Identification of sequence element in the <i>BcNTR1</i> promoter region to which AtBBD1 binds.

<p>(A) Structures of reporter and activator genes used in Y1H assays. The promoter region of <i>BcNTR1</i>, −3518 to −3390, was divided into 3 segments and each segment was used as bait for Y1H assays. The control does not contain any of those segments. AtBBD1 was fused with the GAL4 activating domain (AD) as an activator. The position of the putative JARE is shown (▾). (B) The segment a was divided further into 8 subsegments (6 nt each) and each subsegment, a1 to a8, was mutated into 6 adenines. Each mutant segment was tested as bait in Y1H assays. (C) Subsegments a6 and a7 to which AtBBD1 bound, were dissected further by mutation in overlapping frames. In each mutant, 6 nucleotides were mutated into 6 adenines. Each mutant subsegment, M0–M5, was tested by Y1H assays. The sequence motif to which AtBBD1 binds is shown in bold. (D) Mutation analysis of the AtBBD1 binding element. Mutant series (CM1 to CMR) of JARE was created by changing a single nucleotide from purine to pyrimidine, or <i>vice versa</i>, in the fragment −2305 to −2278 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055482#pone-0055482-g004" target="_blank">Fig. 4A</a> as a bait and Y1H assays were carried out with AD-AtBBD1. CMR is a JARE in reverse orientation_.</p

Proposed model for transcription repression by AtBBD1.

<p>In the absence of signal, AtBBD1 represses <i>AtJMT</i> gene expression by recruiting corepressor or HDAc through AtJAZ. In the presence of signal, JA-Ile is released and the SCF^COI1 complex degrades JAZ proteins. A putative activator (+) that binds to the JARE competes with AtBBD1 (repressor). In knockout plants, the putative activator dominantly occupies the JARE and <i>AtJMT</i> gene expression is activated higher than wild type. In the AtBBD1-overexpressing plant, AtBBD1(repressor) competes with the putative activator and dominantly occupies the JARE; therefore, <i>AtJMT</i> gene expression is repressed more than in wild type. Size of each circle represents relative abundance.</p

AtBBD1 interacts with AtJAZ proteins.

<p>(A) Y2H assay between AtBBD1 and each of 12 AtJAZs. Full length CDS of AtBBD1 was fused to GAL4 DNA binding domain (BD) and each full length CDSs of 12 AtJAZs was fused to AD. (B) The pull-down assay between AtBBD1 and AtJAZ1. <i>35S:6xmyc-AtBBD1</i> plant extract (input) was incubated with amylose resin bound recombinant MBP-AtJAZ1 protein. Pulled-down protein complex was detected by immunoblotting using anti-MYC antibody (left). MBP protein was used as a pull-down control. The panel on the right shows input recombinant MBP and MBP-AtJAZ1 proteins in the pull-down assay. (C) Immunodetection of the AtBBD1 and AtJAZ1 complex <i>in vivo.</i> 35S:6xMYC-AtBBD1 and 35S:3xHA-AtJAZ1 constructs were transiently coexpressed in tobacco leaves by agroinfiltration. The expressed proteins were immunoprecipitated (IP) using anti-HA antibody (+/+) and immunoblotting was carried out with anti-myc antibody. Left lane (−/−) is control leaf extract that was not agroinfiltrated. MYC-AtBBD1 and HA-AtJAZ1 proteins were detected in input coexpressed leaf extracts by each antibody (right). (D) Each truncated AtBBD1 protein was fused to AD as a prey for Y2H assay with AtJAZ1. AtJAZ1 was fused to BD as bait. Numbers indicates amino acid residues, and putative domains were represented. (E) Each truncated AtJAZ1 protein was fused to AD as a prey for Y2H assay with AtBBD1 protein. AtBBD1 was fused to BD as bait.</p

AtBBD1 and AtBBD2 bind to promoter sequences containing the JARE.

<p>Promoter segments, P<i>_NTR1,</i> (−3497 to −3470 of <i>BcNTR1</i> promoter) or P<i>_JMT</i> (−2305 to −2278 of <i>AtJMT</i> promoter) were used as bait in Y1H assays. A mutated segment, P<i>_G-box</i>, which contains G-box sequence in P<i>_NTR1</i> was used as a bait and an empty vector (pHIS2) was used as a control. AtBBD1 and AtBBD2 were fused with AD. G-box is AtMYC2 binding element (CACGTG) (Boter et al., 2004).</p

Acetylation of chromatin histones associated with <i>P_JMT</i> is enhanced by MeJA.

<p>(A) Chromatin immunoprecipitation was carried out with antibodies recognizing acetylated histone H3 (AcH3K14) or H4 (AcH4K12). Precipitated DNA was amplified by primers corresponding to sequences adjacent to the AtBBD1 binding sites in the <i>AtJMT</i> promoter (<i>P_JMT</i>). PCR product was analyzed by agarose gel electrophoresis. Actin was used as a control. Input indicates samples before immunoprecipitation. (B) qPCR analysis of ChIP assay with Col-0, <i>atbbd1 atbbd2</i>, and OX-4. Open bar is without MeJA treatment and closed bar is with MeJA treatment for 3 hours. Relative fold difference is represented. Statistical significance of the measurements was determined using a t-test (P≤0.05) by comparison with the value for Col-0 (*). Comparison between indicated values is also shown by (**). Data represent the mean values of 3 independent experiments and error bars represent standard deviation.</p

Localization of the JA-responsive <i>cis</i>-element (JARE) in the <i>AtJMT</i> promoter.

<p>(A) A series of 5’ deleted promoters (closed bar) was cloned upstream of <i>GUS</i> coding region (open bar) and transformed into <i>Arabidopsis</i>. RT-PCR analysis of each transgenic plant was carried out after 1 hour of MeJA treatment. The JARE is located in the region between −2500 and −2000. (B) Additional promoter deletion constructs between −2500 and −2000 are shown and their <i>GUS</i> gene expression in response to MeJA treatment is shown. The putative JARE is located in the region between −2294 and −2280 (gray bar). The positions of the G-boxes are shown at −2529, −2406 and −2342 (▾).</p

JARE-containing transgenic plants show MeJA response.

<p>(A) Schematic representation of multimerized JARE- containing construct (4xJARE:GUS), and its mutant version (4xmJARE:GUS). The DNA fragment from the <i>AtJMT</i> promoter (−2305 to −2278) containing the JARE was repeated 4 times and recombined to the GUS reporter containing a minimal promoter (TATA) from CaMV 35S. JARE(TCCTGA) and its mutant version are shown in bold. (B) Histochemical staining of 4xJARE:GUS (#17) and 4xmJARE:GUS (#12) transgenic plants with (+) or without (−) MeJA treatment for 4h. (C) RT-PCR analysis of <i>GUS</i> in each transgenic line was carried out after MeJA treatment.</p

<i>AtJMT</i> and <i>BcNTR1</i> promoters contain JA-responsive transcriptional regulatory elements.

<p>Northern blot analysis of the recombinant <i>GUS</i> gene after MeJA treatment of transgenic <i>Arabidopsis</i>. <i>BcNTR1</i> (4.4 kb length, A) and <i>AtJMT</i> (4.5 kb length, B) promoters fused to the <i>GUS</i> gene are shown. Nucleotide sequence of the promoter is numbered from translation initiation site.</p