CaWRKY22 Acts as a Positive Regulator in Pepper Response to Ralstonia Solanacearum by

19 Pages·2017·6.86 MB·English

International Journal o f
Molecular Sciences
Article
CaWRKY22 Acts as a Positive Regulator in Pepper
Response to Ralstonia Solanacearum by Constituting
Networks with CaWRKY6, CaWRKY27, CaWRKY40,
and CaWRKY58
AnsarHussain1,2,3,†,XiaLi1,2,3,†,YahongWeng1,2,3,ZhiqinLiu1,2,3,MuhammadFurqanAshraf1,2,3,
AliNoman1,2,3,4 ID,ShengYang1,2,3,MuhammadIfnan1,2,3,ShanshanQiu1,2,3,
YingjieYang1,2,3,DeyiGuan1,2,3andShuilinHe1,2,3,*
1 MinistryofEducationKeyLaboratoryofPlantGeneticImprovementandComprehensiveUtilization,Fujian
AgricultureandForestryUniversity,Fuzhou350002,China;[email protected](A.H.);
[email protected](X.L.);[email protected](Y.W.);[email protected](Z.L.);
[email protected](M.F.A.);[email protected](A.N.);[email protected](S.Y.);
[email protected](M.I.);[email protected](S.Q.);[email protected](Y.Y.);
[email protected](D.G.)
2 CollegeofCropScience,FujianAgricultureandForestryUniversity,Fuzhou350002,China
3 KeyLaboratoryofAppliedGeneticsofUniversitiesinFujianProvince,FujianAgricultureandForestry
University,Fuzhou350002,China
4 DepartmentofBotany,GovernmentCollegeUniversity,Faisalabad38040,Pakistan
* Correspondence:[email protected]
† Theseauthorscontributedequallytothepaper.
(cid:1)(cid:2)(cid:3)(cid:1)(cid:4)(cid:5)(cid:6)(cid:7)(cid:8)(cid:1)
(cid:1)(cid:2)(cid:3)(cid:4)(cid:5)(cid:6)(cid:7)
Received:26March2018;Accepted:1May2018;Published:10May2018
Abstract: TheWRKYweb,whichiscomprisedofasubsetofWRKYtranscriptionfactors(TFs),plays
acrucialroleintheregulationofplantimmunity,however,themodeoforganizationandoperation
ofthisnetworkremainsobscure,especiallyinnon-modelplantssuchaspepper(Capsicumannuum).
Herein,CaWRKY22,amemberofasubgroupofIIeWRKYproteinsfrompepper,wasfunctionally
characterizedinpepperimmunityagainstRalstoniaSolanacearum. CaWRKY22wasfoundtotarget
thenuclei,anditstranscriptlevelwassignificantlyupregulatedbyRalstoniaSolanacearuminoculation
(RSI)andexogenouslyappliedsalicylicacid(SA),Methyljasmonate(MeJA),orethephon(ETH).
Loss-of-functionCaWRKY22,causedbyvirus-inducedgenesilencing(VIGS),enhancedpepper’s
susceptibilitytoRSI.Inaddition,thesilencingofCaWRKY22perturbedthehypersensitiveresponse
(HR)-likecelldeathelicitedbyRSIanddownregulateddefense-relatedgenesincludingCaPO2,CaPR4,
CaACC,CaBPR1,CaDEF1,CaHIR1,andCaWRKY40.CaWRKY22wasfoundtodirectlybindtothe
promotersofCaPR1,CaDEF1,andCaWRKY40bychromatinimmuno-precipitation(ChIP)analysis.
Contrastingly,transientoverexpressionofCaWRKY22inpepperleavestriggeredsignificantHR-like
cell death and upregulated the tested immunity associated maker genes. Moreover, the transient
overexpression of CaWRKY22 upregulated the expression of CaWRKY6 and CaWRKY27 while it
downregulatedoftheexpressionofCaWRKY58.Conversely,thetransientoverexpressionofCaWRKY6,
CaWRKY27,andCaWRKY40upregulatedtheexpressionofCaWRKY22,whiletransientoverexpression
ofCaWRKY58downregulatedthetranscriptlevelsofCaWRKY22.Thesedatacollectivelyrecommend
theroleofCaWRKY22asapositiveregulatorofpepperimmunityagainstR.Solanacearum,whichis
regulatedbysignalingsynergisticallymediatedbySA,jasmonicacid(JA),andethylene(ET),integrating
intoWRKYnetworkswithWRKYTFsincludingCaWRKY6,CaWRKY27,CaWRKY40,andCaWRKY58.
Keywords: Capsicumannuum;CaWRKY22;immunity;RalstoniaSolanacearum;WRKYnetworks
Int.J.Mol.Sci.2018,19,1426;doi:10.3390/ijms19051426 www.mdpi.com/journal/ijms
Int.J.Mol.Sci.2018,19,1426 2of19
1. Introduction
Beingsessile,plantsfrequentlyencountervariousbioticandabioticstressesindividually,andin
somecases,collectively[1]. Toprotectagainststresses,plantshaveevolvedasophisticateddefense
system developed under frequent selection pressures from the main environmental constraints.
This system is largely regulated at the transcriptional level by the action of different transcription
factors(TFs)interconnectedtomakeacomplicatedtranscriptionalnetworks[2]. Defenseresponses
to different stresses need to be appropriately coordinated and strictly controlled since they are
costlytotheplantsintermsofenergyexpenditureanddevelopment[3]. Thedefensesystemmight
differindifferentplantspeciesduetodiverseecologicalconditionsaffectingtheirevolutioninthe
regions/habitats[4].Therefore,defensemechanismsfoundinmodelplantscannotbetotallysuggested
forothernon-modelplantsdirectly.Plantdefensemechanismshavebeenintensivelystudiedinthepast
decades,butthemajorityofthesestudieshavefocusedonmodelplantssuchasArabidopsisandrice.
However,theorganizationoftranscriptionalnetworksandtheirfunctionalcoordinationtoregulate
plantresponsestodifferentstresses,especiallyinnon-modelplants,remainspoorlyunderstood.
WRKYproteinsconstituteoneofthelargestTFfamiliesinplants. WRKYTFsarecharacterized
by one or two conserved WRKY domains and the almost invariant WRKYGQK sequence at the
N-terminus followed by a C2H2 or C2HC zinc-finger motif [5]. Based on the number of WRKY
domainsandthestructureofzinc-fingermotif,WRKYproteinsarephylogeneticallyclassifiedinto
three major groups (groups I–III). Group II is further divided into five subgroups (IIa, IIb, IIc, IId,
andIIe)[6,7]. Byformingauniquewedgeshapethatinsertsperpendicularlyintothemajorgroove
oftheDNA[8],WRKYTFsprimarilybindW-boxes[TTGAC(C/T)]presentinthepromoterregions
oftargetgenesthroughtheWRKYGQKmotifonthesecondb-strand,andthereby,transcriptionally
modulatetheexpressionofthesetargetgenes. Byactivatingorrepressingthetranscriptionoftheir
target genes, WRKY TFs have been implicated in plant biological processes like senescence, seed
development,dormancy,andgerminationaswellasbioticandabioticstressresponses[9]. Ithasbeen
foundthatasubsetofWRKYgeneswastranscriptionallymodifiedbyasinglestress,orasingleWRKY
TFparticipatesinmultiplestresses. TheW-boxesareenrichedwithinthepromoterregions[10,11].
