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Structure, DNA binding and subcellular localization
Arabidopsis WRKY25 (At2g30250) encodes a protein of 394 amino acids with a molecular weight of 44.134 kD and an isolelectric point of 6.43 (Figure 1A). Based on the presence of two WRKY domains, WRKY25 is classified as a group I WRKY protein. The N-terminus and the region between the WRKY domains are rich in serine and/or threonine residues (Figure 1A). Thus, WRKY25 may be regulated, at least in part, via protein phosphorylation by a protein kinase(s) such as MPK4 .
WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box cis-elements in the promoter regions of target genes and activating or repressing their expression . A number of isolated WRKY proteins have been shown to bind W-box sequences [8,12,20]. To examine the DNA-binding activity of WRKY25, we expressed the gene in E. coli, purified the recombinant protein, and assayed its binding to an oligonucleotide that contains two direct TTGACC repeats (Pchn5; Figure 1B) using EMSA. Several WRKY25/DNA complexes with differing mobility were detected when purified recombinant WRKY25 protein was incubated with the Pchn5 probe (Figure 1C). Whether the different complexes represent probes in which one or both of the W boxes are bound by WRKY25, or whether they are caused by formation of monomeric and oligomeric WRKY25 complexes is unclear. Alternatively, some of these complexes might result from protein degradation or incompletely translation of the WRKY25 gene. Binding of WRKY25 was not detected with a mutant probe (mPchn5) in which both TTGACC sequences were changed to TTGAAC (Figure 1B and 1C). Thus, binding of WRKY25 to the TTGACC W-box sequence is highly specific.
If WRKY25 is a transcription factor, it is likely to be localized in the nucleus. The presence of putative nuclear localization signal predicted by the PSORT II program is consistent with this possibility. To determine the subcellular location of WRKY25, we constructed a GFP protein fusion of WRKY25. The fusion construct, driven by the Cauliflower mosaic virus (CaMV)35S promoter, was directly bombarded into onion (Allium cepa) epidermal cells. As shown in Figure 2, the transiently expressed WRKY25-GFP fusion protein was localized exclusively to the nucleus. By contrast, GFP was found in both the nucleus and cytoplasm due to its small size (Figure 2).
Expression of WRKY25
A possible role for WRKY25 during defense signaling was further investigated by analyzing its expression in Arabidopsis after inoculation with PsmES4326. As shown in Figure 3A, WRKY25 mRNA levels increase in wild-type plants after infiltration with either the control MgCl2 solution (mock inoculation) or the bacterial suspension. However, WRKY25 expression was prolonged in pathogen-infected plants, as transcript levels remained elevated at 24 hours post infiltration (hpi), whereas they were nearly undetectable in MgCl2-treated plant at this time (Figure 3A).
To determine whether WRKY25 expression is influenced by the SA, ET and/or JA signaling pathways, WRKY25 expression was monitored in various signaling mutants. Induced WRKY25 expression was modestly reduced in the npr1-3 and sid2 mutants, which are defective in SA signaling and biosynthesis, respectively [35,36] (Figure 3A). By contrast, no significant difference was observed in WRKY25 expression between the wild-type plants and the ET-insensitive ein2 mutant plants following mock or pathogen inoculation. Analysis of the JA-insensitive coi1 mutant revealed a delay in WRKY25 expression following mock inoculation; however, it was significantly enhanced after pathogen infiltration, as compared with that observed in wild-type plants (Figure 3A). These results suggest that WRKY25 expression is sensitive to environmental cues and it appears to be positively regulated by the SA signaling pathway but negatively regulated by the JA pathway.
We also analyzed WRK25 induction in wild-type plants sprayed with water, SA, 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate precursor of ET) or methyl JA. WRKY25 expression was rapidly induced in water-treated plants (Figure 3B), underscoring that the gene is very responsive to environmental stimuli. Plants sprayed with SA or ACC accumulated greater levels of WRKY25 transcripts than water-sprayed plants, whereas JA-treated plants accumulated less (Figure 3B). Thus, both SA and ET regulate WRKY25 expression in a positive manner, whereas JA has a negative effect on WRKY25 expression.
