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| <?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article PUBLIC "-//NLM//DTD JATS (Z39.96) Journal Archiving and Interchange DTD v1.1d1 20130915//EN" "JATS-archivearticle1.dtd"><article article-type="research-article" dtd-version="1.1d1" xmlns:xlink="http://www.w3.org/1999/xlink"><front><journal-meta><journal-id journal-id-type="nlm-ta">elife</journal-id><journal-id journal-id-type="hwp">eLife</journal-id><journal-id journal-id-type="publisher-id">eLife</journal-id><journal-title-group><journal-title>eLife</journal-title></journal-title-group><issn publication-format="electronic">2050-084X</issn><publisher><publisher-name>eLife Sciences Publications, Ltd</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="publisher-id">00983</article-id><article-id pub-id-type="doi">10.7554/eLife.00983</article-id><article-categories><subj-group subj-group-type="display-channel"><subject>Research article</subject></subj-group><subj-group subj-group-type="heading"><subject>Plant biology</subject></subj-group></article-categories><title-group><article-title>The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth</article-title></title-group><contrib-group><contrib contrib-type="author" id="author-5548"><name><surname>Lozano-Durán</surname><given-names>Rosa</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con1"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5550"><name><surname>Macho</surname><given-names>Alberto P</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con2"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5549"><name><surname>Boutrot</surname><given-names>Freddy</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="con3"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5551"><name><surname>Segonzac</surname><given-names>Cécile</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="fn" rid="pa1">†</xref><xref ref-type="fn" rid="con4"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" id="author-5552"><name><surname>Somssich</surname><given-names>Imre E</given-names></name><xref ref-type="aff" rid="aff2"/><xref ref-type="other" rid="par-3"/><xref ref-type="fn" rid="con5"/><xref ref-type="fn" rid="conf1"/></contrib><contrib contrib-type="author" corresp="yes" id="author-5450"><name><surname>Zipfel</surname><given-names>Cyril</given-names></name><xref ref-type="aff" rid="aff1"/><xref ref-type="corresp" rid="cor1">*</xref><xref ref-type="other" rid="par-1"/><xref ref-type="other" rid="par-2"/><xref ref-type="fn" rid="con6"/><xref ref-type="fn" rid="conf1"/></contrib><aff id="aff1"><institution>The Sainsbury Laboratory</institution>, <addr-line><named-content content-type="city">Norwich</named-content></addr-line>, <country>United Kingdom</country></aff><aff id="aff2"><institution>Max Planck Institute for Plant Breeding Research</institution>, <addr-line><named-content content-type="city">Köln</named-content></addr-line>, <country>Germany</country></aff></contrib-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Nürnberger</surname><given-names>Thorsten</given-names></name><role>Reviewing editor</role><aff><institution>University of Tübingen</institution>, <country>Germany</country></aff></contrib></contrib-group><author-notes><corresp id="cor1"><label>*</label>For correspondence: <email>cyril.zipfel@tsl.ac.uk</email></corresp><fn fn-type="present-address" id="pa1"><label>†</label><p>Institute of Agriculture and Environment, Massey University Manawatu, Palmerston North, New Zealand</p></fn></author-notes><pub-date date-type="pub" publication-format="electronic"><day>31</day><month>12</month><year>2013</year></pub-date><pub-date pub-type="collection"><year>2013</year></pub-date><volume>2</volume><elocation-id>e00983</elocation-id><history><date date-type="received"><day>22</day><month>05</month><year>2013</year></date><date date-type="accepted"><day>18</day><month>11</month><year>2013</year></date></history><permissions><copyright-statement>© 2013, Lozano-Durán et al</copyright-statement><copyright-year>2013</copyright-year><copyright-holder>Lozano-Durán et al</copyright-holder><license xlink:href="http://creativecommons.org/licenses/by/3.0/"><license-p>This article is distributed under the terms of the <ext-link ext-link-type="uri" xlink:href="http://creativecommons.org/licenses/by/3.0/">Creative Commons Attribution License</ext-link>, which permits unrestricted use and redistribution provided that the original author and source are credited.</license-p></license></permissions><self-uri content-type="pdf" xlink:href="elife00983.pdf"/><abstract><object-id pub-id-type="doi">10.7554/eLife.00983.001</object-id><p>The molecular mechanisms underlying the trade-off between plant innate immunity and steroid-mediated growth are controversial. Here, we report that activation of the transcription factor BZR1 is required and sufficient for suppression of immune signaling by brassinosteroids (BR). BZR1 induces the expression of several WRKY transcription factors that negatively control early immune responses. In addition, BZR1 associates with WRKY40 to mediate the antagonism between BR and immune signaling. We reveal that BZR1-mediated inhibition of immunity is particularly relevant when plant fast growth is required, such as during etiolation. Thus, BZR1 acts as an important regulator mediating the trade-off between growth and immunity upon integration of environmental cues.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.001">http://dx.doi.org/10.7554/eLife.00983.001</ext-link></p></abstract><abstract abstract-type="executive-summary"><object-id pub-id-type="doi">10.7554/eLife.00983.002</object-id><title>eLife digest</title><p>Like all organisms, plants must perform a careful balancing act with their resources. Investing in the growth of new roots or leaves can allow a plant to better exploit its environment—but it must not be at the expense of leaving the plant vulnerable to attack by pests and pathogens. As such, there is an obvious trade-off between allocating resources to growth or defense against disease. This trade-off must be finely balanced, and must also be responsive to different cues in the environment that would favor either growth or defense.</p><p>The plant’s immune system is able to detect invading microbes, and trigger a defensive response against them. At the surface of plant cells, proteins called pattern recognition receptors are able to recognize specific molecules that are the tell-tale signs of microbes and pathogens—such as the proteins in the molecular tails that bacteria use to move around.</p><p>For many pattern recognition receptors, signaling that they have recognized a potential invading microbe requires the actions of a co-receptor called BAK1. Interestingly, BAK1 also interacts with the receptor that identifies brassinosteroids—hormones that stimulate plant growth. Since growth and a functioning immune system are both reliant on BAK1, it was hypothesized that competition for this co-receptor could have a role in the trade-off between the two processes in plants. However, this explanation was controversial and the mechanisms underlying the trade-off still required clarification.</p><p>Now, Lozano-Durán et al. have debunked the idea that competition for BAK1 is directly responsible for the trade-off between growth and immunity. By examining how BAK1 interacts with immune receptors in the plant model species <italic>Arabidopsis thaliana</italic>, the trade-off was actually shown to be independent of BAK1. Instead, it was discovered that activation of a protein, called BZR1, reprogramed gene expression to ‘switch off’ immune signaling in response to brassinosteroids.</p><p>Lozano-Durán et al. also show that BZR1 allows the balance of the trade-off between growth and immunity to be shifted in response to cues from the environment. The suppression of the immune system by BZR1 was particularly pronounced when the conditions required fast plant growth—for example, when they mimicked the conditions experienced by seedlings before they emerge from the soil, and must grow swiftly to reach the light before they starve.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.002">http://dx.doi.org/10.7554/eLife.00983.002</ext-link></p></abstract><kwd-group kwd-group-type="author-keywords"><title>Author keywords</title><kwd>brassinosteroids</kwd><kwd>PAMPs</kwd><kwd>innate immunity</kwd><kwd>growth</kwd><kwd>transcription factors</kwd></kwd-group><kwd-group kwd-group-type="research-organism"><title>Research organism</title><kwd>Arabidopsis</kwd></kwd-group><funding-group><award-group id="par-1"><funding-source><institution-wrap><institution>The Gatsby Charitable Foundation</institution></institution-wrap></funding-source><principal-award-recipient><name><surname>Zipfel</surname><given-names>Cyril</given-names></name></principal-award-recipient></award-group><award-group id="par-2"><funding-source><institution-wrap><institution>UK Biotechnology and Biological Sciences Research Council</institution></institution-wrap></funding-source><award-id>BB/G024936/1; BB/G024944/1</award-id><principal-award-recipient><name><surname>Zipfel</surname><given-names>Cyril</given-names></name></principal-award-recipient></award-group><award-group id="par-3"><funding-source><institution-wrap><institution>Deutsche Forschungsgemeinschaft</institution></institution-wrap></funding-source><award-id>SFB 670</award-id><principal-award-recipient><name><surname>Somssich</surname><given-names>Imre E</given-names></name></principal-award-recipient></award-group><funding-statement>The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.</funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>elife-xml-version</meta-name><meta-value>2</meta-value></custom-meta><custom-meta specific-use="meta-only"><meta-name>Author impact statement</meta-name><meta-value>The transcriptional regulator BZR1 acts as a molecular integrator of environmental cues regulating the trade-off between growth and innate immunity.