TheseresultsindicatetheexistenceofWRKYnetworksinvolvedinplantresponsestoaspecificstress
orcombinedstresses. AlthoughtherolesofWRKYproteinsinplantresponsestobioticandabiotic
stressesandtheirunderlyingmechanismshavebeenintensivelystudiedovertheyears,themajority
ofthesestudieshavemainlyfocusedonasinglegeneintheresponseofplantstoasinglestressin
modelplantssuchasArabidopsisandrice. Asignificantfunctionaldivergenceamongclosestructural
homologsofWRKYproteinsfromdifferentplantspecieswasrecentlysuggested[12]. Therolesof
WRKYTFsandtheirnetworksintheresponseofnon-modelplantstodifferentsinglestresses,orto
closelyrelatedstresscombinations,remainpoorlyunderstood.
Pepper (Capsicum annuum) is a vegetable of great economic importance and is a Solanaceae
distributedorplantedinuplandsduringwarmseasons,whereitisconfrontedwithvarioussoil-borne
pathogens,suchasPhytophthoracapsiciandRalstoniaSolanacearum,thecausalagentsforpepperblight
andbacterialwiltdiseases,respectively[13,14]. Thecoexistenceofthesesoil-bornepathogenscauses
severedestruction,oncethepepperplantsexperiencethecombinedstressofhightemperatureandhigh
humidity(HTHH)whichattenuatesRproteinmediatedimmunityandacceleratesthedevelopment
ofpathogens. Ontheotherhand,thecombinationofpathogenattackandHTHHconstitutesmost
naturalselectionpressuresonpepperthathavehistoricallyaffecteditsevolution[15]. However,this
pressuremightnotactintheevolutionofriceandArabidopsis,whichgrowinpaddyfieldsduring
warmseasonsordrylandsduringcoolseasonswithfewersoil-bornepathogens. Thus,pepperseems
moresuitableasaplantforinvestigationonthecoordinationofresistanceortoleranceagainstabiotic
or biotic stresses, for example, Ralstonia Solanacearum inoculation (RSI) and HTHH. Some native
peppervarietiesfromsubtropicalregionsspecificallyshowaugmenteddiseaseresistanceevenunder
HTHH[15]. HSE,ahightemperatureresponsivecis-element, wasubiquitouslyfoundtoco-occur
with Salicylic acid (SA)-, Jasmonic acid (JA)-, Ethylene (ET)-, or pathogen-responsive elements in
Int.J.Mol.Sci.2018,19,1426 3of19
thepromotersofthemajorityofCDPKsandMAPKs,whichhavebeenfrequentlyinvolvedinplant
immunity [10,11]. This suggests the existence of cross-talk mechanisms between immunity, high
temperatureandhumidity.
Since the genome of pepper is about 27 and 7.5 times larger than that of Arabidopsis and rice,
respectively,atotalof73WRKYgeneswerefoundinthegenomeofpepper[16],whichismuchlessthan
whatweexpectedwhencomparedtothe72WRKYgenesinArabidopsisand122inrice[3,6].Ourprevious
studiesindicatedthatCaWRKY6[17],CaWRKY27[18],CaWRKY40[19],andCaWRKY58[20]havebeen
implicatedinthepepperresponsetoRSI.Ofthese,CaWRKY6,CaWRKY27,andCaWRKY40actas
positive regulators, while CaWRKY58 acts as a negative regulator. A subset of W-boxes and HSE
elements were found in the promoters of these genes as well as that of other WRKY promoters,
implyingWRKYnetworksareinvolvedinpepper’sresponsetoRSI.Inaddition,CaWRKY40wasalso
foundtoberegulateddirectlybyCaWRKY6[17]andCabZIP63[21]andindirectlybyCaCDPK15[22].
However,themajorityofpepperWRKYTFshavenotbeencharacterizedintermsofpepper’sresponse
to pathogen infection. In the present study, we report that CaWRKY22, a new IIe WRKY TF of
pepper, acts as a positive regulator in pepper’s response to Ralstonia Solanacearum inoculation by
directlytargetingCaWRKY40andincorporatingaWRKYnetworkincludingCaWRKY6,CaWRKY27,
CaWRKY40,andCaWRKY58.
2. Results
2.1. CloningandSequenceAnalysisofCaWRKY22cDNA
By cis-element scanning within the promoters of WRKY genes in the genome sequence of
Capsicumannuum(http://peppergenome.snu.ac.kr),CaWRKY22wasselectedforfurtherfunctional
characterization. The presence of HSE and immunity associated cis-elements such as the TCA,
TGACG-motif, and W-box in the CaWRKY22 promoter region imply its potential role in pepper
immunity(FigureS1).Byusinggenespecificprimers(TableS1),weclonedacDNAfragmentofCaWRKY22
(CA08g07730)of1500bpinlengththatcontaineda1122bpopen-readingframe(ORF).Itsdeducedamino
acidsequencewas373aminoacidresiduesinlength,containingoneconservedWRKY-domainandwas
classified into subgroup IIe [23] (Figure 1). The size and theoretical pI of the predicted protein were
41.29kDaand5.87,respectively.CaWRKY22shares91%,91%,88%,and55%ofaminoacididentitieswith
SpWRKY22,StWRKY22,NsWRKY22,andGrWRKY22,respectively(FigureS2).
2.2. TheTranscriptionalExpressionofCaWRKY22IsUpregulatedbyR.SolanacearumInfectionand
ExogenousAppliedPhytohormonesIncludingSA,MeJA,andETH
The presence of a subset of putative immunity responsive cis-elements in the promoter of
CaWRKY22impliesitsinducibleexpressionuponpathogenattack. Totestthispossibility,qRT-PCR
was performed to examine the expression pattern of CaWRKY22 in response to inoculation of
R.Solanacearum. TheresultsshowedthatthetranscriptionallevelsofCaWRKY22wereupregulatedin
pepperleavesinoculatedwithR.Solanacearum,comparedtothatinthemocktreatedleaves(Figure2A).
TheincreasedCaWRKY22transcriptionallevelsweremaintainedbetween6and24hpi(hourspost
inoculation)andexhibitedmaximallevelsat6hpi,implyingtheinvolvementofCaWRKY22inthe
responseofpeppertowardR.Solanacearum(Figure2A).
