Submit a Revision

*your email :**
page name* : such as "Introduction", "Conclusion"..etc
<p>Structure, DNA binding and subcellular localization</p> <p><em>Arabidopsis WRKY25 </em>(At2g30250) encodes a protein of 394 amino acids with a molecular weight of 44.134 kD and an isolelectric point of 6.43 (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1A</a>). 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 <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1A</a>). Thus, WRKY25 may be regulated, at least in part, via protein phosphorylation by a protein kinase(s) such as MPK4 <a></a>[<a href="http://www.biomedcentral.com/1471-2229/7/2#B31">31</a>].</p> <p>WRKY transcription factors are thought to function by binding their cognate TTGACC/T W-box <em>cis</em>-elements in the promoter regions of target genes and activating or repressing their expression <a></a>[<a href="http://www.biomedcentral.com/1471-2229/7/2#B6">6</a>]. A number of isolated WRKY proteins have been shown to bind W-box sequences <a></a><a></a><a></a>[<a href="http://www.biomedcentral.com/1471-2229/7/2#B8">8</a>,<a href="http://www.biomedcentral.com/1471-2229/7/2#B12">12</a>,<a href="http://www.biomedcentral.com/1471-2229/7/2#B20">20</a>]. To examine the DNA-binding activity of WRKY25, we expressed the gene in <em>E. coli</em>, purified the recombinant protein, and assayed its binding to an oligonucleotide that contains two direct TTGACC repeats (Pchn5; Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1B</a>) using EMSA. Several WRKY25/DNA complexes with differing mobility were detected when purified recombinant WRKY25 protein was incubated with the Pchn5 probe (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1C</a>). 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 <em>WRKY25 </em>gene. Binding of WRKY25 was not detected with a mutant probe (mPchn5) in which both TTGACC sequences were changed to TTGAAC (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1B</a> and <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F1">1C</a>). Thus, binding of WRKY25 to the TTGACC W-box sequence is highly specific.</p> <p>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 <em>Cauliflower mosaic virus </em>(CaMV)<em>35S </em>promoter, was directly bombarded into onion (<em>Allium cepa</em>) epidermal cells. As shown in Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F2">2</a>, 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 <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F2">2</a>).</p> <p>Expression of <em>WRKY25</em></p> <p>A possible role for WRKY25 during defense signaling was further investigated by analyzing its expression in <em>Arabidopsis </em>after inoculation with <em>Psm</em>ES4326. As shown in Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3A</a>, <em>WRKY25 </em>mRNA levels increase in wild-type plants after infiltration with either the control MgCl<sub>2 </sub>solution (mock inoculation) or the bacterial suspension. However, <em>WRKY25 </em>expression was prolonged in pathogen-infected plants, as transcript levels remained elevated at 24 hours post infiltration (hpi), whereas they were nearly undetectable in MgCl<sub>2</sub>-treated plant at this time (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3A</a>).</p> <p>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 <em>WRKY25 </em>expression was modestly reduced in the <em>npr1-3 </em>and <em>sid2 </em>mutants, which are defective in SA signaling and biosynthesis, respectively <a></a><a></a>[<a href="http://www.biomedcentral.com/1471-2229/7/2#B35">35</a>,<a href="http://www.biomedcentral.com/1471-2229/7/2#B36">36</a>] (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3A</a>). By contrast, no significant difference was observed in <em>WRKY25 </em>expression between the wild-type plants and the ET-insensitive <em>ein2 </em>mutant plants following mock or pathogen inoculation. Analysis of the JA-insensitive <em>coi1 </em>mutant revealed a delay in <em>WRKY25 </em>expression following mock inoculation; however, it was significantly enhanced after pathogen infiltration, as compared with that observed in wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3A</a>). These results suggest that <em>WRKY25 </em>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.</p> <p>We also analyzed <em>WRK25 </em>induction in wild-type plants sprayed with water, SA, 1-aminocyclopropane-1-carboxylic acid (ACC, the immediate precursor of ET) or methyl JA. <em>WRKY25 </em>expression was rapidly induced in water-treated plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3B</a>), 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 <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3B</a>). Thus, both SA and ET regulate <em>WRKY25 </em>expression in a positive manner, whereas JA has a negative effect on <em>WRKY25 </em>expression.