</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="s1" sec-type="intro"><title>Introduction</title><p>The trade-off between plant growth and immunity needs to be finely regulated to ensure proper allocation of resources in an efficient and timely manner upon effective integration of environmental cues (<xref ref-type="bibr" rid="bib34">Pieterse et al., 2012</xref>). A key aspect of plant immunity is the perception of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern-recognition receptors (PRRs), leading to PAMP-triggered immunity (PTI) (<xref ref-type="bibr" rid="bib11">Dodds and Rathjen, 2010</xref>). PRRs of the leucine-rich repeat receptor kinases (LRR-RKs) class rely on the regulatory LRR-RK BAK1 (BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1) for signaling (<xref ref-type="bibr" rid="bib23">Monaghan and Zipfel, 2012</xref>); that is the case of FLS2 (FLAGELLIN SENSITIVE 2) and EFR (EF-TU RECEPTOR), which perceive bacterial flagellin (or the active peptide flg22) and EF-Tu (or the active peptide elf18) respectively. BAK1 also interacts with the LRR-RK BRI1 (BRASSINOSTEROID INSENSITIVE 1), the main receptor for the growth-promoting steroid hormones brassinosteroids (BR), and is a positive regulator of BR-mediated growth (<xref ref-type="bibr" rid="bib20">Kim and Wang, 2010</xref>). Hence, a crosstalk between the BR- and PAMP-triggered signaling pathways resulting from competition for BAK1 was hypothesized. While a unidirectional antagonism between BR and PTI signaling has been recently described in Arabidopsis (<xref ref-type="bibr" rid="bib1">Albrecht et al., 2012</xref>; <xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>), the exact underlying mechanisms are still controversial. Activation of the BR signaling pathway via either transgenic overexpression of <italic>BRI1</italic> or the BR biosynthetic gene <italic>DWF4</italic> or expression of the activated BRI1 allele <italic>BRI1</italic><sup><italic>sud</italic></sup> suppresses PTI outputs (<xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>). One such output, the PAMP-triggered callose deposition, could be restored by over-expression of <italic>BAK1-HA</italic>, suggesting that BAK1 is a limiting factor (<xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>). However, exogenous BR treatment of wild-type plants does not affect the FLS2-BAK1 complex formation upon FLS2 activation, while it results in decreased PTI responses (<xref ref-type="bibr" rid="bib1">Albrecht et al., 2012</xref>).</p></sec><sec id="s2" sec-type="results|discussion"><title>Results and discussion</title><p>In order to clarify the role of BAK1 in the BR-PTI crosstalk, we investigated FLS2-BAK1 complex formation in the transgenic Arabidopsis lines overexpressing <italic>BRI1</italic> or <italic>DWF4</italic> or expressing <italic>BRI1</italic><sup><italic>sud</italic></sup> (<xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>). Upon treatment with flg22, FLS2 associated normally with BAK1 in these transgenic plants, and neither FLS2 nor BAK1 accumulation was altered (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1A</xref>). Moreover, these plants displayed a weaker reactive oxygen species (ROS) burst in response to chitin (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1B</xref>), whose signaling pathway is BAK1-independent (<xref ref-type="bibr" rid="bib41">Shan et al., 2008</xref>; <xref ref-type="bibr" rid="bib35">Ranf et al., 2011</xref>). This result is consistent with the previous finding that exogenous BR treatment can also inhibit the chitin-induced ROS burst (<xref ref-type="bibr" rid="bib1">Albrecht et al., 2012</xref>). BAK1-HA is not fully functional in BR signaling and exerts a dominant-negative effect on the endogenous BAK1 (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1C</xref>), which may explain that introduction of the <italic>BAK1-HA</italic> transgene can override the suppression of immunity triggered by overexpression of <italic>BRI1</italic> (<xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>); BAK1-HA does not exert such a dominant negative effect, however, on PTI signaling (<xref ref-type="fig" rid="fig1s1">Figure 1—figure supplement 1D</xref>). Taken together, these results indicate that the BR-mediated suppression of PTI is triggered independently of a competition between BRI1 and PRRs for BAK1.</p><p>We sought to determine at which level of the BR signaling pathway the antagonism initiates. After BR perception by BRI1 and activation of the BRI1-BAK1 complex, the BR signal transduction cascade includes inactivation of BIN2 (BR INSENSITIVE 2) and BIN2-like kinases, a family of GSK3-like kinases acting as negative regulators of the pathway (<xref ref-type="bibr" rid="bib44">Vert and Chory, 2006</xref>). This leads to dephosphorylation of BZR1 (BRASSINAZOLE RESISTANT 1) and BES1/BZR2 (BRI1-EMS-SUPPRESSOR 1/BRASSINAZOLE RESISTANT 2), two bHLH transcription factors acting as major regulators of BR-induced transcriptional changes, which then become active (<xref ref-type="bibr" rid="bib46">Wang et al., 2002</xref>; <xref ref-type="bibr" rid="bib52">Yin et al., 2002</xref>). Treatment with the chemicals LiCl and bikinin, which inhibit GSK3-like kinases (<xref ref-type="bibr" rid="bib10">De Rybel et al., 2009</xref>; <xref ref-type="bibr" rid="bib51">Yan et al., 2009</xref>), resulted in impaired flg22-triggered ROS burst (<xref ref-type="fig" rid="fig1">Figure 1A,B</xref>), as observed upon genetic or ligand-induced activation of the BR pathway. Furthermore, a triple mutant in <italic>BIN2</italic> and the two closest related GSK3-like kinases, <italic>BIL1</italic> (<italic>BIN2-LIKE 1</italic>) and <italic>BIL2</italic> (triple GSK3 mutant; <xref ref-type="bibr" rid="bib44">Vert and Chory, 2006</xref>), shows a similar impairment in response to either flg22 or chitin (<xref ref-type="fig" rid="fig1">Figure 1C</xref>). Interestingly, in spite of regulating MAPKs involved in stomata development (<xref ref-type="bibr" rid="bib19">Kim et al., 2012</xref>; <xref ref-type="bibr" rid="bib18">Khan et al., 2013</xref>), neither BR treatment nor loss of function of BIN2 affect flg22-triggered MAPK activation (<xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>), contrary to what has been recently suggested (<xref ref-type="bibr" rid="bib9">Choudhary et al., 2012</xref>; <xref ref-type="bibr" rid="bib55">Zhu et al., 2013</xref>). These results indicate that the BR-PTI crosstalk occurs downstream of BIN2.<fig-group><fig id="fig1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.003</object-id><label>Figure 1.</label><caption><title>Activation of BZR1 is sufficient to inhibit the PAMP-triggered ROS burst.</title><p>(<bold>A</bold>) and (<bold>B</bold>) Flg22-triggered ROS burst after LiCl (<bold>A</bold>) or bikinin (<bold>B</bold>) treatment. Leaf discs were pre-treated with a 10 mM LiCl solution for 5 hr or with a 50 μM bikinin solution for 16 hr. (<bold>C</bold>) Flg22- or chitin-triggered ROS burst in Col-0 and the triple GSK3 mutant plants. (<bold>D</bold>) Flg22- or chitin-induced ROS burst in Col-0 and <italic>BZR1Δ</italic> plants. (<bold>E</bold>) Elf18-triggered ROS burst in <italic>bri1-5</italic> and <italic>bri1-5</italic>/BZR1Δ plants. In all cases, bars represent SE of n = 28 rosette leaf discs. Asterisks indicate a statistically significant difference compared to the corresponding control (mock treatment [<bold>A</bold> and <bold>B</bold>], Col-0 [<bold>C</bold> and <bold>D</bold>] or <italic>bri1-5</italic> [<bold>E</bold>]), according to a Student’s <italic>t</italic>-test (p<0.05). Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 and elf18 were used at a concentration of 50 nM; chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment. All experiments were repeated at least three times with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.003">http://dx.doi.org/10.7554/eLife.00983.003</ext-link></p></caption><graphic xlink:href="elife00983f001"/></fig><fig id="fig1s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.004</object-id><label>Figure 1—figure supplement 1.</label><caption><title>The BR-mediated suppression of PTI can be triggered independently of a competition for BAK1.</title><p>(<bold>A</bold>) Co-IP of BAK1 and FLS2 in Col-0, <italic>35S:BRI1-cit</italic>, <italic>35S:BRI1</italic><sup><italic>sud</italic></sup><italic>-cit</italic> and <italic>35S:DWF4</italic> seedlings treated with 1 μM flg22 for 10 min. Coimmunoprecipitated proteins were analyzed by using anti-FLS2 or anti-BAK1 antibodies. (<bold>B</bold>) Chitin-triggered ROS burst in Col-0, <italic>35S:BRI1-cit</italic> and <italic>35S:DWF4</italic> plants. Chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28 rosette leaf discs. (<bold>C</bold>) Root length of seven-day-old Col-0, <italic>BAK1p:BAK1-HA</italic> (in Col-0 WT background) or <italic>bak1-3</italic> seedlings grown on medium supplemented or not with 10 nM BL. Bars represent SE of 12 ≤ n ≤ 17. Asterisks indicate a statistically significant difference between treatments according to a Student's <italic>t</italic>-test (p<0.05). (<bold>D</bold>) Flg22-triggered ROS burst in Col-0, <italic>BAK1p:BAK1-HA</italic> (in Col-0 WT background) or <italic>bak1-3</italic> plants. Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28 rosette leaf discs. All experiments were repeated at least twice with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.004">http://dx.doi.org/10.7554/eLife.00983.004</ext-link></p></caption><graphic xlink:href="elife00983fs001"/></fig><fig id="fig1s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.005</object-id><label>Figure 1—figure supplement 2.</label><caption><title>PAMP-triggered MAPK activation is not impaired upon activation of BR signaling.</title><p>(<bold>A</bold>) MAPK activation in Col-0 seedlings upon treatment with 1 μM flg22 (<bold>F</bold>) and/or epiBL (<bold>B</bold>) for 10 min (with or without a 90-min or 5-hr BL pre-treatment). (<bold>B</bold>) Quantification of total MAPK activation in the experiment shown in (<bold>A</bold>), measured as pixel intensity using ImageJ. Results are the average of two independent blots, corresponding to two independent biological replicated. (<bold>C</bold>) MAPK activation in Col-0 and Triple GSK3 mutant seedlings upon treatment with 1 μM flg22. (<bold>D</bold>) Quantification of total MAPK activation in the experiment shown in (<bold>C</bold>), measured as pixel intensity using ImageJ. Results are the average of two independent blots, corresponding to two independent biological replicated. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. Bars represent SD.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.005">http://dx.doi.org/10.7554/eLife.00983.005</ext-link></p></caption><graphic xlink:href="elife00983fs002"/></fig><fig id="fig1s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.006</object-id><label>Figure 1—figure supplement 3.</label><caption><title>Activation of BZR1, but not BES1, is sufficient to inhibit the PAMP-triggered ROS burst.