Rice WRKY13 Regulates Cross Talk between Abiotic and Biotic

15 Pages·2013·1.15 MB·English

Rice WRKY13 Regulates Cross Talk between Abiotic and
Biotic Stress Signaling Pathways by Selective Binding to
Different cis-Elements1[C][W][OPEN]
Jun Xiao2, Hongtao Cheng2, Xianghua Li, Jinghua Xiao, Caiguo Xu, and Shiping Wang*
National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research (Wuhan),
Huazhong Agricultural University, Wuhan 430070, China
Plants use a complex signal transduction network to regulate their adaptation to the ever-changing environment. Rice (Oryza
sativa)WRKY13playsavitalroleinthecrosstalkbetweenabioticandbioticstresssignalingpathwaysbysuppressingabiotic
stressresistanceandactivatingdiseaseresistance.However,itisnotclearhowWRKY13directlyregulatesthiscrosstalk.Here,
we show that WRKY13 is a transcriptional repressor. During the rice responses to drought stress and bacterial infection,
WRKY13 selectively bound to certain site- and sequence-specific cis-elements on the promoters of SNAC1 (for STRESS
RESPONSIVENOAPICALMERISTEM,ARABIDOPSISTRANSCRIPTIONACTIVATIONFACTOR1/2,CUP-SHAPEDCOTYLEDON),
theoverexpressionofwhichincreasesdroughtresistance,andWRKY45-1,theknockoutofwhichincreasesbothbacterialdisease
anddroughtresistance.WRKY13alsoboundtotwocis-elementsofitsnativepromotertoautoregulatethebalanceofitsgene
expressionindifferentphysiologicalactivities.WRKY13wasinducedinleafvasculartissue,wherebacteriaproliferate,during
infection,andinguardcells,wherethetranscriptionalfactorSNAC1enhancesdroughtresistance,duringbothbacterialinfection
and drought stress. These results suggest that WRKY13 regulates the antagonistic cross talk between drought and disease
resistance pathways by directly suppressing SNAC1 and WRKY45-1 and autoregulating its own expression via site- and
sequence-specific cis-elements on the promoters of these genes in vascular tissue where bacteria proliferate and guard cells
wherethetranscriptionalfactor SNAC1mediatesdroughtresistance bypromoting stomatalclosure.
Plants face various abiotic and biotic stresses. To rapid stress response cis-acting regulatory elements
survive,plants haveevolvedcomplexmechanisms for (Walley and Dehesh, 2010), reactive oxygen species
rapidly reallocating metabolic resources and changing (Møller et al., 2007; Wong and Shimamoto, 2009), cal-
thetranscriptomeamongdifferentphysiologicalactivities ciumions(Galonetal.,2010),thephytohormonesabscisic
to adapt to the ever-changing environment (Matyssek acid (Ton et al., 2009) and jasmonic acid (Fonseca et al.,
et al., 2005). Although biotic interactions may diminish 2009), and mitogen-activated protein kinase cascades
the effects of abiotic stress on the allocation of various (Pitzschke et al., 2009). The common features have re-
resources(Matysseketal.,2005),aprolongedmetabolic vealedsomeconvergentnodesofabioticandbioticstress
and transcriptional adaptation to one type of stress is response pathways, and plants can rapidly adapt to a
more likely to influence the adaptation of plants to an- changed environment by synergistically or antagonis-
othertypeofstressduetosystemicresourcereallocation ticallyregulatingthesignalingcrosstalkthroughthese
(Liuetal.,2012).However,thecost-benefitrelationshipin nodes (Fujita et al., 2006; Park et al., 2007; Qiu et al.,
balancing resource allocation between abiotic and biotic 2008). Although research is clarifying the cross talk
stresses remains unclear. nodesbetweenabioticandbioticstresssignaling,onlya
Thedefensesignalingpathwaysforabioticandbiotic fewexamplesforrice(Oryzasativa)havebeenpublished.
stressessharesomecommoncomponents,includingthe In rice, mitogen-activated protein kinase5 (MPK5) ki-
naseenhancesresistancetodrought,salt,andcoldstresses
1ThisworkwassupportedbytheNationalProgramofHighTech- and to Cochliobolus miyabeanus fungal infection but de-
nology Development of China (grant no. 2012AA10A303) and the creases resistance to the fungal pathogen Magnaporthe
National Program on the Development of Basic Research in China oryzaeandthebacterialpathogenXanthomonasoryzaepv
(grantno.2012CB114005). oryzae (Xoo; Xiong and Yang, 2003; De Vleesschauwer
2Theseauthorscontributedequallytothearticle. et al.,2010; Xu et al., 2013). The WRKY89 transcription
*[email protected]. factor increases rice resistance to fungalpathogensand
Theauthorresponsiblefordistributionofmaterialsintegraltothe UV-B irradiation (Wang et al., 2007). The WRKY45-2
findings presented in this article in accordancewith the policy de-
transcription factor mediates broad-spectrum disease
scribed in the Instructions for Authors (www.plantphysiol.org) is:
resistance but negatively regulates adaptation to salt,
ShipingWang([email protected]).