Int.J.Mol.Sci.2018,19,1426 4of19
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 4 of 19

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o(fFSigAu,rter e2aCtm). Seinmtiolfarth toe ptheep appeprlpiclaantitown iotfh S1A00, tµreMatmMeenJAt oof rth1e0 0pµepMpeErT pHla(nett hweipthh o1n00) uµpMre MguelJaAte odr t1h0e0
tµraMns cErTipHti o(enthoefpChaoWnR) Yu2p2refrgoumlat1edto t2h4e htpratncsocmrippatiroend otof tChaeWmRoYc2k2t rferoatmm e1n tto( F2i4g uhrpet 2cDom,Ep).ared to the
mock treatment (Figure 2D,E).
Int.J.Mol.Sci.2018,19,1426 5of19
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2.5 2.5 1.6
Relative expresssion level 112....0505 A BA** BA **BA MRBSoA*c*k Relative expression level 112....0505 B BA BA BA MSBAoAck Relative expression level 111.......0242468 C AB AB AB AMABBoAck
0.0 0.0 0.0
6 12 24 36(hpi) 1 6 12 24 (hpt) 1 6 12 24 (hpt)
3.0 3.0
D E
e expression level 1122....0505 BA BA MMboecJakA BA e expression level 1122....0505 BA BA BA BMETAoHck
v v
ati ati
el .5 el .5
R R
0.0 0.0
1 6 12 24 (hpt) 1 6 12 24 (hpt)

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2
control plants (mock), whose relative expression level was set to “1”. (B–E) The transcriptional levels
whoserelativeexpressionlevelwassetto“1”. (B–E)Thetranscriptionallevelsinhormone-treated
in hormone-treated pepper leaves were compared with those in ddH2O-treated plants (mock), whose
pepperleaveswerecomparedwiththoseinddH O-treatedplants(mock),whoseexpressionlevelwas
expression level was set to “1”. Error bars indic2ated the standard error. Different letters above the bar
setto“1”.Errorbarsindicatedthestandarderror.Differentlettersabovethebarshowasignificant
show a significant difference between the means of the three biological replicates based on the
differencebetweenthemeansofthethreebiologicalreplicatesbasedontheFisher’sprotectedLSDtest:
Fisher’s protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.
uppercaseletters,p<0.01;lowercaseletters,p<0.05.
2.3. CaWRKY22 Is Located in the Nuclei
2.3. CaWRKY22IsLocatedintheNuclei
Sequence analysis using WoLFPSORT (“http://www.genscript.com/psort/wolf_psort.html”
olf_psort.html) showed that the predicted CaWRKY22 amino acid sequence contains a putative
SequenceanalysisusingWoLFPSORT(“http://www.genscript.com/psort/wolf_psort.html\T1\
nuclear localization signal (Figure 1), indicating its potential nucleus targeting. To confirm this
textquotedblrightolf_psort.html)showedthatthepredictedCaWRKY22aminoacidsequencecontains
speculation, we constructed a CaWRKY22–GFP fusion construct driven by the constitutive promoter
aputativenuclearlocalizationsignal(Figure1),indicatingitspotentialnucleustargeting. Toconfirm
of CaMV35S, and the generated vector was transformed into Agrobacterium tumefaciens strain
this speculation, we constructed a CaWRKY22–GFP fusion construct driven by the constitutive
GV3101. CaWRKY22–GFP was transiently overexpressed in N. benthamiana leaves by Agrobacterium
promoterofCaMV35S,andthegeneratedvectorwastransformedintoAgrobacteriumtumefaciensstrain
infiltration, and the GFP signals were observed using a confocal fluorescence microscope. The result
GV31s0h1o.wCeadW thRaKt tYh2e 2G–FGPF sPigwnaals otfr tahnes CieanWtRlyKoYv2e2r–eGxFpPr ewsasse dexicnluNsi.vbeleyn tfohuamndia inna thleea nvuecslebi,y wAhgerroebaas cttheer ium
infiltrGaFtiPo nco,natnrdol twheasG fFoPunsdig inna mlsuwltieprlee osbusbecrevlleudlaru scionmgpaarctomnefnotcsa, linflculuodreinsgc etnhcee cymtoicprloassmco paned. Tnhueclreei sult
show(eFdigtuhrea t3tAh)e. SGeFvePrasli gstnuadlieosf hthaveeC maWenRtioKnYe2d2 t–hGe FbPindwinags eoxf cWluRsKivYe lpyroftoeuinnsd toi nthteh eWn-buocxl emi,awnnheerr eas
the G[FTPTGcAoCn(tCro/Tl)w] apsrefsoeuntn dini nthme uplrtoipmloetesru brecgeilolun laorf ctohme pdaerfetmnsee natsss,oicniactleudd itnagrgteht egceynetos,p lwahsmicha nd
nuclefir(eFqiugeunrtely3 sAer)v.eS eavse praalthsotugdenie rsehspavonesmiveen rteiognuleadtotrhye ebleinmdeinntgs.o TfoW cRheKcYk wprhoettehiners tthoist haelsWo r-beloaxtems taon ner
CaWRKY22, we conducted a transient coexpression experiment with an effector vector carrying the
[TTGAC(C/T)]presentinthepromoterregionofthedefenseassociatedtargetgenes,whichfrequentlyserve
full length CaWRKY22 cDNA controlled by a CaMV35S promoter (35S:CaWRK22-HA), and a reporter
aspathogenresponsiveregulatoryelements.TocheckwhetherthisalsorelatestoCaWRKY22,weconducted
vector having GUS-control driven by the CaMV35S core promoter (−46 to +8 bp), with two copies of
atransientcoexpressionexperimentwithaneffectorvectorcarryingthefulllengthCaWRKY22cDNA
W-box (2xW-p35Score:GUS) or muted W-box (2xW-m-p35Score:GUS) in its proximal upstream region
controlledbyaCaMV35Spromoter(35S:CaWRK22-HA),andareportervectorhavingGUS-controldriven
(Figure 3B). Reporter vectors were either transformed individually or co-transformed with the
bytheCaMV35Scorepromoter(−46to+8bp),withtwocopiesofW-box(2xW-p35Score:GUS)ormuted
effector construct into N. benthamiana leaves by infiltration, and the co-infected leaves were sampled
W-bofxo(r2 GxWUS-m a-cpti3v5iStyc omree:GasUurSe)minenitts. Tphroe xGimUaSl quupasntrtiefaicmatiroeng iroensu(lFt igreuvreea3leBd) .tRhaetp Nor. tbeernvtheacmtoirasnaw leeraeveesit her
transfcoorimnfeecdteidn dwivitihd 3u5aSll:CyaoWrRcKo2-t2r-aHnAsf oanrmd e2dxWw-pit3h5Stchoeree:GffUecSt oexrhciobnitsetdr usctrtoinngt oGNU.S baecntitvhiatmy,i acnoma pleaarveeds by
infiltration,andtheco-infectedleavesweresampledforGUSactivitymeasurement.TheGUSquantification
result revealed that N. benthamiana leaves coinfected with 35S:CaWRK22-HA and 2xW-p35Score:GUS

exhibitedstrongGUSactivity,comparedtothemockleaves,suggestingthatCaWRKY22iscapableof
activetranscriptionexpressionofthedownstreamtargetgene(Figure3C).
