cotton wrky1 mediates the plant defense-to- development ... · chao li, xin he, xiangyin luo, li...

Cotton WRKY1 Mediates the Plant Defense-to-Development Transition during Infection of Cotton byVerticillium dahliae by Activating JASMONATEZIM-DOMAIN1 Expression1[C][W]

Chao Li, Xin He, Xiangyin Luo, Li Xu, Linlin Liu, Ling Min, Li Jin, Longfu Zhu*, and Xianlong Zhang

National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, Hubei430070, China

Plants have evolved an elaborate signaling network to ensure an appropriate level of immune response to meet the differingdemands of developmental processes. Previous research has demonstrated that DELLA proteins physically interact withJASMONATE ZIM-DOMAIN1 (JAZ1) and dynamically regulate the interaction of the gibberellin (GA) and jasmonate (JA)signaling pathways. However, whether and how the JAZ1-DELLA regulatory node is regulated at the transcriptional level inplants under normal growth conditions or during pathogen infection is not known. Here, we demonstrate multiple functionsof cotton (Gossypium barbadense) GbWRKY1 in the plant defense response and during development. Although GbWRKY1expression is induced rapidly by methyl jasmonate and infection by Verticillium dahliae, our results show that GbWRKY1 is anegative regulator of the JA-mediated defense response and plant resistance to the pathogens Botrytis cinerea and V. dahliae.Under normal growth conditions, GbWRKY1-overexpressing lines displayed GA-associated phenotypes, including organ elongationand early flowering, coupled with the down-regulation of the putative targets of DELLA. We show that the GA-related phenotypesof GbWRKY1-overexpressing plants depend on the constitutive expression of Gossypium hirsutum GhJAZ1. We also show thatGhJAZ1 can be transactivated by GbWRKY1 through TGAC core sequences, and the adjacent sequences of this binding site areessential for binding specificity and affinity to GbWRKY1, as revealed by dual-luciferase reporter assays and electrophoretic mobilityshift assays. In summary, our data suggest that GbWRKY1 is a critical regulator mediating the plant defense-to-developmenttransition during V. dahliae infection by activating JAZ1 expression.

To survive and thrive, plants have evolved sophisti-cated mechanisms to allocate limited resources to rap-idly activate both local and systemic immune responses(Robert-Seilaniantz et al., 2011). It has been stated thatthe activation of defense pathways occurs at the expenseof plant growth (Kazan and Manners, 2009, 2012). How-ever, vegetative growth and reproductive success inplants are directly related to population development,so plants require optimal immune activation to maintaingrowth and development (Walters and Heil, 2007;Pieterse et al., 2009).

Jasmonate (JA) is a fatty acid-derived plant hormonethat is perceived by an F-box protein, CORONATINE

INSENSITIVE1 (COI1), a component of the SKIP-CULLIN-F-box-type (SCF) E3 ubiquitin ligase complex (Katsir et al.,2008; Yan et al., 2009; Sheard et al., 2010). JASMONATEZIM-DOMAIN (JAZ) family proteins are transcriptionalrepressors that negatively regulate JA signaling via directinteraction with several transcription factors, such as basichelix-loop-helix (bHLH) subgroup IIIe transcription factors(myelocytomatosis viral oncogene2 [MYC2], MYC3, andMYC4) and myeloblastosis (MYB) transcription factors(MYB21, MYB24, and MYB57; Cheng et al., 2009, 2011;Fernández-Calvo et al., 2011; Niu et al., 2011; Qi et al., 2011;Song et al., 2011, 2013). In the presence of JA, JAZ proteinsare targeted by the SCFCOI1 complex for ubiquitination anddegradation, which consequently relieves the repressionand rapid activation of JA responses (Chini et al., 2007;Thines et al., 2007; Howe, 2010). Moreover, a recent studyshowed that JAZ1 physically interacts with DELLAproteins, repressors of GA signaling, to dynamicallycoordinate the balance between GA signaling and JAsignaling (Hou et al., 2010; Robert-Seilaniantz et al., 2011).The attenuation of JA signaling via the overexpression ofseveral JAZ proteins can activate the GA signaling path-way, which is consistent with the common phenomenonof the so-called growth-defense conflict (Kazan andManners, 2009, 2012; Yang et al., 2012). However, one ofthe open questions is, which regulators are involved in theorchestration of development and disease resistance?

1 This work was supported by the National High-Tech Program863 (grant no. 2013AA102601–4), the Ministry of Agriculture of China(grant no. 2014ZX0800503B), and the Program of Introducing Talentsof Discipline to Universities in China (grant no. B14032).

* 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:Longfu Zhu ([email protected]).

[C] Some figures in this article are displayed in color online but inblack and white in the print edition.

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.114.246694

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WRKY transcription factors have been implicated invarious transcriptional programs, including biotic andabiotic stress responses, growth, and development (Pandeyand Somssich, 2009; Rushton et al., 2010, 2012; Van Akenet al., 2013). WRKY family members are known for theirconserved DNA-binding region, which is called theWRKYdomain, usually found at the N terminus (Rushton et al.,1995, 2010). Based on the number of WRKY domainsand the structural features of the zinc-finger motifs atthe C terminus, WRKY transcription factors are classifiedinto three groups (Eulgem et al., 2000). Intensive studieshave demonstrated that most WRKY transcription factorsspecifically recognize the DNA-binding site termed theW-box (TTGACC/T; Rushton et al., 1995, 2010). However,a recent study demonstrated that the TGAC core sequenceis sufficient to be targeted for binding by the AtWRKY28protein (van Verk et al., 2011). Some other WRKY trans-cription factors, such as rice (Oryza sativa) OsWRKY13 andHordeum vulgare HvWRKY46, can bind to non-W-box se-quences (Cai et al., 2008; Mangelsen et al., 2008). Gel-shiftexperiments also suggest that different groups of WRKYtranscription factors show different binding site preferencesto recognize TTGACC and TTGACT (Ciolkowski et al.,2008). Despite the W-box being required for the binding ofthe WRKY protein, the sequences that are adjacent to theW-box also play an important role in binding selectivity(Ciolkowski et al., 2008).

Accumulating evidence indicates that some WRKYtranscription factors exhibit multiple regulatory rolesand are involved in several seemingly disparate processes.HvWRKY1/38 is a key regulator of cold and drought re-sponses, immune response, and seed germination (Marèet al., 2004; Shen et al., 2007; Zou et al., 2008). OsWRKY13is involved in plant resistance to bacterial infection andresponds to drought stress by selectively binding to dif-ferent target genes (Deng et al., 2012b; Xiao et al., 2013).AtWRKY75 was first identified as a critical modulatorof phosphate uptake and phosphate stress responses(Devaiah et al., 2007). Subsequently, AtWRKY75 has beenimplicated in plant defense to Pseudomonas syringae, andwrky75 mutant plants display increased susceptibility todifferent P. syringae strains (Encinas-Villarejo et al., 2009).Moreover, the overexpression of AtWRKY75 and its riceorthologOsWRKY72 causes early flowering in Arabidopsis(Arabidopsis thaliana; Yu et al., 2010). In addition toAtWRKY75, other WRKY transcription factors, such asWRKY6, WRKY7, WRKY18, and WRKY70, also exhibitmultiple regulatory roles in immune response and flow-ering time control (Chen and Chen, 2002; Robatzek andSomssich, 2002; Li et al., 2004; Kim et al., 2006, 2008),although the molecular mechanism of such a transitionremains unclear.

With the limitation of energy resources, plants haveevolved an elaborate signaling network to prioritize bioticand abiotic stress responses and growth development forsurvival (Kazan and Manners, 2009, 2012; Pauwels et al.,2009; Alcázar et al., 2011; Claeys and Inzé, 2013). There-fore, new information on how the multiple regulatoryfunctions of WRKY transcription factors are integratedinto a dynamic web is fundamentally important for our

understanding of how plants coordinate growth anddevelopment in response to environmental cues.

To identify genes that are differentially expressed incotton (Gossypium barbadense) resistance to Verticilliumdahliae, we constructed a suppression subtractive hybrid-ization complementary DNA (cDNA) library of cottonafter inoculation with V. dahliae, from which GbWRKY1was isolated and characterized as having similar regulatoryfunctions in phosphate stress responses to its Arabidopsishomolog AtWRKY75 (Xu et al., 2012b). In this study, wereport the characterization of multiple roles for GbWRKY1in JA-mediated disease resistance and GA-mediateddevelopment. Our results indicate that the dual regu-latory roles of GbWRKY1 depend on the expression ofGossypium hirsutum GhJAZ1, which we propose is trans-activated by GbWRKY1 in vivo. We further demonstratethat the TGAC core sequence of the GhJAZ1 promoterand its adjacent DNA sequences are essential for thebinding specificity and affinity for GbWRKY1.

RESULTS

Down-Regulation of Cotton GbWRKY1 Enhances PlantResistance to V. dahliae and Botrytis cinerea

We previously identified a subset of genes that areinvolved in the cotton immune response to V. dahliaeby screening a cDNA library from the cotton ‘7124’ (Xuet al., 2011a). Among these genes,GbWRKY1was inducedby V. dahliae strain V991 at the early stage of infection(Supplemental Fig. S1). To elucidate the putative role ofGbWRKY1 during cotton defense to V. dahliae, tobaccorattle virus (TRV)-based virus-induced gene silencing(VIGS) was employed to knock down the transcript ofGbWRKY1 in cotton. Two weeks after inoculation withthe recombinant viruses, the expression of GbWRKY1was efficiently suppressed compared with the TRV:00control through reverse transcription (RT)-PCR analysis(Fig. 1A). Subsequently, the plants were either inoculatedwith the V. dahliae strain V991 or received a mock treat-ment. Typical disease symptoms caused by V. dahliae,including extensive chlorosis, necrosis of leaves, and darkbrown streaks in the stem, were more severe in TRV:00plants compared with those in TRV:GbWRKY1 plants(Fig. 1A; Supplemental Fig. S2A). The lower disease in-dex also indicated that the down-regulation ofGbWRKY1through VIGS could increase the resistance of cotton toV. dahliae (Supplemental Fig. S2C). We also infected withthe typical necrotroph B. cinerea to examine the role ofGbWRKY1 in plant immunity. Cotton leaves from thesame stem nodes were detached and infected with 8 mLof B. cinerea spore suspension (2 3 105 spores mL21). Asshown in Supplemental Figure S2, B and C, the diseaselesions in the TRV:00 leaves extended more rapidlycompared with those in the TRV:GbWRKY1 leaves.