[C]Somefiguresinthisarticlearedisplayedincoloronlinebutin cold,anddroughtstressesinrice(Taoetal.,2009,2011).
Interestingly, the allele of WRKY45-2, the WRKY45-1
blackandwhiteintheprintedition.
[W]TheonlineversionofthisarticlecontainsWeb-onlydata. that also encodes a transcription factor but with a dif-
[OPEN]Articlescanbeviewedonlinewithoutasubscription. ference of 10 amino acids from WRKY45-2, enhances
www.plantphysiol.org/cgi/doi/10.1104/pp.113.226019 rice resistance to M. oryzae but decreases resistance to
1868 Plant Physiology(cid:1), December 2013, Vol. 163,pp. 1868–1882, www.plantphysiol.org (cid:3)2013AmericanSociety ofPlant Biologists.All RightsReserved.
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Selective Binding of Rice WRKY13 to cis-Elements
the bacterialpathogens Xooand Xanthomonasoryzaepv and the tissue- and cell-specific expression patterns of
oryzicola and to cold and drought stresses (Tao et al., WRKY13inabioticandbioticstressresponses.Ourresults
2009, 2011). Overexpression of the rice WRKY30 tran- suggest that WRKY13 is a negative regulator of tran-
scription factor increases rice resistance to fungal path- scription.Ittranscriptionallyregulatesriceresponsesto
ogenanddroughtstresses(Pengetal.,2012;Shenetal., both drought stress and bacterial infection by specifi-
2012). However, our understanding of the molecular cally binding to certain cis-acting elements on the pro-
mechanisms underlying the cross talk through these motersofthedroughtresistance-relatedgeneSNAC1,the
convergent nodes remains preliminary, and the direct defense-responsivegeneWRKY45-1,anditsowngene.
downstreamcomponentsofthesenodesinbothabiotic
and biotic stress signaling are largely unknown.
RESULTS
The function of the rice WRKY13 gene in the cross
talk between disease resistance and abiotic stress re- AbioticStressesInfluenced WRKY13Expression
sistance pathways has been more intensively studied.
WRKY13enhancesriceresistancetoXooandM.oryzae WRKY13 (also named EI12I1 for its complementary
and decreases rice resistance to cold and salt stresses DNA) is transcriptionally induced in rice-pathogen
(Qiuetal.,2007,2008).OverexpressionofWRKY13tran- interactions. The increased transcript level is closely
scriptionally influenced more than 500 genes, including associated with the presence of a major disease resis-
thoseknowntofunctionindiseaseresistanceandabiotic tance gene, Bacterial Blight Resistance3/Bacterial Blight
stress responses (Qiu et al., 2008). The rice SNAC1 Resistance26 (Xa3/Xa26), against Xoo and positively
(for STRESS RESPONSIVE NO APICAL MERISTEM, correlated with the transcript level of Xa3/Xa26 (Wen
ARABIDOPSIS TRANSCRIPTION ACTIVATION FAC- et al., 2003; Cao et al., 2007; Cai et al., 2008). To de-
TOR1/2,CUP-SHAPEDCOTYLEDON)gene,encodinga termine whether WRKY13 was also transcriptionally
NAC-type transcription factor, was induced predomi- responsive to abiotic stress, its expression in the rice
nantly by drought in guard cells that constitute the varietiesMudanjiang8andMinghui63wasexamined
stomata;overexpressionofSNAC1significantlyenhanced afterdroughtandhigh-salinity(150mMNaCl)stresses.
drought resistance in transgenic rice, which isassociated WRKY13expressionwassignificantlysuppressed(P,0.05)
with increased stomatal closure (Hu et al., 2006). Over- at12hto5dafterdroughtstressinbothricevarieties
expressingWRKY13suppressedSNAC1;genome-wide (Fig. 1; Supplemental Fig. S1). Salt stress also signifi-
analysis of the expression profiles of WRKY13 trans- cantlysuppressed(P,0.05)WRKY13at3to36hafter
geniclinessuggestedthattheWRKY13-associateddis- treatmentinthetworicevarieties(Fig.1;Supplemental
easeresistancepathwayinteractsantagonisticallywith
theSNAC1-mediatedabioticstressresponses(Qiuetal.,
2008).FurtherresearchsuggestedthatWRKY13-mediated
signalingpathwaysarepartitionedbydifferenttranscrip-
tionfactors.WhenWRKY13wasactivated,theexpression
of at least 39 transcription factor genes was influenced,
and 30 of them were down-regulated (Qiu et al., 2009).
Analysisofthecis-regulatoryelementsofalltheWRKY13-
influenced genes suggested that WRKY13-influenced
WRKY transcription factors may play important roles
inthetranscriptionalregulationofWRKY13-up-regulated
genesandpathogen-associateddefense-responsivegenes.
OneoftheWRKY13-regulatedWRKYgenesisWRKY45.
