Transcription Factor OsWRKY53 Positively Regulates Brassinosteroid Signaling … · Transcription Factor OsWRKY53 Positively Regulates Brassinosteroid Signaling and Plant Architecture1

Transcription Factor OsWRKY53 Positively RegulatesBrassinosteroid Signaling and Plant Architecture1

Xiaojie Tian,a,b Xiufeng Li,a Wenjia Zhou,a,b Yuekun Ren,a,b Zhenyu Wang,a Zhiqi Liu,c Jiaqi Tang,a,b

Hongning Tong,d Jun Fang,a and Qingyun Bua,2

aNortheast Institute of Geography and Agroecology, Key Laboratory of Soybean Molecular Design Breeding,Chinese Academy of Sciences, Harbin 150081, ChinabGraduate University of Chinese Academy of Sciences, Beijing 100049, ChinacCollege of Life Science, Northeast Forestry University, Harbin 150040, ChinadInstitute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China

ORCID IDs: 0000-0001-9040-5156 (X.T.); 0000-0003-3107-1525 (Z.L.); 0000-0002-5386-3577 (Q.B.).

Brassinosteroids (BRs) are a class of steroid hormones regulating multiple aspects of plant growth, development, and adaptation.Compared with extensive studies in Arabidopsis (Arabidopsis thaliana), the mechanism of BR signaling in rice (Oryza sativa) is lessunderstood. Here, we identified OsWRKY53, a transcription factor involved in defense responses, as an important regulator ofrice BR signaling. Phenotypic analyses showed that OsWRKY53 overexpression led to enlarged leaf angles and increased grainsize, in contrast to the erect leaves and smaller seeds in oswrky53 mutant. In addition, the oswrky53 exhibited decreased BRsensitivity, whereas OsWRKY53 overexpression plants were hypersensitive to BR, suggesting that OsWRKY53 positivelyregulates rice BR signaling. Moreover, we show that OsWRKY53 can interact with and be phosphorylated by the OsMAPKK4-OsMAPK6 cascade, and the phosphorylation is required for the biological function of OsWRKY53 in regulating BR responses.Furthermore, we found that BR promotes OsWRKY53 protein accumulation but represses OsWRKY53 transcript level. Takentogether, this study revealed the novel role of OsWRKY53 as a regulator of rice BR signaling and also suggested a potential roleof OsWRKY53 in mediating the cross talk between the hormone and other signaling pathways.

Brassinosteroids (BRs) are a group of plant-specificsteroidal hormones that play various roles in plantgrowth, development, and stress responses. In the pastdecades, extensive studies have identified numerousBR-signaling components to establish a signaling net-work and further provided a global view of BR functionin the model plant Arabidopsis (Arabidopsis thaliana;Kim and Wang, 2010). In brief, BR is recognized by themembrane-localized receptorBR insensitive1 (BRI1) and itscoreceptor BRI1-associated receptor kinase1 (BAK1) andforms the BRI1-BR-BAK1 complex (Li and Chory, 1997;Li et al., 2002; Sun et al., 2013). Transphosphorylation

between BRI1 and BAK1 activates BRI1, which thenphosphorylates cytoplasmic kinase BSKs (BR signalingkinases) and Constitutive differential growth1, andactivation of BSKs/Constitutive differential growth1leads to phosphorylation and activation of the proteinphosphatase bri1-suppressor1 (Tang et al., 2008; Wanget al., 2008; Kim et al., 2011). bri1-suppressor1 dephos-phorylates and inactivates the GSK3/Shaggy-like ki-nase BR insensitive2 (BIN2; Kim et al., 2009). Therefore,in the presence of BR, BIN2 is inhibited, which allowsBrassinazole-resistant1 (BZR1) and BRI1 EMS suppres-sor1 (BES1) to be dephosphorylated by Protein phos-phatase 2A (Tang et al., 2011). Finally, dephosphorylatedBZR1 and BES1 regulate the expression of numerousBR-responsive genes through binding to a BR responseelement or E-box cis-element (Yin et al., 2005; Sun et al.,2010; Yu et al., 2011).

In contrast to the tremendous progress of BR signal-ing in Arabidopsis, relatively fewer components havebeen characterized in rice (Zhang et al., 2014). So far,many known rice BR signaling components (e.g.OsBRI1, OsBAK1, OsGSK2, OsBZR1, and OsBSK) haveorthologs in Arabidopsis and served conserved func-tions (Yamamuro et al., 2000; Bai et al., 2007; Li et al.,2009; Tong et al., 2012; Zhang et al., 2016). However,some components, including Oryza sativa Dwarf andlow tillering (OsDLT), Taihu dwarf1, and Leaf and tillerangle increased controller have no counterpart identifiedin Arabidopsis, implying that a rice-specific BR-signaling

1 This studywas supported byNationalNatural Science Foundationof China (grant no. 31671653), the Strategic Priority ResearchProgram of Chinese Academy of Sciences (grant no. XDA08040101),the Natural Science Foundation of Heilongjiang (grant no.ZD2015005), and the Hundred-Talent-Program of Chinese Academyof Sciences to Q.B.

2 Address correspondence to [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policy de-scribed in the Instructions for Authors (www.plantphysiol.org) is:Qingyun Bu ([email protected]).

Q.B. conceived and supervised the project; X.T. performed most ofexperiments; X.L., W.Z., Y.R., Z.W., Z.L., and J.T. assisted the exper-iments; Q.B. and X.T. analyzed the data and wrote the article with thecontributions from H.T. and J.F.

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pathwaymight exist (Tong et al., 2009; Zhang et al., 2012;Hu et al., 2013). In addition, helix-loop-helix proteins suchas Oryza sativa Brassinsteroid upregulated1 (OsBU1),Brassinsteroid upregulated like1, and Increased laminarinclination1 can positively regulate rice BR signaling(Tanaka et al., 2009; Zhang et al., 2009; Jang et al., 2017).Reduced leaf angle1 (RLA1)/Small organ size1 wascharacterized as a positive regulator of BR signaling,which can form a complex with OsBZR1 and OsDLTto coregulate the expression of downstream genes(Hirano et al., 2017; Qiao et al., 2017). Very recently,Enhanced leaf inclination and tiller number1, a receptor-like protein, was shown to promote BR signaling throughinteracting with and suppressing the degradation ofOsBRI1 (Yang et al., 2017).