We further generated stable transgenic cotton lines toup-regulate or down-regulate the expression of GbWRKY1via an overexpression or RNA interference (RNAi) strat-egy, respectively, via Agrobacterium tumefaciens-mediatedtransformation. After two generations of selfing, we

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identified two GbWRKY1-silencing lines (Ci2 and Ci3)and two GbWRKY1-overexpressing lines (COV4 andCOV7), which were from independent transgenic events,for further experiments (Fig. 1B). Similar to the resultsthrough VIGS, silencing of GbWRKY1 improved cottonresistance to B. cinerea and V. dahliae, while the over-expression of GbWRKY1 compromised this resistance(Fig. 1, C and D). The corresponding disease index andaverage lesion diameter data also support these results(Supplemental Fig. S2D).To better understand the molecular mechanisms

of GbWRKY1 in the immune response, GbWRKY1-overexpressing Arabidopsis plants were also generated.Northern blotting indicated that the GbWRKY1 transcriptwas significantly up-regulated in the two independentlines AOV4 and AOV9 (Fig. 1E). Similar to the cotton dis-ease assay, the overexpression of GbWRKY1 in Arabidopsisalso compromised resistance to B. cinerea and V. dahliae

(Fig. 1, F and G; Supplemental Fig. S2E). Meanwhile,GbWRKY1-overexpressing plants showed an early-floweringphenotype, whether inoculated with V. dahliae or not(Fig. 1G). This indicates that GbWRKY1 is involved notonly in the regulation of plant immunity but also in plantdevelopment.

GbWRKY1 Negatively Regulates the JA Signaling Pathway

The central role of JA in plant responses to necrotrophicpathogens has been demonstrated in a number of plantspecies (Veronese et al., 2006; Mengiste, 2012). Moreover,Verticillium wilt Ve1-mediated resistance is compromisedin the Arabidopsis JA response mutant jar1-1 (Fradinet al., 2011), and the JA signaling pathway can be activatedin cotton by inoculation with V. dahliae (Gao et al., 2013),suggesting that JA plays an important role in cotton re-sistance to V. dahliae.

Figure 1. GbWRKY1 is a negative regulator of cotton resistance to V. dahliae and B. cinerea. A, TRV:00 plants and TRV:GbWRKY1plants 7 d after inoculation with V. dahliae. The GbWRKY1 knockdown plants exhibited enhanced resistance to V. dahliae. B,Northern-blot analysis to examine the expression of GbWRKY1 in two independent RNAi lines (Ci2 and Ci3) and two inde-pendent overexpression lines (COV4 and COV7). rRNA, Ribosomal RNA. C, Wild-type (WT) and transgenic plants 4 d afterinoculation with B. cinerea. D, Wild-type and transgenic plants 9 d after inoculation with V. dahliae. E, Northern-blot analysisto examine the expression of GbWRKY1 in two independent overexpression lines (AOV4 and AOV9) in Arabidopsis. F and G,Plants at 5 and 15 d after inoculating B. cinerea and V. dahliae spore suspension (2 3 105 spores mL21) on 20-d-old soil-grownArabidopsis, respectively, by spraying or dipping. Disease assays were repeated at least three times. [See online article for colorversion of this figure.]

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GbWRKY1 Prioritizes Development over Defense





To further evaluate the role of JA in cotton’s defenseagainst V. dahliae, cotton seedlings were treated withmethyl jasmonate (MeJA) 2 d before inoculation withV. dahliae. Typical chlorosis and necrosis of infected leaveswere delayed in MeJA-treated plants compared withcontrol plants, suggesting that the JA signaling pathwaypositively contributes to cotton resistance to V. dahliae(Fig. 2, A and B). To test the potential involvement ofGbWRKY1 in JA signaling, we analyzed the expres-sion of GbWRKY1 in response to MeJA and measuredthe content of JA in GbWRKY1-silencing and -over-expressing cotton plants. Interestingly, the GbWRKY1transcript was significantly and rapidly induced byMeJA (Supplemental Fig. S3), while no significantdifferences in the endogenous level of JA were foundbetween the wild type and GbWRKY1 transgenic cottonlines (Supplemental Fig. S4).

To determine whetherGbWRKY1 could negatively affectthe JA-mediated defense response, we checked the ex-pression of cotton pathogenesis-related protein GbPR4,which is a molecular marker of the JA signaling path-way in cotton (Xu et al., 2011b; Gao et al., 2013).Quantitative reverse transcription (qRT)-PCR analysisdemonstrated that the expression of GbPR4 was highlyinduced by MeJA in the GbWRKY1-silencing cotton lineCi2, while only a slight up-regulation of GbPR4 tran-script could be found in the GbWRKY1-overexpressingcotton line COV4 after MeJA treatment (Fig. 2C), indicatingthat overexpression of GbWRKY1 suppressed the MeJA in-duction of GbPR4. Meanwhile, overexpression of GbWRKY1appeared to repress the expression of plant defensin PDF1.2in Arabidopsis plants (Fig. 2D).

To further verify the role of GbWRKY1 in the JA sig-naling pathway, JA-induced anthocyanin accumulationwas compared between wild-type and GbWRKY1-overexpressing Arabidopsis lines. Anthocyanin accu-mulated nearly 19-fold in wild-type plants and only 8-foldin GbWRKY1-overexpressing plants in the presence ofMeJA compared with mock treatments (Fig. 2, E and F).JA-regulated anthocyanin accumulation was mediatedby JAZ proteins marked by the down-regulation of threeanthocyanin biosynthetic genes (UDP-Glc:flavonoid3-Oglucosyltransferase [UF3GT], leucoanthocyanidin dioxy-genase [LDOX], and dihydroflavonol reductase [DFR]) inthe dominant-negative transgenic plant JAZ1△3A (Qiet al., 2011). Consistent with this result, the expressionsof UF3GT, LDOX, and DFR were also down-regulatedby GbWRKY1 after MeJA treatment (Fig. 2G). These re-sults demonstrate that GbWRKY1 might be an importantnegative regulator in the JA signaling pathway.

GbWRKY1-Overexpressing Plants Display GA-RelatedPhenotypes Coupled with the Down-Regulation ofPutative DELLA Target Genes

Although the transcript ofGbWRKY1 barely respondedto GA treatment (data not shown), the overexpressioncotton lines exhibited morphological phenotypes similarto those of GA-overdose cotton plants under normalgrowth conditions as described by Xiao et al. (2010),

including petiole elongation and pale green leaves (Fig. 3,A, C, and E). GA-mediated elongation and the lossof green color rely on the alteration of cell length andchlorophyll accumulation, respectively (Cheminant et al.,2011; de Saint Germain et al., 2013). As shown in Figure 3,B and D, GbWRKY1-overexpressing cotton plants displaylonger petiole epidermal cells than those of the wild typeas observed with a scanning electron microscope. More-over, the chlorophyll level was reduced in GbWRKY1-overexpressing plants (Fig. 3F), indicating that the effectsof GbWRKY1 on the petiole length and leaf green colorare likely to be due to the changes in cell length andchlorophyll accumulation.

To verify this result, the sensitivity to the GA biosyn-thesis inhibitor paclobutrazol (PAC) was evaluated bycomparing the hypocotyl length in the dark as describedpreviously (Zhang et al., 2011). The results demonstratethat the up-regulation of GbWRKY1 dramatically pro-motes cotton hypocotyl elongation not only under mockconditions but also under PAC treatment conditionscompared with wild-type and GbWRKY1-RNAi plants(Fig. 3G). The wild-type hypocotyl length was signifi-cantly inhibited by PAC treatment, with an inhibitionratio of up to 75%, compared with a ratio of 58% in theGbWRKY1-overexpressing plants (Fig. 3H). This suggeststhat overexpression of GbWRKY1 leads to a reducedsensitivity to PAC in cotton. Interestingly, no observabledifferences in terms of plant morphology and sensitivityto PAC could be found betweenwild-type andGbWRKY1-RNAi plants (Fig. 3, G and H; Supplemental Fig. S5),possibly due to the indirect involvement of GbWRKY1in the GA pathway. Similarly, the positive role ofGbWRKY1in the GA pathway was further confirmed from thephenotypic investigation of transgenic Arabidopsis plants.The GbWRKY1-overexpressing Arabidopsis lines AOV4and AOV9 also displayed GA-related phenotypes, suchas early flowering and longer hypocotyls (Fig. 4A;Supplemental Figure S6, A–C).

Given the consistent GA-associated effects in GbWRKY1-overexpressing Arabidopsis plants and cotton plants, thetranscripts of genes that are involved in GA biosynthesisand catabolism were analyzed in Arabidopsis seedlingsto determine whether GbWRKY1 contributes to GA me-tabolism. The results indicate that the GA catabolic geneAtGA2ox2 was up-regulated in 35S:GbWRKY1 seedlings(Supplemental Fig. S7A).

GA is known to promote GA signaling transductionby destabilizing a number of GA repressors, the DELLAproteins (GIBBERELLICACID INSENSITIVE, REPRESSOROF GIBBERELLIC ACID INSENSITIVE3 [RGA], RGA-LIKE1 [RGL1], RGL2, and RGL3), in Arabidopsis (Harberdet al., 2009). Recently, chromatin immunoprecipitation andmicroarray analyses have identified a group of putativeRGA targets (GIBBERELLIC ACID INSENSITIVE DWARF1[GID1a], GID1b, XERICO, SCARECROW-LIKE3 [SCL3],bHLH137, LOB DOMAIN-CONTAINING PROTEIN40[LBD40], andMYB; Zentella et al., 2007), which are down-regulated in quadruple-DELLAmutant plants (Josse et al.,2011). To determine whether the GA signaling pathway isinfluenced by GbWRKY1, the transcripts of these putative


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DELLA targets (GID1a, GID1b, XERICO, SCL3, bHLH137,LBD40, and MYB) were detected in Arabidopsis throughquantitative PCR. Strikingly, most of their transcript levelswere repressed by GbWRKY1, and the expression ofAtGID1a and AtGID1b was repressed approximately2- to 4.5-fold in GbWRKY1-overexpressing plants (Fig.4B; Supplemental Fig. S7B). The homologs of AtGID1aand AtGID1b in cotton, GhGID1-a and GhGID1-b, havebeen isolated and are strongly inhibited by GA treatmentin cotton ovules (Aleman et al., 2008). Consistent with a

previous study, our qRT-PCR analysis demonstrated thatthe expression of GhGID1-a/GhGID1-b was strongly sup-pressed in cotton leaves after GA treatment (SupplementalFig. S7C), similar to AtGID1a and AtGID1b in Arabidopsis(Zentella et al., 2007). In addition, the transcript levels ofGhGID1-a and GhGID1-b were also significantly inhibitedvia the overexpression of GbWRKY1 (Fig. 4C). This im-plies that the positive regulatory role of GbWRKY1 in theGA pathway is probably via an activation of the GA-responsive pathway downstream of DELLA.