WRKY13 appears to suppress both alleles of WRKY45,
WRKY45-1andWRKY45-2.Inrice,WRKY13physically
binds to the promoter regions of these alleles, which
harborWorW-likebox-typecis-regulatoryelementsfor
the binding of WRKY transcription factors (Qiu et al.,
2009; Tao et al., 2009). In addition, WRKY13 can also
bind to its own gene promoter via a W-like box and a
non-Wornon-W-likeboxcis-elementinvitro(Qiuetal.,
2007; Cai et al., 2008). However, it remains to be deter-
mined whether WRKY13 is a transcriptional repressor
and whether it directly regulates SNAC1 and its own
gene expression in rice. It is also unclear which cis-
Figure1. DroughtandsaltstressesinfluencedWRKY13expressionin
elements on the promoter of WRKY45 are essential for
rice varieties Mudanjiang 8 and Minghui 63 analyzed by qRT-PCR.
WRKY13 binding during rice responses to abiotic and Values represent means (three technical replicates) 6 SD. The letters
biotic stresses.
“a”and“b”indicatethatasignificantdifferencewasdetectedbetween
To address these questions, we analyzed the inter- thetreatedplantsandtheuntreatedcontrolatP,0.01andP,0.05,
actions of WRKY13 with candidate promoters in rice respectively.
Plant Physiol. Vol. 163, 2013 1869
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Xiao et al.
Fig. S1). These results suggest that WRKY13 is involved
in rice responses to abiotic stresses. The suppressed
expression of WRKY13 in response to salt stress in
Mudanjiang 8 is consistent with the phenotype of
WRKY13transgenicplantsafterstress.Overexpression
of WRKY13 in the genetic background of Mudanjiang
8 resulted in growth retardation of these transgenic
plants after salt stress (Qiu et al., 2008).
WRKY13 TransgenicPlantsShowedanAlteredResponse
toDroughtStress
To test the putative role of WRKY13 in drought
stress, we examined the phenotype of WRKY13 trans-
genic plants after drought stress. The survival rates of
the two WRKY13-oe lines were 22% and 18%, which
aresignificantlylower(P,0.05)thanthat(34%)forthe
corresponding control (wild-type 1 with the genetic
backgroundofjaponica ricevarietyMudanjiang 8);the
survival rates of the two WRKY13-RNAi lines were
19% and 20% compared with 14% for the corre-
sponding control (wild-type 2 with the genetic back-
ground of indica rice variety Minghui 63; Fig. 2A). We
then measured water loss rates of detached leaves
from the transgenic plants. Consistent with the phe-
notypes of these transgenic plants, WRKY13-oe plants
had a significantly higher (P , 0.05) water loss rate
than the corresponding control at 1 to 1.5 h after
detaching the leaves, whereas WRKY13-RNAi plants
had a significantly lower (P , 0.05) water loss rate
than the corresponding control at 0.5 to 2 h after
detaching the leaves (Fig. 2B). These results suggest
that WRKY13 negativelyregulates the rice responseto
drought stress.
WRKY13 Functionedasa TranscriptionalRepressor
ThericeSNAC1transcriptionfactorenhancesdrought
resistance by promoting stomatal closure (Hu et al.,
2006).WRKY13-oeplantsshowedmarkedlysuppressed Figure 2. Modulating WRKY13 expression influenced the rice re-
expressionofSNAC1withoutpathogeninfectionandin sponse to dehydration. WRKY13-oe, WRKY13-overexpressing lines
the rice-Xoo interaction (Qiu et al., 2008). This study D11UM1-1 and D11UM7-2; WRKY13-RNAi, WRKY13-suppressing
showed that WRKY13-RNAi plants have significantly lines WRKY13S4-10 and WRKY13S12-4. The letters “a” and “b” in-
enhanced (P , 0.05) expression of SNAC1 compared dicate that a significant difference was detected between WRKY13
transgenic plants and corresponding control plants at P , 0.01 and
with control plants without infection (Fig. 3A). The P , 0.05, respectively. A, WRKY13-oe plants and WRKY13-RNAi
oppositeexpressionpatternsofSNAC1inWRKY13-oe
plantsshowedoppositeresponsestodroughtstress.Waterwaswith-
and WRKY13-RNAi plants as compared with its ex- heldfromriceseedlingsatthethree-tofour-leafstagefor6d.After5d
pression in control plants suggest that WRKY13 neg- ofrecovery,thesurvivalrateswererecorded.Valuesrepresentmeans
atively and transcriptionally regulates SNAC1. (45–50plants)6SD.B,Waterlossofdetachedleavesintransgenicrice
TofurtherexaminewhetherWRKY13wasanegative plantsafterdroughtstress.Eachdatapointrepresentsthemean(three
transcriptionalregulator,weanalyzedtheinteractionof replicates, with each replicate consisting of approximately 30 de-
the WRKY13 protein and the SNAC1 promoter by tachedleaves)6SD.[Seeonlinearticleforcolorversionofthisfigure.]