Plants WRKY transcription factors contain one ortwo conserved WRKYGQK sequences followed by aC2H2 or C2HC zinc finger motif. Accumulating evi-dence revealed that the WRKY proteins play diverseroles in responses to biotic and abiotic stresses and areinvolved in various processes of plant growth and de-velopment by regulating the expression of target genesvia binding to the W-box cis-element (Rushton et al.,2010). In rice, WRKY family has at least 102 members,and only a few members have been functionally char-acterized (Xie et al., 2005; Sun et al., 2014). Most of theidentified rice WRKY members are involved in plantbiotic stress response, including OsWRKY70, OsWRKY53,OsWRKY13, OsWRKY45, and OsWRKY28 (Chujo et al.,2007; Qiu et al., 2007; Shimono et al., 2007; Tao et al., 2009;Chujo et al., 2013; Hu et al., 2015;Ma et al., 2015). Recently,OsWRKY70 and OsWRKY53 were proposed to func-tion in tradeoff mechanisms between biotic stress re-sponse and growth; however, the mechanism by whichthey regulate plant growth remains elusive (Hu et al.,2015; Ma et al., 2015).

MAPK cascade is comprised of three components,MAPK kinase kinases, MAPK kinases, and MAPKs, andhas pivotal roles in plant innate immunity (Ishihama et al.,2011; Adachi et al., 2015). Several group Ia members ofWRKY proteins have been shown as direct downstreamtargets of MAPK cascade. MAPK can interact with andphosphorylate group Ia WRKYs via five conserved Serin SP clusters (Clustered Prodirected Ser residues), andphosphorylation of WRKY proteins by MAPK can en-hance theDNAbinding activity or transcriptional activityof WRKY proteins (Qiu et al., 2008; Ishihama et al., 2011;Shen et al., 2012; Chujo et al., 2014; Yoo et al., 2014). In rice,the mutation of either OsMAPKK4 or OsMAPK6 leadsto BR-deficient phenotypes (dwarfism, erect leaf, andsmaller grain size), decreasedBRsensitivity, anddisruptedexpression of BR-related genes, which suggested thatthe OsMAPKK4-OsMAPK6 cascade is involved in riceBR signaling (Duan et al., 2014; Liu et al., 2015). How-ever, their downstream targets remain elusive.

Here, we show that OsWRKY53 plays a positive role inrice BR signaling and acts downstreamof theOsMAPKK4-MAPK6 cascade. Overexpression and knockout ofOsWRKY53 led to contrasting BR-related phenotypes.Genetic analysis showed thatOsWRKY53acts downstream

of OsBRI1 receptor. In addition, we show that thephosphorylation of OsWRKY53 by OsMAPKK4-MAPK6 is critical for its biological function. Moreover,OsWRKY53 protein is enhanced by BR and can repressits own expression. Taken together, our results suggestthatOsWRKY53 is a novelfine tuner of rice BR signaling.

RESULTS

Characterization of OsWRKY53 Overexpression Lines

We previously generated a rice mutant library over-expressing different transcription factors directed bythe maize (Zea mays) Ubiquitin promoter. A number oflines carrying OsWRKY53 (Os05g0343400) showed ob-viously increased leaf angles (described in detail below)and were chosen for further study.

Generally,OsWRKY53 overexpression lines (OsWRKY53-OEs) showed greatly enlarged leaf angles and dwarfism(Fig. 1, A–C). At the three-leaf stage, the angles of the firstleaf in the wild type were about 30 degrees, while theangles of the parallel leaf in OsWRKY53-OE reached al-most 60 degrees (Supplemental Fig. S1,A andB).With thegrowth of plant, the larger leaf angles of OsWRKY53-OEappeared to bemore evident. At heading stage, the anglesof flag leaf in OsWRKY53-OE were about 2120 degrees,which is significantly larger than that of wild type at thesame stage (Fig. 1, B and C). It has been shown that en-larged leaf inclination is mainly associatedwith abnormaldevelopment of lamina joint (Sun et al., 2015). Morpho-logical observation revealed that the collar length of ad-axial surface in OsWRKY53-OE is significantly increasedcomparedwith that inwild type (Supplemental Fig. S1C).Scanning electronmicroscopic observation of lamina jointshowed that the adaxial surface in OsWRKY53-OE is notsmooth and contains some protuberance comparedwith that in wild type (Supplemental Fig. S1D).Further cytological observation showed that the celllength of adaxial surface inOsWRKY53-OE is markedlylonger than that in wild type (Supplemental Fig. S1, Eand F), which contributed to the enlarged leaf angles ofOsWRKY53-OE.

In addition, compared with wild type, both the grainlength and grain width of OsWRKY53-OE increasedsignificantly (Fig. 1, D–F; Supplemental Fig. S11, A–C).Scanning electron microscopic observation of the spikelethull showed that the epidermal cells of both palea andlemma in theOsWRKY53-OE aremuch longer than that inwild type, indicating that increased grain size is mainlyattributed to the cell enlargement (Fig. 1, G–I). In agree-ment with this result, a number of genes associated withcell expansion were up-regulated in the panicles of theOsWRKY53-OE (Supplemental Fig. S2). Moreover, com-pared with wild type, the leaves of OsWRKY53-OEappeared to be pale green (Supplemental Fig. S3), and theplant height was also reduced (Supplemental Fig. S4D).

Most of the independent OsWRKY53-OEs showedsimilar phenotypes as described above (SupplementalFig. S4, A and B), thus excluding the possibility that

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the phenotypes are caused by mutations of other en-dogenous genes coming from T-DNA insertion or tissueculture. We also showed the expression of OsWRKY53 isindeed overexpressed in three representative OsWRKY53-OE plants (Supplemental Fig. S4B). Further immunoblot-ting analyses confirmed the accumulation of OsWRKY53proteins in theseOsWRKY53-OEs (Supplemental Fig. S4C).Taken together, we concluded that the overexpression ofOsWRKY53 leads to enlarged leaf angles and increasedgrain size in rice.

OsWRKY53 Positively Regulates BR Signaling in Rice

The increased leaf angles and enlarged grain size inOsWRKY53-OEs resembledmany of the typical enhanced-BR-signaling mutants, such as osbzr1-D, GSK2-RNAi,OsBU1-OE, and mRLA1-OE (Tanaka et al., 2009; Tonget al., 2012; Qiao et al., 2017). Therefore, we hypothe-sized that OsWRKY53 might be involved in rice BRsignaling. We then performed the BR-induced laminainclination assay to test the BR sensitivity ofOsWRKY53-OEin response to different concentrations of 24-epibrassinolide(24-epiBL), an active form of BRs (Tanabe et al., 2005). Theresults showed that lamina bending ofOsWRKY53-OE

was obviously much more sensitive than that of wildtype (Fig. 2, A and B). After incubation in 10 nM 24-epiBLfor 3 d, leaf angles in OsWRKY53-OE reached around;140 degrees, dramatically higher than those in wildtype, which were about ;70 degrees (Fig. 2, A and B).This result strongly suggested that overexpression ofOsWRKY53 leads to enhanced BR responses.