Figure 2. Evaluation of the role ofGbWRKY1 in JA signaling. A, MeJA-induced resistance to V. dahliae. Wild-type (WT) plants weregrown for 15 to 20 d and then infected with V. dahliae. Two days before infection, the roots of each cotton plant were drenchedeach day with 3 mL of MeJA solution or 3 mL of mock solution. B, The disease index of mock- and MeJA-treated cotton plants wascalculated 9 d after V. dahliae infection. C, qRT-PCR analysis of JA-responsive gene (GbPR4) expression in cotton lines with orwithout (Mock) 10 mM MeJA treatment for 5 h. Values are means 6 SD; n = 3. D, qRT-PCR analysis of AtPDF1.2 in GbWRKY1-overexpressing Arabidopsis seedlings (AOV4 and AOV9). Values are means 6 SD; n = 3. E, Arabidopsis seedlings of wild-type,COV4, and COV9 plants grown on 0.53 Murashige and Skoog (MS) medium with or without (Mock) 8 mM MeJA treatment.F, Anthocyanin content of the Arabidopsis seedlings in E. G, qRT-PCR analysis of UF3GT, LDOX, and DFR in wild-type andGbWRKY1-overexpressing Arabidopsis plants with or without (Mock) 10 mM MeJA treatment for 8 h. Values are means6 SD; n = 3.The values are given relative to the housekeeping genes ACTIN in Arabidopsis and ubiquitin (UB7 ) in cotton. Statistical analyseswere performed using Student’s t test: *P, 0.05, **P, 0.01, and ***P, 0.001. [See online article for color version of this figure.]








A recent study demonstrated that DELLA proteinsmodulate flowering time by a direct binding of RGA toSQUAMOSA PROMOTER BINDING-LIKE (SPL) toattenuate the SPL transcriptional activities of SUPPRESSOROF OVEREXPRESSION OF CONSTANS1 (SOC1; Yu et al.,2012). Our results demonstrate that the expression ofSOC1 is consistently up-regulated by the overexpressionof GbWRKY1 during seedling development (Fig. 4D). Toexamine the genetic relationship between GbWRKY1 andSOC1, we employed an F2 population generated bycrossing AOV9with a soc1mutant. The results demonstratethat the early-flowering phenotype of AOV9 in the soc1background is similar to that in the soc1 mutant (Fig. 4E),

indicating that the early-flowering phenotype from theoverexpression of GbWRKY1 depends on the activationof SOC1.

GbWRKY1-Orchestrated Balance between the JA and GASignaling Pathways Depends on the ConstitutiveActivation of G. hirsutum GhJAZ1

In accordance with the reports that an antagonisticinteraction of JA and GA exists in defense and devel-opment (Navarro et al., 2008; Kazan and Manners, 2012),our results demonstrate that GbWRKY1 contributes op-posite functions to JA-mediated immune responses and

Figure 3. Morphological phenotypes of GbWRKY1-overexpressing cotton. A and C, Image and quantification of the petiolelength of wild-type (WT) and GbWRKY1-overexpressing plants, respectively. B and D, Image and quantification of the petioleepidermal cell lengths in A, respectively. Bars = 50 mm. E, Image of cotton with two true leaves. F, Quantification of chlorophyllcontents in E. G, PAC susceptibility of wild-type and GbWRKY1 transgenic seedlings. H, The susceptibility of wild-type andGbWRKY1 transgenic cotton to PAC was scored as relative inhibition ratios (%). The assays were repeated at least three times.The data in C, D, F, and H represent means 6 SD from a minimum of 16 plants. Statistical analyses were performed usingStudent’s t test: **P , 0.01. [See online article for color version of this figure.]


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GA-mediated development. To examine how GbWRKY1coordinates the cross talk between the JA/GA signalingpathways, flowering time was investigated after treat-ment with MeJA. The early-flowering phenotype byGbWRKY1, which is mediated possibly through the GApathway, could be partially suppressed by MeJA (Fig.5A; Supplemental Fig. S8), suggesting that the GA-related phenotypes of GbWRKY1-overexpressing plantsmight be indirectly regulated by JA.To validate the participation of GbWRKY1 in the cross

talk between the JA and GA signaling pathways, theexpression of GhGID1-a/GhGID1-b was examined intransgenic cotton plants under MeJA treatment. In wild-type plants, the expression levels of GhGID1-a andGhGID1-b increased by 2.7- and 2.4-fold after JA treat-ment, respectively (Fig. 5B). Meanwhile, the transcriptlevels of these two genes were up-regulated by 4.5-and 7.6-fold in GbWRKY1 knockdown cotton plants(Fig. 5B), respectively. However, the overexpression of

GbWRKY1 appears to attenuate the induction of GhGID1-aand GhGID1-b in response to MeJA (Fig. 5B). Reciprocally,the expression of the JA-responsive gene GbPR4 was alsoexamined in GbWRKY1 transgenic plants after GA treat-ment (Supplemental Fig. S9) and showed that the GA-mediated change in expression of GbPR4 in wild-type andGbWRKY1 transgenic plants was similar to both mocktreatment and the effect of JA (Fig. 2C), although the ex-pression of GbPR4 showed an opposite regulatory patternafter GA treatment and JA treatment. These expressionanalyses indicate that GbWRKY1 most likely functions inthe JA pathway to indirectly regulate the GA pathway,not the other way around.

Recent studies have demonstrated that the physicalinteraction between JAZ1-DELLA and their dynamicbalance contributes to the antagonism between JA andGA (Hou et al., 2010). The up-regulation of a selectedgroup of JAZ genes (including AtJAZ1, AtJAZ3, AtJAZ4,AtJAZ9, AtJAZ10, and AtJAZ11) in Arabidopsis can

Figure 4. Evaluation of the role ofGbWRKY1 in GA signaling. A, Quantification of the flowering time ofGbWRKY1-overexpressinglines (AOV4 and AOV9) in Arabidopsis. Values are means 6 SD; n = 24 plants. B, qRT-PCR analysis of a set of putative RGA targetgenes in 5-d-old Arabidopsis seedlings. Values are means 6 SD; n = 3. C, qRT-PCR analysis of the expression of GhGID1-a andGhGID1-b inGbWRKY1-overexpressing cotton lines. Values are means6 SD; n = 3. D, qRT-PCR analysis of the expression of SOC1in GbWRKY1-overexpressing Arabidopsis seedlings. Values are means 6 SD; n = 3. E, Flowering phenotypes of AOV9:soc1 F2progeny. Phenotype was evaluated from three replicates, and each replicate contained at least 160 AOV9:soc1 F2 plants. Twoindividual plants from the F2 population were imaged as representatives. PCR amplification of the genomic DNA from the wild type(WT), GbWRKY1-overexpressing Arabidopsis plants (AOV9), the Arabidopsis soc1 mutant, and AOV9:soc1 F2 progeny is shown.The primer sets that were used in the PCR tests are listed in Supplemental Table S1. The assays were repeated three times. Statisticalanalyses were performed using Student’s t test: *P , 0.05, **P , 0.01, ***P , 0.001. The values are given relative to the house-keeping genes ACTIN in Arabidopsis and UB7 in cotton. [See online article for color version of this figure.]








promote GA-mediated development, such as hypocotylelongation and flowering time (Yang et al., 2012). Toexamine whether there is a similar mechanism in reg-ulating the GbWRKY1-mediated orchestration betweendefense and development, the expression of a series ofJAZ genes (AtJAZ1, AtJAZ3, AtJAZ4, AtJAZ9, AtJAZ10,and AtJAZ11) was characterized. Interestingly, onlyAtJAZ1 was up-regulated via the overexpression ofGbWRKY1 in Arabidopsis, and similar results were alsofound in cotton, in which GhJAZ1 showed approximately

3.7- and 1.7-fold activation in two independentGbWRKY1-overexpressing cotton lines (Fig. 5C; Supplemental Fig.S10). We then used GhJAZ1-overexpressing lines (J92and J131) and GhJAZ1-RNAi transgenic cotton lines (JR1and JR3) to assess the interaction between GbWRKY1 andGhJAZ1 in cotton (Supplemental Fig. S11). The resultsdemonstrate that the transcript level of GbWRKY1 wasnot significantly influenced in the GhJAZ1 overexpressionlines and was slightly up-regulated in the GhJAZ1-RNAilines (Fig. 5, D and E). This suggests that GbWRKY1

Figure 5. Evaluation of the role ofGbWRKY1 in coordinating the interaction of GA and JA. A, Flowering time of wild-type (WT)and GbWRKY1-overexpressing Arabidopsis plants (AOV4 and AOV9) after MeJA treatment. B, Analysis of GA-responsive geneexpression in wild-type and GbWRKY1 transgenic cotton plants with or without (Mock) 10 mM MeJA treatment for 5 h. Valuesare means 6 SD; n = 3. C, qRT-PCR analysis of AtJAZ1 and GhJAZ1 in GbWRKY1 transgenic Arabidopsis plants and cottonplants, respectively. Values are means6 SD; n = 3. D and E, qRT-PCR analysis ofGbWRKY1 inGhJAZ1 overexpression lines (J92and J131) and RNAi lines (JR1 and JR3), respectively. Values are means 6 SD; n = 3. F, Petiole length of the wild-type plant,GhJAZ1 RNAi plant (JR3), GbWRKY1 overexpression cotton plant (COV4), F1 hybrid plant of the wild type and COV4(COV4&WT), and F1 hybrid plant of COV4 and JR3 (COV4&JR3). G, Flowering phenotypes of AOV9 and amiRNAJAZ1 hybridprogeny. HY-AOV9, HY-amiRNAJAZ1, and AOV9/amiRNAJAZ1 are three homozygous lines from the AOV9 and amiRNAJAZ1hybrid population. Data were from three replicates, and each replicate contained at least 24 plants. RT-PCR analysis to examinethe expression of GbWRKY1 and AtJAZ1 in HY-AOV9, HY-amiRNAJAZ1, and AOV9/amiRNAJAZ1 plants is shown. The assayswere repeated three times. Statistical analyses were performed using Student’s t test: *P , 0.05, **P , 0.01, and ***P , 0.001.[See online article for color version of this figure.]


