Identification of Arabidopsis Candidate Genes in Response to Biotic and Abiotic Stresses Using ...

21 Pages·2015·2.09 MB·English

RESEARCHARTICLE
Identification of Arabidopsis Candidate
Genes in Response to Biotic and Abiotic
Stresses Using Comparative Microarrays
ArjunSham1,KhaledMoustafa2,SalmaAl-Ameri1,AhmedAl-Azzawi1,RabahIratni1,
SynanAbuQamar1*
1 DepartmentofBiology,UnitedArabEmiratesUniversity,Al-Ain,UnitedArabEmirates,2 Conservatoire
NationaldesArtsetMétiers(CNAM),Paris,France
* [email protected]
Abstract
Plantshaveevolvedwithintricatemechanismstocopewithmultipleenvironmental
stresses.Toadaptwithbioticandabioticstresses,plantresponsesinvolvechangesatthe
cellularandmolecularlevels.Thecurrentstudywasdesignedtoinvestigatetheeffectsof
OPENACCESS
combinationsofdifferentenvironmentalstressesonthetranscriptomelevelofArabidopsis
Citation:ShamA,MoustafaK,Al-AmeriS,Al-
genomeusingpublicmicroarraydatabases.Weinvestigatedtheroleofcyclopentenonesin
AzzawiA,IratniR,AbuQamarS(2015)Identification
mediatingplantresponsestoenvironmentalstressthroughTGA(TGACGmotif-bindingfac-
ofArabidopsisCandidateGenesinResponseto
BioticandAbioticStressesUsingComparative tor)transcriptionfactor,independentlyfromjasmonicacid.Candidategeneswereidentified
Microarrays.PLoSONE10(5):e0125666. bycomparingplantsinoculatedwithBotrytiscinereaortreatedwithheat,saltorosmotic
doi:10.1371/journal.pone.0125666
stresswithnon-inoculatedornon-treatedtissues.About2.5%heat-,19%salinity-and41%
AcademicEditor:TzvetankaD.Dinkova, osmoticstress-inducedgeneswerecommonlyupregulatedbyB.cinerea-treatment;and
UniversidadNacionalAutonomadeMexico,MEXICO
7.6%,19%and48%ofgeneswerecommonlydownregulatedbyB.cinerea-treatment,re-
Received:October31,2014 spectively.Ourresultsindicatethatplantresponsestobioticandabioticstressesaremedi-
Accepted:March23,2015 atedbyseveralcommonregulatorygenes.Comparisonsbetweentranscriptomedatafrom
Arabidopsisstressed-plantssupportourhypothesisthatsomemolecularandbiologicalpro-
Published:May1,2015
cessesinvolvedinbioticandabioticstressresponseareconserved.Thirteenofthecom-
Copyright:©2015Shametal.Thisisanopen
monregulatedgenestoabioticandbioticstresseswerestudiedindetailtodeterminetheir
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,whichpermits roleinplantresistancetoB.cinerea.Moreover,aT-DNAinsertionmutantoftheResponsive
unrestricteduse,distribution,andreproductioninany toDehydrationgene(rd20),encodingforamemberofthecaleosin(lipidsurfaceprotein)
medium,providedtheoriginalauthorandsourceare
family,showedanenhancedsensitivitytoB.cinereainfectionanddrought.Overall,the
credited.
overlappingofplantresponsestoabioticandbioticstresses,coupledwiththesensitivityof
DataAvailabilityStatement:Allrelevantdataare
therd20mutant,mayprovidenewinterestingprogramsforincreasedplantresistanceto
withinthepaperanditsSupportingInformationfiles.
multipleenvironmentalstresses,andultimatelyincreasesitschancestosurvive.Futurere-
Funding:ThisprojectwasfundedbytheUAEU
searchdirectionstowardsabetterdissectionofthepotentialcrosstalkbetweenB.cinerea,
[COS/IRG-05/14];andtheKhalifaCenterfor
BiotechnologyandGeneticEngineering-UAEU abioticstress,andoxylipinsignalingareofourparticularinterest.