It is well known that expressions of BR biosynthesisgenes are usually negatively feedback regulated by BRsignaling in both rice and Arabidopsis (Tong et al.,2012; Zhang et al., 2016; Qiao et al., 2017). The en-hancement of BR signaling inOsWRKY53-OE promptedus to investigate whether OsWRKY53 is involved inthis process. We analyzed the expression of BR bio-synthesis genes, including D2, OsDWF4, and D11, andfound all of them have significantly decreased expres-sion inOsWRKY53-OE plants compared with wild type(Fig. 2C), indicating that OsWRKY53 is involved infeedback inhibition of BR biosynthesis genes. In addition,we checked the expression of several BR-responsivegenes and found that the expression of OsBU1 andOsXTR1was indeed increased inOsWRKY53-OE plants(Fig. 2D). Taken together, these results provided strongevidence that OsWRKY53 positively regulates BR sig-naling in rice.

Figure1. OverexpressionofOsWRKY53leads to enlarged leaf angle and in-creased grain size. A, The grossmorphology of OsWRKY53-OE andwild type (WT) at heading stage. B,The lamina joint of flag leaf in WTand OsWRKY53-OE. C, Quantifi-cation of lamina angle of the flagleaf. Data aremeans6 SE (n= 20). (D)The grain phenotype of OsWRKY53-OE andWT (scale bar = 5mm). E andF, Quantification of grain length (E)and grain width (F), respectively. Dataare means6 SE (n = 50). G, Scanningelectron microscopic observation ofspikelet of OsWRKY53-OE and WT.The squares are positions for ob-servation. The palea and lemma ofOsWRKY53-OE and WT were ob-served, respectively (scale bar =50 mm). H and I, Quantification ofcell length (H) and cell width (I) inG were shown as means 6 SE (n =50). P values were calculated byStudent’s t test; *P , 0.05, and**P , 0.01.

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Characterization of oswrky53 Mutant

To further confirm the function of OsWRKY53 inregulating BR signaling, we generated oswrky53mutantvia CRISPR/Cas9-mediated genome-editing technology.Two independent oswrky53mutant alleles, oswrky53-1 andoswrky53-2, were identified, and the mutation sites werecharacterized by DNA sequencing (Fig. 3A; SupplementalFig. S5). In contrast to OsWRKY53-OE, the leaves ofoswrky53 were more erect than those of wild type (Fig. 3,B–D). The plant height of oswrky53 was also slightly de-creased (Fig. 3E). In addition, the mutants produced ob-viously smaller grains, with reduced seed length and seedwidth (Fig. 3, F–H, Supplemental Fig. S11,D–F).Moreover,oswrky53 were dark green compared with wild type(Supplemental Fig. S6). Thesephenotypes of oswrky53weresimilar to those of typical BR-deficient or BR-defectivemutants, such as d11, d61, andGSK2-OE (Yamamuro et al.,2000; Tanabe et al., 2005; Tong et al., 2009; Tong et al., 2012;Qiao et al., 2017). Furthermore, a lamina-bending assayshowed that oswrky53 is hyposensitive to BR, which isopposite to OsWRKY53-OE (Fig. 3, I and J). Together,oswrky53 exhibited BR-deficient phenotypes anddecreasedBR response, which further supported our hypothesis thatOsWRKY53 plays positive roles in regulating BR signalingin rice.

OsWRKY53 Acts Downstream of OsBRI1

To analyze whether OsWRKY53 functions in the pri-mary BR-signaling pathway, we crossed OsWRKY53-OEwith d61-2, a weak allele of the BR receptor OsBRI1 mu-tant (Yamamuro et al., 2000), and identified the doublemutant (Fig. 4A). Compared with wild type,OsWRKY53-OE exhibited larger leaf angles and increased seed size,

while d61-2 showed smaller leaf angles and decreasedseed size (Fig. 1A; Yamamuro et al., 2000). However, forboth leaf angles and seed size, d61-2 OsWRKY53-OEdouble mutant was remarkably larger than d61-2 (Fig. 4,B–F; Supplemental Figure S11, G–I), suggesting thatoverexpression of OsWRKY53 can largely rescue BR sig-naling deficiency phenotypes of d61-2. This result sug-gested that OsWRKY53 is involved in BR signaling andmight act downstream of BR receptor.

OsWRKY53 Interacts with and Is Phosphorylatedby OsMAPK6

OsWRKY53 belongs to the group Ia subset of WRKYfamily and contains twoWRKY domains followed by aC2H2-type zinc finger motif (Xie et al., 2005; Chujoet al., 2007; Hu et al., 2015). It has been shown thatgroup Ia WRKY proteins can be phosphorylated by theMAPK cascade, and the phosphorylation can affectthe DNA binding activity or transcription activity ofWRKYproteins (Ishihama et al., 2011; Chujo et al., 2014;Adachi et al., 2015). Interestingly, previous studies alsoshowed that OsWRKY53 can interact with and bephosphorylated by OsMAPK3 and OsMAPK6, and thephosphorylated sites were located in the SP cluster atthe amino terminal domain of OsWRKY53 (Chujo et al.,2014; Hu et al., 2015). Consistent with these results, weconfirmed that OsWRKY53 can interact with OsMAPK6in yeast two-hybrid assays (Fig. 5A). Further bi-molecular fluorescence complementation (BiFC) andLUC complementation imaging (LCI) assays confirmedthat OsWRKY53 can interact with the OsMAPK6 inplanta system (Fig. 5, B and C). In addition, we showedthat the OsWRKY53 was weakly phosphorylated

Figure 2. OsWRKY53-OE is hypersensitive to24-epiBL treatment. A The leaf inclination ofOsWRKY53-OE and wild type (WT) in the pres-ence of indicated concentration of 24-epiBL. B,Statistical analysis of leaf inclination in A, data aremeans 6 SE (n = 20). C, The relative expression ofBR biosynthesis genes D2, OsDWF4, and D11 inWT and OsWRKY53-OE. The expression level inWTwas set as “1”; data were shown as means6 SE(n = 3). D, The relative expression of BR-signalingpathway genes OsBU1 and OsXTR1 in WT andOsWRKY53-OE. The expression level in WT wasset as “1”; data were shown as means6 SE (n = 3).