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promotes GA-related growth, potentially via the activationof JAZ1.To further elucidate the genetic interaction between

GbWRKY1 and GhJAZ1, a GbWRKY1-overexpressing line(COV4) was crossed with wild-type cotton and aGhJAZ1-RNAi line (JR3), respectively, and the cotyledon petiolelength was compared in the F1 hybrid seedlings. Asshown in Figure 5F, GbWRKY1-mediated petiole elon-gation was rescued in the GhJAZ1 knockdown back-ground. These data indicate that GhJAZ1 is geneticallyepistatic to GbWRKY1 in cotton. This interaction was fur-ther confirmed in Arabidopsis by introducing GbWRKY1into amiRNATIFY10A (an AtJAZ1 knockdown line;Grunewald et al., 2009) via cross-pollination, and the flow-ering time was investigated in AOV9 and amiRNAJAZ1hybrid progeny. The flowering time of AOV9 was res-cued following down-regulation of the expression ofAtJAZ1 (Fig. 5G). Our genetic analyses indicate that theGbWRKY1-orchestrated tradeoff between the JA and GAsignaling pathways depends on the constitutive activa-tion of JAZ1 both in cotton and in Arabidopsis.

GbWRKY1 Transactivates the Expression of GhJAZ1 andAtJAZ1 in Vivo

WRKY proteins specifically bind to the W-box(TTGACC/T) in most instances (Rushton et al., 2010).To examine the possibility that GbWRKY1 can bind tothe promoter of GhJAZ1, the promoter region ofGhJAZ1 was analyzed. Unexpectedly, only one typicalW-box (TTGACC) was found within 2 kb upstream ofthe GhJAZ1 start codon. However, four TTGACT se-quences were found within the 2-kb promoter sequenceof AtJAZ1 according to the Arabidopsis cis-regulatoryelement database (AtcisDB).To test the interaction between GbWRKY1 and the

promoters of JAZ1 genes, the GhGS1 (G. hirsutum sequencefor gel-shift assay, from2663 to2698) sequence from theGhJAZ1 promoter and the AtGS1 (Arabidopsis sequencefor gel-shift assay, from2665 to2721) sequence from theAtJAZ1 promoter were synthesized for an electrophoreticmobility shift assay (EMSA; Fig. 6A). Surprisingly,GbWRKY1 binding to the AtJAZ1 promoter was detected(Fig. 6C), whereas no detectable binding was found in thecase of the GhJAZ1 promoter (Fig. 6B).A recent study demonstrated that the TGAC core

sequence is sufficient to bind to the AtWRKY28 protein(van Verk et al., 2011); we found nine core sequenceswithin the GhJAZ1 promoter in the 2711 to 21,649region. The fragment containing two core sequences(GhGS2, from 21,599 to 21,654) was synthesized as atarget probe to assay the interaction between GbWRKY1and the GhJAZ1 promoter (Fig. 6A), and a shifted bandfor GbWRKY1 binding to the GhJAZ1 promoter wasdetected (Fig. 6D). A competitive experiment and aTGAC-mutated EMSA experiment further demonstratedthat GbWRKY1 could specifically bind to the TGAC coresequences of GhJAZ1 (Fig. 6D).However, the binding selectivity of GbWRKY1 with

regard to GhGS1 and AtGS1 led us to speculate that

the sequences that are adjacent to the binding site maydetermine whether the site can be bound. To test thishypothesis, we synthesized a modified version of GhGS1(designated as GhGS3), for which the W-box adjacentsequences were replaced by the partial sequences adjacentto the GhGS2 TGAC core (Fig. 6A). After replacement,however, the GhGS3 probe was bound by GbWRKY1,and this binding could be competed by nonlabeled probes(Fig. 6E). Next, we synthesized several variants of GhGS3(Fig. 6A). As expected, a mutation within the TTGACTsequence (designated as GhGS3-M1) could completelyblock binding of GbWRKY1 (Fig. 6E). However, thebinding affinity of GhGS3 significantly decreased afterthe mutation of GhGS3 oligonucleotide at positions 21/22/23/24 from the TTGACT sequence (designated asGhGS3-M2). The mutation at positions +1/+2/+3/+4from the TTGACT sequence (designated as GhGS3-M3)did not affect the binding capability of GhGS3. BecauseGhGS1 and GhGS3 had the same nucleotides at positions21/22 from the binding site (Fig. 6A), we next deter-mined the effect of positions 23/24 on binding toGbWRKY1, and a similar binding abundance betweenGhGS3-M2 and GhGS3-M4 suggested that positions23/24 were responsible for the decreased binding ofGhGS3-M2 (Fig. 6E). Our results, therefore, demonstratethat GbWRKY1 not only targets the W-box but alsoTGAC core sequences and that the adjacent DNA se-quences of the binding site are also important for thebinding selectivity of GbWRKY1.

To validate the possibility that the TGAC core se-quences are required for binding with GbWRKY1, the419-bp promoter sequence (see “Materials andMethods”)of GhJAZ1 containing four TGAC sequences was clonedinto the pHisi-1 yeast (Saccharomyces cerevisiae) one-hybridbait vector and the full-length cDNA of GbWRKY1 wascloned into the pDEST22 prey vector. As shown in Figure6F, GbWRKY1 was also able to bind to the wild-typeGhJAZ1 promoter in yeast, whereas mutations withinthe four TGAC core sequences abolished GbWRKY1binding to the GhJAZ1 promoter in the presence of 15 mM

3-amino-1,2,4-triazole, demonstrating that the TGAC se-quence is required for GbWRKY1 binding.

We also employed the dual-luciferase reporter systemin tobacco (Nicotiana tabacum) to examine the tran-scriptional activity of GbWRKY1 in vivo. The wild-typeor mutated GhJAZ1 promoters (see “Materials andMethods”) were cloned and fused with the firefly lu-ciferase (LUC) reporter gene, and the relative LUC ac-tivity was measured after transfection. Figure 6G showsthat the overexpression of GbWRKY1 transactivated theactivity of the LUC reporter under the control of theGhJAZ1 promoters, whereas this activity was abolishedwhen the TGAC cores in the GhJAZ1 promoter weredisrupted.

To further assess the role of W-boxes (TTGACT) andthe TGAC motif in GbWRKY1 transactivation, the1,229-kb wild-type AtJAZ1 promoter (containing fourW-boxes and five TGAC core sequences) and a modi-fied AtJAZ1 promoter (the four W-boxes were mutatedbut not the TGAC core sequences) were fused with the





LUC reporter gene. These were used to examine the effectof GbWRKY1 on AtJAZ1 transcription. The data showthat the wild-type AtJAZ1 promoter was transactivatedby approximately 3.2-fold after cotransfection with theeffector (35S:GbWRKY1; Fig. 6G). With theW-box-mutatedversion of the promoter, overexpression of GbWRKY1only caused a 1.7-fold increase in the activity of the LUCreporter (Fig. 6G). These results show that GbWRKY1 iscapable of transactivating GhJAZ1 via binding to theTGAC core sequences, but AtJAZ1 binds via the W-box(TTGACT) and the TGAC core sequence.

DISCUSSION

Due to the sessile lifestyle of plants and the limitationof available energy, plants have evolved an elaborateregulatory network to effect a tradeoff between growthand immune responses to ensure plant survival underfluctuating environments (Kazan and Manners, 2009;Fan et al., 2014). V. dahliae is notorious for its infectionstrategies and great genetic plasticity, and it causessignificant economic losses for a broad range of crops,such as cotton, strawberry (Fragaria 3 ananassa), oilseedrape (Brassica napus), tomato (Solanum lycopersicum), and

Figure 6. Assessment of the transactivation activity of GbWRKY1. A, Promoter sequences of GhJAZ1, AtJAZ1, and differentmutated and replaced versions as indicated in the text. The W-box element, TGAC core, and mutated positions are indicated byshort underlines. B to E, EMSA of the binding of GbWRKY1 protein to the digoxigenin-labeled oligonucleotide as shown in A. F,GbWRKY1 bound to the GhJAZ1 promoter in the yeast one-hybrid assay. 3AT, 3-Amino-1,2,4-triazole. G, GbWRKY1 trans-activates the promoter ofGhJAZ1 and AtJAZ1 in vivo. The assays were repeated three times. The data in G represent means6 SD

from three replicates. Statistical analyses were performed using Student’s t test: *P, 0.05 and **P, 0.01. [See online article forcolor version of this figure.]


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potato (Solanum tuberosum; Zhou et al., 2006; Klostermanet al., 2009). Typical symptoms caused byV. dahliae includeleaf necrosis, premature defoliation, altered flowering time,and severe growth stunting (Veronese et al., 2003; Fradinet al., 2009; Xu et al., 2011b). Recent studies have identifiedsignaling pathways that play essential roles in cottonresistance to V. dahliae (Xu et al., 2011b; Gao et al., 2013;Zhang et al., 2013), but how plants orchestrate devel-opment under V. dahliae infection remains elusive. Bymining genes that are expressed differentially in re-sponse to V. dahliae from the cotton ‘7124’, the WRKYtranscription factor gene GbWRKY1 was identified asputatively functioning in the phosphate starvation andresistance response (Xu et al., 2011a, 2012b). In thisstudy, we provide molecular and genetic evidence todemonstrate that GbWRKY1 might be a key regulatorin the orchestration of plant defense and development.To determine the precise role of GbWRKY1 in the

resistance response in cotton, we generated GbWRKY1-overexpressing transgenic cotton and Arabidopsis plantsand GbWRKY1 knockdown cotton plants through VIGSand RNAi, respectively. All the inoculation assays con-sistently demonstrated that overexpressing GbWRKY1 incotton and Arabidopsis attenuated resistance to B. cinereaand V. dahliae (Fig. 1; Supplemental Fig. S2). However,when GbWRKY1 was knocked down through VIGS orRNAi, the plants showed more resistance to B. cinereaand V. dahliae (Fig. 1; Supplemental Fig. S2). Generally,the JA signaling pathway plays a central role in plantresistance to necrotrophic pathogens. Several lines ofevidence have suggested that JA signaling is also requiredfor plant resistance to V. dahliae, as revealed by the ex-ogenous application of MeJA (Fig. 2, A and B) and bygenetic and molecular biology analyses in Arabidopsisand cotton (Fradin et al., 2011; Gao et al., 2013). Fur-thermore, GbWRKY1 showed clear negative regulationin JA-mediated defense gene expression and anthocyaninaccumulation (Fig. 2). These results suggest thatGbWRKY1acts as a negative regulator in the plant disease resis-tance response, possibly by attenuating the JA signalingpathway.Previous studies have demonstrated that the transition

from vegetative to reproductive growth is a critical phasefor Verticillium spp. invasion in oilseed rape (Zhou et al.,2006). This is in agreement with the common observationthat the prevalence of cotton Verticillium spp. wilt is mostsevere after flowering in the field (Ma et al., 2000; Wanget al., 2012). Meanwhile, the regulation of flowering timeseems to be a crucial strategy that plants select for sur-vival under pathogen infection. Susceptible ecotypesexhibit a pathogen-induced early-flowering phenotype,whereas more tolerant ecotypes exhibit delayed flower-ing after Verticillium spp. inoculation (Veronese et al.,2003; Häffner et al., 2010). It is reported that VET1 (forV. dahliae tolerance) plays a negative role in floweringtime and a positive role in pathogen resistance (Veroneseet al., 2003). By contrast, GbWRKY1 appears to possessfunctions antagonistic to VET1, as its overexpression inArabidopsis resulted in early flowering and the at-tenuation of resistance to V. dahliae. Moreover, the

overexpression of other defense-related WRKY tran-scription factors, such asWRKY6,WRKY7, andWRKY18,also can alter flowering time (Chen and Chen, 2002;Robatzek and Somssich, 2002; Kim et al., 2006, 2008).Therefore, these WRKY transcription factors may havemultifaceted functions in the coordination of plant de-velopment and immune response.