transient expression of WRKY13 driven by the consti-
tutive 35S promoter in rice calli generated from trans-
genic lines carrying a SNAC1 promoter-reporter (GUS) (2 h after the transformation of WRKY13) compared
gene(P :GUS)constructviaAgrobacteriumtumefaciens- with that in the calli immediately before the transfor-
SNAC1
mediatedtransformation.TheexpressionofGUSdriven mation (0 h; Fig. 3B). GUS expression in the trans-
bytheSNAC1promoterinthecalliwasrapidlysuppressed formed calli reached its lowest level at 12 h after the
1870 Plant Physiol. Vol. 163, 2013
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Selective Binding of Rice WRKY13 to cis-Elements
transformation of WRKY13. However, the expression
of GUS in the mock-transformed (control) calli was main-
tained at a relatively high and stable level throughout
thetimecoursesexamined.TheGUSlevelinthecontrol
calliwasmorethan6-foldhigherthanthatinWRKY13-
transformed calli at 12 h after transformation.
WRKY13proteincanalsobindtoitsnativepromoter
in vitro (Qiu et al., 2007; Cai et al., 2008). To ascertain
whetherWRKY13couldalsosuppressitsownexpression,
wetransientlyexpressedWRKY13in ricecallicarryinga
P :GUS construct via A. tumefaciens-mediated trans-
WRKY13
formation.TheexpressionofGUSdrivenbytheWRKY13
promoterinthecalliwasmarkedlysuppressedcompared
with that in untransformed calli (Fig. 3B). Mock trans-
formation (control) did not markedly influence GUS
expression driven by the WRKY13 promoter. The GUS
levelinthecontrolcalliwasapproximately5-foldhigher
than that in WRKY13-transformed calli at 12 h after
transformation.
WRKY13 can also bind to the WRKY45-1 promoter
inrice(Taoetal.,2009).ToexaminewhetherWRKY13
couldalsosuppressWRKY45-1expression,wetransiently
expressed a WRKY45-1 promoter-reporter (GFP) gene
(P :GFP) in rice calli overexpressing WRKY13
WRKY45-1
via A. tumefaciens-mediated transformation. The tran-
script level of GFP driven by the WRKY45-1 promoter
in the calli was increased at 12 h after transformation
(Fig.3C).However,theGFPtranscriptlevelinthewild-
type calli (control) was significantly higher (P , 0.05)
than that in WRKY13-oe calli. These results suggest
that WRKY13 is a transcriptional repressor that not
only suppresses SNAC1 and WRKY45-1 but perhaps it
own gene as well.
WRKY13SelectivelyBoundtoDNA SegmentsHarboring
Knowncis-Elements onSNAC1,WRKY13,and
WRKY45-1Promoters duringAbioticandBiotic
StressesinVivo
Figure3. WRKY13regulatedSNAC1,WRKY13,andWRKY45-1ex- Sequenceanalysisrevealedthatthepromoterregion
pression.Eachdata point representsthe mean(three technical repli- of SNAC1 harbored five cis-acting elements putative
cates)6SDforgeneexpressionanalyzedbyqRT-PCR(AandC);0h for WRKY protein binding, the W-like boxes. The five
indicatesimmediatelyaftertransformation(B and C). A,Suppressing boxesincludetwoTTGACT-typeW-likeboxes(named
WRKY13(WRKY13-RNAi)increasedtheSNAC1transcriptlevelatthe WBOX1 and WBOX3 according to their locations in
bootingstageasanalyzedbyqRT-PCR.Theletters“a”and“b”indicate the promoter region) and three TGACC-type W-like
that a significant difference was detected between WRKY13-RNAi
boxes (named WBOX2, WBOX4, and WBOX5; Fig. 4A;
plants and the wild-type control at P , 0.01 and P , 0.05, respec-
tively.B,TransientoverexpressionofWRKY13inricecallisuppressed Supplemental Fig. S2). To evaluate whether WRKY13
GUSexpressiondrivenbytheSNAC1(PSNAC1)orWRKY13(PWRKY13) directlyboundtotheSNAC1promoter,weperformed
promoter. Mock, Mock transformation of rice calli carrying PSNAC1: chromatin immunoprecipitation (ChIP) assays using
GUSorPWRKY13:GUS.GUSactivitywasdeterminedbymeasuringthe anti-WRKY13 antibody. Four segments of the SNAC1
amount of4-methylumbelliferone(Mu)producedunderthecataly- promoter, which harbor W-like boxes, were analyzed
sis of GUS in 1 mg of total protein per minute. Each data point by quantitative PCR before immunoprecipitation (in-
represents the mean (three biological replicates, with each repli- put) or after immunoprecipitation with or without anti-
cate containing approximately 20 g of calli) 6 SD. C, Over- WRKY13 antibody (Fig. 4A; Supplemental Fig S2). The
expressing WRKY13 suppressed the transcript level of GFP driven
by the WRKY45-1 promoter (PWRKY45-1) as analyzed by qRT-PCR.