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 6 of 19
to the mock leaves, suggesting that CaWRKY22 is capable of active transcription expression of the
Int.J.Mdoolw.Sncsi.tr2e0a18m,1 t9a,r1g4e2t6 gene (Figure 3C). 6of19

Figure 3. Subcellular localization of CaWRKY22 and its transactivation confirmation experiment.
Figure 3. Subcellular localization of CaWRKY22 and its transactivation confirmation experiment. (A)
(A)CaWRKY22wasexclusivelyfoundinthenucleusofN.benthamianaleaves,thetransientlyexpressed
CaWRKY22 was exclusively found in the nucleus of N. benthamiana leaves, the transiently expressed
35S:C3a5WSR:CKaWY2R2K–GY2F2P–.GGFrPee. nGrceoelonr csohloorw sshoGwFsP .GBFluP.e Bclouleo rcoslhoorw shsoDwAs PDIAstPaIi nstianignionfgn oufc nleuucsle.uCsy. aCnyaclno crlsohr ows
mergsehroowfsg mreeerngeGr FoPf agnredenD GAFPPI satnadin eDdAnPIu cslteauinse.d GnFuPclesuigsn. aGlF(PG rseigenna)l fo(Grrteheen)c ofonrt rtohleN c.onbetrnotlh aNm. iana
leavebsenwthaasmifaonua nledavtehsr wouasg hfoouuntd tthherouceglhl.outI mthea gceelsl. wImeargeest awkeerne tbaykenc obnyf ococnaflocmali cmroicsrcoospcoypya tat4 488 hpi
(hourhsppi o(shtoiunros cpuolastt ioinno)c.uBlaatrison=).2 B5aµrsm =; (2B5) sµcmh;e m(Ba) tiscchdeimagartaicm doiafgtrhaeme foffe cthtoer eafnfedctroerp aonrdte rrecpoonrstetrru cts
usedcfoonrsttrrauncstsie unstecdo feoxr ptrraensssiieonnt; c(oCe)xlperaevsseisono;f (pCe)p lepaevrews oefr epecpoptrearn wsfeercet ceodtrwanitshfetchteedr wepitohr ttehre arenpdoertfefer ctor
plasmanidds eafnfedctothr epilansfimltirdast eadndl etahve einsfwilterraetehda lrevaevsetse wdefroer hGarUvSesatecdti vfoirty GmUeSa ascutirveimty emnet.asTuhreemGeUntS. aTchtei vity
GUS activity of pepper leaves transient coexpressing 2xW-p35Score:GUS and the empty effector
ofpepperleavestransientcoexpressing2xW-p35Score:GUSandtheemptyeffectorvectorwassetto
vector was set to “1”. Error bars indicated the standard error. The data represents the means ± SD
“1”.Errorbarsindicatedthestandarderror.Thedatarepresentsthemeans±SDfromthreebiological
from three biological replicates. The asterisk indicates significant differences, as determined by
replicates.Theasteriskindicatessignificantdifferences,asdeterminedbyFisher’sprotectedLSDtest
Fisher’s protected LSD test (* p < 0.05, ** p < 0.01).
(*p<0.05,**p<0.01).
2.4. Effect of CaWRKY22 Loss-of-Function by VIGS on Response of Pepper to R. Solanacearum Inoculation
2.4. EffectofCaWRKY22Loss-of-FunctionbyVIGSonResponseofPeppertoR.SolanacearumInoculation
The loss-of-function experiment was performed in pepper seedlings by Virus Induced Gene
TSihleenlcoinsgs- (oVfI-GfuSn) ctoti ionnveesxtipgearteim theen rtolwe aosf CpaeWrfRoKrmY2e2d ini nimpmeupnpietyr. s5e0e pdllainntgs sofb TyRVVi:0ru0 sanIdn d50u pceladntGs ene
Silencoifn TgR(VV:ICGaSW)RtoKYin2v2e swtiegrea teactqhueirreodle. SoifxC palWanRtsK Yfr2o2mi nthime mTRuVn:iCtyaW. 5R0KpYl2a2n tpsloanftTsR wVe:0re0 aranndd5o0mplyla nts
ofTRsVe:lCecatWedR tKoY a2s2sewsse rtheeairc qgueinree ds.ilSeinxcipnlga netfsficfrieonmcyt hbey TrRooVt: CinaoWcuRlKatYio2n2 wpliathn tcsewllse roef rtahne dvoimrullyenste Rle.c ted
Solanacearum strain FJC100301. The result showed that in R. Solanacearum challenged
toassesstheirgenesilencingefficiencybyrootinoculationwithcellsofthevirulentR.Solanacearum
TRV:CaWRKY22 pepper plants, transcriptional levels of CaWRKY22 were reduced to ~30% of that in
strainFJC100301. TheresultshowedthatinR.SolanacearumchallengedTRV:CaWRKY22pepperplants,
TRV:00 plants, showing the successful silencing of CaWRKY22 (Figure 4A). After R. Solanacearum
transcriptional levels of CaWRKY22 were reduced to ~30% of that in TRV:00 plants, showing the
inoculation, TRV:CaWRKY22 pepper plants exhibited a significantly enhanced susceptibility to the
successfulsilencingofCaWRKY22(Figure4A).AfterR.Solanacearuminoculation,TRV:CaWRKY22
pathogen compared to TRV:00 (control). This susceptibility was coupled with an increase in the
pepperplantsexhibitedasignificantlyenhancedsusceptibilitytothepathogencomparedtoTRV:00

(control). This susceptibility was coupled with an increase in the growth of R. Solanacearum in
CaWRKY22-silenced pepper plants, manifested by higher cfu values compared with those in the
controlplantsat3dpi(dayspostinoculation)(Figure4B).Histochemicalstainingwasperformedto
assesscelldeathandH O productioninR.Solanacearum-infected,CaWRKY22-silenced,andcontrol
2 2
pepperleaves,anintensiveDAB(darkbrowncolor)staining(indicatorofH O accumulation)and
2 2
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 19
growth of R. Solanacearum in CaWRKY22-silenced pepper plants, manifested by higher cfu values
compared with those in the control plants at 3 dpi (days post inoculation) (Figure 4B). Histochemical
Isntta.iJn.Minogl. Swci.a2s0 18p,e1r9f,o14r2m6ed to assess cell death and H2O2 production in R. Solanacearum-infe7cotfe1d9,
CaWRKY22-silenced, and control pepper leaves, an intensive DAB (dark brown color) staining
(indicator of H2O2 accumulation) and hypersensitive reaction (HR) mimic cell death, manifested by
hypersensitive reaction (HR) mimic cell death, manifested by darker trypan blue staining, were
darker trypan blue staining, were detected in the control leaves at 48 hpi, whereas the intensities of
detected in the control leaves at 48 hpi, whereas the intensities of DAB and trypan blue staining
DAB and trypan blue staining were distinctly reduced in CaWRKY22-silenced leaves (Figure 4C). Ion
were distinctly reduced in CaWRKY22-silenced leaves (Figure 4C). Ion leakage was estimated to
leakage was estimated to analyze the severity of cell death and plasma membrane damage after
analyzetheseverityofcelldeathandplasmamembranedamageafterinoculationbyR.Solanacearum,
inoculation by R. Solanacearum, the results showed that the unsilenced pepper plants exhibited
the results showed that the unsilenced pepper plants exhibited higher ion leakage compared to
higher ion leakage compared to CaWRKY22-silenced pepper plants at 24, 48, and 72 hpi (Figure 4D).