Interestingly, GbWRKY1-overexpressing cotton plantsdid not exhibit observable differences in flowering time(Supplemental Fig. S6D), although the overexpression ofGbWRKY1 appears to promote GA-related developmentin cotton seedlings and GA enhances flowering time inArabidopsis. Additionally, the flowering time of cottonplants did not display any alteration after exogenousapplication of GA but showed severely delayed flower-ing in the presence of its biosynthesis inhibitor PAC(Supplemental Fig. S6E), which is consistent with pre-vious observations that flowering time was delayed bydown-regulation of the cotton GA biosynthesis geneGhGA20ox1 but shows little change when GhGA20ox1 isoverexpressed (Xiao, 2004). Thus, GA may exert a dif-ferent regulatory role in controlling the development ofdifferent plant species.

In agreement with the premise that the activation ofplant immune responses is at the expense of growth(Kazan and Manners, 2012), JA-incubated cotton plantswere slightly smaller than the mock plants after inoc-ulation with V. dahliae (Fig. 2A). Meanwhile, JA-treatedArabidopsis plants showed retarded vegetative growth

Figure 7. Schematic model of how GbWRKY1 prioritizes developmentover defense during V. dahliae infection. When plants detect V. dahliaeinvasion, JA signaling is rapidly activated to allocate energy resources todefense (Gao et al., 2013). However, activation of JA signaling is at theexpense of growth and development. To avoid the hyperactivation of JAsignaling and maintain the growth- and development-related processes,GbWRKY1 is rapidly induced by V. dahliae infection and subsequentlyup-regulates the transcription of a JAZ1-like gene in cotton, whichpromote GA-related phenotypes (such as flowering). This relieves therepression of DELLA and at the same time limits the extent of JA sig-naling activation, thereby prioritizing development and reproduction(flowering) over defense during V. dahliae infection. [See online articlefor color version of this figure.]









and a late-flowering phenotype (Fig. 5A; SupplementalFig. S8). A previous study demonstrated that the JA-mediated inhibitory effect on Arabidopsis hypocotylelongation was partly mediated by DELLA proteins(Hou et al., 2010). We found that two GA-repressedgenes, GhGID1-a and GhGID1-b, that are homologs ofputative DELLA targets in Arabidopsis were dramati-cally up-regulated by JA (Fig. 5B). These results indicatethat JA exerts its suppressive role on plant growth partlyby suppressing GA signaling (Fig. 7). We suggest thatGbWRKY1 may be a pivotal molecular switch in the or-chestration of cotton development and defense duringV. dahliae infection.

Studies in Arabidopsis have demonstrated that changesin GA signaling transduction can affect the expression ofGA biosynthetic genes and GA catabolic genes by feed-back mechanisms and that AtGA2ox is induced in GA-overproducing plants (Zhang et al., 2011). Similarly, theinduction of AtGA2ox-2 was accompanied by the acti-vation of the GA signaling pathway downstream ofDELLA (Supplemental Fig. S7). Further genetic analysissuggests that the early-flowering phenotype of GbWRKY1-overexpressing Arabidopsis depends on the expressionof SOC1 (Fig. 4E), which participates in DELLA-mediatedflowering time control (Yu et al., 2012). Interestingly, wild-type and GbWRKY1-RNAi plants did not show ob-servable differences in terms of plant morphology andsensitivity to PAC (Fig. 3, G andH; Supplemental Fig. S5),but the expression of GhGID1-a and GhGID1-b was in-duced to higher levels by MeJA treatment in RNAi plants(Fig. 5B). In addition, there was no obvious effect on theexpression of GbWRKY1 after GA treatment (data notshown). We can infer that there is cross-regulationbetween defense and growth, and the alteration ofdefense-related processes indirectly affects developmen-tal processes (Alcázar et al., 2011).

A recent study demonstrated that the overexpressionof a selected group of JAZ-encoding genes by the 35Spromoter is able to activate the GA signaling pathwaythrough the physical interaction between JAZ andDELLA proteins (Yang et al., 2012). Molecular and ge-netic analyses indicated that GbWRKY1 is likely to actupstream of GhJAZ1 and transactivate the activity ofGhJAZ1 (Figs. 5F and 6G), suggesting that a preciseinteraction occurs between GbWRKY1 and GhJAZ1.

The Arabidopsis transcription factor WRKY33 nega-tively regulates the JAZ1 and JAZ5 transcripts throughB. cinerea infection-induced binding to their promoterregions (Birkenbihl et al., 2012), although 35S:WRKY33plants exhibit enhanced resistance to B. cinerea and aslightly earlier flowering time compared with the wildtype (Zheng et al., 2006b).

In addition, an expression profile analysis demon-strated that the homolog of GbWRKY1 in Arabidopsis,AtWRKY75, and three JAZ genes (AtJAZ1, AtJAZ5, andAtJAZ10) share the same expression pattern in wild-type and wrky33 mutant plants during B. cinerea in-fection (Birkenbihl et al., 2012). Interestingly, qRT-PCRanalysis showed that only AtJAZ1 was up-regulated in35S:GbWRKY1 Arabidopsis plants, and similar results

were obtained in the transcriptional profiling of grapevine(Vitis vinifera), showing that only VvJAZ1.1 and VvJAZ1.2were up-regulated by VvWRKY1, the grapevine homologgene of AtWRKY75 (Marchive et al., 2013). 35S:VvWRKY1transgenic plants also displayed pale green leaves, similarto 35S:GbWRKY1 plants, indicating that these homologsof AtWRKY75 may share a similar regulatory mechanismto selectively recognize their target promoters.

A previous study suggested that the sequences that areadjacent to the W-box play important roles in bindingselectivity (Rushton et al., 2010). However, our EMSAresults demonstrated that the sequences that are adjacentto the binding site are required not only to determinewhether the binding site can be bound by GbWRKY1 butalso to ensure its high-affinity binding. In addition tothese cis-acting elements, we cannot exclude the possi-bility that other interacting partners of GbWRKY1 werealso involved in determining the specific recognition,which remains a challenging area for further investiga-tion in cotton and in the model plant Arabidopsis.

JA is known to activate its signaling transductionthrough promoting JAZ protein ubiquitination and deg-radation (Chini et al., 2007; Thines et al., 2007). However,once JA signaling is activated, the expression of manyJAZ genes is increased rapidly (Zheng et al., 2006a). Thissuggests that a negative feedback inhibition mechanismmay exist to avoid the hyperactivation of JA signaling(Figueroa and Browse, 2012), although how the expres-sion of JAZ genes is activated via the negative feedbackloop is still unknown. In our study, GbWRKY1 expressionwas rapidly induced by JA treatment and was also in-creased slightly in the GhJAZ1-RNAi lines (Figs. 5E and 7;Supplemental Fig. S3). Through the direct binding ofGbWRKY1 to the JAZ1 promoter and transactivatingexpression of JAZ1, a feedback regulatory loop might beformed to maintain JA homeostasis in the plant (Fig. 7).GbWRKY1 shows early expression after V. dahliae infec-tion and prioritizes development over pathogen defensethrough a known antagonistic interaction of the JA/GApathway (Fig. 7), shedding new light on the interplaybetween cotton and V. dahliae.

MATERIALS AND METHODS

Plant Materials, VIGS Experiments, and Disease Assays

Cotton (Gossypium barbadense) ‘7124’ and Gossypium hirsutum ‘YZ1’ plantswere used in this study. For the VIGS experiments and disease assays, plantswere grown in a growth room with a 16-h-day/8-h-night cycle at 25°C.

The cDNA sequence of GbWRKY1 was cloned into the TRV plasmid usingBamHI and KpnI to construct the TRV:GbWRKY1 VIGS vector and was thentransformed into Agrobacterium tumefaciens GV3101 via electroporation asdescribed previously (Fradin et al., 2009). The TRV:00 (control) and TRV:GbWRKY1 vectors were agroinfiltrated into the cotyledons of 10-d-old cv YZ1plants using a needleless syringe as described previously (Gao et al., 2013).Almost 2 weeks after infiltration, RNAwas extracted from cotton roots to measurethe expression of GbWRKY1.

The Verticillium dahliae strain V991 and Botrytis cinerea were cultured onpotato dextrose agar at 25°C for 4 d and then further incubated on new potatodextrose agar medium for another 7 d. The conidia of V. dahliae and B. cinereawere collected and resuspended in distilled water and 1% (w/v) Sabouraudmaltose broth buffer, respectively. The V. dahliae infection assays were performed byroot dipping with spore suspension (2 3 105 spores mL21) as described previously


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(Xu et al., 2011b; Gao et al., 2013). The disease index (%) was measured to examinethe susceptibility to V. dahliae as described previously (Xu et al., 2012a). B. cinerea in-fection assays were performed using either a spore suspension (23 105 spores mL21)or colonized agar plugs as indicated in the text. The average lesion size(diameter) and plant decay were measured as described previously (Coego et al.,2005; Veronese et al., 2006).