WRKY13-oe,WRKY13-overexpressinglineD11UM1-1;WT,wild-type
Mudanjiang 8. Asterisks indicate that a significant difference was thewild-typebackgroundatP,0.01(**)andP,0.05(*).Eachdata
detectedbetweenGFPexpressionintheWRKY13-oebackgroundand pointwasobtainedbyanalyzingapproximately5gofcalli.
Plant Physiol. Vol. 163, 2013 1871
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Xiao et al.
Figure4. WRKY13proteinboundtothepromoterregionsofSNAC1(A),WRKY13(B),andWRKY45-1(C)asanalyzedbyChIP
assays.SampleswerefromricevarietyMudanjiang8thatwasuntreated(control),2dafterinoculationwithXoo(strainPXO61),
or2dafterdroughtstress(withholdingwater).ThequantitativePCRwasconductedbeforeimmunoprecipitation(input),after
immunoprecipitation(IP)usinganti-WRKY13antibody(whitebars),orwithmockimmunoprecipitation(withoutusinganti-
WRKY13antibody;blackbars).ThepresentedpercentageofPCRproductfromafterimmunoprecipitationisrelativetothat
from the input. Values represent means (three replicates) 6 SD. Asterisks indicate that a significant difference was detected
betweenthePCRproductsfromafterimmunoprecipitationwithandwithoutusinganti-WRKY13antibodyat P,0.01(**)
1872 Plant Physiol. Vol. 163, 2013
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Selective Binding of Rice WRKY13 to cis-Elements
immunoprecipitationpercentage(IP%),whichwasthe WRKY13bindstoitsnativepromoterinvivo;itappears
percentage of PCR product from after immunoprecipi- to bind selectively and preferentially to different seg-
tation relative to that from input, was used to evaluate ments under different physiological conditions.
the binding intensity of WRKY13 to the target DNA ActivationofWRKY13suppressedtheexpressionof
segment.Afterimmunoprecipitationwithanti-WRKY13 the defense-responsive gene WRKY45-1, and this sup-
antibody, we detected 4.6-, 3.4-, and 7.7-fold increased pression was reduced after Xoo infection (Qiu et al.,
IP% of the DNA segment harboring WBOX1 from un- 2009).Inaddition,WRKY13canbindtotheWRKY45-1
treated, Xoo-inoculated, and drought-stressed samples, promoter in vivo without stress (Tao et al., 2009). To
respectively, as compared with their corresponding con- ascertain whether WRKY13 also bound to site-specific
trols (samples after precipitation without anti-WRKY13 WorW-likeboxesontheWRKY45-1promoterduring
antibody; Fig. 4A). Compared with untreated sample, riceresponsestoabioticandbioticstresses,foursegments
there was less of an increase in IP% of the DNA seg- of the WRKY45-1 promoter, which harbored W-like
ment harboring WBOX1 in the Xoo-inoculated sample boxes WBOXa (TTGAC) and WBOXb (TGACC), W box
and more of an increase in IP% of this segment in the WBOXd (TTGACC), and the complementary strand
drought-stressed sample. No significant or obvious in- (ACTGA; named WBOXc-C in this paper) of a W-like
creased IP% of the DNA segments harboring WBOX2, box WBOXc (TGACT), were analyzed (Supplemental
WBOX3, or WBOX4 and WBOX5 was detected; thus, Fig. S4). Consistent with previous results (Tao et al.,
WBOX2,WBOX3,WBOX4,andWBOX5couldserveas 2009), we detected 4.2- and 5.2-fold increased IP% of
controlsinthisassay.WBOX1andWBOX3arethesame DNA segments harboring WBOXa and WBOXc-C, re-
type of cis-elements but with different flanking se- spectively,butnotWBOXbandWBOXdfromuntreated
quences (Supplemental Fig. S2), indicating the binding controls after immunoprecipitation with anti-WRKY13
specificity of WRKY13 to the DNA segment harboring antibody (Fig. 4C). Increased IP% of the DNA seg-
WBOX1. These results suggest that WRKY13 binds to ments harboring WBOXa and WBOXc-C was also
the promoter of SNAC1 in vivo; it may preferentially detected in Xoo-inoculated and drought-stressed sam-
bindtotheWBOX1cis-elementoftheSNAC1promoter ples. However, compared with the untreated control,
both with and without abiotic and biotic stresses. there was less of an increase in IP% of the DNA seg-
WRKY13 can bind to its native promoter at two ments harboring WBOXa and WBOXc-C in treated
sites, a novel cis-element, PATHOGEN RESPONSIVE samples. These results suggest that WRKY13 prefer-
ELEMENT4 (PRE4; TACTGCGCTTAGT), and a DNA entiallybindstoW-likeboxesotherthantheWboxon
probe harboring the complementary strand (TCTA- the WRKY45-1 promoter under both stressed and un-
GAACGTCAAATAAAA;namedWBOX-Cinthispaper) stressed conditions.
