expression pattern raises the possibility that the three WRKY proteins are part of a regulatory network that modulates gene expression in the ABA signaling pathway. Upon ABA induction, WRKY18 and WRKY40 are first induced and their products could act as early transcriptional effectors to regulate expression of additional ABA-induced genes including WRKY60 (Figure 10). Induced WRKY60 would then act with WRKY40 to repress WRKY18, forming a negative feedback loop. The prolonged expression and the transcription-activating activity of WRKY60 would allow it to have a relatively sustained effect on ABA-regulated gene expression. This interpretation is consistent with the relatively strong phenotypes of the wrky60 mutant in ABA and stress tolerance when compared to those of the wrky18 mutant (Figure 1, 2 and 3).
Roles of WRKY18, WRKY40 and WRKY60 in crosstalk between abiotic and biotic responses
We have previously shown that single wrky18, wrky40 and wrky60 mutants exhibited no or small alterations in response to the hemibiotrophic bacterial pathogen P. syringae or the necrotrophic fungal pathogen B. cinerea . However, wrky18 wrky40 and wrky18 wrky60 double mutants and the wrky18 wrky40 wrky60 triple mutant were substantially more resistant to P. syringae but more susceptible to B. cinerea than wild-type plants . These phenotypes and additional analysis of SA- and JA-regulated gene expression suggest that these WRKY proteins have a partially redundant negative effect on SA-mediated defense but exerted a positive role in JA-mediated defense. Likewise, we have shown in this report that WRKY18 and WRKY60 positively regulate while WRKY40 negatively regulates plant ABA response (Figure 1, 2 and 3). As ABA is known to counteract SA-defense  but function as a signal in JA-mediated defense against necrotrophic pathogens , the roles of these three WRKY proteins in plant defense and ABA and stress responses might be mechanistically linked. This notion is particularly attractive for WRKY18 and WRKY60, which might negatively impact SA-dependent defense through positively modulating ABA signaling. On the other hand, WRKY40 antagonized WRKY18 and WRKY60 in ABA response but functions partially redundantly with WRKY18 and WRKY60 in SA-dependent defense. As will be discussed later, WRKY18, WRKY40 and WRKY60 interact with themselves and with each other to form distinct complexes that may differ in both DNA-binding and transcription-regulating activities. The interacting partners of WRKY40 formed during pathogen infection might not be the same as those in ABA-treated plants and, therefore, may function in distinct manners during plant defense and stress responses.
Molecular basis of functional interactions among WRKY18, WRKY40 and WRKY60
We have previously shown that through a leucine-zipper motif present at the N-terminus of the three proteins, WRKY18, WRKY40 and WRKY60 interacts with themselves and with each other to form both homo-complexes and hetero-complexes with altered DNA binding activities . In the present study, we have shown that WRKY18 and WRKY60 act as weak transcriptional activators and WRKY40 is a transcriptional repressor in plant cells (Figure 8). Furthermore, we have shown that SA treatment can diminish or reduce the transcription-activating activity of WRKY18 and WRKY60 (Figure 8). Thus, the three WRKY proteins may form a range of protein complexes with distinct DNA-binding and transcription-activating or -repressing activities. The complex pattern of DNA binding and transcription regulatory activities of the three WRKY proteins may explain their complex biological roles in plant defense and stress responses.
In plant defense responses, analysis of T-DNA insertion mutants indicated that WRKY18, WRKY40 and WRKY60 have redundant repressor function in plant defense against virulent hemibiotrophic P. syringae and biotrophic Golovinomyces orontii [27, 39]. Genome-wide gene expression profiling experiments also showed that WRKY18 and WRKY40 have a redundant role in repressing a subset of 23 genes associated with PAMP-triggered immunity . The redundant roles of WRKY18 and WRKY40 as repressors of plant defense genes are consistent with the demonstrated repressing activity of WRKY40 but not with the transcription-activating activity of WRKY18. However, we have also shown that after treatment with SA, which is elevated in pathogen-infected plants, the transcription-activating activity of WRKY18 is largely diminished (Figure 8). Under such conditions WRKY18 may compete for binding to promoter sequences with other pathogen-induced WRKY proteins with stronger transcription-activating activities, thereby preventing strong expression of the target genes. In the absence of SA treatment or pathogen infection, on the other hand, WRKY18 may function as a positive regulator of plant disease resistance by acting as an activator of plant defense genes as observed in transgenic WRKY18-overexpressing plants . The positive role of WRKY18 as a positive regulator of disease resistance and activator of defense gene would be antagonized by the transcription-repressing WRKY40 if they are co-exppressed. Indeed, we have previously observed that potentiated defense responses in WRKY18-overexpressing Arabidopsis plants are abolished by co-overexpression of WRKY40 in the same transgenic plants .