Gene Cloning, Vector Construction, andPlant Transformation

The EST of GbWRKY1 was isolated from the cotton ‘7124’ using suppres-sion subtractive hybridization (Xu et al., 2011a). The full-length sequence wasobtained through RACE-PCR according to the SMART RACE cDNA ampli-fication kit user manual (Clontech) and using V. dahliae-infected cotton rootcDNA as the template. The full-length coding sequence of GbWRKY1 wasinserted into the modified binary plant vector pCAMBIA2300 (Cambia) usingXbaI and SacI to construct the vector 35S:GbWRKY1 for overexpression (Xuet al., 2012b). The RNAi region containing a partially conserved domain andthe specific 39 region of GbWRKY1was cloned into the RNAi vector pHellsgate4 through recombination reaction (Tan et al., 2013). The overexpression vectorand RNAi vectors were introduced into G. hirsutum ‘YZ1’ plants by A. tumefaciens(strain EHA105)-mediated transformation as described previously (Jin et al., 2006).The overexpression vector was transferred into A. tumefaciens strain GV3101to transform the Arabidopsis (Arabidopsis thaliana) ecotype Columbia-0 usingthe floral dip method (Hao et al., 2012).

Nucleic Acid Extraction and Expression Analysis

Total RNA was isolated as described previously (Tan et al., 2013). Fornorthern blotting, 20 mg of total RNA per lane was separated on 1.2% agarose-formaldehyde gels and transferred onto a nylon membrane. Blots were hybridizedwith 32P-labeled GbWRKY1-specific probes. Hybridization was performed in Per-fectHyb hybridization solution (Toyobo). The procedures for detecting the signalwere performed as described previously (Tu et al., 2007). For RT-PCR and qRT-PCR analyses, RNAwas reverse transcribed to cDNA using SuperScript III reversetranscriptase (Invitrogen). qRT-PCR was performed using the ABI Prism 7000system (Applied Biosystems). The values are given relative to the housekeepinggenes ACTIN in Arabidopsis and UB7 in cotton. The primers that were used in thenorthern blotting, RT-PCR, and qRT-PCR are listed in Supplemental Table S1.

Scanning Electron Microscopy andChlorophyll Measurement

Seeds of the wild-type and GbWRKY1 transgenic lines were sown intoindividual pots (8 cm in diameter). At least 5 cm of space was allowed betweenthe plants to avoid mutual shading. After the second true leaves were fullyexpanded, cotyledon petioles were collected and fixed in 2.5% (v/v) glutaral-dehyde. The samples were pretreated as described previously (Deng et al.,2012a) and photographed with a JSM-6390/LV scanning electron microscope(JEOL). At the same time, the second true leaves were harvested for chlorophyllmeasurement. Briefly, the leaves were weighed and ground into powder inliquid nitrogen. Total chlorophyll was then extracted with 80% (v/v) acetoneand measured using a Beckman Coulter DU800 spectrophotometer. Totalchlorophyll content was calculated as described previously (Kim and Kim, 2013).

Protoplast Transient Transfection for Dual-LuciferaseReporter Assays

The transient dual-luciferase reporter assays were performed as describedpreviously (Hellens et al., 2005). The wild-type promoter sequence (from 21,307to 21,726) of GhJAZ1 containing five TGAC core sequences was amplified byPCR using cv YZ1 genomic DNA, and the relevant TGAC mutated version(mutation at positions 21,333, 21,375, 21,521, 21,612, and 21,645) was syn-thesized by GenScript (http://www.genscript.com/). The 1,229-kb wild-typeAtJAZ1 promoter (from 26 to 21,235 containing four W-boxes [TTGACT]and five TGAC core sequences) was amplified by PCR using Arabidopsis genomicDNA, and the TTGACT-mutated version (mutation at positions2428,2672,2679,and 2938) was obtained using overlap extension PCR. These fragmentswere cloned into pGreenII 0800-LUC at the PstI and BamHI sites to generateGhJAZ1PRO:LUC, GhJAZ1PRO-M-LUC, AtJAZ1PRO:LUC, and AtJAZ1PRO-M-LUC. The coding region of GbWRKY1 was amplified by PCR and cloned

into pGreenII 62-SK at the PstI and BamHI sites to generate 35:GbWRKY1. Pro-toplasts were isolated from tobacco (Nicotiana tabacum ‘Petite Havana’) leavesaccording to Yoo et al. (2007). After transformation, protoplasts were culturedfor 16 h and collected. Firefly luciferase and Renilla spp. luciferase activities werequantified using the dual-luciferase assay reagents (Promega) using a Multi-mode Plate Reader (PerkinElmer). The primers that were used in dual-luciferasereporter assays are listed in Supplemental Table S1.

Yeast One-Hybrid Assay

A yeast (Saccharomyces cerevisiae) one-hybrid assay was performed as de-scribed by Ou et al. (2011). To generate reporter strains, the 419-bp wild-typepromoter sequence (from 21,307 to 21,726) and the relevant TGAC-mutatedversion were amplified by PCR using GhJAZ1PRO:LUC and GhJAZ1PRO-M-LUC plasmids as the template, respectively. Then, the PCR-amplified frag-ments were cloned into the pHisi-1 yeast one-hybrid bait vector to generateGhJAZ1-PRO and GhJAZ1W-box-m PRO. After linearization by XhoI, the baitvectors were integrated into the yeast strain YM4271 (MAT a) and selected onsynthetic dropout (SD) medium without His. In addition, the full-lengthcDNA of GbWRKY1 was cloned into the pDEST22 prey vector to generateAD-GbWRKY1 and then transferred to yeast strain Y187 (MAT a) followed byselection on SD medium without Trp. The positive transformants of the baitplasmid and prey plasmid were transferred to yeast extract-peptone-adenine-dextrose medium and mated for 24 h at 30°C. A total of 8 mL of 1:10 dilutionwas dripped onto SD-Leu-Trp-His plates that were supplemented with 15 mM

3-amino-1,2,4-triazole and incubated for 4 to 6 d at 30°C to determine theinteraction between GbWRKY1 and the GhJAZ1 promoter.

The primers that were used in the yeast one-hybrid assay are listed inSupplemental Table S1.

EMSA

GbWRKY1was cloned into a pET-28a-inducible expression vector and expressedin the Escherichia coli BL21 strain. The GbWRKY1 recombinant fusion protein waspurified using the MagneHis Protein Purification System (Promega). Synthesizedoligonucleotides (Fig. 6A) were labeled using the Roche DIG gel shift kit. DNA-protein-binding reactions were performed by incubating 100 ng of purifiedGbWRKY1 recombinant protein with digoxigenin-labeled GhJAZ1 or AtJAZ1 pro-moter fragment as described in the Roche protocol manual. The protein-DNAmixture was incubated at room temperature for 30 min and separated on a 12%polyacrylamide gel in Tris-Gly buffer (25 mM Tris, 2 mM EDTA, and 380 mM Gly)as described by Deng et al. (2012a). The probes were detected using the C-DiGitBlot Scanner (LI-COR Biosciences).

Sensitivity to Plant Hormones andAnthocyanin Measurement

Cotton seeds were surface sterilized with 0.1% (w/v) HgCl2 for 8 min andwashed three times with sterile distilled water. Seeds were then sown in asterile plant culture box containing 0.53 agar MS medium (Murashige andSkoog, 1962). After germination at 28°C in the dark, approximately 16 seedsfrom the wild-type and GbWRKY1 transgenic lines were transferred to new0.53 agar MS medium with or without 0.6 mM PAC and allowed to grow for5 d before scoring.

The anthocyanin content of seedlings wasmeasured as described previouslywith minor modifications (Qi et al., 2011). Surface-sterilized Arabidopsis seedswere sown onto 0.53 agar MS medium with or without 8 mM MeJA for 10 d andcollected directly in liquid nitrogen. The anthocyanin content was calculated asdescribed previously (Xu et al., 2012b).

Sequence data from this article can be found in the GenBank/EMBL data librariesunder accession numbers. GbWRKY1, JQ640573.1; GhGID1-a, DQ829776;GhGID1-b, EF607794; UF3GT, AT5G54060; LDOX, AT4G22880; DFR, AT5G42800;GID1a, AT3G05120; GID1b, AT3G63010; XERICO, AT2G04240; SCL3, AT1G50420;bHLH137, AT5G50915; LBD40, AT1G67100; and MYB, AT3G11280.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Response of GbWRKY1 to V. dahliae infection incotton.





http://www.genscript.com/





Supplemental Figure S2. Responses of GbWRKY1 transgenic plants toV. dahliae and the necrotrophic pathogen B. cinerea.

Supplemental Figure S3. Response of GbWRKY1 to MeJA.

Supplemental Figure S4. Levels of JA in wild-type and GbWRKY1 trans-genic cotton plants.

Supplemental Figure S5. Morphological phenotypes of GbWRKY1-RNAiplants.

Supplemental Figure S6. Morphological phenotypes of GbWRKY1 trans-genic lines.

Supplemental Figure S7. Gene expression analyses of GA-related genes.

Supplemental Figure S8. Flowering phenotypes of wild-type, AOV4, andAOV9 plants after MeJA treatment.

Supplemental Figure S9. Evaluation of the expression of a JA-responsivegene (GbPR4) after GA treatment.

Supplemental Figure S10. qRT-PCR analysis of a set of JAZs in GbWRKY1transgenic plants.

Supplemental Figure S11. Examination of GhJAZ1 expression in GhJAZ1transgenic cotton lines.

Supplemental Table S1. Primers that were used in this study.

ACKNOWLEDGMENTS

We thank Wim Grunewald (Department of Plant Systems Biology, FlandersInstitute for Biotechnology, Ghent University) and Tao Huang (School of LifeSciences, Xiamen University) for sharing Arabidopsis research materials andKeith Lindsey (School of Biological and Biomedical Sciences, University of Durham)for revising the article.

Received July 12, 2014; accepted October 7, 2014; published October 9, 2014.