of a W-like box WBOX (TTTTATTTGACGTTCTAGA;
the W-like box is underlined), as analyzed using elec-
WRKY13Preferentially BoundtoW-LikeBoxesand
trophoreticmobilityshiftassay(EMSA;Qiuetal.,2007;
thePRE4ElementtoRegulateGeneExpressionduring
Caietal.,2008).TodeterminewhethertheWRKY13
AbioticandBiotic Stresses
protein physically bound to its native promoter
(Supplemental Fig. S3) in vivo, we performed ChIP To determine whether the positive segments for
assays using the same samples used for studying the WRKY13 binding in the promoter regions of target
interaction of WRKY13 and the SNAC1 promoter. Af- genes identified by ChIP assays were due to known
ter immunoprecipitation with anti-WRKY13 antibody, cis-elements, we performed EMSA. Total nuclear pro-
we detected 1.8- and 4.2-fold increased IP% of the tein samples from untreated control, Xoo-inoculated,
DNA segments harboring PRE4 and WBOX-C from and drought-stressed rice plants were used for the bind-
untreatedsamples,respectively,ascomparedwiththeir inganalysis.Theproteinsfrombothuntreatedandtreated
corresponding controls (Fig. 4B). Approximately 3-fold plants bound to the probe harboring WBOX1 but not
increased IP% of the segments harboring PRE4 and to the probe harboring WBOX2 from the SNAC1 pro-
WBOX-CfromtheXoo-inoculatedsamplewasdetected. moter (Fig. 5A). The protein binding to the WBOX1
However, 5.1-fold increased IP% of the segment har- probewasreducedorabolishedbyaddinganti-WRKY13
boringWBOX-CbutnotPRE4fromthedrought-stressed antibody. The binding of protein from drought-stressed
sample was detected. No increased IP% of the segment andXoo-inoculatedplantswasabolishedbymutatingthe
harboring the cis-element PRE2 (ACGCTGCCG), which TTGACTsequenceoftheWBOX1elementintheprobeto
is specific for the binding of other proteins (Cai et al., TTGACC(M7probe)thatwasatypicalWbox.However,
2008), was detected, indicating the specificity of the proteinfromuntreatedplantsshowedaweakbindingto
bindingintheChIPassays.Theseresultssuggestthat theM7probe,andthisbindingwasabolishedbyadding
Figure4. (Continued.)
and P , 0.05 (*). The numbers in the white bars are fold differences compared with samples without using anti-WRKY13
antibodyforimmunoprecipitation.Boxes1to5,cis-elementsWBOX1toWBOX5ontheSNAC1promoter.Theamplification
oftheDNAfragmentharboringanon-WRKY13-binding element,PRE2,servedasa samplequantitycontrol.Boxesatod,
cis-elementsWBOXa,WBOXb,WBOXc-C,andWBOXdontheWRKY45-1promoter.
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Xiao et al.
Figure5. WRKY13selectivelyboundtothecis-elementsonSNAC1(A),WRKY13(B),andWRKY45-1(C)promotersunder
differentphysiologicalconditionsanalyzedbyEMSA.ThesequencesofDNAprobesharboringdifferentcis-elements(under-
lined) and their corresponding point-mutated probes are presented. WBOX1 and WBOX2 are cis-elements on the SNAC1
promoter;WBOX-C,PRE4,andPRE2arecis-elementsontheWRKY13promoter;andWBOXaandWBOXc-Carecis-elements
ontheWRKY45-1promoter.SampleswerefromricevarietyMudanjiang8thatwasuntreated(control),2dafterinoculation
with Xoo (strain PXO61), or 2 d after drought stress (withholding water). The cis-elements WBOX2 and PRE2 are negative
controls.+,Plus100ngofanti-WRKY13antibody;–,withoutusinganti-WRKY13antibody.
1874 Plant Physiol. Vol. 163, 2013
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Selective Binding of Rice WRKY13 to cis-Elements
anti-WRKY13 antibody (Fig. 5, A and C). Consistent slightlyinducedinricecalliafterXootreatment(Fig.6).