CaWRKY22-silenced pepper plants at 24, 48, and 72 hpi (Figure 4D). A fluorescent modulation
A fluorescent modulation meter was used to take the photos of leaf cell death in TRV:CaWRKY22
meterwasusedtotakethephotosofleafcelldeathinTRV:CaWRKY22andTRV:00afterinfection
and TRV:00 after infection with R. Solanacearum FJC100301. The cell death detected in the unsilenced
withR.SolanacearumFJC100301. Thecelldeathdetectedintheunsilencedpepperleaveswasvery
pepper leaves was very noticeable and strong, while very weak and negligible cell death was
noticeableandstrong,whileveryweakandnegligiblecelldeathwasdetectedinCaWRKY22-silenced
detected in CaWRKY22-silenced plants leaves (Figure 4E). 10 pepper plants of TRV:CaWRKY22 and
plantsleaves(Figure4E).10pepperplantsofTRV:CaWRKY22andTRV:00wererandomlyselected
TRV:00 were randomly selected and root inoculated with R. Solanacearum. At 7 dpi, definite wilting
and root inoculated with R. Solanacearum. At 7 dpi, definite wilting symptoms were observed in
symptoms were observed in CaWRKY22-silenced pepper plants, whereas unsilenced plants
CaWRKY22-silencedpepperplants,whereasunsilencedplantsexhibitedonlyfaintwiltingsymptoms
exhibited only faint wilting symptoms (Figure 4F). QRT-PCR was used to check the transcriptional
(Figure4F).QRT-PCRwasusedtocheckthetranscriptionalexpressionlevelsofknowndefense-related
expression levels of known defense-related genes, the results showed that transcriptional levels of
genes,theresultsshowedthattranscriptionallevelsofthedefense-relatedpeppergenes,including
the defense-related pepper genes, including CaPO2, CaPR4, CaACC, CaBPR1, CaDEF1, and CaHIR1,
CaPO2,CaPR4,CaACC,CaBPR1,CaDEF1,andCaHIR1,werelessenedinCaWRKY22-silencedpepper
were lessened in CaWRKY22-silenced pepper plant leaves compared to that in control pepper plants
plantleavescomparedtothatincontrolpepperplantsat24hpi(Figure4G).
at 24 hpi (Figure 4G).
A 12 B 11Xe1+055 C TRV:CaWRKY22 TRV:00 D TRV::00-Mock TRV::CaWRKY22-Mock
TRV:00 TRV::00-RSI TRV::CaWRKY22-RSI
TRV:CaWRKY22 400
Relative expression level102468 A A A B -2cfu (cm)024688642XXXXeeee1111++++000044444444 A B DABanblue -1Conductivity (S cm)123000000 CA C B C A C B C A C B
0 Mock RSI TRV::CaWRKY22TRV::00 Tryp 0 24 48 72 (hpi)
E TRV:00 TRV:CaWRKY22 F TRV:CaWRKY22 G 3 A TTRRVV::::0000--MRSocIk TTRRVV::::CCaaWWRRKKYY2222--MRSocIk
RSI Relative expression level 12 B CC BADC BACC BACC BADC BACC
0
CaPO2 CaPR4 CaACC CaBPR1 CaDEF1 CaHIR1
TRV:00
Figure 4. Distinctive responses of CaWRKY22-knockout attenuates the pepper’s resistance to RSI.
Figure 4. Distinctive responses of CaWRKY22-knockout attenuates the pepper’s resistance to RSI. (A)
(A)QRT-PCRanalysisofCaWRKY22expressioninR.Solanacearum-inoculated,mock(inoculatedwith
QRT-PCR analysis of CaWRKY22 expression in R. Solanacearum-inoculated, mock (inoculated with
MgCl solution)CaWRKY22-silencedpepperplants(TRV:CaWRKY22),andcontrolplants(TRV:00);
MgCl22 solution) CaWRKY22-silenced pepper plants (TRV:CaWRKY22), and control plants (TRV:00);
(B) difference in R. Solanacearum growth between CaWRKY22-silenced and control pepper plants
(B) difference in R. Solanacearum growth between CaWRKY22-silenced and control pepper plants
inoculatedwithR.Solanacearumat3dpi(dayspostinoculation);(C)DABandtrypanbluestaining
inoculated with R. Solanacearum at 3 dpi (days post inoculation); (C) DAB and trypan blue staining in
inR.Solanacearum-inoculatedCaWRKY22-silenced(TRV:CaWRKY22)andcontrol(TRV:00)pepper
R. Solanacearum-inoculated CaWRKY22-silenced (TRV:CaWRKY22) and control (TRV:00) pepper
leavesat48hpi.Scalebar=50µm;(D)electrolyteleakageasionconductivitytoassessthecelldeath
leaves at 48 hpi. Scale bar = 50 µm; (D) electrolyte leakage as ion conductivity to assess the cell death
responsesintheleafdiscsofCaWRKY22-silenced(TRV:CaWRKY22)andcontrol(TRV:00)pepperafter
responses in the leaf discs of CaWRKY22-silenced (TRV:CaWRKY22) and control (TRV:00) pepper
24,48,and72hofinoculationwithandwithoutR.Solanacearum; (E)celldeathinR.Solanacearum
after 24, 48, and 72 h of inoculation with and without R. Solanacearum; (E) cell death in R.
inoculatedCaWRKY22-silenced(TRV:CaWRKY22)andcontrol(TRV:00)pepperleavesunderfluorescent
Solanacearum inoculated CaWRKY22-silenced (TRV:CaWRKY22) and control (TRV:00) pepper leaves
modulation meter; (F) phenotypic effect of R. Solanacearum treatment on CaWRKY22-silenced
under fluorescent modulation meter; (F) phenotypic effect of R. Solanacearum treatment on
(TRV:CaWRKY22)andcontrol(TRV:00)pepperplantsat7dpi;(G)qRT-PCRanalysisoftranscriptional
CaWRKY22-silenced (TRV:CaWRKY22) and control (TRV:00) pepper plants at 7 dpi; (G) qRT-PCR
levelsofdefense-relatedmarkergenesinCaWRKY22-silenced(TRV:CaWRKY22)andcontrol(TRV:00)
analysis of transcriptional levels of defense-related marker genes in CaWRKY22-silenced
pepper plants 24 h post inoculation with R. Solanacearum. The relative expression level of mock
(TRV:CaWRKY22) and control (TRV:00) pepper plants 24 h post inoculation with R. Solanacearum.
treatedunsilencedplantswassetto“1”. Errorbarsindicatedthestandarderror. Datarepresents
The relative expression level of mock treated unsilenced plants was set to “1”. Error bars indicated
the means ± SD from four biological replicates. Different letters indicate significant differences,

asdeterminedbyFisher’sprotectedLSDtest:uppercaseletters,p<0.01;lowercaseletters,p<0.05.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 8 of 19
the standard error. Data represents the means ± SD from four biological replicates. Different letters
Int.J.Mol.Sci.2018,19,1426 8of19
indicate significant differences, as determined by Fisher’s protected LSD test: uppercase letters, p <
0.01; lower case letters, p < 0.05.