LITERATURE CITED

Alcázar R, Reymond M, Schmitz G, de Meaux J (2011) Genetic and evolutionaryperspectives on the interplay between plant immunity and development. CurrOpin Plant Biol 14: 378–384

Aleman L, Kitamura J, Abdel-Mageed H, Lee J, Sun Y, Nakajima M,Ueguchi-Tanaka M, Matsuoka M, Allen RD (2008) Functional analysisof cotton orthologs of GA signal transduction factors GID1 and SLR1.Plant Mol Biol 68: 1–16

Birkenbihl RP, Diezel C, Somssich IE (2012) Arabidopsis WRKY33 is a keytranscriptional regulator of hormonal and metabolic responses toward Botrytiscinerea infection. Plant Physiol 159: 266–285

Cai M, Qiu D, Yuan T, Ding X, Li H, Duan L, Xu C, Li X, Wang S (2008)Identification of novel pathogen-responsive cis-elements and theirbinding proteins in the promoter of OsWRKY13, a gene regulating ricedisease resistance. Plant Cell Environ 31: 86–96

Cheminant S, Wild M, Bouvier F, Pelletier S, Renou JP, Erhardt M, HayesS, Terry MJ, Genschik P, Achard P (2011) DELLAs regulate chlorophylland carotenoid biosynthesis to prevent photooxidative damage duringseedling deetiolation in Arabidopsis. Plant Cell 23: 1849–1860

Chen C, Chen Z (2002) Potentiation of developmentally regulated plantdefense response by AtWRKY18, a pathogen-induced Arabidopsistranscription factor. Plant Physiol 129: 706–716

Cheng H, Song S, Xiao L, Soo HM, Cheng Z, Xie D, Peng J (2009) Gibberellinacts through jasmonate to control the expression of MYB21, MYB24, andMYB57 to promote stamen filament growth in Arabidopsis. PLoS Genet 5:e1000440

Cheng Z, Sun L, Qi T, Zhang B, Peng W, Liu Y, Xie D (2011) The bHLHtranscription factor MYC3 interacts with the jasmonate ZIM-domain proteinsto mediate jasmonate response in Arabidopsis. Mol Plant 4: 279–288

Chini A, Fonseca S, Fernández G, Adie B, Chico JM, Lorenzo O, García-CasadoG, López-Vidriero I, Lozano FM, Ponce MR, et al (2007) The JAZ family ofrepressors is the missing link in jasmonate signalling. Nature 448: 666–671

Ciolkowski I, Wanke D, Birkenbihl RP, Somssich IE (2008) Studies onDNA-binding selectivity of WRKY transcription factors lend structuralclues into WRKY-domain function. Plant Mol Biol 68: 81–92

Claeys H, Inzé D (2013) The agony of choice: how plants balance growthand survival under water-limiting conditions. Plant Physiol 162: 1768–1779

Coego A, Ramirez V, Gil MJ, Flors V, Mauch-Mani B, Vera P (2005) AnArabidopsis homeodomain transcription factor, OVEREXPRESSOR OFCATIONIC PEROXIDASE 3, mediates resistance to infection by necrotrophicpathogens. Plant Cell 17: 2123–2137

Deng F, Tu L, Tan J, Li Y, Nie Y, Zhang X (2012a) GbPDF1 is involved incotton fiber initiation via the core cis-element HDZIP2ATATHB2. PlantPhysiol 158: 890–904

Deng H, Liu H, Li X, Xiao J, Wang S (2012b) A CCCH-type zinc fingernucleic acid-binding protein quantitatively confers resistance againstrice bacterial blight disease. Plant Physiol 158: 876–889

de Saint Germain A, Ligerot Y, Dun EA, Pillot JP, Ross JJ, Beveridge CA,Rameau C (2013) Strigolactones stimulate internode elongation inde-pendently of gibberellins. Plant Physiol 163: 1012–1025

Devaiah BN, Karthikeyan AS, Raghothama KG (2007) WRKY75 transcriptionfactor is a modulator of phosphate acquisition and root development inArabidopsis. Plant Physiol 143: 1789–1801

Encinas-Villarejo S, Maldonado AM, Amil-Ruiz F, de los Santos B, Romero F,Pliego-Alfaro F, Muñoz-Blanco J, Caballero JL (2009) Evidence for a posi-tive regulatory role of strawberry (Fragaria 3 ananassa) Fa WRKY1 andArabidopsis At WRKY75 proteins in resistance. J Exp Bot 60: 3043–3065

Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY su-perfamily of plant transcription factors. Trends Plant Sci 5: 199–206

Fan M, Bai MY, Kim JG, Wang T, Oh E, Chen L, Park CH, Son SH, Kim SK,Mudgett MB, et al (2014) The bHLH transcription factor HBI1 mediates thetrade-off between growth and pathogen-associated molecular pattern-triggeredimmunity in Arabidopsis. Plant Cell 26: 828–841

Fernández-Calvo P, Chini A, Fernández-Barbero G, Chico JM, Gimenez-Ibanez S, Geerinck J, Eeckhout D, Schweizer F, Godoy M, Franco-ZorrillaJM, et al (2011) The Arabidopsis bHLH transcription factors MYC3 and MYC4are targets of JAZ repressors and act additively with MYC2 in the activationof jasmonate responses. Plant Cell 23: 701–715

Figueroa P, Browse J (2012) The Arabidopsis JAZ2 promoter contains a G-boxand thymidine-rich module that are necessary and sufficient for jasmonate-dependent activation by MYC transcription factors and repression by JAZproteins. Plant Cell Physiol 53: 330–343

Fradin EF, Abd-El-Haliem A, Masini L, van den Berg GCM, JoostenMHAJ, Thomma BPHJ (2011) Interfamily transfer of tomato Ve1 mediatesVerticillium resistance in Arabidopsis. Plant Physiol 156: 2255–2265

Fradin EF, Zhang Z, Juarez Ayala JC, Castroverde CD, Nazar RN, Robb J, LiuCM, Thomma BP (2009) Genetic dissection of Verticillium wilt resistancemediated by tomato Ve1. Plant Physiol 150: 320–332

Gao W, Long L, Zhu LF, Xu L, Gao WH, Sun LQ, Liu LL, Zhang XL (2013)Proteomic and virus-induced gene silencing (VIGS) analyses reveal thatgossypol, brassinosteroids, and jasmonic acid contribute to the resistance ofcotton to Verticillium dahliae. Mol Cell Proteomics 12: 3690–3703

Grunewald W, Vanholme B, Pauwels L, Plovie E, Inzé D, Gheysen G,Goossens A (2009) Expression of the Arabidopsis jasmonate signallingrepressor JAZ1/TIFY10A is stimulated by auxin. EMBO Rep 10: 923–928

Häffner E, Karlovsky P, Diederichsen E (2010) Genetic and environmentalcontrol of the Verticillium syndrome in Arabidopsis thaliana. BMC PlantBiol 10: 235

Hao J, Tu L, Hu H, Tan J, Deng F, Tang W, Nie Y, Zhang X (2012) GbTCP,a cotton TCP transcription factor, confers fibre elongation and root hairdevelopment by a complex regulating system. J Exp Bot 63: 6267–6281

Harberd NP, Belfield E, Yasumura Y (2009) The angiosperm gibberellin-GID1-DELLA growth regulatory mechanism: how an “inhibitor of aninhibitor” enables flexible response to fluctuating environments. PlantCell 21: 1328–1339

Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD,Karunairetnam S, Gleave AP, Laing WA (2005) Transient expressionvectors for functional genomics, quantification of promoter activity andRNA silencing in plants. Plant Methods 1: 13

Hou X, Lee LYC, Xia K, Yan Y, Yu H (2010) DELLAs modulate jasmonatesignaling via competitive binding to JAZs. Dev Cell 19: 884–894

Howe GA (2010) Ubiquitin ligase-coupled receptors extend their reach tojasmonate. Plant Physiol 154: 471–474

Jin S, Zhang X, Nie Y, Guo X, Liang S, Zhu H (2006) Identification of anovel elite genotype for in vitro culture and genetic transformation ofcotton. Biol Plant 50: 519–524

Josse EM, Gan Y, Bou-Torrent J, Stewart KL, Gilday AD, Jeffree CE,Vaistij FE, Martínez-García JF, Nagy F, Graham IA, et al (2011) A


Li et al.














DELLA in disguise: SPATULA restrains the growth of the developingArabidopsis seedling. Plant Cell 23: 1337–1351

Katsir L, Schilmiller AL, Staswick PE, He SY, Howe GA (2008) COI1 is acritical component of a receptor for jasmonate and the bacterial virulencefactor coronatine. Proc Natl Acad Sci USA 105: 7100–7105

Kazan K, Manners JM (2009) Linking development to defense: auxin inplant-pathogen interactions. Trends Plant Sci 14: 373–382

Kazan K, Manners JM (2012) JAZ repressors and the orchestration ofphytohormone crosstalk. Trends Plant Sci 17: 22–31

Kim JH, Kim WT (2013) The Arabidopsis RING E3 ubiquitin ligaseAtAIRP3/LOG2 participates in positive regulation of high-salt anddrought stress responses. Plant Physiol 162: 1733–1749

Kim KC, Fan B, Chen Z (2006) Pathogen-induced Arabidopsis WRKY7 is atranscriptional repressor and enhances plant susceptibility to Pseudomonassyringae. Plant Physiol 142: 1180–1192

Kim KC, Lai Z, Fan B, Chen Z (2008) Arabidopsis WRKY38 and WRKY62transcription factors interact with histone deacetylase 19 in basal de-fense. Plant Cell 20: 2357–2371

Klosterman SJ, Atallah ZK, Vallad GE, Subbarao KV (2009) Diversity,pathogenicity, and management of Verticillium species. Annu RevPhytopathol 47: 39–62

Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node ofconvergence for jasmonate-mediated and salicylate-mediated signals inplant defense. Plant Cell 16: 319–331

Ma P, Huang HC, Kokko EG, Tang WH (2000) Infection of cotton pollen byVerticillium dahliae. Plant Pathol Bull 9: 93–98

Mangelsen E, Kilian J, Berendzen KW, Kolukisaoglu UH, Harter K,Jansson C, Wanke D (2008) Phylogenetic and comparative gene expressionanalysis of barley (Hordeum vulgare) WRKY transcription factor familyreveals putatively retained functions between monocots and dicots. BMCGenomics 9: 194

Marchive C, Léon C, Kappel C, Coutos-Thévenot P, Corio-Costet MF,Delrot S, Lauvergeat V (2013) Over-expression of VvWRKY1 in grapevinesinduces expression of jasmonic acid pathway-related genes and confershigher tolerance to the downy mildew. PLoS ONE 8: e54185

Marè C, Mazzucotelli E, Crosatti C, Francia E, Stanca AM, Cattivelli L (2004)Hv-WRKY38: a new transcription factor involved in cold- and drought-response in barley. Plant Mol Biol 55: 399–416

Mengiste T (2012) Plant immunity to necrotrophs. Annu Rev Phytopathol50: 267–294

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473–497

Navarro L, Bari R, Achard P, Lisón P, Nemri A, Harberd NP, Jones JD(2008) DELLAs control plant immune responses by modulating thebalance of jasmonic acid and salicylic acid signaling. Curr Biol 18: 650–655