[KCGEB-2-2013]toSAQ.Thefundershadnorolein
studydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 1/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Introduction
Plantsareimmobileorganismsconvictedtofacenumerousenvironmentalstressesduring
theirlifetime.Bioticandabioticstressesoftenoccursuddenlyand/orsimultaneously;and,im-
mediateplantresponsesarethereforecriticaltoensurecellsurvival[1].Afundamentalstrategy
forplantstoadapttoenvironmentalchallengesimposedbybioticandabioticthreatsisthe
modulationofgeneexpression.Atthecellularlevel,plantstunegeneexpressionalongwith
theirphysiologicalneedstopromoteadaptationtoshort-aswellaslong-termenvironmental
changes.Now,thereisgrowingevidencethatplantsreprogramtheirresponsesundercontinu-
ouslychangingenvironmentalfactorsindividually,ormorefrequently,incombination.De-
pendingontheenvironmentalconditionsencountered,plantsactivateaspecificprogramof
geneexpression[2].Thespecificityofresponseisfurthercontrolledbyarangeofmolecular
mechanismsthat“crosstalk”inacomplexregulatorynetwork,includingtranscriptionfactors,
kinasecascades,reactiveoxygenspecies,heatshockfactorsandsmallRNAsthatmayinteract
witheachother[3].Theinteractionbetweenbioticandabioticstressesisorchestratedbyhor-
moneandnon-hormonesignalingpathwaysthatmayregulateoneanotherpositivelyornega-
tively.Inresponsetobioticorabioticstress,geneexpressionstudiesfoundthatdisease
resistance-relatedgenesincorncouldbeinducedorrepressedbyabioticstresses[4].
SeveralstudieshaveidentifiedtheregulationofsinglegenesinresponsetoB.cinereaand
abioticstress.ArabidopsisBotrytisSusceptible1(BOS1),Botrytis-inducedKinase1(BIK1),
WRKY33geneswerepreviouslyidentified[5–7].Incomparisonwithwild-typeplants,the
threemutantsbos1,bik1andwrky33wereextremelysusceptibletoB.cinerea.TheMYBtran-
scriptionfactor,BOS1,playsamajorroleinplantdefenseresponsetoB.cinereathatisregulat-
edbyjamonate(JA)[5].Thesusceptibilityofbos1mutanttoB.cinereawasalsolinkedto
alteredplantsensitivitytooxidativestress.BIK1gene,inturn,encodesamembrane-associated
kinaseproteininwhichbik1mutantshowedhighsalicylate(SA)levelsbeforeandaccumulated
afterB.cinereainoculation[6].WhileWRKY33transcriptionfactorshowedacrosstalkbe-
tweenJA-andSA-regulateddiseaseresponsepathways,bothBIK1andWRKY33playanan-
tagonisticroleinplantdefenseaspositiveandnegativeregulatorstoresistancetoB.cinerea
andPseudomonassyringaepvtomato,respectively[5,6].Effortstowardstheidentificationof
ArabidopsisBOS1interactors(BOI)andBIK1regulatorshaveledtouncoverthefunctionof
someinteractorsandregulatorsinplantresponsestopathogeninfectionandabioticstress[8,
9].Recently,theArabidopsismutationexpansin-likeA2(EXLA2)enhancedresistanceto
necrotrophicfungi,butcausedhypersensitivitytosaltandcoldstresses[10].UponB.cinerea
attack,anaccumulationofcyclopentenonesresultedintherepressionofEXLA2;whereas
EXLA2inductionwasdependentonabscisicacid(ABA)responses[10,11].