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by OsMAPK6, whereas this phosphorylation wasgreatly enhanced in the presence of constitutivelyactive form of OsMAPKK4 (Fig. 5E). However, thephosphorylation of OsWRKY53 by OsMAPKK4-OsMAPK6 was markedly decreased when the fiveconserved Ser residues in the SP cluster were mu-tated to Ala, indicating that the five conserved Serresidues are the critical phosphorylation sites ofOsWRKY53 by OsMAPKK4-OsMAPK6 cascade (Fig.5, D and E).To examine the effect of OsWRKY53 phosphoryl-

ation by OsMAPK6, EMSA was performed, and theresult indicated that phosphorylation of OsWRKY53by the OsMAPKK4-OsMAPK6 cascade can markedlyenhance the DNA binding activity of OsWRKY53protein to the W-box containing the DNA sequence(Supplemental Fig. S7A). In addition, we coexpressedOsWRKY53 and the OsMAPKK4-OsMAPK6 cascade inrice protoplast transient-expression assay and foundthat the addition of OsMAPKK4 and OsMAPK6 doesnot change the protein level of OsWRKY53, implyingthat phosphorylation by the OsMAPKK4-OsMAPK6cascade cannot affect OsWRKY53 stability (SupplementalFig. S7B).

Phosphorylation of OsWRKY53 by the OsMAPKK4-OsMAPK6Cascade Is Critical for the Function of OsWRKY53 inRegulating BR Response

To test biological significance of phosphorylation ofOsWRKY53 by the OsMAPKK4-OsMAPK6 cascade,we mutated the five conserved Ser in SP cluster toAla (generating OsWRKY53 [SA]) or Asp (generatingOsWRKY53 [SD]) to mimic the inactive or active phos-phorylated forms of OsWRKY53, respectively (Fig. 5D).We then generated the transgenic linesOsWRKY53 (SA)-OEs and OsWRKY53 (SD)-OEs in which OsWRKY53(SA) and OsWRKY53 (SD) were overexpressed, respec-tively. Interestingly,we found thatOsWRKY53 (SD)-OEsshowed much stronger phenotypes than OsWRKY53-OEs, including larger leaf angles, increased grain size,and dwarfism (Fig. 6, A–E; Supplemental Figure S11, Jand K). In addition, OsWRKY53 (SD)-OEs were morehypersensitive to BR thanOsWRKY53-OEs, as shown bylamina-bending assay (Fig. 6, F and G). Furthermore,compared with that in OsWRKY53-OEs, expression ofthe three BR biosynthesis genes including D2, OsDWF4,and D11 was decreased more significantly in OsWRKY53(SD)-OEs (Fig. 6H). Together, these results suggestedthat constitutive phosphorylation of OsWRKY53 by the

Figure 3. oswrky53 mutant showdecreased sensitive to BR. A, Iden-tification of oswrky53mutants. ATGand TGA are initiation codon andstop codon, respectively. B, Thegross morphology of oswrky53mutants and wild type (WT) atheading stage. C and D, The phe-notype (C) and statistical analysis(D) of leaf angle; the flag leaf wasthe first leaf. Data were shown asmeans 6 SE (n = 20). E, The plantheight of oswrky53mutants andWT(scale bar = 10 cm). Data wereshown as means6 SE (n = 20). F, Thegrain phenotype of oswrky53 mu-tants and WT (scale bar = 5 mm). Gand H, Quantification of grainlength (G) and grain width (H) ofoswrky53 mutants and WT. Datawere shown as means6 SE (n = 50).I, The leaf inclination of oswrky53andWTin the presence of indicatedconcentration of 24-epiBL. J, Sta-tistical analysis of leaf inclination inI. Data are means 6 SE (n = 20). Pvalues were calculated by Student’st test; *P , 0.05, and **P , 0.01).



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OsMAPKK4-OsMAPK6 cascade can enhance thefunction of OsWRKY53 in BR signaling. In contrast,OsWRKY53 (SA)-OEs failed to produce any obvious phe-notypes; even the expression level of OsWRKY53 wasmuch higher than that in OsWRKY53-OE (SupplementalFigs. S8 and S11, L and M), suggesting that these phos-phorylation sites are critical for OsWRKY53 functionsin regulating BR-related phenotypes. Collectively, theseresults indicated that OsMAPKK4-OsMAPK6-mediatedphosphorylation of OsWRKY53 is indispensable forfunction of OsWRKY53 in regulating BR signaling.

OsWRKY53 Is Promoted by BR and Negatively FeedbackRegulated by Itself

The fact that OsWRKY53 is involved in BR responsesprompted us to test whether expression of OsWRKY53is regulated by BR. For this purpose, 2-week-old seed-lings of wild type were treated with 1 mM 24epi-BL, andthen the transcript levels ofOsWRKY53were examinedby reverse transcription (RT)-qPCR assay. As shown inFigure 7A, OsWRKY53 transcripts were decreased uponBR treatment, similar to the expression ofOsDWF4 gene.We also investigated the BR effects on OsWRKY53protein stability using MYC-OsWRKY53-OE plants inwhich OsWRKY53 fused with MYC tag was overex-pressed.MYC-OsWRKY53-OE lineswere characterized andshow similar phenotypes toOsWRKY53-OE (SupplementalFig. S9). Interestingly, unlike OsWRKY53 transcript,OsWRKY53 protein level was enhanced by BR

treatment (Fig. 7B). By contrast, when the MYC-OsWRKY53-OE plants were grown on medium con-taining brassinozole (BRZ), a BR biosynthesis inhibitor,the MYC-OsWRKY53 protein level decreased (Fig. 7C),whereas the RNA level of OsWRKY53 did not change(Fig. 7D). These results indicated that BR can promoteOsWRKY53 protein stability. It has been reported thatthe transcript and protein levels of several key compo-nents in BR signaling (e.g. DLT, BZR1, RLA1) also showa similar BR-responsive pattern, which tends to repre-sent a commonnegative feedback regulationmechanismin fine-tuning BR signaling (Tong et al., 2009; Qiao etal., 2017).

Considering that BR oppositely regulates the expres-sion pattern of OsWRKY53 transcript and OsWRKY53protein level (Fig. 7, A and B), we asked if OsWRKY53can regulate its own expression. RT-qPCR assay wasperformed to check the expression of nativeOsWRKY53in oswrky53 mutants. We showed that the expression ofOsWRKY53 in both of these two mutants was increased(Fig. 7F). Then, we checked the expression of nativeOsWRKY53 in OsWRKY53-OE lines using the 59-UTR-and 39-UTR-specific primers, respectively (Fig. 7E). Asshown in Figure 7G, in OsWRKY53-OE lines with in-creased expression of ectopic OsWRKY53, the nativeOsWRKY53 expression was reduced. This result wasfurther supported by the transient expression assay inrice protoplast, in which 35Spro:OsWRKY53 can suppressthe expression of LUC fused with the nativeOsWRKY53promoter (Fig. 7,H and I). There are twoW-box elementsin OsWRKY53 promoter that could be bound by WRKY

Figure 4. OsWRKY53-OE can partiallyrescue phenotype of d61-2. A, The grossphenotype of OsWRKY53-OE, d61-2,and d61-2 OsWRKY53-OE. CK is WTcontrol that separated from the F2 pop-ulation. B andC, The leaf angle phenotype(B) and statistical analysis of OsWRKY53-OE, d61-2, and d61-2 OsWRKY53-OE.Data are means 6 SE (n = 20). D to F, Thegrain phenotype (scale bar = 5 mm).D, and the statistical analysis (E and F)of OsWRKY53-OE, d61-2, and d61-2OsWRKY53-OE. Data are means 6 SE(n = 50). P values were calculated byStudent’s t test; *P, 0.05, and **P, 0.01.