</title><p>(<bold>A</bold>) Flg22- or chitin-triggered ROS burst in <italic>BZR1</italic><sup><italic>S173A</italic></sup> plants. (<bold>B</bold>) Flg22-triggered ROS burst in <italic>BES1</italic><sup><italic>S171A</italic></sup> plants. (<bold>C</bold>) Flg22-triggered ROS burst in mock- or bikinin-treated Col-0 or <italic>bri1-5</italic> plants. Leaf discs were pre-treated with a 50 μM bikinin solution for 16 hr. (<bold>D</bold>) Flg22-triggered ROS burst in mock- or BRZ-treated Col-0 or <italic>BZR1Δ</italic> plants. Leaf discs were pre-treated with a 2.5 μM BRZ solution for 16 hr. In all cases, bars represent SE of 21 ≤ n ≤ 28. Asterisks indicate a statistically significant difference compared to Col-0 (<bold>A</bold> and <bold>B</bold>) or mock-treatment (<bold>C</bold> and <bold>D</bold>) according to a Student's <italic>t</italic>-test (p<0.05). Flg22 was used at a concentration of 50 nM; chitin was used at a concentration of 1 mg/ml. Total photon counts were integrated between minutes two and 40 after PAMP treatment.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.006">http://dx.doi.org/10.7554/eLife.00983.006</ext-link></p></caption><graphic xlink:href="elife00983fs003"/></fig></fig-group></p><p>Transgenic expression of two different constitutively active versions of BZR1, BZR1Δ (<xref ref-type="bibr" rid="bib13">Gampala et al., 2007</xref>) and BZR1<sup>S173A</sup> (<xref ref-type="bibr" rid="bib37">Ryu et al., 2007</xref>), results in impaired flg22- or chitin-triggered ROS burst (<xref ref-type="fig" rid="fig1">Figure 1D</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3A</xref>). Consistent with previous results (<xref ref-type="bibr" rid="bib1">Albrecht et al., 2012</xref>; <xref ref-type="fig" rid="fig1s1 fig1s2">Figure 1—figure supplements 1A and 2</xref>), plants expressing <italic>BZR1Δ</italic> or <italic>BZR1</italic><sup><italic>S173A</italic></sup> display normal FLS2-BAK1 complex formation and MAPK activation upon flg22 treatment (<xref ref-type="fig" rid="fig2">Figure 2A,B</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1A,B</xref>), but are impaired in PAMP-triggered marker gene expression, seedling growth inhibition (SGI) (<xref ref-type="fig" rid="fig2">Figure 2C–E</xref>) and induced resistance to <italic>P. syringae</italic> pv. <italic>tomato</italic> (<italic>Pto</italic>) DC3000 (<xref ref-type="fig" rid="fig2">Figure 2F</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1C</xref>), and are more susceptible to the non-host strain <italic>Pseudomonas syringae</italic> pv. <italic>cilantro</italic> (<italic>Pci</italic>) 0788-9 (<xref ref-type="bibr" rid="bib21">Lewis et al., 2010</xref>) (<xref ref-type="fig" rid="fig2">Figure 2G</xref>). Notably, transgenic expression of a constitutively active form of BES1, BES1<sup>S171A</sup> (<xref ref-type="bibr" rid="bib13">Gampala et al., 2007</xref>), does not impact the flg22-triggered ROS burst (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3B</xref>). We then tested if activation of BZR1 is sufficient to inhibit PTI signaling. Induction of BR signaling by bikinin treatment still represses elf18-induced ROS burst in the BRI1 mutant <italic>bri1-5</italic> (we used elf18 in this experiment because <italic>bri1-5</italic> is in the Ws-2 background, which is a natural <italic>fls2</italic> mutant) (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3C</xref>). <italic>bri1-5/BZR1Δ</italic> plants (<xref ref-type="bibr" rid="bib13">Gampala et al., 2007</xref>) still exhibited reduced PAMP-triggered ROS burst (<xref ref-type="fig" rid="fig1">Figure 1E</xref>), and treatment with the BR biosynthetic inhibitor brassinazole (BRZ) did not affect the <italic>BZR1Δ</italic> effect (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3D</xref>). Interestingly, BRZ treatment of wild-type Col-0 plants results in increased ROS production (<xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3D</xref>), which is consistent with the fact that BR inhibits PTI responses and suggests that endogenous concentrations of the hormone exert this effect. These results demonstrate that activation of BZR1 affects PTI signaling independently of BR perception or synthesis.<fig-group><fig id="fig2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.007</object-id><label>Figure 2.</label><caption><title>Activation of BZR1 results in the suppression of specific PTI outputs.</title><p>(<bold>A</bold>) Co-immunoprecipitation (Co-IP) of BAK1 and FLS2 in Col-0 and <italic>BZR1Δ</italic> seedlings after 10 min mock (−) or 1 μM flg22 (+) treatment. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-FLS2 or anti-BAK1 antibodies. (<bold>B</bold>) MAPK activation in Col-0 and <italic>BZR1Δ</italic> seedlings upon 1 μM flg22 treatment. Proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. CBB: Coomassie brilliant blue. (<bold>C</bold>) Marker gene (<italic>At2g17700</italic> and <italic>NHL10</italic>) expression in Col-0 and <italic>BZR1Δ</italic> seedlings after 1 hr mock (−) or 1 μM flg22 (+) treatment, as determined by qPCR. Bars represent SE of n = 3. (<bold>D</bold>) and (<bold>E</bold>) Seedling growth inhibition of 10-day-old Col-0 or <italic>BZR1Δ</italic> seedlings induced by increasing concentrations of flg22, as indicated. Scale bar (<bold>D</bold>), 1 cm. Bars (<bold>E</bold>) represent SE of 8 ≤ n ≤ 16. (<bold>F</bold>) Flg22-induced resistance to <italic>P. syringae</italic> pv. <italic>tomato</italic> DC3000 in Col-0 and <italic>BZR1Δ</italic> plants. Plants were pre-treated with 1 μM flg22 or water 24 hr prior to bacterial infiltration. Bars represent SE of n = 4. This experiment was repeated seven times with similar results. (<bold>G</bold>) Susceptibility of Col-0 and <italic>BZR1Δ</italic> plants to <italic>P. syringae</italic> pv. <italic>cilantro</italic> 0788-9. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s <italic>t</italic>-test (p<0.05); ns = not significant. All experiments were repeated at least twice with similar results unless otherwise stated.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.007">http://dx.doi.org/10.7554/eLife.00983.007</ext-link></p></caption><graphic xlink:href="elife00983f002"/></fig><fig id="fig2s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.008</object-id><label>Figure 2—figure supplement 1.</label><caption><title>Expression of the constitutively active BZR1<sup>S173A</sup> results in the suppression of specific PTI outputs.</title><p>(<bold>A</bold>) Co-IP of BAK1 and FLS2 in Col-0 and <italic>BZR1</italic><sup><italic>S173A</italic></sup> seedlings treated with 1 μM flg22 for 10 min. Co-immunoprecipitated proteins were analyzed by using anti-FLS2 or anti-BAK1 antibodies. (<bold>B</bold>) MAPK activation in Col-0 and <italic>BZR1</italic><sup><italic>S173A</italic></sup> seedlings upon treatment with 1 μM flg22. Membranes were blotted with phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies. (<bold>C</bold>) Flg22-induced resistance to <italic>Pto</italic> DC3000 in <italic>BZR1</italic><sup><italic>S173A</italic></sup> plants. Plants were pre-treated with 1 μM flg22 or water 24 hr prior to bacterial inoculation. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to mock-treated plants according to a Student's <italic>t</italic>-test (p<0.05); ns = not significant. All experiments were repeated at least twice with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.008">http://dx.doi.org/10.7554/eLife.00983.008</ext-link></p></caption><graphic xlink:href="elife00983fs004"/></fig></fig-group></p><p>To understand how BZR1 mediates the BR-PTI crosstalk, we performed meta-analysis of microarray and ChIP-chip data containing BR-regulated and BZR1 or BES1 target genes (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>; <xref ref-type="bibr" rid="bib53">Yu et al., 2011</xref>). Functional enrichment of BR-regulated genes unveiled a statistically significant over-representation of defense-related GO terms of the Biological Process ontology (<xref ref-type="table" rid="tbl1">Table 1</xref>), indicating that BR signaling regulates the expression of defense-related genes. Independent analysis of BR-regulated BZR1 or BES1 targets confirmed BZR1 as the main transcription factor involved in the regulation of defense gene expression (<xref ref-type="table" rid="tbl1">Table 1</xref>). Two out of four over-represented GO terms of the Molecular Function ontology among the BR-regulated BZR1 targets are transcription factor and transcription repressor activity (<xref ref-type="table" rid="tbl2">Table 2</xref>). Interestingly, several defense-related GO terms are also over-represented in the subset of BR-regulated BZR1-targeted transcription factors (<xref ref-type="table" rid="tbl3">Table 3</xref>), pointing at a BZR1-mediated secondary transcriptional wave of defense-related genes.<table-wrap id="tbl1" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.009</object-id><label>Table 1.</label><caption><p>Defense-related Gene Ontology terms (Biological Process ontology) over-represented among all BR-regulated genes, BR-regulated BZR1 targets and BR-regulated BES1 targets</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.009">http://dx.doi.org/10.7554/eLife.00983.009</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Defense-related GO term</th><th>Observed frequency (%)</th><th>Expected frequency (%)</th><th>p-value</th></tr></thead><tbody><tr><td colspan="4">BR-Regulated genes</td></tr><tr><td> response to bacterium</td><td align="char" char=".">2.2</td><td align="char" char=".">1</td><td>3.31 × 10<sup>−08</sup></td></tr><tr><td> defense response to bacterium</td><td align="char" char=".">1.9</td><td align="char" char=".">0.8</td><td>3.31 × 10<sup>−08</sup></td></tr><tr><td> response to chitin</td><td align="char" char=".">1.4</td><td align="char" char=".">0.5</td><td>1.78 × 10<sup>−07</sup></td></tr><tr><td> defense response</td><td align="char" char=".">4.7</td><td align="char" char=".">3</td><td>3.32 × 10<sup>−07</sup></td></tr><tr><td> response to fungus</td><td align="char" char=".">1.5</td><td align="char" char=".">0.7</td><td>3.4 × 10<sup>−06</sup></td></tr><tr><td> response to nematode</td><td align="char" char=".">0.7</td><td align="char" char=".">0.2</td><td>0.000532</td></tr><tr><td> defense response to fungus</td><td align="char" char=".">1</td><td align="char" char=".">0.5</td><td>0.0035</td></tr><tr><td colspan="4">BR-regulated BZR1 targets</td></tr><tr><td> response to chitin</td><td align="char" char=".">2.6</td><td align="char" char=".">0.5</td><td>9.13 × 10<sup>−13</sup></td></tr><tr><td> response to bacterium</td><td align="char" char=".">2.3</td><td align="char" char=".">1</td><td>0.00112</td></tr><tr><td> defense response to bacterium</td><td align="char" char=".">1.9</td><td align="char" char=".">0.8</td><td>0.00154</td></tr><tr><td> response to fungus</td><td align="char" char=".">1.6</td><td align="char" char=".">0.7</td><td>0.00495</td></tr><tr><td colspan="4">BR-regulated BES1 targets</td></tr><tr><td> response to chitin</td><td align="char" char=".">2.4</td><td align="char" char=".">0.5</td><td>0.00439</td></tr></tbody></table></table-wrap><table-wrap id="tbl2" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.010</object-id><label>Table 2.