Theimpactofanabioticstresscanalsoleadtoincreasedresistanceorsusceptibilitytoa
pathogen,orviceversa.Theplant-parasiticnematodeMeloidogynegraminicolareducedthe
damageofdroughtonrice(Oryzasativa)growth[3].Bycontrast,drought-stressedsorghum
(Sorghumbicolor)andcommonbean(Phaseolusvulgaris)showedincreasedsusceptibilityto
thesamefungusMacrophominaphaseolina[12,13].InArabidopsis,drought-stressedplants
showedseveresusceptibilitytothebacterialpathogenP.syringae[14].Ontheotherhand,in
tomato(Solanumlycopersicum)andbarley(Hordeumvulgare),itwasfoundthatincreasingthe
toleranceleveltodrought,saltandosmoticstressalsoenhancedtheresistancetoBlumeriagra-
minisandB.cinerea[15,16].Thesefindingssuggestthatbioticandabioticstressesmayinter-
actwitheachotherpositivelyornegativelyandsomemicroorganismscanthusbeemployedto
efficientlyenhancecropstresstolerance[17].Infact,thecombinationofbioticandabiotic
stressesactivatestheexpressionofuniqueand/orcommonsetsofgenesthatareorchestrated
byhormonal,mainlyABA,ornon-hormonalpathways.
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 2/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Sofar,limitedattemptshavebeenmadetoanalyzegeneexpressionchangesinplantsin-
fectedwithpathogensandexposedtoabioticstresses.InArabidopsis,atranscriptomeprofiling
bymicroarraywasperformedinresponsetodehydrationandtheplantparasitic-nematode
Heteroderaschachtii[18].AnalysisoftranscriptprofilesinArabidopsistreatedwithflagellin,
cold,heat,highlightintensityandsaltconcentrationsdetectsspecificandsharedresponsesbe-
tweenbioticandabioticstressesandcombinationsofthem[19].Arecentreportontranscrip-
tomeanalysisinArabidopsisidentifiedpotentialregulatorygenesafterinfectionwithB.
cinereaandtreatmentswithcold,droughtandoxidativestressesindividuallyandincombina-
tion[20].Here,wecompareandanalysemicroarraydataemanatingfromgeneexpressionpro-
filinginArabidopsisinresponsetoB.cinerea(bioticstress)andheat,saltandosmoticstresses
(abioticstresses).Weanalyzedplantresponsestothesestressestakenindividually,andidenti-
fiedtranscriptionalregulatorynetworksatasingletimepointofgeneexpression.Arabidopsis
plantsweredeliberatelysubjectedtofourindividualstresstreatments(onebioticandthreeabi-
oticstresses).Inlarge,wecombinedtheexpressionofB.cinereaupregulatedgenes(BUGs)
withthatofheat,saltorosmoticstresses;about2.5%,19%or41%ofthetranscriptsresponded
respectively,albeitthemodepredictedfromanindividualstresstreatment.Withaminorin-
creaseinthefractionofthetranscriptsaftercombiningB.cinereadownregulatedgenes
(BDGs)withthoseofabioticstresstreatments,atranscriptionalbalancebetweenplantre-
sponsestoenvironmentalstressesissuggested.
MaterialsandMethods
Plantgrowthandstressassays
WeanalyzeddatafromapreviousstudyonArabidopsisplants(ecotypeCol-0)infectedwithB.
cinerea[21].Inthatstudy,theexperimentalconditionswereconductedasfollows:Five-week-
oldArabidopsisplantswereinoculatedbyplacingfour5μldropsofa5x105sporemL-1solu-
tiononeachleaf.Controlleaveswerespottedwithdropletsof24gL-1potatodextrosebroth
medium.ResponsestoB.cinereainfectionwereassayedat18and48hpiofadultleaves.
FortheqRT-PCRandfunctionalanalyses,B.cinereastrainBO5-10,wasgrownon2xV8
agar(36%V8juice,0.2%CaCO3,2%Bacto-agar).Fungalcultureswereinitiatedbytransfer-
ringpiecesofagarcontainingmyceliumtofresh2xV8agarandincubatedat20–25°C.Collec-
tionofconidiafrom10-day-oldculturesandinoculationwerecarriedoutaspreviously
described[6].Diseaseassayswereperformedonwholeplantsordetachedleaves(five-week-
oldplants)growninsoilwerespray-inoculatedordrop-inoculated(3μL)withB.cinereaspore
suspension(3x105sporesmL-1)respectively,asdescribedpreviously[10].Controlplantswere
sprayedwith1%SabouraudmaltosebrothbufferusingaPrevalsprayer(ValveCorp.,Yonkers,
NY,USA).Plantswerefurtherkeptunderasealedtransparentcovertomaintainhighhumidi-
tyinagrowthchamberwith21°Cday/18°Cnighttemperatureanda12-hlight/12-hdarkpho-
toperiodcycle.ResponsestoB.cinereainfectionwereassayedat18hpiofleaves,unless
otherwisestated.