with the results from ChIP assays (Fig. 4A), there was Unexpectedly, WRKY13 expression in rice calli was
more protein from untreated and drought-stressed obviously induced after drought treatment (Fig. 6;
plants bound to the WBOX1 probe compared with the SupplementalFig.S5), but its expression in rice plants
binding intensity of the protein from Xoo-inoculated was suppressed after drought stress (Fig. 1), suggest-
plants (Fig. 5A). These results suggest that the binding ing that the drought treatment on rice calli may not
proteinisprimarilyWRKY13,andWRKY13specifically fully represent drought stress in rice plants under our
andpreferentiallybindstotheW-likeboxWBOX1onthe conditions. However, consistent with the finding that
SNAC1promoterunderdifferentphysiologicalconditions. WRKY13isatranscriptionalrepressorthatbindstothe
The nuclear proteins also bound to DNA probes promoters of SNAC1 and WRKY45-1 (Figs. 3–5), the
harboringthecis-elementsWBOX-CandPRE4butnot transcriptlevelsofGFPdrivenbythenativepromoters
PRE2ontheWRKY13promoter(Fig.5B).Anti-WRKY13 of SNAC1 (P ) and WRKY45-1 (P ) were neg-
SNAC1 WRKY45-1
antibody reduced or abolished this binding. The muta- atively correlated with the transcript level of WRKY13
tion of WBOX-C (M8 probe) or PRE4 (Mc probe) abol- (Fig. 6, A and B; Supplemental Fig. S5, A and B). Muta-
ished this binding, which is consistent with previous tionoftheWBOX1elementontheSNAC1promotertoa
reports (Qiu et al., 2007; Cai et al., 2008). Consistent typical W box (TTGACC; P ) resulted in sig-
SNAC1△152C
with the results from ChIP assays (Fig. 4B), there was nificantly increased (P , 0.01) GFP transcripts com-
moreproteinfromuntreatedanddrought-stressedplants paredwiththatdrivenbyP (Fig.6A;Supplemental
SNAC1
binding to the WBOX-C probe compared with the Fig. S5A). However, mutation of WBOX2 (P )
SNAC1△298G
bindingintensityofproteinfromXoo-inoculatedplants or WBOX3 (P ) on the SNAC1 promoter did
SNAC1△645G
(Fig. 5B). Only protein from Xoo-inoculated plants notchangethetranscriptlevelofGFP.TheGFPtranscripts
bound to PRE4, and anti-WRKY13 antibody reduced wasalsosignificantlyincreased(P,0.05)aftermutation
this binding. Mutation of PRE4 (Mc probe) abolished of WBOXa (P ) and WBOXc-C (P ),
WRKY45△30G WRKY45△408C
proteinbinding.TheseresultssuggestthattheWRKY13 but not WBOXb (P ), on the WRKY45-1 pro-
WRKY45△93G
protein also specifically and preferentially binds to moter (Fig. 6B; Supplemental Fig. S5B). These results
WBOX-CandPRE4elementsontheWRKY13promoter providefurtherevidencethatWRKY13maysuppress
under different physiological conditions. SNAC1 through the WBOX1 element and suppress
The nuclear proteins also bound to DNA probes har- WRKY45-1throughtheWBOXaandWBOXc-Celements.
boring the cis-element WBOX-a or WBOXc-C from the Mutation of WBOX-C (P ), but not PRE2
WRKY13△541T
WRKY45-1 promoter (Fig. 5C). Anti-WRKY13 antibody (P ),ontheWRKY13promoteralsoresulted
WRKY13△371G
reducedthisbinding.MutationofWBOXa(M9probe)or in significantly increased (P , 0.01) GFP transcript
WBOXc-C (Mh probe) abolished this binding (Fig. 5, levels in untreated and Xoo- or drought-treated calli
AandC).TheseresultssuggestthattheWRKY13protein compared with the GFP transcript level regulated by
alsospecificallyandpreferentiallybindstoWBOXaand the native promoter (P ; Fig. 6C; Supplemental
WRKY13
WBOXc-C elements on the WRKY45 promoter under Fig. S5C). Consistent with the results of ChIP and
different physiological conditions. EMSA,thatWRKY13preferentiallyboundtothePRE4
To determine whether the differential binding of element on the WRKY13 promoter only after Xoo inoc-
WRKY13 to different cis-elements affected the transcrip- ulation(Figs.4and5),mutationofPRE4influencedthe
tionalregulationofthesepromoters,weconstructedfive GFP transcript level only in Xoo-treated but not un-
site-mutated promoters, in which the core sequences of treated or drought-treated calli (Fig. 6C; Supplemental
the cis-elements WBOX1 on the SNAC1 promoter, PRE4 Fig. S5C). However, mutation of PRE4 (P )
WRKY13△158T
and WBOX-C on the WRKY13 promoter, and WBOXa resulted in a slightly decreased GFP transcript level in
and WBOXc-C on the WRKY45-1 promoter were mu- Xoo-treated calli. This result is consistent with a previ-
tatedin thesamewayasDNA probes used forEMSA ous report that PRE4 positively regulated marker gene
(Fig. 6; Supplemental Fig. S5). Four site-mutated pro- expression after Xoo challenge (Cai et al., 2008). In ad-
moters, in which the core sequences of WBOX2 and dition to WRKY13, several other nuclear proteins can
WBOX3ontheSNAC1promoter,PRE2ontheWRKY13 also bind to PRE4, but with different binding core se-
promoter, and WBOXb on the WRKY45-1 promoter quences (Cai et al., 2008). These results suggest that
were mutated, were used as negative controls (Fig. 6; WRKY13maysuppressitsowngeneexpressionviathe
Supplemental Fig. S5). Rice callus has been proved to WBOX-C element and may cooperate with other pro-
be a reliable material for studying the rice-Xoo interac- teins to maintain or increase its gene expression via
tion(Yuanetal.,2011),anditisalsofrequentlyusedfor PRE4 in rice-Xoo interactions.
studyingabioticstresses,suchassaltandwaterstresses
(Ahmad et al., 2007; Wani et al., 2010; Maragathamani
and Khurana, 2012). These mutated promoters were WRKY13WasTranscriptionallyInducedinVascular
fused with GFP and transiently expressed in the calli TissueandGuardCellsduringXooInfectionand
generated from the WRKY13-oe line and wild-type
DroughtStress
Mudanjiang 8.