</label><caption><p>Gene Ontology terms (Molecular Function ontology) over-represented among all BR-regulated BZR1 targets</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.010">http://dx.doi.org/10.7554/eLife.00983.010</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Over-represented GO term</th><th>Observed frequency (%)</th><th>Expected frequency (%)</th><th>p value</th></tr></thead><tbody><tr><td colspan="4">BR-regulated BZR1 targets</td></tr><tr><td> nucleic acid binding transcription factor activity</td><td align="char" char=".">14.8</td><td align="char" char=".">10.2</td><td align="char" char=".">0.000223</td></tr><tr><td> transferase activity</td><td align="char" char=".">21.6</td><td align="char" char=".">16.8</td><td align="char" char=".">0.00333</td></tr><tr><td> kinase activity</td><td align="char" char=".">11.6</td><td align="char" char=".">8.1</td><td align="char" char=".">0.00702</td></tr><tr><td> transcription repressor activity</td><td align="char" char=".">1.1</td><td align="char" char=".">0.3</td><td align="char" char=".">0.01</td></tr></tbody></table></table-wrap><table-wrap id="tbl3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.011</object-id><label>Table 3.</label><caption><p>Defense-related Gene Ontology terms (Biological Process ontology) over-represented among the BZR1-target BR-regulated transcription factors</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.011">http://dx.doi.org/10.7554/eLife.00983.011</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Defense-related GO Term</th><th>Observed frequency (%)</th><th>Expected frequency (%)</th><th>p value</th></tr></thead><tbody><tr><td colspan="4">BZR1-target BR-regulated TFs</td></tr><tr><td> response to chitin</td><td align="char" char=".">16.6</td><td align="char" char=".">0.5</td><td>1.36 × 10<sup>−26</sup></td></tr><tr><td> defense response to bacterium</td><td align="char" char=".">7.6</td><td align="char" char=".">0.8</td><td>4.71 × 10<sup>−07</sup></td></tr><tr><td> response to bacterium</td><td align="char" char=".">7.6</td><td align="char" char=".">1</td><td>4.51 × 10<sup>−06</sup></td></tr><tr><td> regulation of defense response to virus by host</td><td align="char" char=".">1.4</td><td align="char" char=".">0</td><td>0.000964</td></tr><tr><td> regulation of immune effector process</td><td align="char" char=".">1.4</td><td align="char" char=".">0</td><td>0.00151</td></tr><tr><td> regulation of defense response to virus</td><td align="char" char=".">1.4</td><td align="char" char=".">0</td><td>0.00151</td></tr><tr><td> regulation of defense response</td><td align="char" char=".">2.8</td><td align="char" char=".">0.3</td><td>0.00484</td></tr><tr><td> defense response</td><td align="char" char=".">8.3</td><td align="char" char=".">3</td><td>0.00603</td></tr><tr><td> response to fungus</td><td align="char" char=".">3.4</td><td align="char" char=".">0.7</td><td>0.01</td></tr><tr><td> defense response to fungus</td><td align="char" char=".">2.8</td><td align="char" char=".">0.5</td><td>0.02</td></tr></tbody></table></table-wrap></p><p>To identify BZR1-regulated transcription factors with a prominent role in defense, we performed promoter enrichment analysis on the subset of defense-related BR-regulated genes, and found the W-box motif as the only significantly over-represented motif (<xref ref-type="table" rid="tbl4">Table 4</xref>). The W-box motif is the binding site for the WRKY family of transcription factors (<xref ref-type="bibr" rid="bib36">Rushton et al., 2010</xref>), and several members of this family are BR-regulated BZR1-targets (<xref ref-type="table" rid="tbl5">Table 5</xref>). We hypothesized that WRKYs that are BR-induced and BZR1 targets may be involved in PTI signaling. Notably, <italic>wrky11</italic>, <italic>wrky15</italic>, <italic>wrky18</italic> and <italic>wrky70</italic> mutants displayed enhanced PAMP-triggered ROS (<xref ref-type="fig" rid="fig3">Figure 3A</xref>), suggesting that these transcription factors act as negative regulators of early PTI signaling. This is in accordance with their role as negative regulators of immunity (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1A</xref>; <xref ref-type="bibr" rid="bib16">Journot-Catalino et al., 2006</xref>). Therefore, the BZR1-mediated inhibition of PTI might be partially explained by the up-regulation of genes encoding WRKY transcription factors that negatively control the expression of genes involved in early PTI signaling.<table-wrap id="tbl4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.012</object-id><label>Table 4.</label><caption><p>Over-represented <italic>cis</italic>-acting promoter elements among the defense-related BR-regulated genes according to Athena (<ext-link ext-link-type="uri" xlink:href="http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl">http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl</ext-link>)</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.012">http://dx.doi.org/10.7554/eLife.00983.012</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Enriched TF site</th><th>% promoters</th><th>p value</th></tr></thead><tbody><tr><td colspan="3">Defense-related BR-regulated genes</td></tr><tr><td> W-box</td><td>72.4</td><td><10<sup>−6</sup></td></tr></tbody></table></table-wrap><table-wrap id="tbl5" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.013</object-id><label>Table 5.</label><caption><p>BR-regulated BZR1-target <italic>WRKY</italic> genes</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.013">http://dx.doi.org/10.7554/eLife.00983.013</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>AGI number</th><th>WRKY TF</th></tr></thead><tbody><tr><td colspan="2">BR-Induced BZR1 targets</td></tr><tr><td> AT4G31800</td><td>WRKY18</td></tr><tr><td> AT4G31550</td><td>WRKY11</td></tr><tr><td> AT4G23810</td><td>WRKY53</td></tr><tr><td> AT3G56400</td><td>WRKY70</td></tr><tr><td> AT5G49520</td><td>WRKY48</td></tr><tr><td> AT5G52830</td><td>WRKY27</td></tr><tr><td> AT1G69310</td><td>WRKY57</td></tr><tr><td> AT2G23320</td><td>WRKY15 (<xref ref-type="bibr" rid="bib53">Yu et al., 2011</xref>)</td></tr><tr><td colspan="2">BR-repressed BZR1 targets</td></tr><tr><td> AT4G01250</td><td>WRKY22</td></tr><tr><td> AT1G80840</td><td>WRKY40</td></tr><tr><td> AT2G24570</td><td>WRKY17</td></tr><tr><td> AT2G23320</td><td>WRKY15 (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>)</td></tr><tr><td> AT2G30590</td><td>WRKY21</td></tr></tbody></table></table-wrap><fig-group><fig id="fig3" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.014</object-id><label>Figure 3.</label><caption><title>WRKY transcription factors play a dual role on the BR-mediated regulation of PTI signaling.</title><p>(<bold>A</bold>) Flg22-triggered ROS burst in mutants in each BR-induced BZR1-targeted <italic>WRKY</italic>. Leaf discs of four- to five-week-old Arabidopsis plants were used in these assays. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 28. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s <italic>t</italic>-test (p<0.05). (<bold>B</bold>) Flg22-triggered ROS burst in epiBL (BL)- or mock- pre-treated <italic>wrky40</italic> mutant or wild-type plants. Leaf discs of four- to five-week-old plants were pre-treated with a 1 μM BL solution or mock solution for 8 hr. Flg22 was used at a concentration of 50 nM. Total photon counts were integrated between minutes two and 40 after PAMP treatment. Bars represent SE of n = 21. Asterisks indicate a statistically significant difference compared to Col-0 according to a Student’s <italic>t</italic>-test (p<0.05). (<bold>C</bold>) Co-IP of BZR1-GFP transiently expressed in <italic>N. benthamiana</italic>, alone or together with WRKY40-HA or WRKY6-HA. BZR1-GFP was immunoprecipitated with an anti-GFP antibody. Immuniprecipitated or total proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-HA or anti-GFP antibodies. CBB: Coomassie brilliant blue. (<bold>D</bold>) Co-IP of BZR1-GFP transiently expressed in Arabidopsis protoplasts, alone or together with WRKY40-HA. BZR1-GFP was immunoprecipitated with an anti-GFP antibody. Immuniprecipitated or total proteins were separated in a 10% acrylamide gel and transferred to PVDF membranes. Membranes were blotted with anti-HA or anti-GFP antibodies. CBB: Coomassie brilliant blue. All experiments were repeated at least twice with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.014">http://dx.doi.org/10.7554/eLife.00983.014</ext-link></p></caption><graphic xlink:href="elife00983f003"/></fig><fig id="fig3s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.015</object-id><label>Figure 3—figure supplement 1.</label><caption><title>Mutants in <italic>WRKY11</italic>, <italic>WRKY15</italic>, <italic>WRKY18</italic> and <italic>WRKY40</italic> are more resistant to <italic>Pto</italic> DC3000.</title><p>(<bold>A</bold>) and (<bold>B</bold>) <italic>Pto</italic> DC3000 infections in Col-0, <italic>wrky11</italic>, <italic>wrky15</italic>, and <italic>wrky18</italic> (<bold>A</bold>) and in Col-0 and <italic>wrky40</italic> (<bold>B</bold>) plants. Bars represent SE of n = 4. Asterisks indicate a statistically significant difference compared to Col-0 plants according to a Student's <italic>t</italic>-test (p<0.05); ns = not significant. All experiments were repeated at least three times with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.015">http://dx.doi.org/10.7554/eLife.00983.015</ext-link></p></caption><graphic xlink:href="elife00983fs005"/></fig></fig-group></p><p>One of the <italic>WRKY</italic> genes targeted by BZR1 is <italic>WRKY40</italic> (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>). Interestingly, all described targets of WRKY40 (<xref ref-type="bibr" rid="bib32">Pandey et al., 2010</xref>) are also targets of BZR1 (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>) (<xref ref-type="table" rid="tbl6">Table 6</xref>). The over-representation of the W-box motif among BZR1 targets (<xref ref-type="table" rid="tbl7">Table 7</xref>) suggests that BZR1 may interact with WRKY transcription factors (such as WRKY40) to cooperatively regulate transcription. <italic>WRKY40</italic> has been described as a negative regulator of defense against biotrophic pathogens and insects (<xref ref-type="bibr" rid="bib50">Xu et al., 2006</xref>; <xref ref-type="bibr" rid="bib32">Pandey et al., 2010</xref>; <xref ref-type="bibr" rid="bib7">Brotman et al., 2013</xref>; <xref ref-type="bibr" rid="bib38">Schon et al., 2013</xref>; <xref ref-type="bibr" rid="bib39">Schweizer et al., 2013</xref>). In agreement with this, we found that a null <italic>wrky40</italic> mutant is more resistant to <italic>Pto</italic> DC3000 (<xref ref-type="fig" rid="fig3s1">Figure 3—figure supplement 1B</xref>). Strikingly, <italic>wrky40</italic> plants are partially impaired in the BR-mediated suppression of PAMP-triggered ROS (<xref ref-type="fig" rid="fig3">Figure 3B</xref>), suggesting that WRKY40 may act coordinately with BZR1 to suppress immunity. Indeed, we found that BZR1 associates with WRKY40, but not WRKY6, in co-immunoprecipitation experiments when transiently co-expressed in <italic>Nicotiana benthamiana</italic> leaves (<xref ref-type="fig" rid="fig3">Figure 3C</xref>) or Arabidopsis protoplasts (<xref ref-type="fig" rid="fig3">Figure 3D</xref>). Collectively, these results indicate that BZR1 and WRKY40 form a protein complex that may participate in the transcriptional inhibition of PTI signaling.