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transcription factors (Fig. 7E).We then performedEMSAto test if OsWRKY53 binds to its native promoter. Asshown in Figure 7J, the MBP-OsWRKY53 fusion proteinbound to its own promoter in a W-box-dependent man-ner. Furthermore, chromatin immunoprecipitation (ChIP)assay also indicated that OsWRKY53was associatedwiththe W-box region of its own promoter (Fig. 7K). Takentogether, these results suggested that OsWRKY53 pro-tein can directly suppress its own expression, whichpartially explained the negative feedback regulation ofOsWRKY53 and implied that OsWRKY53 might be afine tuner in rice BR-signaling pathway.In addition,weanalyzed the expressionofOsWRKY53 in

different tissues by RT-qPCR.We showed thatOsWRKY53transcripts can be detected in various tissues and havethe highest expression in lamina joint (Supplemental Fig.S10A). In addition, we also generated OsWRKY53 pro:GUS transgenic plants. In agreement with RT-qPCR re-sults, GUS staining of transgenic plants showed thatactivity of the promoter could be detected in differentorgans, preferentially higher in lamina joint comparedwith other tissues (Supplemental Fig. S10B). Altogether,

the spatial expression pattern of OsWRKY53 was corre-lated with one of its physiological functions that con-trolled the leaf angles.

DISCUSSION

Function of OsWRKY53 in Regulating BR Signaling

In this study, we provide substantial physiological,genetic, and biochemical evidence for the involvementof OsWRKY53 in rice BR-signaling pathway. First,OsWRKY53-OE showed enhanced BR-signaling pheno-types, including enlarged leaf angles and increased seedsize (Fig. 1; Supplemental Figs. S1–S4), whereas oswrky53mutant exhibited BR-deficient phenotypes, such as erectleaves, smaller seed size, and dark green leaves (Fig. 3;Supplemental Fig. S6). Second, BR-sensitivity assay oflamina bending in OsWRKY53-OE and oswrky53 dem-onstrated that OsWRKY53 is a positive regulator of BRsignaling (Figs. 2 and 3). Third, genetic analysis revealedthat OsWRKY53 overexpression can largely rescue BR-deficiency phenotypes of BR receptormutant d61-2 (Fig. 4).

Figure 5. OsMAPK6 can interact with and phosphorylate OsWRKY53. A, Interactions between OsMAPK6 and OsWRKY53 inyeast two-hybrid assays. B, Interaction between OsMAPK6 and OsWRKY53 in BiFC assays. C, Interaction between OsMAPK6and OsWRKY53 in LCI assays. D, Schematic diagram of OsWRKY53. Five Ser amino acids in conserved SP cluster and WRKYdomain were indicated, respectively. E, In vitro phosphorylation of OsWRKY53 by OsMAPK6. Top, western blot detected byPhostag Biotin BTL-104. Bottom, the equal protein loading in top was monitored by Coomassie blue staining.



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Forth, OsWRKY53 interacts with and is phosphorylatedby OsMAPK6, and the phosphorylation is required forBR-related function of OsWRKY53 (Figs. 5 and 6). Finally,we showed that the protein level of OsWRKY53 ispromoted by BR treatment, and the transcript level ofOsWRKY53 is suppressed by BR, possibly through anegative autofeedback regulation (Fig. 7). Taken together,these results demonstrated an important biologicalrole of OsWRKY53 in regulating rice BR signaling(Supplemental Fig. S12).

Functional Diversity of OsWRKY53

WRKY family transcription factors play a variety ofdevelopmental and physiological roles in plants. Ricegenome contains more than 102WRKY genes that were

divided into four subgroups (Xie et al., 2005; Sun et al.,2014). Up to now, several rice WRKY genes have beenfunctionally characterized, and most of them were in-volved in stress responses. For instance, OsWRKY31,OsWRKY33, and OsWRKY53 can positively regulatepathogen-infection response (Chujo et al., 2007; Zhanget al., 2008; Koo et al., 2009), while OsWRKY53 andOsWRKY70 negatively regulate herbivore resistance(Hu et al., 2015; Li et al., 2015). Besides, OsWRKY30 andOsWRKY42 play roles in drought response and senes-cence process, respectively (Shen et al., 2012; Han et al.,2014). By contrast, a role of WRKY genes in controllinggrowth and development is less known. Similar toOsWRKY53-OE plants, overexpression of OsWRKY70led to a severe reduction in plant height (Li et al., 2015).In addition, a decreased expression of OsWRKY78resulted in semidwarfism and small grain due to the

Figure 6. OsMAPK6 mediated phosphorylation of OsWRKY53 is required for BR-related function of OsWRKY53. A, The grossmorphology ofOsWRKY53-OE-1 andOsWRKY53(SD)-OE-3. The picture at top right is phenotype of the corresponding flag leafangle. B, The relative expression ofOsWRKY53 inOsWRKY53-OE-1,OsWRKY53(SD)-OE-3, and wild type (WT). The expressionlevel inWTwas set as “1”; datawere shown asmeans6 SE (n= 3). C, The statistical analysis of leaf angles inOsWRKY53-OE-1 andOsWRKY53(SD)-OE-3. Data are means6 SE (n = 20). D and E, The grain phenotype (scale bar = 5mm;D) and the quantification ofgrain length and grain width of OsWRKY53-OE-1, OsWRKY53(SD)-OE-3, and WT (E). Data are means 6 SE (n = 50). F, The leafinclination of OsWRKY53-OE-1, OsWRKY53(SD)-OE-3, and WT in the presence of indicated concentration of 24-epiBL. G,Statistical analysis of leaf inclination in F; data are means6 SE (n = 20). H, The relative expression of D2, OsDWF4, and D11 inWT,OsWRKY53-OE-1, andOsWRKY53(SD)-OE-3. The expression level inWTwas set as “1.”Datawere shown asmeans6 SE (n=3). P values were calculated by Student’s t test; *P , 0.05, and **P , 0.01.