Thedroughtsensitivityassaywasperformedon3-week-oldwell-wateredplantsthatwere
plantedinsoil.Seedlingswerekeptinagrowthchamberunderthesameconditionsmentioned
abovewithoutwatering(droughtstress)for10days.Survivalrateswerescored3daysafter
rewatering.Controlplantswerewell-wateredandkeptunderthesameconditions.
IdentificationofT-DNAinsertionlines
T-DNAinsertionlineswereidentifiedasdescribedpreviously[22].PCRprimerswerede-
signedtotheArabidopsisgenomicsequenceflankingtheT-DNAinsertionsite.Theseprimers
wereusedtoanalyze12siblingplantsfromeachT-DNAlinetoconfirmtheT-DNAinsertion
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 3/21
MicroarrayAnalysisofArabidopsis-StressedPlants
cosegregatedwiththemutantphenotype.Theprimerswerealsousedforgenotypingindividual
lineswithinasegregatingpopulationtoidentifyindividualshomozygousfortheinsertional-
lele.AcombinationofonegenomicprimerplusaT-DNAinsertprimerwasusedtodetectthe
insertionallele.Twogenomicprimerswereusedtogethertodetectthewild-typeallele.rd20
(SAIL_737_G01;stocknumberN876376)wasobtainedfromtheNottinghamArabidopsis
StockCentre(NASC,Nottingham,UK).TheT-DNAinsertionintherd20mutantwascon-
0
firmedbyPCRusingaT-DNA-specificprimer(LB2,5-GCTTCCTATTATATCTTCCC
0 0
AAATTACCAATACA-3)andanRD20-specificprimer(RP,5-AAGTACGGAACGATTTG
0
GAGG-3).Homozygousrd20mutantplantswereidentifiedbyPCRusingapairofprimers
0
correspondingtosequencesflankingtheT-DNAinsertion(LP,5-TTAACCGTTAGCGCG
0
TATTTG-3;RP).
RNAextractionandexpressionanalysis
RNAextractionandqRT-PCRexpressionanalyseswereperformedasdescribedpreviously
[10].TheqRT-PCRwasperformedusinggene-specificprimers,withArabidopsisActin2
(AtActin2)asanendogenousreferencefornormalization.Expressionlevelswerecalculatedby
thecomparativecyclethresholdmethod,andnormalizationtothecontrolwasperformedas
described[23].PrimersequencesarefoundinS1Table.
Statisticalanalysis
Foreachsample,threetechnicalreplicatesoftheqRT-PCRassaywereusedwithaminimum
ofthreebiologicalreplicates.Resultswereexpressedasmeans±standarddeviation(SD)ofthe
numberofexperiments.AStudent’st-testforthevalueswasperformedatP<0.05.
DataofB.cinereagrowthininoculatedplantsrepresentthemean±SDfromaminimumof
16plants.Dataofdroughtsensitivityassayperformedonplantsrepresentthemean±SD
(n=12).AnalysisofvarianceandDuncan’smultiplerangetestwereperformedtodetermine
thestatisticalsignificance[24].Meanvaluesfollowedbyanasteriskaresignificantlydifferent
fromthecorrespondingcontrol(P<0.05).Allexperimentswerecarriedoutintriplicatewith
similarresults.
Heat,salinityandosmoticstresstreatments
WeanalyzeddatafromapreviousstudyontheresponsesofArabidopsistovariousstresscon-
ditions[21].Inthatstudy,seeds(ecotypeCol-0)weresurface-sterilizedbytreatingthemse-
quentiallyin70%ethanolfor2min,then30%Cloroxsolutioncontaining0.01%Tweenfor10
min,andrinsedseveraltimesinsterilewater.SeedswereplatedonmediacontainingtheMura-
shigeandSkoog(MS)growthmedium,2%sucrose,0.7%(w/v)purifiedagar,unlessotherwise
stated.Plateswerekeptat4°Cfor48htosynchronizegermination,transferredtogrowth
chamberswithfluorescentlights,andmaintainedundertheenvironmentalconditionsasde-
scribedin[25]withsomemodifications.
