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
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 . For production of recombinant WRKY3 and WRKY4 proteins, full-length WRKY3 and WRKY4 coding
sequences were amplified by PCR using gene-specific primers
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 .
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'-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 
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
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  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.
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.
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.