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reduced cell length (Zhang et al., 2011), which is similarto the oswrky53 mutant. Interestingly, OsWRKY53,OsWRKY70, and OsWRKY78 belong to the same sub-group Ia of WRKY genes and show high sequencesimilarity (Xie et al., 2005), implying that this subgroup

of WRKY genes may play common roles in regulatinggrowth and development. However, the underlyingmechanismsmight be diverse.OsWRKY70 overexpressionlines showed strong dwarfism, but no observable effecton leaf angles and seed size, while OsWRKY70 RNAi

Figure 7. BR promotes the accumulation of OsWRKY53 protein but represses its transcription level. A Time course of relativeexpression ofOsWRKY53 in response to BR treatment. The expression level before treatment was set as “1”; data were shown asmeans6 SE (n = 3). The expression of OsDWF4was examined as control. B, Protein gel blot ofMYC-OsWRKY53-OE treatedwith1 mM BR. The heat shock protein was used as internal control. C, Protein gel blot ofMYC-OsWRKY53-OE treated with 5 mM BRZ.The HSP was used as internal control. D, The relative expression of OsWRKY53 in response to BRZ treatment. The expressionlevel in dimethyl sulphoxide treatment was set as “1”; data were shown as means 6 SE (n = 3). E, The schematic diagrams ofOsWRKY53. The position of PCR primers (P1, P2, and P3) used for detecting the relative expression of internal and ectopicOsWRKY53 was indicated. DNA fragments (a and b) were used for ChIP; the black square is the conserved W-box region. F,Relative expression levels of internal OsWRKY53 in oswrky53 mutant plants. The expression level in wild type (WT) was set as“1”; data were shown as means6 SE (n = 3). G, Relative expression levels of internal and ectopicOsWRKY53 inOsWRKY53-OEplants. The expression level in WTwas set as “1”; data were shown as means6 SE (n = 3). H, Schematic diagrams of the effectorand reporter plasmids used in the transient assay in rice protoplasts. REN, Renilla luciferase; LUC, firefly luciferase. I, The relativeLUC activity expressed with reporter 35S:REN-OsWRKY53Pro:LUC together with control (empty vector) or 35Spro-OsWRKY53effector. Data were shown as means6 SE (n = 5). J, OsWRKY53 binds to the conserved W-box of theOsWRKY53 promoter. TheW-box close to ATG was shown as reprehensive. The W-box mutated to AAAAAA was used as competitive probes; maltose-binding protein was used as a negative control. K, ChIP assays showing that OsWRKY53 binds to the promoter of OsWRKY53in vivo. Immunoprecipitation was performed with anti-OsWRKY53 antibody. Immunoprecipitated chromatin was analyzed byRT-qPCR using primers indicated in (D). RT-qPCR enrichment was calculated by normalizing to actin and to the total input of eachsample. Data were shown as means 6 SE (n = 3). P values were calculated by Student’s t test; *P , 0.05, and **P , 0.01.



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lines were similar to wild type, and OsWRKY70 wassuggested as growth suppressor by inhibiting GA bio-synthesis (Li et al., 2015). In contrast, in this study, bothOsWRKY53-OE and oswrky53 showed opposite BR-related phenotypes and BR sensitivities. Therefore, weproposed that OsWRKY53 is a positive regulator of riceBR signaling. It is worthy to note that two previousstudies also generated the OsWRKY53 overexpres-sion lines; however, while one study showed thatOsWRKY53-OE plants have dwarfism and larger leafangles (Hu et al., 2015), another study showed thenormal growth of the OsWRKY53-OE plants (Chujoet al., 2007). We speculated that these differences mayeither result from different rice varieties used for theanalyses or different expression levels of OsWRKY53 intransgenic lines.

Interestingly, it had been shown that OsWRKY53 canenhance the defense response to pathogen (Chujo et al.,2007; Chujo et al., 2014), and our data show thatOsWRKY53 positively regulates BR signaling as well. Itis reasonable to speculate that active OsWRKY53 canpositively regulate both BR signaling and defense re-sponse, which greatly support the previous conclusionthat BR can enhance the pathogen response in rice(Nakashita et al., 2003) and also suggest thatOsWRKY53might be a new node mediating the crosstalk betweenthe growth versus defense response.

Moreover, OsWRKY53 transcription is induced bypathogen infection, wounding, and herbivore attack(Chujo et al., 2007; Hu et al., 2015), whereas is repressedby BR (this study). Nevertheless, OsWRKY53 positivelyregulated BR (this study) and pathogen response(Chujo et al., 2007, 2014), but negatively regulatedherbivore-induced defenses (Hu et al., 2015), suggest-ing that OsWRKY53 might mediate cross talk amongdiverse signaling pathways and play diverse roles incontext-dependent conditions (Supplemental Fig. S12).

MAPK Module in Regulating Rice BR Signaling

Plant MAPK plays crucial roles in multiple signal-transduction pathways, such as plant immunity re-sponse and hormone signaling (Tena et al., 2001;Ishihama et al., 2011; Shen et al., 2012; Chujo et al., 2014;Adachi et al., 2015). Rice dwarf and small grains1 andsmall grains1 were caused by mutations in OsMAPK6and OsMAPKK4, respectively, and both showed small-grain phenotype (Duan et al., 2014; Liu et al., 2015).OsMAPK6 interacts with OsMAPKK4, implying thatthe OsMAPKK4-OsMAPK6 cascade was also involvedin regulating seed development (Liu et al., 2015). Fur-ther studies indicated that the mechanism of OsMAPK6and OsMKK4 in controlling seed size is through regu-lating BR response and expression of BR-related genes(Duan et al., 2014; Liu et al., 2015). However, thedownstream target of the OsMAPKK4-OsMAPK6 cas-cade involved in controlling seed size and BR signalinghas not been identified. In rice, OsMAPK6 interactswith and can phosphorylate OsWRKY53, and the

OsMAPKK4-OsMAPK6-OsWRKY53 module playskey roles in pathogen, wounding, and herbivore-induced defense response (Chujo et al., 2007, 2014;Yoo et al., 2014; Hu et al., 2015). In this study, weshowed that OsMAPK6 can phosphorylate OsWRKY53in an OsMAPKK4-dependent manner, and this phos-phorylation is required for the biological function ofOsWRKY53 in regulating BR-related responses. Col-lectively, we proposed that OsWRKY53 is one of thedownstream targets of the OsMAPKK4-OsMAPK6cascade in mediating BR signaling as well (SupplementalFig. S12). Very interestingly, during preparation of thismanuscript, it was reported that the ArabidopsisWRKY46/54/70 are positively involved in BR-regulatedgrowth and negatively involved in drought responses;WRKY54 can be phosphorylated by BIN2 and interactwith BES1 to cooperatively regulate the expression oftarget genes (Chen et al., 2017). These findings in Ara-bidopsis greatly support our conclusion that OsWRKY53can positively regulate BR signaling in rice, which impliedthat the some WRKY family members may play conserveroles in regulating plant BR signaling to some extent.However, their functionmechanismsmight not be similar.First, AtWRKY46/54/70 are not the OsWRKY53’s homo-logswith the highest sequence similarity. AtWRKY46/54/70 belong to group IIIWRKY proteins, which contains oneWRKYdomain,whereasOsWRKY53 contains twoWRKYdomains and belongs to group I WRKY proteins. Second,we showed that phosphorylation of OsWRKY53 byOsMAPKK4/OsMAPK6 is required for function ofOsWRKY53, and the five Ser residues in the SP clusterof OsWRKY53 are the critical phosphorylation sites.But there is no SP cluster in AtWRKY46/54/70, andwhether AtWRKY46/54/70 can be phosphorylated byMAPK is not known. Third, we need to test the physicaland genetic interaction of OsWRKY53 with rice BRsignaling components (OsBZR1 and OsGSK2) to fur-ther investigate the function mechanism of OsWRKY53in rice BR-signaling pathway. Collectively, there is stillmuch work to be done to prove whether functionmechanisms of OsWRKY53 and AtWRKY46/54/70 aresimilar or not.

