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Supporting Information legends
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Figure S1. MeJA Response of AtMYB44 overexpressing and atmyb44 knockout
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plants.
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(a) Root lengths of Arabidopsis Col-0, AtMYB44 overexpression plants (OX18) and
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atmyb44 knockout plants were measured. Data represent the mean root length from
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fifty seedlings. Error bars represent standard deviation.
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(b) Root hair density was quantified from Arabidopsis Col-0, AtMYB44
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overexpression plants (OX18, OX21) and atmyb44 knockout plants. The values
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represent the mean of fifteen seedlings. Error bars represent standard deviation.
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Statistical significance of the measurements was determined using a t-test (*P<0.01)
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by comparison with the value of Col-0.
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Figure S2. Resistance of npr1-1 and NahG plants against the biotrophic pathogen
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Pst DC3000.
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Four-week-old Arabidopsis Col-0, npr1-1 and NahG plants were inoculated with Pst
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DC3000. Bacterial growth in leaves was determined one (open bars) or three days
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(closed bars) after inoculation. Statistical significance of the measurements was
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determined using a t-test (*P<0.05) by comparison with the value of Col-0. Data
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represent the mean values of eight independent plants and error bars represent
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standard deviation.
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Figure S3. Expression of SA signaling genes in AtMYB44 overexpressing and
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atmyb44 knockout plants.
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Expression of SA signaling genes was determined by RT-PCR with a limited number
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of cycles (25 cycles, Method S1) and PCR product was visualized by ethidium
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bromide staining.
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Figure S4. Transcriptional activation domain assay of AtMYB44.
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(a) Various fragments of AtMYB44 were recombined with the GAL4 binding domain
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as shown in the diagram.
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(b) Transcriptional activity was measured by –galactosidase assay (Method S1).
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Data represent mean values of three independent measurements. Error bars
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represent standard deviation.
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Figure S5. Direct binding of AtMYB44 to the WRKY70 promoter in vivo.
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(a) Interaction of AtMYB44 with WRKY70 promoter was tested by yeast one-hybrid
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(Y1H) assay. WRKY70 promoter sequence of -328 to -299 containing the core
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binding sequence (RE44) or its mutant version (mRE44) was repeated 4 times and
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combined with HIS3 for the Y1H assay. A cDNA encoding the whole AtMYB44
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protein was fused with the activation domain as bait.
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(b) Structure of WRKY70 indicating regions amplified by PCR (top). Open boxes at
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sites 1, 2 and 4 represent the core AtMYB44 binding sequence. PCR primer sets
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binding to another promoter region (Site 3), CDS region (Site 5) or 3’-UTR region of
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WRKY70 (Site 6) were also employed for negative controls. Fragmented chromatin
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DNA of Arabidopsis Col-0 and AtMYB44-GFP overexpressing plants were
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immunoprecipitated with anti-GFP and analyzed by PCR (bottom left). Chromatin
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DNA before immunoprecipitation was PCR amplified as an input control.
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Figure S6. SA-mediated suppression of JA response in atmyb44 knockout mutant
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plants.
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Two-week-old Arabidopsis plants were treated with combination of SA and JA.
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Treated seedlings were harvested 6 hour after treatment. Total RNA was analyzed by
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Northern blot and rRNA was visualized by ethidium bromide staining as a loading
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control.
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Figure S7. Expression of AtMYB44 to Pst DC3000.
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Four-week-old Arabidopsis Col-0 plants were treated with Pst DC3000. rRNA is
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shown for checking equal loading.
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Figure S8. Induction of WRKY70 and PR1 by MeJA.
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Arabidopsis Col-0 and coi1-1 mutants were treated with 50 μM of MeJA for the
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indicated number of hours and JA-responsive genes were analyzed by Northern blot.
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rRNA was visualized by ethidium bromide staining as a loading control.
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Table S1. Primers used in Northern blot analysis, ChIP PCR and subcloning.
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Methods S1. Supplemental experimental procedures.
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