<table-wrap id="tbl6" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.016</object-id><label>Table 6.</label><caption><p>Overlap between the targets of WRKY40 and BZR1</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.016">http://dx.doi.org/10.7554/eLife.00983.016</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>Known targets of WRKY40 (<xref ref-type="bibr" rid="bib32">Pandey et al., 2010</xref>)</th><th>Targets of BZR1 (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>)</th></tr></thead><tbody><tr><td>Confirmed by ChIP</td><td/></tr><tr><td> <italic>EDS1</italic></td><td>Yes</td></tr><tr><td> <italic>RRTF1</italic></td><td>Yes</td></tr><tr><td> <italic>JAZ8</italic></td><td>Yes</td></tr><tr><td>Putative (according to expression analyses)</td><td/></tr><tr><td> <italic>LOX2</italic></td><td>Yes</td></tr><tr><td> <italic>AOS</italic></td><td>Yes</td></tr><tr><td> <italic>JAZ7</italic></td><td>Yes</td></tr><tr><td> <italic>JAZ10</italic></td><td>Yes</td></tr></tbody></table></table-wrap><table-wrap id="tbl7" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.017</object-id><label>Table 7.</label><caption><p>Representation of the W-box motif among the BR-regulated BZR1 targets according to Athena (<ext-link ext-link-type="uri" xlink:href="http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl">http://www.bioinformatics2.wsu.edu/cgi-bin/Athena/cgi/home.pl</ext-link>)</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.017">http://dx.doi.org/10.7554/eLife.00983.017</ext-link></p></caption><table frame="hsides" rules="groups"><thead><tr><th>BZR1 targets</th><th>% of promoters with W-box motif(s)</th><th>p value</th></tr></thead><tbody><tr><td>BR-induced</td><td>66</td><td><10<sup>−10</sup></td></tr><tr><td>BR-repressed</td><td>72</td><td><10<sup>−4</sup></td></tr></tbody></table></table-wrap></p><p>BZR1, together with DELLAs and PIF4, is part of a core transcription module that integrates hormonal (gibberellin [GA] and BR) and environmental (light) signals (<xref ref-type="bibr" rid="bib12">Gallego-Bartolome et al., 2012</xref>; <xref ref-type="bibr" rid="bib22">Li et al., 2012</xref>; <xref ref-type="bibr" rid="bib30">Oh et al., 2012</xref>; <xref ref-type="bibr" rid="bib4">Bai et al., 2012b</xref>). In the dark, BZR1 is activated by endogenous BR and GA to promote growth, partially through the synergistic interaction with PIF4 (<xref ref-type="bibr" rid="bib15">Jaillais and Vert, 2012</xref>). Because etiolation requires rapid growth, we hypothesized that plants may prioritize growth over immunity in dark conditions. In fact, we found that PAMP-triggered SGI was partially impaired in dark-grown seedlings (<xref ref-type="fig" rid="fig4">Figure 4A–D</xref>). This impairment was abolished in the BR-insensitive mutants <italic>bri1-301</italic> and <italic>bin2-1</italic> (<xref ref-type="fig" rid="fig4">Figure 4A</xref>, <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2A</xref>), indicating that BR signaling is responsible for the dark-induced suppression of this PTI response. Activation of BZR1 in the <italic>BZR1Δ</italic> line mimicked the dark-induced suppression of SGI in both light and dark (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). However, activation of BES1 in the <italic>BES1</italic><sup><italic>S171A</italic></sup> line did not impact SGI (<xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3A</xref>). Consistent with the previous results, exogenous BR treatment suppressed SGI in both light and dark (<xref ref-type="fig" rid="fig4">Figure 4C</xref>, <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2B,C</xref>). While treatment with GA alone did not have a dramatic effect on SGI, co-treatment with BL and GA resulted in an enhancement of the BR-mediated suppression of SGI (<xref ref-type="fig" rid="fig4">Figure 4C</xref>, <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2B</xref>), indicating an additive effect of these two hormones when applied together. Moreover, treatment with the GA synthesis inhibitors paclobutrazol (PAC) or uniconazole (Uni) abolished the effect of BL on SGI (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A,B</xref> and <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2B,C</xref>), and this effect was reduced in the GA biosynthetic mutant <italic>ga1-3</italic> (<xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1C</xref>). Taken together, these results demonstrate that BR suppress at least one PTI output, SGI, in the dark in a GA-dependent manner, most likely through activation of BZR1. Notably, although the <italic>wrky40</italic> mutant undergoes etiolation normally (<xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2D</xref>), it shows a diminished suppression of SGI in the dark (<xref ref-type="fig" rid="fig4">Figure 4D</xref>, <xref ref-type="fig" rid="fig4s2">Figure 4—figure supplement 2D</xref>), supporting the idea that WRKY40 is required for the BZR1-mediated inhibition of PTI.<fig-group><fig id="fig4" position="float"><object-id pub-id-type="doi">10.7554/eLife.00983.018</object-id><label>Figure 4.</label><caption><title>Activation of BR signaling and BZR1 prioritizes growth over immunity in the dark.</title><p>(<bold>A</bold>) and (<bold>B</bold>) Relative seedling growth inhibition of 10-day-old (<bold>A</bold>) Col-0, <italic>bri1-301</italic> and <italic>bin2-1</italic> or (<bold>B</bold>) Col-0 and <italic>BZR1Δ</italic> seedlings induced by increasing concentrations of flg22 in either light or dark. (<bold>C</bold>) Relative seedling growth inhibition of 10-day-old Col-0 seedlings grown on medium supplemented or not with BL (1 μM), GA (1 μM), BL+GA (1 μM + 1 μM) or mock solution in light or dark. (<bold>D</bold>) Relative seedling growth inhibition of Col-0 or <italic>wrky40</italic> seedlings induced by increasing concentrations of flg22 in either light or dark. Bars represent SE of n = 16 (<bold>A</bold>, <bold>B</bold> and <bold>D</bold>) or n = 8 (<bold>C</bold>) Asterisks indicate a statistically significant difference compared to Col-0 in the same condition (light or dark and same concentration of flg22), according to a Student’s <italic>t</italic>-test (p<0.05); ‘a’ indicates a statistically significant difference compared to the same genotype/treatment and concentration of flg22 in light, according to a Student’s <italic>t</italic>-test (p<0.05). All experiments were repeated at least three times with similar results. Values are relative to Col-0 (<bold>A</bold>, <bold>B</bold> and <bold>D</bold>) or mock-treated seedlings (<bold>C</bold>) (set to 100). Absolute values of these experiments are shown in <xref ref-type="fig" rid="fig4s3">Figure 4—figure supplement 3</xref>. (<bold>E</bold>) Schematic model depicting the BZR1-mediated inhibition of PTI. Upon BR- and DELLA-dependent activation, BZR1 induces the expression of negative regulators of PTI, such as <italic>WRKY11</italic>, <italic>WRKY15</italic>, <italic>WRKY18,</italic> or <italic>HBI1</italic>. In addition, BZR1 also inhibits the expression of immune genes, acting cooperatively with WRKY40 and possibly other WRKYs. Ultimately, the BZR1-mediated changes in transcription would lead to the suppression of PTI signaling. The PTI signaling pathway is shadowed in violet; the BR signaling pathway is shadowed in green.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.018">http://dx.doi.org/10.7554/eLife.00983.018</ext-link></p></caption><graphic xlink:href="elife00983f004"/></fig><fig id="fig4s1" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.019</object-id><label>Figure 4—figure supplement 1.</label><caption><title>The BR-mediated suppression of seedling growth inhibition in the dark requires GA synthesis.</title><p>Seedling growth inhibition of 10-day-old Col-0 seedlings grown on medium supplemented or not with (<bold>A</bold>) BL (1 μM), paclobutrazol (PAC; 1 μM), BL+PAC (1 μM + 1 μM) and PAC+GA (1 μM + 1 μM), or (<bold>B</bold>) uniconazole (Uni; 100 μM), BL (1 μM) and Uni+BL (100 μM + 1 μM) induced by increasing concentrations of flg22 in light or dark. (<bold>C</bold>) Seedling growth inhibition of 10-day-old Ler and <italic>ga1-3</italic> seedlings grown on medium supplemented or not with BL (1 μM). Bars represent SE of n = 8. Asterisks indicate a statistically significant difference compared to Col-0 in the same condition (light or dark and same concentration of flg22), according to a Student's <italic>t</italic>-test (p<0.05); ‘a’ indicates a statistically significant difference compared to the same genotype/treatment and concentration of flg22 in light, according to a Student's <italic>t</italic>-test (p<0.05). Values are relative to mock-treated seedlings (set to 100). All experiments were repeated at least twice with similar results.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.019">http://dx.doi.org/10.7554/eLife.00983.019</ext-link></p></caption><graphic xlink:href="elife00983fs006"/></fig><fig id="fig4s2" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.020</object-id><label>Figure 4—figure supplement 2.</label><caption><title>Phenotype of the light- or dark-grown seedlings used in the seedling growth inhibition assays (<xref ref-type="fig" rid="fig4">Figure 4</xref> and <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>).</title><p>Representative seedlings of the seedling growth inhibition experiments depicted in: (<bold>A</bold>) <xref ref-type="fig" rid="fig4">Figure 4A</xref>; (<bold>B</bold>) <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>; (<bold>C</bold>) <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1B</xref>; (<bold>D</bold>) <xref ref-type="fig" rid="fig4">Figure 4D</xref>. Scale bar, 1 cm.