Fortheheatstressexperiment,sixteen-day-oldseedlingsweretreatedwitheitherliquid-MS
mediaat25°C(control)orexposedto38°Cfor24h.Forthesaltandosmoticstressexperi-
ments,sixteen-day-oldplantsweretreatedwitheitherliquid-MSmedia(control)orstressedby
150mMNaCl(saltstress)or300mMMannitol(osmoticstress)for24h.Alltreatmentsand
preparationsweredoneonthesamebatchofseedlings,asdescribedin[21].
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 4/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Datasourceandanalysis
RawmicroarraydatasetsweredownloadedfromNASCArrays[affy.arabidopsis.info/link_to_
iplant.shtml][21]foreachstress.Dataof“shoots”classwereanalyzedusingRStatisticalCom-
puting[26],whichusesAffyandMAS5packagesfordatanormalization.Affycomputesthe
probesetsignalintensity;whereasMAS5computesthedetectioncallsofeachprobeIDdis-
playedasPresent(P),Absent(A)andMarginal(M).Thereferencenumbersare:control(for
allabioticstresses),NASCArrays-137;osmoticstress,NASCArrays-139;saltstress,NASCAr-
rays-140;heatstress;NASCArrays-146;andB.cinerea,NASCArrays-167(includingnon-inoc-
ulatedcontrol).Thenumberoftestedsamples(n)foreachtreatmentis8(control;andheat
stress),6(salt;andosmoticstresses),and2(B.cinereaanditscontrol);with22810genesper
array.Log -transformedexpressionleveldatawereusedtogeneratescatterplotstodetectthe
2
effectofB.cinereainfectionat18hpiorabioticstresstreatmentat24hourspost-treatment
(hpt)onplantgeneexpression.Comparisonsofthreereplicatesforeachsetofexperiment
wereperformed.Inallsamples,probeswithexpressionlabelledas‘A’or‘M’acrossallsamples
wereremovedfromthedataset.Atthetestedtimepoint,theoverallgeneexpressiondifference
betweencontrol(non-treated/non-inoculated)andtreated/inoculatedsampleswasdetermined
bypairwisecomparison.Thenormalized-foldchangevalueforeachgenewascalculatedbydi-
vidingtheexpressionlevelofatreated/inoculatedsamplebytheexpressionlevelofanon-treat-
ed/non-inoculatedsample.Atwofoldorhalf-fold(unlessotherwisestated)differencein
expressionlevelbetweentreated/inoculatedandnon-treated/non-inoculatedsamplesat
P<0.05wassetasthethresholdforconsideringagenetobeup-ordown-regulated,respec-
tively.Thecutoffsofthefoldchangewerechosentofilterfalsepositivesandtocompareour
dataanalyseswiththoseinthemicroarrayliteratures.Allgenesacrossthemicroarraysdata
wereidentifiedusingtheArabidopsisInformationResources(TAIR;www.arabidopsis.org).
WeusedmicroarraysdataoftreatedseedlingswithB.cinerea,cold,droughtandoxidative
stressasdescribed[20];and12-oxo-phytodienoicacid(OPDA)andphytoprostaneA (PPA )
1 1
aspreviouslydescribed[11,27].
Results
Identificationofdifferentiallyexpressedgenestoabioticstresses
Inthisstudy,weaimedtoidentifycomponentsoftheregulatorynetworksinvolvedinArabi-
dopsisresponsestoB.cinereainfectionandabioticstresses(heat,salinityandosmoticstress).