WRKY13 expression is induced by Xoo invasion in To ascertain in which tissue WRKY13 regulated the
rice plants (Cai et al., 2008). Its expression was also cross talk between abiotic and biotic stress signaling
Plant Physiol. Vol. 163, 2013 1875
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Xiao et al.
Figure6. TheexpressionofGFPregulatedbynativeandmutatedSNAC1(A),WRKY45-1(B),andWRKY13(C)promotersin
ricecalliwasanalyzedbyqRT-PCR.CalliweregeneratedfromtheWRKY13-overexpressinglineD11UM1-1.Thecalliwere
untreated(control)ortreatedwithXoo(strainPXO61)ordroughtfor2h.Asterisksindicatethatasignificantdifferencewas
detectedbetweennativeandmutatedpromoterswithinthesametreatmentatP,0.01(**)andP,0.05(*).
pathways, we expressed GFP driven by the promoter obviousdifferencefromthatincontrolplants(Fig.7A).
of WRKY13 (P ) in rice variety Mudanjiang 8. Xooinvadesriceplantsthroughhydathodesorwounds
WRKY13
Without biotic and abiotic stress (control), a strong and spreads via the vascular system. Thus, the tissue-
GFPsignalwasobservedinvarioustissues(i.e.callus, specific induction of WRKY13 by Xoo infection is asso-
node, stem, collar, ligule, root, pistil, lemma, and pa- ciated with its function in bacterial resistance.
lea) in the transgenic plants carrying P :GFP, but Enhanced SNAC1 expression specifically in guard
WRKY13
no obvious GFP signal was observed in the leaf tissue cells is associated with increased stomatal closure in
(Fig. 7A). Two hours after inoculation with Xoo, a de- SNAC1-mediated drought resistance (Hu et al., 2006).
tectableGFPsignalwasobservedinthevasculartissue ToascertainwhetherWRKY13hadasimilarcell-specific
of leaf immediately next to the infection site, whereas expression pattern to SNAC1 in the rice response to
no obvious GFP signal difference was observed in drought stress, we analyzed GFP expression in guard
other tissues compared with the tissues from unin- cells of transgenic plants carrying P :GFP. No
WRKY13
fected plants. However, 2 h after drought stress, the GFP expression was detected in untreated (control)
GFP expression pattern in various tissues showed no plants, whereas a strong GFP signal was observed in
1876 Plant Physiol. Vol. 163, 2013
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.
Selective Binding of Rice WRKY13 to cis-Elements
Figure 7. WRKY13 showed specific tissue and
cell expression patterns. Control, Before stress
treatment;Xoo,2h(fortissueexpressionpattern)
or 30 min (for cell expression pattern) after in-
oculationofXoostrainPXO61;drought,2hafter
drought stress (withholding water). A, GFP was
expressedindifferentricetissuesinT1transgenic
plants carrying PWRKY13:GFP. The green fluores-
cence is GFP, and the red fluorescence is auto-
fluorescence of tissues. Part of leaf and stem
tissues, entire node, collar, ligule, and a root
branchwerecollectedatthebootingstage;entire
pistil,lemma,andpaleawerecollectedatflow-
ering and grain-filling stages. B, Expression of
GFPinguardcellsofstomataafterXooinfection
ordroughtstressinT1transgenicplantscarrying
PWRKY13:GFPatthefour-leafstage.Bars=15mm.
[Seeonlinearticleforcolorversionofthisfigure.]
guard cells 2 h after drought stress (Fig. 7B). Expres- 2008). Although WRKY13 possesses the structural
sionofP :GFPinguardcellswasalsoobservedat characteristicsofWRKY-typetranscriptionfactorsand
WRKY13
30 min after Xoo infection. These results suggest that hasDNA-bindingability,itstranscriptionalregulation
theexpressionofWRKY13inguardcellsisspecifically functionhasnotbeenexaminedpreviously(Qiuetal.,
induced by both biotic and abiotic stresses. 2007,2009;Caietal.,2008;Taoetal.,2009).Ourresults
heresuggestthatWRKY13isatranscriptionalrepressor.
This conclusion is also supported by a previous report
that WRKY13 preferentially bound to the promoters
DISCUSSION
of genes whose expression was down-regulated in
WRKY13 plays multiple roles in rice biological ac- WRKY13-oelinesanalyzedbyyeastone-hybridassays
tivities. It is involved in the regulation of antagonistic (Qiuetal.,2009).Thetranscriptionalrepressorfunction
cross talk between the pathways of abiotic stress re- of WRKY13 appears to be associated with its direct
sponse and biotic resistance, and it reduces rice plant binding to site- and sequence-specific W-like box-type
height and delays flowering time (Qiu et al., 2007, and PRE4 cis-elements on the promoters of genes
Plant Physiol. Vol. 163, 2013 1877
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Copyright © 2013 American Society of Plant Biologists. All rights reserved.