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.020">http://dx.doi.org/10.7554/eLife.00983.020</ext-link></p></caption><graphic xlink:href="elife00983fs007"/></fig><fig id="fig4s3" position="float" specific-use="child-fig"><object-id pub-id-type="doi">10.7554/eLife.00983.021</object-id><label>Figure 4—figure supplement 3.</label><caption><title>Absolute fresh weight values of seedling growth inhibition assays.</title><p>(<bold>A</bold>) Seedling growth inhibition of 10-day-old Col-0 or <italic>BES1</italic><sup><italic>S171A</italic></sup> seedlings induced by increasing concentrations of flg22. (<bold>B</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4">Figure 4B</xref>, dark. (<bold>C</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4">Figure 4A</xref>. (<bold>D</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4">Figure 4C</xref>. (<bold>E</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1A</xref>. (<bold>F</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1B</xref>. (<bold>G</bold>) Absolute fresh weight values of the seedling growth inhibition assay depicted in <xref ref-type="fig" rid="fig4">Figure 4D</xref>. Error bars represent SE as indicated in <xref ref-type="fig" rid="fig4">Figure 4</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>.</p><p><bold>DOI:</bold> <ext-link ext-link-type="doi" xlink:href="10.7554/eLife.00983.021">http://dx.doi.org/10.7554/eLife.00983.021</ext-link></p></caption><graphic xlink:href="elife00983fs008"/></fig></fig-group></p><p>Previously, a unidirectional negative crosstalk between the growth-promoting hormone BR and PTI had been described (<xref ref-type="bibr" rid="bib1">Albrecht et al., 2012</xref>; <xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>). In this work, we show that activation of one of two major BR-activated transcription factors, BZR1, is sufficient to suppress PTI, measured as PAMP-triggered ROS production, PAMP-triggered gene expression, SGI or induced resistance (<xref ref-type="fig" rid="fig1 fig2">Figures 1 and 2</xref>, <xref ref-type="fig" rid="fig1s3">Figure 1—figure supplement 3</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1</xref>). Of note, another PTI output, MAPK activation, is not affected by activation of the BR pathway (<xref ref-type="fig" rid="fig2">Figure 2B</xref>, <xref ref-type="fig" rid="fig1s2">Figure 1—figure supplement 2</xref>, <xref ref-type="fig" rid="fig2s1">Figure 2—figure supplement 1B</xref>). BR treatment results in BZR1-dependent changes in the expression of defense-related genes, among which several members of the WRKY family of transcription factors can be found. Because the promoters of BR-regulated defense-related genes are enriched in the W-box motif (<xref ref-type="table" rid="tbl4">Table 4</xref>), BZR1-targeted <italic>WRKY</italic> transcription factors could be responsible for a secondary wave of transcription, ultimately leading to the suppression of PTI. In agreement with this idea, a subset of <italic>WRKY</italic>s induced by BR (<italic>WRKY11</italic>, <italic>WRKY15</italic> and <italic>WRKY18</italic>) (<xref ref-type="fig" rid="fig3">Figure 3A</xref>) act as negative regulators of PAMP-triggered ROS, potentially by controlling the steady-state expression of genes encoding components required for this response. The over-representation of the W-box motif among the BZR1 targets (<xref ref-type="table" rid="tbl7">Table 7</xref>) raises the possibility that, additionally, WRKY transcription factors could also act together with BZR1 to cooperatively regulate gene expression. We found that WRKY40 associates with BZR1 directly or indirectly in planta (<xref ref-type="fig" rid="fig3">Figure 3C,D</xref>); in the absence of WRKY40, the BR-mediated suppression of PAMP-triggered ROS burst is partially impaired (<xref ref-type="fig" rid="fig3">Figure 3B</xref>). Therefore, WRKYs may play a dual role in the BZR1-mediated suppression of defense, as both co- and secondary regulators of defense gene expression. Given that the loss of BR-mediated suppression of PAMP-triggered ROS burst in the <italic>wrky40</italic> mutant is only partial, BZR1 may interact with other members of the WRKY family, such as WRKY18 or WRKY60, to repress immunity.</p><p>Furthermore, we recently described that the bHLH transcription factor HBI1, which is a BRZ1 target (<xref ref-type="bibr" rid="bib42">Sun et al., 2010</xref>; <xref ref-type="bibr" rid="bib3">Bai et al., 2012a</xref>), negatively regulates PTI (Malinovsky et al., under revision). All together, these results illustrate that BZR1 controls the expression of transcription factors (e.g. WRKY11, WRKY15, WRKY18 and HBI1), which themselves might control the expression of PTI components (see model in <xref ref-type="fig" rid="fig4">Figure 4E</xref>) whose identities remain to be identified.</p><p>Plants need to finely regulate allocation of resources upon integration of environmental cues, both biotic and abiotic, in order to rapidly and readily adapt to changing conditions and ensure survival in a cost-efficient manner. Dark conditions impose an energetic limitation due to lack of photo-assimilates; in this situation, the restoration of normal photosynthesis by reaching light is an essential requirement to guarantee perpetuation, and as such must be given priority (<xref ref-type="bibr" rid="bib8">Casal, 2013</xref>). We hypothesize that when plants face conditions that require rapid growth, such as when germinating in soil or when under a canopy, limited resources are invested in this developmental process at the expense of immunity in what must be a quantitative choice. Indeed, we show that etiolated seedlings do not arrest their growth in response to PAMPs as light-grown seedlings do, as measured by total fresh weight (<xref ref-type="fig" rid="fig4">Figure 4A–D</xref>). In addition, BR signaling, acting cooperatively with GA signaling, is required for the dark-induced suppression of this PTI response (<xref ref-type="fig" rid="fig4">Figure 4C</xref>, <xref ref-type="fig" rid="fig4s1">Figure 4—figure supplement 1</xref>), and activation of BZR1 is sufficient to exert this effect regardless of light conditions (<xref ref-type="fig" rid="fig4">Figure 4B</xref>). Although seedlings were used in these experiments due to technical reasons, BR also regulate growth at later developmental stages, so this phenomenon may be more general. Based on these findings, we propose a model in which BZR1 regulates the expression of defense genes, assisted by WRKY40 (and potentially other WRKYs), ultimately resulting in a quantitative suppression of immunity (<xref ref-type="fig" rid="fig4">Figure 4E</xref>). Because the activation status of BZR1 depends on BR, GA and light signaling, BZR1 would act as a molecular integrator of these inputs to effectively regulate the trade-off between growth and immunity.</p></sec><sec id="s3" sec-type="materials|methods"><title>Materials and methods</title><sec id="s3-1"><title>Plant materials and growth conditions</title><p>Col-0 plants were used as control. The transgenic lines <italic>BZR1Δ</italic>, <italic>bri1-5/BZR1Δ</italic> and <italic>BES1</italic><sup><italic>S171A</italic></sup> (<xref ref-type="bibr" rid="bib13">Gampala et al., 2007</xref>), <italic>BZR1</italic><sup><italic>S173A</italic></sup> and <italic>BZR1-CFP</italic> (<xref ref-type="bibr" rid="bib37">Ryu et al., 2007</xref>), <italic>35S:BRI1-cit</italic>, <italic>BRI1p:BRI1</italic><sup><italic>sud</italic></sup><italic>-cit</italic>, <italic>35S:DWF4</italic> and <italic>BAK1-HA</italic> (<xref ref-type="bibr" rid="bib5">Belkhadir et al., 2012</xref>) are published. The mutant lines Triple GSK3 mutant (<xref ref-type="bibr" rid="bib44">Vert and Chory, 2006</xref>), <italic>bri1-5</italic> (<xref ref-type="bibr" rid="bib28">Noguchi et al., 1999</xref>), <italic>bri1-301</italic> (<xref ref-type="bibr" rid="bib49">Xu et al., 2008</xref>), <italic>bin2-1</italic> (<xref ref-type="bibr" rid="bib33">Peng et al., 2008</xref>), <italic>wrky11</italic> (<xref ref-type="bibr" rid="bib16">Journot-Catalino et al., 2006</xref>), <italic>wrky18, wrky53</italic> and <italic>wrky70</italic> (<xref ref-type="bibr" rid="bib45">Wang et al., 2006</xref>) <italic>wrky27</italic> (<xref ref-type="bibr" rid="bib24">Mukhtar et al., 2008</xref>), <italic>wrky40</italic> (<xref ref-type="bibr" rid="bib32">Pandey et al., 2010</xref>) and <italic>ga1-3</italic> (<xref ref-type="bibr" rid="bib26">Navarro et al., 2008</xref>) are published. <italic>wrky15</italic> mutant was identified in the ZIGIA population (<xref ref-type="bibr" rid="bib47">Wisman et al., 1998a</xref>, <xref ref-type="bibr" rid="bib48">1998b</xref>); <italic>wrky48</italic> and <italic>wrky57</italic> are from the SALK collection (<xref ref-type="bibr" rid="bib2">Alonso et al., 2003</xref>).</p><p>Arabidopsis plants and seedlings were grown as described in <xref ref-type="bibr" rid="bib1">Albrecht et al. (2012)</xref>.</p></sec><sec id="s3-2"><title>Chemicals</title><p>Flg22 and elf18 peptides were purchased from Peptron, and chitin oligosaccharide from Yaizu Suisankagaku. epiBL was purchased from Xiamen Topusing Chemical. LiCl, bikinin, brassinazole and GA were purchased from Sigma (St Louis, MO, USA). Paclobutrazol was purchased from Duchefa (Haarlem, NL). Uniconazole was purchased from Sigma.</p></sec><sec id="s3-3"><title>ROS assays</title><p>The measurement of ROS generation was performed as described in <xref ref-type="bibr" rid="bib1">Albrecht et al. (2012)</xref>. Leaf discs from five-week-old Arabidopsis plants were used in each experiment, as indicated in the figure legends. Total photon counts were measured over 40 min by using a high-resolution photon counting system (HRPCS218) (Photek, St Leonards on Sea, UK) coupled to an aspherical wide lens (Sigma Imaging, Welwyn Garden City, UK).</p></sec><sec id="s3-4"><title>Protein extraction and IP experiments</title><p>Protein extraction and immunoprecipitation of Arabidopsis was performed as described in <xref ref-type="bibr" rid="bib40">Schwessinger et al. (2011)</xref>. Arabidopsis mesophyll protoplasts were prepared from 4 to 5-week-old plants, transfected with the indicated constructs and incubated for 16 hr prior to BL treatment. Protein extraction of <italic>N. benthamiana</italic> was performed as described in <xref ref-type="bibr" rid="bib40">Schwessinger et al. (2011)</xref>; immunoprecipitations were performed using the μMACS GFP Isolation Kit (Miltenyi Biotec, Church Lane Bisley, UK), following the manufacturer’s instructions. In <italic>N. benthamiana</italic>, BZR1-GFP was expressed from the pUb-cYFP-Dest vector (<xref ref-type="bibr" rid="bib14">Grefen et al., 2010</xref>); WRKY40-HA and WRKY6-HA were expressed from the pAM-PAT vector (AY436765; GeneBank). In protoplasts, WRKY40-HA was expressed from the pGWB414 vector (<xref ref-type="bibr" rid="bib25">Nakagawa et al., 2007</xref>); the construct to express BZR1-GFP has been described elsewhere (<xref ref-type="bibr" rid="bib37">Ryu et al., 2007</xref>). In both cases, samples were treated with 1 μM epiBL solution for 1 hr prior to protein extraction.