WRKY family genes function as transcriptional acti-vator or repressor by bindingW-boxmotif in promotersof target genes (Han et al., 2014; Adachi et al., 2015).Previously, it was reported that OsWRKY53 can func-tion as transcriptional activator in pathogen response(Chujo et al., 2007, 2014), but as a suppressor in herbi-vore response (Hu et al., 2015), although the directtarget genes and the underlying mechanism remainedunknown. In this study, we showed that OsWRKY53could function as a transcription repressor to inhibit itsown expression via directly binding to W-box elementsin promoter. Considering the functional diversity ofOsWRKY53, it is reasonable to speculate that OsWRKY53might regulate diverse target genes in response to differ-ential environmental stimuli, and it is of interest to identifythe direct target genes that are involved in specific func-tions of OsWRKY53.


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MATERIALS AND METHODS

Plant Materials and Growth Conditions

Rice (Oryza sativa) cultivar Longjing 11 (Oryza sativa ssp. japonica) was usedfor generate OsWRKY53 transgenic plants. d61-2 (Yamamuro et al., 2000) wasused to develop double mutant. Plants were grown in the field (natural long-day conditions) or growth chamber at 30°C for 14 h (day) and 24°C for 10 h(night). For transient induction analysis, 2-week-old seedlings were sprayedwith 1 mM 24-epiBL (Sigma, E1641), and then whole plants were harvested atvarious time points. For BRZ treatment, the 3-d-old plants were moved tomedium with 5 mM BRZ for 7 d (Sigma, SML 1406). For BR-related gene ex-pression, 2-week-old seedlings were sampled. Materials were collected andfrozen in liquid nitrogen immediately or stored at 280°C for RNA extraction.

Lamina Joint Assay

The lamina joint assay using excised leaf segmentswas performed as describedpreviously (Tong et al., 2009). Simply, uniformgerminated seedswere selected andgrown in the dark for 8 d at 30°C. The entire segments comprising 1 cm of thesecond leaf blade, the lamina joint, and 1 cm of the leaf sheath were incubated invarious concentrations of 24-epiBL for 48 h in dark. The angles of lamina jointbending were measured using ImageJ software (http://rsbweb.nih.gov/ij/).

Generation of Transgenic Plants and Double Mutant

To generate the OsWRKY53 overexpression plant, the coding regions ofOsWRKY53 (Os05g0343400) was amplified by PCR and cloned into p1390U, andUbiquitinpro: OsWRKY53 construct in pCAMBIA1300 backbone was made. Forcreating 35Spro: MYC-OsWRKY53, OsWRKY53 in pENTRY vector was clonedinto pGWB18 through LR recombination reaction (Nakagawa et al., 2007). Forthe generation of oswrky53mutant, the target sequence (Supplemental Table S1)was synthesized and ligated with respective sgRNA catastases and then weresequentially ligated into the CRISPR/Cas9 binary vectors pYLCRISPR/Cas9Pubi-Has described (Ma et al., 2015). To generateOsWRKY53(SA) andOsWRKY53(SD),the mutation was introduced by point mutation kit following the manufac-turer’s instructions. Primers used to generate the above-mentioned constructsare listed in Supplemental Table S1, and all of the constructs were confirmed bysequencing. The constructs were introduced into Agrobacterium tumefaciensstrain EHA105. Cultivar Longjing11 was used as the recipients for Agrobacterium-mediated transformation as described previously (Hiei et al., 1994). HomozygousT2 transgenic rice seedlings were used for the phenotype analysis.

To generate d61-2 OsWRKY53-OE, we crossed d61-2 (Yamamuro et al., 2000)with OsWRKY53-OE#1 (Fig. 1A). The mutation site and expression level ofcorresponding genes was examined by DNA sequence and RT-qPCR.

Total RNA Isolation and RT-qPCR Analysis

Total RNAwas extracted usingTRIzol (Invitrogen) and treatedwithDNaseI.cDNA was synthesized from 2 mg of total RNA using SuperscriptII ReverseTranscriptase (Invitrogen). Real-time PCR was performed with SYBR GreenPCR master mix (Takara). Data were collected using Bio-Rad chromo 4 real-time PCR detector. All expressions were normalized against the Actin gene(Os03g0718100). The primers used are listed in Supplemental Table S1. Threebiological repeats were performed for each analysis. Values are means 6 SE ofthree biological repeats.

Yeast Two-Hybrid Assay

The full-length coding sequence of OsWRKY53 and the OsMAPK6 werecloned into pGADT7 and pGBKT7, respectively. These two vectors and corre-sponding empty vectors were cotransformed into the yeast strain Y2H gold. Theyeast two-hybrid assays was performed as per the kit’s instructions.

BIFC Assay

For BiFC assays, theOsMAPK6 andOsWRKY53were fusedwith partial GFPand generated nGFP-OsMAPK6 and cGFP-OsWRKY53. These vectors weretransformed into Agrobacterium strain GV3101 and coinjected into young leavesof Nicotiana benthamiana. The fluorescence was observed by confocal micros-copy (Leica) after 2 d growth.

LCI Assay

The LCI assays for the interaction between OsMAPK6 andOsWRKY53wereperformed in N. benthamiana leaves. The full-length OsMAPK6 and OsWRKY53coding region were fused with the N-terminal part and C-terminal part of theluciferase reporter gene, respectively. Agrobacteria harboring nLUC-OsMAPK6and cLUC-OsWRKY53 constructs were coinfiltrated into N. benthamiana, andthe infiltrated leaves were analyzed for LUC activity 48 h after infiltration usingChemiluminescence imaging (Tanon 5200).