The differential roles of the three WRKY proteins in plant responses to ABA and abiotic stress conditions are correlated with their distinct transcriptional regulatory activities. WRKY18 and WRKY60 act as transcriptional activators and functional as positive regulators of plant ABA and abiotic stress responses. By contrast, WRKY40 acts as a transcriptional repressor and functional as a negative regulator of plant ABA responses. Thus, it is mostly likely that the roles of the three WRKY proteins in plant ABA and stress responses are mediated by their activities in activating or repressing plant genes involved in ABA and stress signaling.
ABA-induced expression of WRKY60 is severely compromised in both the wrky18 and wrky40 single mutants (Figure 6C). Thus, both WRKY18 and WRKY40 are important for ABA-induced WRKY60 expression. In the promoter of WRKY60, there is a cluster of three W boxes within a 19 bp region (Figure 6A), which are important for ABA-induced expression of WRKY60 in protoplasts (Figure 7A). Using EMSA, we have shown that the cluster of W boxes in the WRKY60 gene promoter is recognized by both WRKY18 and WRKY40 (Figure 6C). Protoplast transfection assays further showed that only co-overexpression of WRKY18 and WRKY40 but not WRKY18 or WRKY40 alone led to activation of the WRKY60 gene promoter and this activation of WRKY60 was dependent on the cluster of three W boxes in its promoter (Figure 7). It is possible that upon ABA treatment, WRKY18 and WRKY40 are first induced and cooperative binding of induced WRKY18 and WRKY40 or binding of a WRKY18/WRKY40 heterocomplex to the cluster of W boxes in the WRKY60 promoter is necessary for the subsequent induction of WRKY60 (Figure 10).
We have found that mutants and overexpression lines for Arabidopsis WRKY18, WRKY40 and WRKY60 genes have altered phenotypes in plant sensitivity to ABA, salt and osmotic stress. Thus, the three WRKY transcription factors play roles in both plant biotic and abiotic stress responses. Additional studies of their expression, DNA binding and transcription-regulating activities strongly suggest that the three WRKY transcription factors form a highly interacting regulatory network that modulates gene expression in both plant defense and stress responses.
Materials and Growth Conditions
The Arabidopsis knockout mutants and overexpression lines for WRKY18, WRKY40 and WRKY60 have been previously described. The plants were grown in mixture of peat/forest soil (purchased from Pingstrup Substrate) and vermiculite (3:1) in a green house at 23°C with 150 μE m-2 s-1 light on a photoperiod of 12 h light and 12 h dark.
Assays of Sensitivity to ABA and Stress
Seeds (100 seeds for each replicate) of wild type, mutants and overexpression lines were surface sterilized by treating for 5 min in 15% bleach and 0.5% Tween-20. The sterilized seeds were placed on 1/2 Murashige and Skoog medium (Gibcol) and 0.3% phytagel (Sigma) and stratified at 4°C for 4 days before transfer to 23°C for germination and growth. For tests of the ABA effect on germination, seeds were plated directly onto media containing various concentrations of ABA. For testing root elongation under ABA or abiotic stress treatments, seeds were firstly germinated on MS media. Four-days-old seedlings were then transferred to media containing ABA, mannitol, PEG or NaCl. Root length was measured 7 days after transfer using the NIH ImageJ1.41 program.
