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Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic …


Biology Articles » Botany » Plant Pathology » Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens » Methods

Methods
- Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens

Materials

32P-dATP (>3000 Ci/mmol) was obtained from DuPont-New England Nuclear; other common chemicals were purchased from Sigma. Arabidopsis thaliana plants were grown in a growth chamber at 24°C under 100 μE·m-2·sec-1 light with 12-hr-light/12-hr-dark photoperiod. PstDC3000 were maintained on King's B medium containing 100 μg/ml of rifampicin and 50 μg/ml kanamycin. The conidiospores of B. cineria isolate B5-10 were collected from 10 days old mycelium growing on V8-agar medium and suspended in 1% maltose for inoculation as previously described [23].

Recombinant protein and DNA-binding

Full-length cDNAs for WRKY3 and WRKY4 were isolated from an Arabidopsis cDNA library constructed from SA-treated Arabidopsis plants as previously described [39]. For production of recombinant WRKY3 and WRKY4 proteins, full-length WRKY3 and WRKY4 coding sequences were amplified by PCR using gene-specific primers (5'-ATCGAATTCATGGCGGAGAAGGAAGAAAAAG-3' and 5'-ATCCTCGAGCTAAGCCATGGTGATTTGCTCTTCTTTAAGCCT-3' for WRKY3 and 5'-ATCGAATTCATGTCGGAAAAGGAAGAAGCTC-3' and 5'-ATCCTCGAGCTAAGCCATGGTTGTTTGCTCTTCTTTAAGCCT-3' for WRKY4). The amplified PCR fragments were digested with EcoRI and XhoI and cloned into the same sites of the pET-32a E. coli expression vector. Preparation of recombinant proteins and DNA-binding assays were performed as previously described [15].

Subcellular localization

Full-length WRKY3 and WRKY4 coding sequences were amplified by PCR with the same gene-specific primers used for generating expression constructs in E. coli as described above. The amplified PCR fragments were digested with EcoRI and NcoI and cloned into the same sites of a GFP fusion expression vector as previously described [23,25]. Onion epidermal cell layers were peeled and placed inside up on the MS plates. Plasmid DNAs of appropriate fusion genes (0.5 μg) were introduced to the onion cells using a pneumatic particle gun (PDS 1000, Du Pont). The condition of bombardment was vacuum of 28 inch Hg, helium pressure of 1100 or 1300 psi, and 6 cm of target distance using 1.1 μm of tungsten microcarriers. After bombardment, tissues were incubated on the MS plates for 24 h at 22°C. Samples were observed directly or transferred to glass slides.

Identification of the wrky3 and wrky4 T-DNA insertion mutants

The wrky3-1 mutant (Salk_107019) contains a T-DNA insertion in the second exon while wrky3-2 (Salk_119051) contains a T-DNAinsertion in the first exon of the WRKY3 gene (Figure 5A). The wrky4-1 (Salk_082016) and wrky4-2 (Salk_073118) mutants both contain a T-DNA insertion in the first exon of the WRKY4 gene. T-DNA insertions were confirmed by PCR using a combination of a T-DNA border primer (5'-GCTTGCTGCAACTCTCTCAG-3') and a gene specific primer (5'-GCTTCATTGACTGAGATTCCATC-3', 5'-CCCGGTGGTTGAGTTATCAT-3', 5'-TCATCGGAATCAGGGAACAT-3' and 5'-TCATCGGAATCAGGGAACAT-3' for wrky3-1, wrky3-2, wrky4-1, and wrky4-2, respectively). The nature and location of the T-DNA insertion was confirmed by sequencing the PCR products. Homozygous T-DNA mutant plants were identified by PCR using primers corresponding to sequences flanking the T-DNA insertion sites (the above four gene-specific primers paired with 5'-ATTCCCAACCTCCTCGCTAT-3' for wrky3-1, 5'-GAGAAACACGACACGAATTTTG-3' for wrky3-2, 5'-AAACACGACACGGATTCACA-3' for wrky4-1 and wrky4-2, respectively). To remove additional T-DNA loci or mutations from the mutants, we backcrossed them to wild-type plants and identified plants homozygous for the T-DNA insertion.

Generation of transgenic WRKY3 and WRKY4 overexpression plants

Full-length WRKY3 and WRKY4 cDNAs were excised from the cloning vectors using BamHI and XhoI and subcloned into the BamHI and SalI sites behind the CaMV 35S promoter in pOCA30 [23,25]. Arabidopsis transformation was performed by the flora-dip procedure [40] and transformants were identified by screening for kanamycin resistance. From the transformants, those with a single copy of T-DNA insertion (based on the 3:1 segregation of antibiotic resistance in T2 progeny) were isolated and homozygote transgenic plants were further identified in the T3 generation based on the segregation in antibiotic resistance.

RNA gel blotting

For RNA gel blot analysis, total RNA was extracted with Trizol reagent (Invitrogen) from leaf tissue, separated on 1.2% agarose-formaldehyde gels and blotted to nylon membranes according to standard procedures. Blots were hybridized with – 32P-dATP-labeled gene-specific probes. Hybridization in PerfectHyb™ Plus hybridization buffer (Sigma) at 68°C and subsequent membrane washing were performed as previously described. Full-length cDNAs were used as probes in Northern blotting for detecting WRKY3 and WRKY4 transcripts. Arabidopsis PR1 gene probe was generated from a PR1 DNA fragment amplified by PCR using two PR1-specific primers (5'-TTCTTCCCTCGAAAGCTCAA-3' and 5'-CGTTCACATAATTCCCACGA-3'). The B. cinerea ActinA gene probe [41] was amplified from the B. cinerea genomic DNA and by PCR using primers 5'-ACTCATATGTTGGAGATGAAGCGCA-3' and 5'-TGTTACCATACAAATCCTTACGGACA-3'.

Pathogen inoculation and disease development

For disease resistance to P. syringae, three mature leaves of each 5-weeks old plant were infiltrated with a virulent strain (OD600 = 0.0001 in 10 mM MgCl2). The bacterial titers were determined immediately after infiltration or after 3 days post-inoculation for bacteria growth analysis. For disease resistance to B. cineria, the fungal spores (5 × 105 spores/ml) were sprayed on 35 day-old plants evenly. The plants were covered with transparent plastic dome to maintain high humidity and disease development was evaluated 5 days later.

 

Authors' contributions

ZL carried out expression analysis of the WRKY genes and analysis of the mutants and overexpression lines. KMV carried out DNA binding assays, determination of subcellular localization, isolation, generation and characterization of the mutants and transgenic overexpression lines. ZZ carried out Botrytis tests. BF carried out isolation of the cDNA clones. ZC conceived of the study, participated in the design and helped to draft and edit the manuscript. All authors read and approved the final manuscript.

Acknowledgements

We thank the ABRC at the Ohio State University (Columbus, OH) for the Arabidopsis mutants. This work was supported by National Science Foundation Grant MCB-0209819. This is Journal Paper 2008-18311 of the Purdue University Agricultural Research Program.


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