2.5. TransientOverexpressionofCaWRKY22-TriggeredHR-LikeCellDeathandAccumulationofH O inthe
2.5. Transient Overexpression of CaWRKY22-Triggered HR-Like Cell Death and Accumulation of H2O2 2in t2he
LeavesofPepperPlants
Leaves of Pepper Plants
TheresultsoftheCaWRKY22silencingexperimentindicatethatCaWRKY22actsasapositive
The results of the CaWRKY22 silencing experiment indicate that CaWRKY22 acts as a positive
regulraetgourlaintorp ienp ppeeprp’serr’ess rpeospnosnesteo toR RSIS.I.T Toof ufurrtthheerr ccoonnffiirrmm tthhisis spspeceuclualtaiotino, na, taratnrsainensite onvteorevxeprreexspsiroens sion
assayaswsaays pwerafso rpmeerfdorimnepde pipne rpleepavpeers toleainvvese sttiog atinevtehsetiegfafteec ttohfeC aeWffeRctK Yo2f 2CtraaWnRsiKenYt22o vterraenxspiernets sion
onthoeveinredxupcretisosinono fopn lathnet iHndRuccteilolnd oefa tphla.nAt HWRe scteellr ndebaltoht.t iAn gWaessstaeryn cbolnofittirnmg eadsstahya tcoCnafiWrmReKd Yth2a2t was
succeCsasWfuRllKyYe2x2p wreasss seudcciensspfuelplyp eexrpprelassnetds i(nF ipgeuprpeer5 Apl)a.nAts n(Fiingtuerne s5ivAe). cAenll idnteeantshivwe caesllm daeantihf ewstaesd by
darkmeratnriyfepsatendb bluy edsatrakienri ntrgy,paannd baluhei gsthaeinrilnegv, ealnodf aa chciugmheur laletvioeln ooff aHccuOmudlaistipolna yoef dHw2Oi2t hdidsaprlakyeerdD AB
2 2
with darker DAB staining was detected in pepper leaves infiltrated with Agrobacterium cells carrying
stainingwasdetectedinpepperleavesinfiltratedwithAgrobacteriumcellscarrying35S:CaWRKY22
35S:CaWRKY22 compared to the control plant leaves infiltrated with Agrobacterium cells carrying
comparedtothecontrolplantleavesinfiltratedwithAgrobacteriumcellscarrying35S:00(Figure5B).
35S:00 (Figure 5B). Consistently, the pepper leaves transiently overexpressing CaWRKY22 exhibited
Consistently,thepepperleavestransientlyoverexpressingCaWRKY22exhibitedsignificantlyhigherion
significantly higher ion leakage at 24, 48, and 72 h post inoculation compared to the leaves
leakageat24,48,and72hpostinoculationcomparedtotheleavesexpressingtheemptyvector(Figure5C).
expressing the empty vector (Figure 5C). QRT-PCR was performed to examine the relative
QRT-PCRwasperformedtoexaminetherelativetranscriptionallevelsofdefense-relatedgenesincluding
transcriptional levels of defense-related genes including the SA-responsive CaPR4 and CaBPR1,
theSJAA--rreessppoonnssiivvee CCaaPDRE4F1a,n dECT aBbPioRs1y,nJtAhe-sriess-pasosnosciivateedC aDCaEAFC1,CE, THbRio smynatrhkeesr is-CaasHsoIRci1a, teadnCd aARCOCS, HR
markdeertCoxaiHfiIcRat1io,ann-adssRoOciaStedde tCoxaPifOic2a.t iTohne-a rsessouclitast esdhoCwaPedO 2th.aTt hCearWesRuKltYs2s2h otrwanesdietnhta toCvearWexRpKreYs2s2iotnr ainns ient
overepxepprpeesrs iloeanveins spigenpipfiecranletlayv ienscrseiagsneidfi cthane ttlryaninsccrriepatisoendalt hleevetlrsa nofs cCraiBpPtiRo1n,a ClalPevRe4l,s CoaDf ECFa1B,P CRa1A,CCCa, PR4,
CaDECFa1H,ICRa1A, aCnCd, CCaaPHOI2R a1s, aconmdpCaarPedO 2toa tshcaot minp paerpepdetro letahvaetsi ntrapnespiepnetrlyle oavveersextrparnesssieinngt lyemopvteyr evxepctroers sing
empt(yFivgeucrteo r5D(F)i.g Aurlle t5hDes).e Adlalttah edseemdoantsatrdaetem tohnastt rCaateWtRhKatYC2a2W acRtsK aYs2 a2 apcotssitaivsea rpeogsuitlaivtoerr eogf upllaatnotr coefllp lant
death.
celldeath.

Figure5.TransientoverexpressionofCaWRKY22inpepperleavestriggeredintenseHR-likecelldeath
Figure 5. Transient over expression of CaWRKY22 in pepper leaves triggered intense HR-like cell
andROSaccumulation.(A)Westernblottingwasperformedtoconfirmthesuccessfuloverexpression
death and ROS accumulation. (A) Western blotting was performed to confirm the successful
of CaWRKY22-Flag; (B) HR caused by transient overexpression of 35S:CaWRKY22, confirmed by
overexpression of CaWRKY22-Flag; (B) HR caused by transient overexpression of 35S:CaWRKY22,
phenotypedetection,UVlightexposure,andDABandTrypanBluestainingat4dpi,respectively;
confirmed by phenotype detection, UV light exposure, and DAB and Trypan Blue staining at 4 dpi,
(C)measurementofelectrolyteleakage(ionconductivity)toevaluatethecelldeathresponseinleaf
respectively; (C) measurement of electrolyte leakage (ion conductivity) to evaluate the cell death
discsreastp2o4n,s4e 8in, alenadf d7i2schs apto 2s4t, a4g8,r oan-idn fi72lt hra ptioosnt ,argersop-iencfitlitvraetliyo;n(, Dre)spqeRcTti-vPeClyR; (Dan) aqlRyTsi-sPCofRt ahneaelyxspisr eosf sion
ofimtmheu nexitpyr-ersesliaotne dofm iamrkmeurngiteyn-eresl,aitnedcl umdainrkgerC agPeOne2s,, CinaPclRu4d,inCga ACCaPCO,2C,a BCPaPRR14,,C CaDaAECFC1,, aCnadBPCRa1H, IR1,
in35S:CaWRKY22expressedpepperleavesat24hpi,respectively. Therelativeexpressionlevelof

markergenesinpepperleavestransientlyexpressingtheemptyvectorweresetto“1”.Datarepresent
themeans±SDfromfourbiologicalreplicates.Errorbarsindicatedthestandarderror.Differentletters
abovethebarsshowssignificantdifferencesbetweenthemeans,asanalyzedbyFisher’sprotectedLSD
test:uppercaseletters,p<0.01;lowercaseletters,p<0.05.