</p></sec><sec id="s3-5"><title>MAP kinase activation assays</title><p>MAP kinase activation assays were performed as described in <xref ref-type="bibr" rid="bib40">Schwessinger et al. (2011)</xref>. Phospho-p44/42 MAPK (Erk1/2; Thr202/Tyr204) rabbit monoclonal antibodies (Cell Signaling Technologies, Hitchin, UK) were used according to the manufacturer’s protocol.</p></sec><sec id="s3-6"><title>RNA isolation and qPCR assays</title><p>RNA isolation was performed from ten-day-old seedling following the protocol described in <xref ref-type="bibr" rid="bib31">Onate-Sanchez and Vicente-Carbajosa (2008)</xref>. First-strand cDNA synthesis was performed with the SuperScript III RNA transcriptase (Invitrogen, Paisley, UK) and oligo(dT) primer, according to the manufacturer’s instructions. For qPCR reactions, the reaction mixture consisted of cDNA first-strand template, primers (5 nmol each) and SYBR Green JumpStart Taq ReadyMix (Sigma). qPCR was performed in a BioRad CFX96 real-time system. <italic>UBQ10</italic> was used as the internal control; expression in mock-treated Col-0 seedlings was used as the calibrator, with the expression level set to one. Relative expression was determined using the comparative Ct method (2-ΔΔCt). Each data point is the mean value of three experimental replicate determinations. Primers for <italic>At2g17740</italic> are described in <xref ref-type="bibr" rid="bib1">Albrecht et al. (2012)</xref>; for <italic>NHL10</italic> are described in <xref ref-type="bibr" rid="bib6">Boudsocq et al. (2010)</xref>; for <italic>LOX2</italic> are described in <xref ref-type="bibr" rid="bib32">Pandey et al. (2010)</xref>; for <italic>UBQ10</italic> (<italic>U-box</italic>) are described in <xref ref-type="bibr" rid="bib1">Albrecht et al. (2012)</xref>.</p></sec><sec id="s3-7"><title>Seedling growth inhibition assay</title><p>Seedling growth inhibition assays were performed as described in <xref ref-type="bibr" rid="bib27">Nekrasov et al. (2009)</xref>. In brief, four-day-old Arabidopsis seedlings were grown in liquid Murashige–Skoog medium containing 1% sucrose supplemented with flg22 and the appropriate chemicals. Seedlings were weighed between 6 and 10 days after treatment.</p></sec><sec id="s3-8"><title>Bacterial infections</title><p>Induced resistance assays were performed as described previously (<xref ref-type="bibr" rid="bib56">Zipfel et al., 2004</xref>). In brief, water or a 1 μM flg22 solution were infiltrated with a needleless syringe into leaves of four-week-old Arabidopsis plants 24 hr prior to bacterial inoculation (<italic>Pto</italic> DC3000, 10<sup>5</sup> cfu/ml). Bacterial growth was determined 2 days after inoculation by plating serial dilutions of leaf extracts.</p><p>Spray inoculation of <italic>P. syringae</italic> pv. <italic>cilantro</italic> (<italic>Pci</italic>) 0788-9 was performed as described in <xref ref-type="bibr" rid="bib40">Schwessinger et al. (2011)</xref>. In brief, bacteria were grown in an overnight culture in LB medium, cells were harvested by centrifugation, and pellets were re-suspended to OD600 = 0.02 in 10 mM MgCl<sub>2</sub> with 0.04% Silwet L-77. Bacterial suspensions were sprayed onto leaf surfaces and plants were kept uncovered. Bacterial growth was determined 3 days after inoculation by plating serial dilutions of leaf extracts.</p></sec><sec id="s3-9"><title>Meta-analysis</title><p>Functional enrichment analyses of the Biological Process ontology were performed using VirtualPlant (<xref ref-type="bibr" rid="bib17">Katari et al., 2010</xref>). Functional enrichment analysis of the Molecular Function ontology was performed using the Classification SuperViewer tool of the Bio-Array Resource for Arabidopsis Functional Genomics, BAR (<xref ref-type="bibr" rid="bib43">Toufighi et al., 2005</xref>). Promoter analyses were performed using Athena (<xref ref-type="bibr" rid="bib29">O’Connor et al., 2005</xref>).</p></sec></sec></body><back><ack id="ack"><title>Acknowledgements</title><p>RL-D is supported by a postdoctoral fellowship from Fundación Ramón Areces; APM is supported by a postdoctoral fellowship from the Federation of European Biochemical Societies. We thank Lena Stransfeld and the horticultural service at the John Innes Centre for excellent technical assistance, Yasuhiro Kadota for technical advice, and Christine Faulkner and all members of the Zipfel laboratory for fruitful discussions and helpful comments. We thank Zhiyong Wang, Joanne Chory, Ildoo Hwang, Dominique Roby, Eugenia Russinova, Xinnian Dong, Kang Chong, Zhiwiang Chen, Jun-Xian He, Jonathan Jones and David Guttman for sharing biological materials, and Sacco de Vries and Ben Scheres for excellent comments on the manuscript.</p></ack><sec sec-type="additional-information"><title>Additional information</title><fn-group content-type="competing-interest"><title>Competing interests</title><fn fn-type="conflict" id="conf1"><p>The authors declare that no competing interests exist.</p></fn></fn-group><fn-group content-type="author-contribution"><title>Author contributions</title><fn fn-type="con" id="con1"><p>RL-D, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article</p></fn><fn fn-type="con" id="con2"><p>APM, Conception and design, Acquisition of data, Analysis and interpretation of data</p></fn><fn fn-type="con" id="con3"><p>FB, Conception and design, Acquisition of data, 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letter</article-title></title-group><contrib-group content-type="section"><contrib contrib-type="editor"><name><surname>Nürnberger</surname><given-names>Thorsten</given-names></name><role>Reviewing editor</role><aff><institution>University of Tübingen</institution>, <country>Germany</country></aff></contrib></contrib-group></front-stub><body><boxed-text><p>eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see <ext-link ext-link-type="uri" xlink:href="http://elife.elifesciences.org/review-process">review process</ext-link>). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.</p></boxed-text><p>Thank you for sending your work entitled “The transcriptional regulator BZR1 mediates trade-off between plant innate immunity and growth” for consideration at <italic>eLife</italic>. Your article has been favorably evaluated by a Senior editor, Detlef Weigel, and 3 reviewers, one of whom, Thorsten Nürnberger, is a member of our Board of Reviewing Editors.</p><p>The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission. </p><p>The manuscript reports on a molecular mechanism underlying opposing plant physiological programs, such as growth and immunity. This is an important yet incompletely understood problem in plant biology. The authors have convincingly demonstrated that two factors, BZR1 and WRKY40, play a crucial role in brassinolide-mediated suppression of immunity. The authors should, however, respond to the referees’ issues listed below prior to final acceptance of their work: </p><p>1) BL treatment reverts PAMP-triggered SGI only partially, but never fully (see <xref ref-type="fig" rid="fig4">Figure 4</xref>). Likewise, constitutively active BZR also results in partial SGI complementation only, suggesting that other factors than BZR (and brassinolide) are implicated in regulation of this trade-off. Thus, calling BZR a master regulator is not fully justified. Similarly, there is the caveat that seedling growth might not always be regulated in the same way as overall vegetative growth. The authors may therefore provide more adequate statements throughout the text. </p><p>2) The synergistic effect of GA and BL (shown in <xref ref-type="fig" rid="fig4">Figure 4</xref> and its figure supplements) is not fully convincing. In fact, in a few cases restoration of SGI by combined hormone treatment seems to be even smaller than that observed with BL alone. As PAC inhibitor specificity is not really clear, it is suggested that the authors either analyze appropriate BL/GA-deficient mutants for a synergistic effect or, alternatively, opt to omit the GA part, which would weaken an otherwise very strong paper.</p><p>3) The long time of pre-incubation with BL (8 hrs) before flg22 treatment raises some concern as it is expected that the system has already shifted to a new steady state equilibrium. Here, the authors have to show that the brassinosteroid signaling is still active at this point and has not been reset through feedbacks. For instance, the authors could check the BZR phosphorylation status in a time course until flg22 challenge, or nuclear target gene transcription.</p><p>4) The authors state that PTI suppression might be particularly important during rapid growth phases, such as de-etiolation. Somewhat surprising here is that they then use fresh weight to measure growth. Given that brassinosteroids mainly regulate cell elongation and considering that during seedling establishment, cell expansion is the by far dominant growth mechanism, it is surprising that the authors have not considered determining hypocotyl elongation. This is an even more rapid growth phase, as compared to the authors’ fresh weight measurements at 10 days after germination. As such experiments can be rapidly conducted it should be tested experimentally whether such data match with those obtained by fresh weight measurements. </p><p>5) The manuscript would substantially benefit if the authors could provide more direct evidence that BZR1 and WRKY40 indeed cooperate in gene regulation. For instance, the authors could conduct relatively straightforward protoplast transfection experiments with BZR1 and WRKY40 alone or together as effectors, and a <italic>LOX2</italic> promoter construct as a reporter. Such an experiment would strongly support the finding of a biologically relevant BZR1-WRKY40 interaction. Alternatively, chromatin-IP assays would be suited to prove that both WRKY40 and BZR1 are associated with the <italic>LOX2</italic> promoter. </p><p>6) The finding that WRKY40 participates in the PTI inhibition is important. While BZR1 activation leads to reduced PTI responses and disease resistance to bacteria, it is not clear if WRKY40 also negatively impact disease resistance. An flg22-protection assay should be performed here. </p></body></sub-article><sub-article article-type="reply" id="SA2"><front-stub><article-id pub-id-type="doi">10.7554/eLife.00983.023</article-id><title-group><article-title>Author response</article-title></title-group></front-stub><body><p><italic>1) BL treatment reverts PAMP-triggered SGI only partially, but never fully (see</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref><italic>). Likewise, constitutively active BZR also results in partial SGI complementation only, suggesting that other factors than BZR (and brassinolide) are implicated in regulation of this trade-off. Thus, calling BZR a master regulator is not fully justified. Similarly, there is the caveat that seedling growth might not always be regulated in the same way as overall vegetative growth. The authors may therefore provide more adequate statements throughout the text</italic>.