</p> <p>Disrupting or altering <em>WRKY25 </em>expression affects disease resistance and symptom severity</p> <p>To analyze the role of WRKY25 in disease resistance, we identified two T-DNA insertion mutants for <em>WRKY25</em>. <em>wrky25-1 </em>(Salk_136966) contains a T-DNA insertion in the promoter region while <em>wrky25-2 </em>(Sail 754_A03) contains a T-DNA insertion in the last intron of the <em>WRKY25 </em>gene (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F4">4A</a>). Homozygous mutant plants were identified by PCR with <em>WRKY25</em>-specific primers. We then compared the wild-type and <em>wrky25 </em>mutants for Induced accumulation of <em>WRKY25 </em>transcripts. Since MgCl<sub>2 </sub>treatment and pathogen infection had almost the same potency in inducing <em>WRKY25 </em>expression (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F3">3</a>), we used only pathogen infection in these experiments. Northern analysis using a full-length <em>WRKY25 </em>cDNA clone as the probe detected <em>WRKY25 </em>transcripts in wild-type plants but not in <em>wrky25-1 </em>plants after pathogen infection. By contrast, a <em>WRKY25 </em>transcript of reduced size was detected in pathogen-infected <em>wrky25-2 </em>plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F4">4B</a>, 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 <em>wrky25-2 </em>(Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F4">4B</a>, lower panel). Thus, the T-DNA insertion in the <em>wrky25-2 </em>mutant results in generation of a truncated <em>WRKY25 </em>transcript that is predicted to generate a truncated WRKY25 protein lacking the C-terminal WRKY domain, which is important for DNA binding <a></a>[<a href="http://www.biomedcentral.com/1471-2229/7/2#B37">37</a>].</p> <p>Analysis of both <em>wrky25 </em>mutants revealed no difference in growth or morphology from that of wild-type plants; flowering also occurred at the normal time. Following inoculation with <em>Psm</em>ES4326, the mutant lines supported similar levels of bacterial growth as wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F5">5A</a>). However, the inoculated leaves of <em>wrky25-1 </em>and <em>wrky25-2 </em>plants consistently displayed less disease symptom than wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F5">5B</a>).</p> <p>To examine the effect of <em>WRKY25 </em>overexpression, we generated plants containing a full-length <em>WRKY25 </em>cDNA driven by the <em>CaMV 35S </em>promoter (<em>35S::W25</em>). Northern blotting identified several transgenic plants that contained elevated levels of <em>WRKY25 </em>transcript constitutively (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F4">4C</a>). Two transgenic lines (#12 and #18 in Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F4">4C</a>) that constitutively expressed <em>WRKY25 </em>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.</p> <p>Analysis of T<sub>3 </sub>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 <em>Psm</em>ES4326, the transgenic 35S:W25 overexpression lines displayed substantially greater bacterial growth (~12 fold) than wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F5">5A</a>). The inoculated leaves of <em>WRKY25</em>-overexpressing plants also developed more severe disease symptoms than those of wild-type plants after infection (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F5">5B</a>).</p> <p><em>PR1 </em>gene expression and SA accumulation</p> <p>To study the molecular basis for the altered responses to <em>Psm</em>ES4326 infection, <em>PR1 </em>gene expression was monitored. Consistent with the enhanced susceptibility phenotype, <em>WRKY25 </em>overexpressing lines contained substantially lower levels of <em>PR1 </em>transcripts than wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F6">6A</a>). In contrast, <em>PR1 </em>transcript levels in the <em>wrky25 </em>mutants were comparable to those in wild-type plants (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F6">6A</a>).</p> <p>To determine whether altered <em>PR1 </em>induction in the <em>WRKY25</em>-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 <em>Psm</em>ES4326 infection (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F6">6B</a>). Free SA levels in <em>WRKY25</em>-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 <em>WRKY25</em>-overexpressing plants also were ~10-fold lower than those in wild-type plants, although they rose to nearly wild-type levels after infection (Figure <a></a><a href="http://www.biomedcentral.com/1471-2229/7/2/figure/F6">6B</a>).</p> <br>
Confirmation code: Enter the code exactly as it appears. All letters are case insensitive, there is no zero.