Niu Y, Figueroa P, Browse J (2011) Characterization of JAZ-interactingbHLH transcription factors that regulate jasmonate responses in Arab-idopsis. J Exp Bot 62: 2143–2154

Ou B, Yin KQ, Liu SN, Yang Y, Gu T, Wing Hui JM, Zhang L, Miao J,Kondou Y, Matsui M, et al (2011) A high-throughput screening systemfor Arabidopsis transcription factors and its application to Med25-dependenttranscriptional regulation. Mol Plant 4: 546–555

Pandey SP, Somssich IE (2009) The role of WRKY transcription factors inplant immunity. Plant Physiol 150: 1648–1655

Pauwels L, Inzé D, Goossens A (2009) Jasmonate-inducible gene: whatdoes it mean? Trends Plant Sci 14: 87–91

Pieterse CM, Leon-Reyes A, Van der Ent S, Van Wees SC (2009) Networkingby small-molecule hormones in plant immunity. Nat Chem Biol 5: 308–316

Qi T, Song S, Ren Q, Wu D, Huang H, Chen Y, Fan M, Peng W, Ren C, Xie D(2011) The jasmonate-ZIM-domain proteins interact with the WD-repeat/bHLH/MYB complexes to regulate jasmonate-mediated anthocyanin accu-mulation and trichome initiation in Arabidopsis thaliana. Plant Cell 23: 1795–1814

Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation duringplant senescence and pathogen defense. Genes Dev 16: 1139–1149

Robert-Seilaniantz A, Grant M, Jones JDG (2011) Hormone crosstalk inplant disease and defense: more than just jasmonate-salicylate antago-nism. Annu Rev Phytopathol 49: 317–343

Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, Langum TJ,Smidt L, Boomsma DD, Emme NJ, et al (2012) WRKY transcription factors:key components in abscisic acid signalling. Plant Biotechnol J 10: 2–11

Rushton PJ, Macdonald H, Huttly AK, Lazarus CM, Hooley R (1995) Membersof a new family of DNA-binding proteins bind to a conserved cis-element inthe promoters of alpha-Amy2 genes. Plant Mol Biol 29: 691–702

Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcriptionfactors. Trends Plant Sci 15: 247–258

Sheard LB, Tan X, Mao H, Withers J, Ben-Nissan G, Hinds TR, Kobayashi Y,Hsu FF, Sharon M, Browse J, et al (2010) Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468: 400–405

Shen QH, Saijo Y, Mauch S, Biskup C, Bieri S, Keller B, Seki H, Ulker B,Somssich IE, Schulze-Lefert P (2007) Nuclear activity of MLA immunereceptors links isolate-specific and basal disease-resistance responses.Science 315: 1098–1103

Song S, Qi T, Fan M, Zhang X, Gao H, Huang H, Wu D, Guo H, Xie D(2013) The bHLH subgroup IIId factors negatively regulate jasmonate-mediated plant defense and development. PLoS Genet 9: e1003653

Song S, Qi T, Huang H, Ren Q, Wu D, Chang C, Peng W, Liu Y, Peng J,Xie D (2011) The jasmonate-ZIM domain proteins interact with theR2R3-MYB transcription factors MYB21 and MYB24 to affect jasmonate-regulated stamen development in Arabidopsis. Plant Cell 23: 1000–1013

Tan J, Tu L, Deng F, Hu H, Nie Y, Zhang X (2013) A genetic and metabolicanalysis revealed that cotton fiber cell development was retarded byflavonoid naringenin. Plant Physiol 162: 86–95

Thines B, Katsir L, Melotto M, Niu Y, Mandaokar A, Liu G, Nomura K,He SY, Howe GA, Browse J (2007) JAZ repressor proteins are targetsof the SCF(COI1) complex during jasmonate signalling. Nature 448:661–665

Tu LL, Zhang XL, Liang SG, Liu DQ, Zhu LF, Zeng FC, Nie YC, Guo XP,Deng FL, Tan JF, et al (2007) Genes expression analyses of sea-islandcotton (Gossypium barbadense L.) during fiber development. Plant CellRep 26: 1309–1320

Van Aken O, Zhang B, Law S, Narsai R, Whelan J (2013) AtWRKY40 andAtWRKY63 modulate the expression of stress-responsive nuclear genesencoding mitochondrial and chloroplast proteins. Plant Physiol 162:254–271

van Verk MC, Bol JF, Linthorst HJ (2011) WRKY transcription factors in-volved in activation of SA biosynthesis genes. BMC Plant Biol 11: 89

Veronese P, Nakagami H, Bluhm B, Abuqamar S, Chen X, Salmeron J,Dietrich RA, Hirt H, Mengiste T (2006) The membrane-anchoredBOTRYTIS-INDUCED KINASE1 plays distinct roles in Arabidopsis resistanceto necrotrophic and biotrophic pathogens. Plant Cell 18: 257–273

Veronese P, Narasimhan ML, Stevenson RA, Zhu JK, Weller SC, SubbaraoKV, Bressan RA (2003) Identification of a locus controlling Verticilliumdisease symptom response in Arabidopsis thaliana. Plant J 35: 574–587

Walters D, Heil M (2007) Costs and trade-offs associated with inducedresistance. Physiol Mol Plant Pathol 71: 3–17

Wang L, Feng HZ, Teng LP, Chen XF, Dan HX, Lu SQ, Xu JM, Zhou LG (2012)Effects of long-term cotton plantations on Fusarium and Verticillium wiltdiseases infection in China. Afr J Agr Res 7: 1562–1565

Xiao J, Cheng H, Li X, Xiao J, Xu C, Wang S (2013) Rice WRKY13 regulatescross talk between abiotic and biotic stress signaling pathways by selectivebinding to different cis-elements. Plant Physiol 163: 1868–1882

Xiao YH (2004) Cloning and function analysis of gibberellin 20-oxidase genes incotton (Gossypium hirsutum L.). PhD thesis. Southwest Agricultural Uni-versity, Chongqing, China

Xiao YH, Li DM, Yin MH, Li XB, Zhang M, Wang YJ, Dong J, Zhao J, LuoM, Luo XY, et al (2010) Gibberellin 20-oxidase promotes initiation andelongation of cotton fibers by regulating gibberellin synthesis. J PlantPhysiol 167: 829–837

Xu F, Yang L, Zhang J, Guo X, Zhang X, Li G (2012a) Prevalence of thedefoliating pathotype of Verticillium dahliae on cotton in central Chinaand virulence on selected cotton cultivars. J Phytopathol 160: 369–376

Xu L, Jin L, Long L, Liu L, He X, Gao W, Zhu L, Zhang X (2012b) Over-expression of GbWRKY1 positively regulates the Pi starvation responseby alteration of auxin sensitivity in Arabidopsis. Plant Cell Rep 31: 2177–2188

Xu L, Zhu L, Tu L, Guo X, Long L, Sun L, Gao W, Zhang X (2011a) Differentialgene expression in cotton defence response to Verticillium dahliae by SSH. JPhytopathol 159: 606–615

Xu L, Zhu L, Tu L, Liu L, Yuan D, Jin L, Long L, Zhang X (2011b) Ligninmetabolism has a central role in the resistance of cotton to the wilt fungusVerticillium dahliae as revealed by RNA-Seq-dependent transcriptional analysisand histochemistry. J Exp Bot 62: 5607–5621

Yan J, Zhang C, Gu M, Bai Z, Zhang W, Qi T, Cheng Z, Peng W, Luo H,Nan F, et al (2009) The Arabidopsis CORONATINE INSENSITIVE1protein is a jasmonate receptor. Plant Cell 21: 2220–2236

Yang DL, Yao J, Mei CS, Tong XH, Zeng LJ, Li Q, Xiao LT, Sun TP, Li J,Deng XW, et al (2012) Plant hormone jasmonate prioritizes defense over





growth by interfering with gibberellin signaling cascade. Proc Natl AcadSci USA 109: E1192–E1200

Yoo SD, Cho YH, Sheen J (2007) Arabidopsis mesophyll protoplasts: aversatile cell system for transient gene expression analysis. Nat Protoc 2:1565–1572

Yu S, Galvão VC, Zhang YC, Horrer D, Zhang TQ, Hao YH, Feng YQ,Wang S, Schmid M, Wang JW (2012) Gibberellin regulates the Arabi-dopsis floral transition through miR156-targeted SQUAMOSA promoterbinding-like transcription factors. Plant Cell 24: 3320–3332

Yu S, Ligang C, Liping Z, Diqiu Y (2010) Overexpression of OsWRKY72gene interferes in the abscisic acid signal and auxin transport pathway ofArabidopsis. J Biosci 35: 459–471

Zentella R, Zhang ZL, Park M, Thomas SG, Endo A, Murase K, Fleet CM,Jikumaru Y, Nambara E, Kamiya Y, et al (2007) Global analysis ofDELLA direct targets in early gibberellin signaling in Arabidopsis. PlantCell 19: 3037–3057

Zhang Y, Wang XF, Ding ZG, Ma Q, Zhang GR, Zhang SL, Li ZK, Wu LQ,Zhang GY, Ma ZY (2013) Transcriptome profiling of Gossypium

barbadense inoculated with Verticillium dahliae provides a resource forcotton improvement. BMC Genomics 14: 637

Zhang ZL, Ogawa M, Fleet CM, Zentella R, Hu J, Heo JO, Lim J, KamiyaY, Yamaguchi S, Sun TP (2011) Scarecrow-like 3 promotes gibberellinsignaling by antagonizing master growth repressor DELLA in Arabi-dopsis. Proc Natl Acad Sci USA 108: 2160–2165

ZhengW, Zhai Q, Sun J, Li CB, Zhang L, Li H, Zhang X, Li S, Xu Y, Jiang H, et al(2006a) Bestatin, an inhibitor of aminopeptidases, provides a chemical geneticsapproach to dissect jasmonate signaling in Arabidopsis. Plant Physiol141: 1400–1413

Zheng Z, Qamar SA, Chen Z, Mengiste T (2006b) Arabidopsis WRKY33transcription factor is required for resistance to necrotrophic fungal pathogens.Plant J 48: 592–605

Zhou L, Hu Q, Johansson A, Dixelius C (2006) Verticillium longisporum andV. dahliae: infection and disease in Brassica napus. Plant Pathol 55: 137–144

Zou X, Neuman D, Shen QJ (2008) Interactions of two transcriptional re-pressors and two transcriptional activators in modulating gibberellinsignaling in aleurone cells. Plant Physiol 148: 176–186


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