</p><p>We have now amended the text to take into account these remarks.</p><p><italic>2) The synergistic effect of GA and BL (shown in</italic> <xref ref-type="fig" rid="fig4"><italic>Figure 4</italic></xref> <italic>and its figure supplements) is not fully convincing. In fact, in a few cases restoration of SGI by combined hormone treatment seems to be even smaller than that observed with BL alone. As PAC inhibitor specificity is not really clear, it is suggested that the authors either analyze appropriate BL/GA-deficient mutants for a synergistic effect or, alternatively, opt to omit the GA part, which would weaken an otherwise very strong paper</italic>.</p><p><xref ref-type="fig" rid="fig4">Figure 4C</xref> shows that combined treatment of BL+GA in the light has a more potent effect than BL or GA alone. In the dark, SGI is already partially suppressed, and this effect remains after exogenous application of the hormones. Thus, we do not fully understand the comment about the additive effect between the hormones being unclear. We have now added statistical analysis of <xref ref-type="fig" rid="fig4">Figure 4C</xref> to make this point clearer.</p><p>Regarding the specificity of PAC, we have now added results (new <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1B</xref>) obtained after treatment with another GA biosynthesis inhibitor, uniconazole, which nicely corroborate those previously obtained with PAC. Furthermore, we report that the BL-induced inhibition of flg22-triggered SGI is hindered in the GA biosynthetic mutant <italic>ga1-3</italic> (new <xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1C</xref>), further confirming that the effect imposed by BL requires GA.</p><p><italic>3) The long time of pre-incubation with BL (8 hrs) before flg22 treatment raises some concern as it is expected that the system has already shifted to a new steady state equilibrium. Here, the authors have to show that the brassinosteroid signaling is still active at this point and has not been reset through feedbacks. For instance, the authors could check the BZR phosphorylation status in a time course until flg22 challenge, or nuclear target gene transcription</italic>.</p><p>Most results presented in this manuscript were obtained using transgenic or mutant lines with constitutive or increased BR responses, or during constant incubation with BL, in the case of seedling growth inhibition assays (e.g., <xref ref-type="fig" rid="fig4">Figure 4</xref>). In <xref ref-type="bibr" rid="bib1">Albrecht et al. (2012)</xref>, we also showed that co-treatment with BL inhibited flg22-induced gene expression. In the current manuscript, we only used pre-treatment in <xref ref-type="fig" rid="fig3">Figure 3B</xref> (8h) and <xref ref-type="fig" rid="fig1s2">Figure 1–figure supplement 2A</xref> (90 min or 5h). Nevertheless, to address the reviewers’ concern, we have tested whether BR signaling was still active after long treatments by looking at the phosphorylation status of BZR1, and found that BR signaling is still active even after an 8-hour BL treatment (this experiment has been repeated three times with similar results) (see <xref ref-type="fig" rid="fig5">Author response image 1</xref> below).<fig id="fig5" position="float"><label>Author response image 1.</label><caption><p>BL-induced dephosphorylation of BZR1 is maintained after an 8-hour BL treatment. Ten-day-old transgenic Arabidopsis seedlings expressing BZR1-YFP were treated with 1μM BL or mock solution for the indicated time. Proteins were detected using an anti-GFP antibody conjugated to HRP.</p></caption><graphic xlink:href="elife00983f005"/></fig></p><p><italic>4) The authors state that PTI suppression might be particularly important during rapid growth phases, such as de-etiolation. Somewhat surprising here is that they then use fresh weight to measure growth. Given that brassinosteroids mainly regulate cell elongation and considering that during seedling establishment, cell expansion is the by far dominant growth mechanism, it is surprising that the authors have not considered determining hypocotyl elongation. This is an even more rapid growth phase, as compared to the authors’ fresh weight measurements at 10 days after germination. As such experiments can be rapidly conducted it should be tested experimentally whether such data match with those obtained by fresh weight measurements</italic>.</p><p>We measured hypocotyl length upon flg22 treatment in light-grown seedlings and did not find any impact) (see <xref ref-type="fig" rid="fig6">Author response image 2</xref> below). This is different to what we observed when measuring total fresh weight, and most likely reflects the fact that hypocotyls are already extremely short in Arabidopsis seedlings grown in light. In contrast, hypocotyls elongate greatly during etiolation; yet, no impact of flg22 treatment could be observed (see <xref ref-type="fig" rid="fig6">Author response image 2</xref> below), which is entirely consistent with the lack of flg22 responsiveness in dark-grown seedlings reported already in this study. Therefore, hypocotyl length measurement does not appear as a suitable assay to compare the impact of BR regulation on PTI between light and dark conditions, which is one of the major aims of our study.<fig id="fig6" position="float"><label>Author response image 2.</label><caption><p>Hypocotyl length of light- or dark-grown Arabidopsis seedlings in increasing concentrations of flg22. Seedlings were grown in MS plates for four days, then transferred to liquid MS supplemented with flg22 at the indicated concentrations. Hypocotyl length was measured four days later. Bars represent SE with n=8.</p></caption><graphic xlink:href="elife00983f006"/></fig></p><p><italic>5) The manuscript would substantially benefit if the authors could provide more direct evidence that BZR1 and WRKY40 indeed cooperate in gene regulation. For instance, the authors could conduct relatively straightforward protoplast transfection experiments with BZR1 and WRKY40 alone or together as effectors, and a</italic> LOX2 <italic>promoter construct as a reporter. Such an experiment would strongly support the finding of a biologically relevant BZR1-WRKY40 interaction. Alternatively, chromatin-IP assays would be suited to prove that both WRKY40 and BZR1 are associated with the</italic> LOX2 <italic>promoter</italic>.</p><p>We now provide additional results to support the reported BZR1-WRKY40 protein interaction (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>). However, while we could previously show that <italic>LOX2</italic> transcript levels were reduced by BL treatment in a WRKY40-dependent manner in seedlings (<xref ref-type="fig" rid="fig3">Figure 3D</xref> in the previous version) (a result that is reproducible), we then encountered difficulties studing <italic>LOX2</italic> gene regulation in other systems (e.g., protoplasts or <italic>N. benthamiana</italic>), systems that would be required to perform the suggested experiments in the allocated time. Furthermore, we requested previously published materials, such as <italic>LOX2::GUS</italic> and <italic>LOX2::LUC</italic> constructs or transgenic lines; but, unfortunately, these tools either are not available anymore or can no longer be used due to severe silencing of both reporter constructs. While the Kombrink lab is currently generating these tools again, we also considered making a <italic>LOX2::LUC</italic> construct ourselves. However, the expression of this reporter transgene has been previously reported to be quasi undetectable in the absence of exogenous MeJA treatment (Jensen et al., Plant J 2002), which would make the suggested experiment (i.e., testing whether BZR1+WRKY40 inhibit LOX2 expression more potently than BZR1 or WRKY40 alone) extremely technically challenging. For these different reasons, we have decided to omit the results on LOX2 expression from the revised manuscript. Nevertheless, the results provided still convincingly show that BZR1 acts together with WRKY40 both genetically (<xref ref-type="fig" rid="fig3">Figure 3B</xref>) and biochemically (<xref ref-type="fig" rid="fig3">Figure 3C–E</xref>) to regulate the BR-mediated inhibition of PTI. The exact mechanisms underlying the latter regulation will be within the scope of future studies.</p><p><italic>6) The finding that WRKY40 participates in the PTI inhibition is important. While BZR1 activation leads to reduced PTI responses and disease resistance to bacteria, it is not clear if WRKY40 also negatively impact disease resistance. An flg22-protection assay should be performed here</italic>.</p><p>Our previous data showed that <italic>wrky40</italic> plants were more resistant to <italic>Pto</italic> DC3000 (<xref ref-type="fig" rid="fig4s1">Figure 4–figure supplement 1B</xref>), indicating that WRKY40 is a negative regulator of immunity. This is consistent with other reports (Xu et al., Plant Cell 2006; Pandey et al., Plant J 2010; Schon et al., MPMI 2013; Brotman et al., PLOS Pathog 2013; Schweizer et al., Front Plant Sci 2013). When we measured flg22-induced resistance, we observed that the flg22 treatment could not further decrease <italic>Pto</italic> DC3000 titers in <italic>wrky40</italic> plants (<xref ref-type="fig" rid="fig7">Author response image 3</xref> below).<fig id="fig7" position="float"><label>Author response image 3.</label><caption><p>Flg22-induced resistance to <italic>P. syringae</italic> pv. <italic>tomato</italic> DC3000 in Col-0 and <italic>wrky40</italic> plants. Plants were pre-treated with 1 μM flg22 or water 24 hours prior to bacterial infiltration. Results are the average of three independent biological replicates; bars represent SE.</p></caption><graphic xlink:href="elife00983f007"/></fig></p></body></sub-article></article> |