Cloning, expression, purification of recombinant proteins and the EMSA
Cloning, expression in E. coli and purification of recombinant WRKY 18 and 40 proteins have been previously described . 5' biotin labeled DNA probes of the WRKY60 promoter was synthesized by Invitrogen. EMSA and detection were performed according to the manual of the Pierce's Lightshift Chemiluminescent EMSA Kit. In each binding assay, 200 fmol recombinant WRKY protein and 20 fmol DNA probe were used.
Gene expression analysis
Total RNA was extracted from plant samples following the instructions in handbook of Trizol (Invitrogen) and treated with RNase-free DNase I (Promega) to remove contaminated DNA. cDNA was synthesized by adding 100 ng total RNA into 10 μl reaction with random hexames and oligo dT primers provided by PrimeScript RT Reagent Kit (Takara). Quantitative-real time PCR was performed in ABI7900 HT machine with SYBR PrimeScript RT-PCR Kit (Takara). The RT-reaction product (2 μl) was used as template in a 25 μl PCR mixture. The following program was used for PCR amplification: Initial denaturation at 95°C 10 sec. followed by 40 cycles of 95°C 5 sec. and 60°C 30 sec. The β-actin gene was used as endogenous reference gene. Data analysis was performed using the ABI SDS 2.0 program. The primers used in real time PCR are listed in Additional file 4.
Protoplast transfection assays
The full-length GUS gene was clone into the XbaI site of pFF19 . The 1.0 kb WRKY60 promoter was PCR-amplified using the following two primers: atgcaagcTTTCTTTGTTTTCTGCCGGTTT and atgcgagctcAAATTTAGGTTCACAGGAGCCA. The amplified promoter DNA was digested with HindIII and SacI and was used to replace the CaMV 35S promoter in pFF19. The mutant WRKY60 promoter in which the cluster of W-box sequences between -791 and -773 relative to the translation start codon was generated by overlapping PCR. The sequences of the promoters were verified by DNA sequencing.
To generate the WRKY18 and WRKY40 effector constructs, their cDNA fragments that contained the full coding sequences and the 3'-untranslated regions were excised from their respective cloning plasmids and subcloned into the same restriction sites of pFF19 in the sense orientation behind the 35S promoter.
Protoplast isolation and transfection were carried out according to the protocols as previously described . Four- to five-weeks old rosette leaves were used for isolation of mesophyll protoplasts. Protoplast transfection was performed using 40% polyethylene glycol with 10 μg reporter plasmid and 15 μg effector plasmid DNA.
Assays of Transcriptional Regulatory Activity
Transgenic Arabidopsis plants containing a GUS reporter gene driven by a synthetic promoter consisting of the -100 minimal CaMV 35S promoter and eight copies of the LexA operator sequence were previously described . To generate effector genes, the DNA fragment for the LexA DBD was digested from the plasmid pEG202 (Clontech) using HindIII and EcoRI and cloned into the same sites in pBluescript. The full-length WRKY18, WRKY40 and WRKY60 cDNA fragments were subsequently subcloned behind the LexA DBD to generate translational fusions. The LexA DBD-WRKY fusion genes were cloned into the XhoI and SpeI site of pTA2002 behind the steroid-inducible promoter . As controls, the unfused LexADBD and WRKY genes were also cloned into the same sites of PTA7002. These effector constructs were directly transformed into the transgenic GUS reporter plants and double transformants were identified through screening for antibiotic (hygromycin) resistance. Determination of activation or repression of GUS reporter gene expression by the effector proteins was performed as previously described . For determining the effect of ABA and SA on the transcription-regulating activity of the WRKY proteins, progeny from 5 independent transgenic lines for each effector gene were divided into three groups (15-20 plants/group) and sprayed with DEX (20 μM), DEX plus ABA (10 μM) or DEX plus SA (1 mM). Leaves were harvested at 0 and 24 hours after the treatment for assays of GUS activities.
We thank Guangdong Natural Science Foundation (grant no. 06023150) and National Science and Technology Major Projects (grant no. 2009ZX08001-016B) (CN) for supporting this research to XX and US National Science Foundation grant MCB-0209819 to ZC.
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