EMSA

Full-length coding sequence ofOsWRKY53was cloned intoPVP13 vector viaan LR recombination reaction to generate MBP-OsWRKY53 fusion protein andtransformed into Escherichia coli strain BL21 (DE3). The recombinant proteinswere affinity purified using Amylose Resin (New England Biobabs, cat. no.E8021S). About 40-bp length oligonucleotide probes containing wide-typeW-box (TTGACC) or mutated W-box (AAAAAA) motifs were synthesizedand labeled with biotin using EMSA Probe Biotin Labeling Kit (Beyotime, cat.no. GS008). For nonlabeled probe competition, nonlabeled probe was addedto the reactions. EMSA was performed using a Chemiluminescent EMSAkit (Beyotime, cat. no. GS009). Probe sequences are shown in SupplementalTable S1.

Transient Transcription Dual-Luciferase Assay

Full-length coding sequence ofOsWRKY53was cloned into KpnI and BamHIsite of pRT107, and effector (35Spro:OsWRKY53) was generated. The promoterregions (upstream of the ATG) of OsWRKY53 were amplified and cloned intoSalI and BamHI site of pGreenII 0800-LUC vector and used as reporter (Hellenset al., 2005). The resulting effector and reporter constructs were cotransfectedinto protoplasts prepared from 2-week-old rice seedlings. Protoplast isolationand polyethylene glycol transformation was carried out as described (Huanget al., 2015). The Renilla luciferase (REN) gene directed by 35S promoter in thepGreenII 0800-LUC vector was used as an internal control. Firefly LUC and RENactivities were measured with a Dual-Luciferase reporter assay kit using aGloMax 20/20 luminometer (Promega). The LUC activity was normalized toREN activity, and LUC/REN ratios were calculated. For each plasmid combi-nation, five independent transformations were performed. Values are means6SE of five biological repeats. Primers used for these constructs are listed inSupplemental Table S1.

ChIP Assay

OsWRKY53-OEwas used for ChIP assay as previously described (Zhu et al.,2012). In brief, approximately 2 g of rice seedlings was cross linked in 1% for-maldehyde under a vacuum, and cross-linking was stopped with 0.125 M Gly.The sample was ground to a powder in liquid nitrogen and used to isolatenuclei. Anti-OsWRKY53 (1:150 dilutions) was used to immunoprecipitate theprotein-DNA complex, and the precipitated DNAwas recovered and analyzedby quantitative PCR. Chromatin precipitated without antibody was used as acontrol. The data are presented as means 6 SE of three biological repeats. Anti-OsWRKY53 (AbP80050-A-SE) was ordered from Beijing Protein innovation.Primers used for ChIP-qPCR are listed in Supplemental Table S1.

In Vitro Kinase Assay

The full-length OsMAPK6 and OsMAPKK4DD was cloned into pDEST15vector via an LR recombination reaction and transformed into E. coli strain BL21(DE3). OsMAPKK4DD was generated as described (Chujo et al., 2014). Primersused for these constructs are listed in Supplemental Table S1. The recombinantproteins were affinity purified using Glutathione Sepharose 4B beads (GEHealthcare). For each kinase assay, 0.5 mg MBP-OsWRKY53 or mutated fusionprotein MBP-OsWRKY53 (SA) and 0.3 mg glutathione S-transferase (GST)-OsMAPK6, GST-OsMAPKK4DD, or GST were incubated with phosphorylationbuffer (25 mM Tris, pH 7.4, 12 mM MgCl2, 1 mM dithiothreitol, and 1 mM ATP).The reactions were incubated at 30°C for 45 min and boiled with 53 sodiumdodecyl sulfate (SDS) loading buffer then separated by SDS-polyacrylamide gelelectrophoresis. The protein was transferred on polyvinylidene difluoridemembrane (Millipore), and phosphorylation signal was detected by Phos-tagBiotin BTL-104 (Wako) according to the manufacturer’s instruction.



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Transient Expression Assay in Rice Protoplast

The coding region ofOsMAPKK4 DD,OsMAPK6, andOsWRKY53 fusedwithMYC tagwere ligated into the pRT107vector to generate the 35Spro: OsMAPKK4

DD,35Spro: OsMAPK6, and 35Spro: MYC-OsWRKY53 constructs. Rice protoplast wasisolated from stem and sheath tissues ofwild-type young seedlings as describedpreviously (Chen et al., 2006). Different combinations of plasmid DNAs (about10 mg DNA of each construct) were transiently expressed in protoplasts byPEG-mediated transfection procedure.

After overnight incubation in dark at 28°C, total proteins were isolated fromrice protoplasts with extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl,0.2%NP-40, 0.1% Triton X-100, 5 mM ethylenediaminetetraacetic acid disodiumsalt, complete protease inhibitor cocktail, and 50 mM MG132), and 53 SDSbuffer was added and boiled for 5 min. The samples were loaded on a 10%SDS-polyacrylamide gel electrophoresis for immunoblotting with anti-MYC antibody (Abmart, M20002L) and anti-HSP antibody (BGI Tech,AbM51099), respectively.

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data-base under the following accession numbers: OsWRKY53 (Os05g0343400);OsMAPKK4 (Os02g0787300); OsMAPK6 (Os06g0154500).

Supplemental Data

The following supplemental materials are available.

Supplemental Figure S1. Enlarged leaf angle of OsWRKY53 overexpres-sion lines.

Supplemental Figure S2. Expression of cell-enlargement-related gene inOsWRKY53-OE.

Supplemental Figure S3. Pale green phenotype of OsWRKY53-OE plant.

Supplemental Figure S4. Identification of OsWRKY53 overexpressionlines.

Supplemental Figure S5. Identification of oswrky53 mutant.

Supplemental Figure S6. The leaf of oswrky53 is dark green.

Supplemental Figure S7. OsWRKY53 phosphorylation by OsMAPK6 en-hances the DNA binding activity of OsWRKY53 protein without affect-ing its stability.

Supplemental Figure S8. Phenotypic analysis of OsWRKY53(SA)-OE.

Supplemental Figure S9. Characterization of MYC-OsWRKY53-OE.

Supplemental Figure S10. Spatial expression pattern of OsWRKY53.

Supplemental Figure S11. Phenotype and quantification of unhuskedgrain of diverse lines.

Supplemental Figure S12. Schematic diagram of working model ofOsWRKY53.

Supplemental Table S1. List of primers.

ACKNOWLEDGMENTS

The authors thank our laboratory members for their helpful comments anddiscussions during the article preparation. They thank Prof. Chengcai Chu,Prof. Wenqiang Tang, and Dr. Jiuyou Tang for their comments on manuscriptand helpful suggestions. They thank Prof. Enamul Huq for his critical readingand revision of the manuscript.

Received July 13, 2017; accepted September 6, 2017; published September 11,2017.

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Transcription Factor OsWRKY53 Positively Regulates Brassinosteroid Signaling … · Transcription Factor OsWRKY53 Positively Regulates Brassinosteroid Signaling and Plant Architecture1