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 9 of 19
CaDEF1, and CaHIR1, in 35S:CaWRKY22 expressed pepper leaves at 24 hpi, respectively. The relative
expression level of marker genes in pepper leaves transiently expressing the empty vector were set
to “1”. Data represent the means ± SD from four biological replicates. Error bars indicated the
Int.J.Mol.Sci.2018,19,1426 9of19
standard error. Different letters above the bars shows significant differences between the means, as
analyzed by Fisher’s protected LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.
2.6. CaWRKY22BindstotheW-BoxContainingPromoterFragmentbutNottheFragmentswithoutW-Box
2.6. CaWRKY22 Binds to the W-Box Containing Promoter Fragment but Not the Fragments without W-Box
It has been generally reported that the majority of members of the WRKY family fulfill their
It has been generally reported that the majority of members of the WRKY family fulfill their
functionsbytargetingandbindingtheconservedW-box[TTGAC(C/T)]presentinthepromotersof
functions by targeting and binding the conserved W-box [TTGAC(C/T)] present in the promoters of
theirtargetgenes[23]. TotestifCaWRKY22canactivatethetranscriptionofitstargetgenesinaW-box
their target genes [23]. To test if CaWRKY22 can activate the transcription of its target genes in a
dependentmanner,achromatinimmuno-precipitation(ChIP)assaywasperformedonchromatins
W-box dependent manner, a chromatin immuno-precipitation (ChIP) assay was performed on
isolatedfromCaWRKY22-HAwhichweretransientlyoverexpressedinpepperleaves. Thechromatin
chromatins isolated from CaWRKY22-HA which were transiently overexpressed in pepper leaves.
was sheared into fragments from 300 to 500 bps in length, and immuno-precipitated (IPed) with
The chromatin was sheared into fragments from 300 to 500 bps in length, and immuno-precipitated
antibodyofHA.TheIPedDNAfragmentswerecollectedasatemplateforPCRwithspecificprimer
(IPed) with antibody of HA. The IPed DNA fragments were collected as a template for PCR with
pairs of the conserved W-box containing promoter fragments of CaPR1, CaDEF1, and CaWRKY40.
specific primer pairs of the conserved W-box containing promoter fragments of CaPR1, CaDEF1, and
TheresultsshowedthatCaWRKY22coulddirectlybindtothe250bpW-box-containingfragments
CaWRKY40. The results showed that CaWRKY22 could directly bind to the 250 bp W-box-containing
withinthepromotersoftestedimmunityassociatedgenes,whereasnobindingsignalwasdetected
fragments within the promoters of tested immunity associated genes, whereas no binding signal was
inthepromoterfragmentswithinthepromoterregionsofthetestedgeneswithoutW-box(Figure6).
detected in the promoter fragments within the promoter regions of the tested genes without W-box
TheseresultsindicatethatCaWRKY22bindstotheW-boxcontainingpromoterregions,butdoesnot
(Figure 6). These results indicate that CaWRKY22 binds to the W-box containing promoter regions,
bindtothepromoterregionswithoutW-box. Inparticular,CaWRKY40,apositiveregulatorinthe
but does not bind to the promoter regions without W-box. In particular, CaWRKY40, a positive
pepperresponsetoRSI,hightemperature,andhighhumiditystresses,asidentifiedinourprevious
regulator in the pepper response to RSI, high temperature, and high humidity stresses, as identified
study[9],isalsofoundtobeatargetofCaWRKY22.
in our previous study [9] , is also found to be a target of CaWRKY22.

Figure 6. CaWRKY22 binds to the W-boxes of different marker genes as detected by chromatin
immunFoigpurercei p6i.t aCtiaoWnR(CKhYI2P2) bainnadlys stios .thCea WWR-bKoYxe2s2 -oHf Adiwffearsentrta mnsaierknetlry goevneerse xaps rdesesteedcteidn pbyep cpherromatin
leaves,itmhemcuhnroopmraetciinpwitaatsioisno l(aCtehdI,Pt)h eanDaNlyAsi–sp. rCotaeWinRcKomY2p2le-HxAw awsiams mtruannospiernectliyp itoavteedreuxspirnegssaendti -iHn Apepper
antibodleiaevseasn, dthade jucshtreodmtoattihne wsaams eiscoolnacteedn,t rathtieo nD.NPCAR–pwroastepine rfcoormmpeldexu swinags pirmimmeurnpoapirrsecbiapsietadteodn using
theseqaunetni-cHeAfla annktiinbgodthieesW an-bdo xadwjuitshteind tthoe tphreo smamoteer csoonfcCenaPtrRa1ti,oCna. DPECFR1 ,waansd pCearWfoRrmKYed4 0u.s“iFn”g sptarinmdesr pairs
forforwbaasreddw ohni ltehe“ Rse”qrueepnrecsee fnlatnrekvinegrs teh.e W-box within the promoters of CaPR1, CaDEF1, and CaWRKY40.
“F” stands for forward while “R” represent reverse.
2.7. TheInter-RelationshipbetweenCaWRKY22andCaWRKY40
2.7. The Inter-Relationship between CaWRKY22 and CaWRKY40
As the promoter of CaWRKY40 was bound by CaWRKY22, we speculated that CaWRKY40
was transcArisp ttihoen palrloymroetgeur loatfe CdaWbyRKCYaW40R wKaYs2 b2.ouTnod tbeys tCtahWisRpKoYss2i2b,i lwitye, sapnedcutloatefudr tthhaetr CaasWsaRyKtYhe40 was
possibtlreanfesecdribpaticoknraellgyu rlaegtiuolnatoefdC bayW CRaKWYR4K0Yb2y2.C TaoW tResKtY th22is, pQoRsTsi-bPiCliRty,w aansde tmo pfulorytheedr taosscahye cthket hpeossible
possibfleeemdboadcukl arteiognuloaftiCoanW oRf KCYaW22RbKyYtr4a0n bsiye nCtaoWveRrKexYp2r2e,s QsioRnT-oPfCCRaW wRaKs Ye4m0,poloryitesds itloe ncchinecgk, atshwe eplolssible
as thempoodsusliabtlieone fofef cCtaoWfRCKaYW2R2 KbYy2 t2ratnrsainensite onvteorvexeprerxepssrieosns ioofn CoarWsRilKeYnc4i0n, goro ints tshileenecxipnrge,s assio wneollf as the
CaWR KY40. TheresultsshowedthattranscriptionofCaWRKY22inCaWRKY40-transiently-expressing
pepper leaves increased at 24 and 48 hpi compared to the control (Figure 7A). On the other hand,
thetranscriptionalabundanceofCaWRKY40wasalsoincreasedinCaWRKY22-overexpressingleaves
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 10 of 19
possible effect of CaWRKY22 transient overexpression or silencing on the expression of CaWRKY40.