Disrupting or altering WRKY25 expression affects disease resistance and symptom severity
To analyze the role of WRKY25 in disease resistance, we identified two T-DNA insertion mutants for WRKY25. wrky25-1 (Salk_136966) contains a T-DNA insertion in the promoter region while wrky25-2 (Sail 754_A03) contains a T-DNA insertion in the last intron of the WRKY25 gene (Figure 4A). Homozygous mutant plants were identified by PCR with WRKY25-specific primers. We then compared the wild-type and wrky25 mutants for Induced accumulation of WRKY25 transcripts. Since MgCl2 treatment and pathogen infection had almost the same potency in inducing WRKY25 expression (Figure 3), we used only pathogen infection in these experiments. Northern analysis using a full-length WRKY25 cDNA clone as the probe detected WRKY25 transcripts in wild-type plants but not in wrky25-1 plants after pathogen infection. By contrast, a WRKY25 transcript of reduced size was detected in pathogen-infected wrky25-2 plants (Figure 4B, upper panel). This transcript was not detected when the same blot was probed with a DNA fragment corresponding to the region downstream of the T-DNA insertion site in wrky25-2 (Figure 4B, lower panel). Thus, the T-DNA insertion in the wrky25-2 mutant results in generation of a truncated WRKY25 transcript that is predicted to generate a truncated WRKY25 protein lacking the C-terminal WRKY domain, which is important for DNA binding .
Analysis of both wrky25 mutants revealed no difference in growth or morphology from that of wild-type plants; flowering also occurred at the normal time. Following inoculation with PsmES4326, the mutant lines supported similar levels of bacterial growth as wild-type plants (Figure 5A). However, the inoculated leaves of wrky25-1 and wrky25-2 plants consistently displayed less disease symptom than wild-type plants (Figure 5B).
To examine the effect of WRKY25 overexpression, we generated plants containing a full-length WRKY25 cDNA driven by the CaMV 35S promoter (35S::W25). Northern blotting identified several transgenic plants that contained elevated levels of WRKY25 transcript constitutively (Figure 4C). Two transgenic lines (#12 and #18 in Figure 4C) that constitutively expressed WRKY25 at elevated levels and contain a single T-DNA locus in their genomes, based on the ratio of antibiotic resistance phenotypes, were chosen for further study.
Analysis of T3 homozygous plants from both lines revealed no difference in growth or development from that of wild-type plants, although their leaf color appear to be slightly paler. Following inoculation with PsmES4326, the transgenic 35S:W25 overexpression lines displayed substantially greater bacterial growth (~12 fold) than wild-type plants (Figure 5A). The inoculated leaves of WRKY25-overexpressing plants also developed more severe disease symptoms than those of wild-type plants after infection (Figure 5B).
PR1 gene expression and SA accumulation
To study the molecular basis for the altered responses to PsmES4326 infection, PR1 gene expression was monitored. Consistent with the enhanced susceptibility phenotype, WRKY25 overexpressing lines contained substantially lower levels of PR1 transcripts than wild-type plants (Figure 6A). In contrast, PR1 transcript levels in the wrky25 mutants were comparable to those in wild-type plants (Figure 6A).
To determine whether altered PR1 induction in the WRKY25-overexpressing plants correlated with reduced SA accumulation, the levels of both free SA and SA-glucoside conjugates (SAG) were monitored. Both wild-type plants and the T-DNA insertion mutants displayed similar levels of free SA and SAG following PsmES4326 infection (Figure 6B). Free SA levels in WRKY25-overexpressing plants were comparable to those in wild-type plants at 0 and 24 hpi; however, the level of free SA at 48 hpi was somewhat lower than in wild-type plants. SAG levels in uninoculated WRKY25-overexpressing plants also were ~10-fold lower than those in wild-type plants, although they rose to nearly wild-type levels after infection (Figure 6B).
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