The results showed that transcription of CaWRKY22 in CaWRKY40-transiently-expressing pepper
Int.J.Mol.Sci.2018,19,1426 10of19
leaves increased at 24 and 48 hpi compared to the control (Figure 7A). On the other hand, the
transcriptional abundance of CaWRKY40 was also increased in CaWRKY22-overexpressing leaves at
at 24 and 48 hpi (Figure 7B). By contrast, it was found that transcriptional levels of CaWRKY40
24 and 48 hpi (Figure 7B). By contrast, it was found that transcriptional levels of CaWRKY40 were
weresignificantlydownregulatedinR.SolanacearuminoculatedCaWRKY22-silencedpepperplants
significantly downregulated in R. Solanacearum inoculated CaWRKY22-silenced pepper plants
comparedtothatinthecontrolplants(Figure7C).Inaddition,CaWRKY22-silencingfullyorpartially
compared to that in the control plants (Figure 7C). In addition, CaWRKY22-silencing fully or
suppressed the upregulation of the tested defense associated marker genes including CaBPR1,
partially suppressed the upregulation of the tested defense associated marker genes including
CaPO2,CaPR4,CaDEF1,andCaHIR1;triggeredbyCaWRKY40transientoverexpression(Figure7D).
CaBPR1, CaPO2, CaPR4, CaDEF1, and CaHIR1; triggered by CaWRKY40 transient overexpression
ThissuggestsdirecttranscriptionalregulationofCaWRKY40byCaWRKY22andthatthereexistsa
(Figure 7D). This suggests direct transcriptional regulation of CaWRKY40 by CaWRKY22 and that
positiveregulatoryloopbetweenCaWRKY22andCaWRKY40.
there exists a positive regulatory loop between CaWRKY22 and CaWRKY40.
A EV CaWRKY22-HA B EV CaWRKY40-HA
2.0 1.8
A
el A el 1.6
on lev401.5 on lev22 11..24 B A
ative expressiof CaWRKY1..05 B B A ative expressiof CaWRKY 1....4680 B
el el
R R .2
0.0 0.0
24 48 (hpi) 24 48 (hpi)
C Y40 2.5 D
K
R TRV:00/EV A
CaW 2.0 A MRSocIk 3.0 TTTRRRVVV:::0CC0aa/WWCaRRWKKRYY22K22Y//E4C0Va-WHARKY40-HA
sion level of 11..05 B 2.0 A A A A A
e expres .5 a b 1.0 B B CC B CC B C C BCB B CC
v D
Relati 0.0 TRV:00 CaW RKY22 0.0 CaPO2 CC CaPR4 CaBPR1 CaDEF1 CaHIR1 CaWRKY22
V:
R
T

Figure7.Theinter-relationshipbetweenCaWRKY22andCaWRKY40.(A)Transcriptionalexpressionof
Figure 7. The inter-relationship between CaWRKY22 and CaWRKY40. (A) Transcriptional expression
CaWRKY40inpepperleavestransientlyoverexpressingCaWRKY22at24and48hpi.Theexpression
of CaWRKY40 in pepper leaves transiently overexpressing CaWRKY22 at 24 and 48 hpi. The
levelofpepperleavestransientlyoverexpressingtheemptyvectorwassetto“1”;(B)transcriptional
expression level of pepper leaves transiently overexpressing the empty vector was set to “1”; (B)
expressionofCaWRKY22inpepperleavestransientlyoverexpressingCaWRKY40at24and48hpi.
transcriptional expression of CaWRKY22 in pepper leaves transiently overexpressing CaWRKY40 at
Theexpressionlevelofpepperleavestransientlyoverexpressingtheemptyvectorwassetto“1”;
24 and 48 hpi. The expression level of pepper leaves transiently overexpressing the empty vector
(C) qRT-PCR analysis of CaWRKY40 expression levels in CaWRKY22-silenced and control pepper
was set to “1”; (C) qRT-PCR analysis of CaWRKY40 expression levels in CaWRKY22-silenced and
plants. Theexpressionlevelofmocktreatedunsilencedpepperleaveswassetto“1”;(D)qRT-PCR
control pepper plants. The expression level of mock treated unsilenced pepper leaves was set to “1”;
analysisofthetranscriptionallevelsofdefense-relatedmarkergenesinCaWRKY22-silencedandcontrol
(D) qRT-PCR analysis of the transcriptional levels of defense-related marker genes in
pepperplantstransientlyoverexpressing35S:CaWRKY40-HAand35S:00.(A–D)Datarepresentsthe
meanCsaW±RSKDYf2r2o-msilfeonucredbi oalnodg iccoanlrtreopll ipcaetpepse.rT hpelarnetlsa ttirvaenesxiepnrtelsys ioovnerleevxeplroesfsminogc k35trSe:CataeWdRuKnYsi4le0n-HceAd and
35S:00. (A–D) Data represents the means ± SD from four biological replicates. The relative expression
plantswassetto“1”. Errorbarsindicatedthestandarderror. Differentlettersindicatesignificant
diffelreevnecle sofb emtwoceke ntrmeaetaends ,uanssdileetnecremdi npeldanbtys Fwisahse rs’est ptroo t“e1c”te. dErLrSoDr bteasrts: uinpdpiecractaesde tlhetet esrtsa,npd<ar0d.0 1e;rror.
loweDricfafesreelnett tleertste,rps< in0d.0i5ca.te significant differences between means, as determined by Fisher’s protected
LSD test: uppercase letters, p < 0.01; lower case letters, p < 0.05.
2.8. TheInter-RelationshipbetweenCaWRKY22andCaWRKY6,CaWRKY27andCaWRKY58
2.8 The Inter-Relationship between CaWRKY22 and CaWRKY6, CaWRKY27 and CaWRKY58
As CaWRKY40 was previously found to be expressionally and functionally related to other
As CaWRKY40 was previously found to be expressionally and functionally related to other
WRKYs,includingCaWRKY6,thecloserelationshipbetweenCaWRKY22andCaWRKY40impliesthat
WRKYs, including CaWRKY6, the close relationship between CaWRKY22 and CaWRKY40 implies
CaWRKY22mightalsobeassociatedwithotherWRKYTFsinpepperimmunityagainstRSI.Totest
that CaWRKY22 might also be associated with other WRKY TFs in pepper immunity against RSI. To
thisspeculation,therelationshipbetweenCaWRKY22andCaWRKY6,CaWRKY27andCaWRKY58,
whichhavebeenimplicatedinpepperimmunityagainstRSIbyourpreviousstudies,wereassayedby

qRT-PCR,theresultsshowedthatthetranscriptionallevelsofCaWRKY22wereincreasedinCaWRKY6