Afullmicroarray-basedanalysisofArabidopsiswhole-genomeAffymetrixgenechip(ATH1)
representingapproximately25,000geneswasdownloadedfromNASC[21]toidentifyregulat-
edgenesbyB.cinereainfectionandtheabioticstress.Todetermineup-anddown-regulated
genesinArabidopsisseedlingsexposedtoheat;salt;andosmoticstresstreatmentsat24hpt,
wefirstidentifieddifferentiallyregulatedgenesbycomparingtheexpressionprofileofuntreat-
ed-(control)ortreatedtissuesinArabidopsiswild-typeplants(Fig1A–1C).Thetranscript
levelforeachgenebeforeandafterthetreatmentwithheat,salinityorosmoticstresswasas-
sessedandcompared.Geneswithexpressionchangesofmorethantwofoldorlessthanhalf-
fold(P<0.05)weredefinedassignificantlystressup-ordown-regulatedgenes,respectively.
Thecompletelistofinducedandrepressedgenestoheat,salinityorosmoticstressesisavail-
able(S2Table).Wealsoinvestigatedwhethertheaccumulatedtranscriptswerefunctionallyin-
volvedinstressresponseanddefense.BasedontheGeneOnology(GO)annotation,we
classifiedthedifferentiallyexpressedgenesaccordingtotheirbiologicalandmolecularactivi-
ties,andcellularcomponents.Ouranalysisshowedthatthedifferentiallyexpressedgenesin
Arabidopsisseedlingsunderheat,salinityandosmoticstressconditionsweremajorlygrouped
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 5/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Fig1.ComparisonsofgeneexpressioninArabidopsisplantsunderbioticandabioticstressconditions.Normalizedexpressionvaluesforeach
probesetinstressedplantswithheat(A);salinity(B);orosmoticstress(C)at24hptisplottedontheY-axis.In(A-C),thevalueinwild-typeplantssampled
beforetheabioticstresstreatment(0hpt;WT-0)isplottedontheX-axis.Numberandtheleveloftranscriptsidentifiedasupregulated(D),ordownregulated
(E)genesinArabidopsisstressedplants.In(D-E),thetreatmentofthetestedabioticstressisplottedontheY-axis;thenumberofdifferentiallyexpressed
genesisplottedontheX-axis.Columnswithdifferentcolorsshowthefoldchangeofcorrespondingdifferentiallyexpressedgenes.*Resultswereobtained
from[20].hpt,hoursposttreatment.
doi:10.1371/journal.pone.0125666.g001
asresponsivetobioticandabioticstimuli/stresses,electrontransport,cellorganizationandde-
velopment,andotherbiologicalprocesses(S1Fig).Thestressup-regulatedgenesencodeforre-
ceptors,transcriptionfactors,transporters,andenzymes(i.e.hydrolyases,kinases,
transferases)correspondingtovariouscellularactivities,mainlylocalizedinthecellwall,Golgi
apparatus,plastidsandplasmamembrane,suggestinganinvolvementofextracellularandin-
tracellularcomponentsinplantresponse/defensetoabioticstressconstraints.
BUGsandBDGshavebeenpreviouslyidentifiedbasedontheirtranscriptionallevelsinre-
sponsetoB.cinereainfectionat18hpianddifferentiallyexpressedgeneswerealsoidentified
inresponsetocold,droughtandoxidativestress[20].Datawereanalyzedtohaveacomplete
setofup-anddown-regulatedgenesofmajorabioticstresscomparedwiththoseofBUGsor
BDGs.Ourmicroarrayanalysisshowedtherewere1498genesconsideredasBUGsand1138
genesconsideredasBDGs(Fig1Dand1E).Inaddition,thegeneexpressionlevelsunderheat,
salinityandosmoticstresstreatmentswerealteredfor660,1649and3905transcripts,respec-
tivelyfromwhich153,799and1695geneswerestress-inducedgenes.Inmostcases,therewere
morerepressedthaninducedgenesexceptforB.cinereatreatment.Theaveragefoldchanges
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 6/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Fig2.Scatter-plotcomparisonsofgeneexpressionandnumberofBUGsandBDGsaffectedbyabioticstress.Normalizedexpressionvalueforeach
probesetinwild-typeplantsinfectedwithB.cinereaat18hpi(B.cinerea-18)isplottedontheX-axis;thevalueinstressedplantswithheat(A);salinity(B);or
osmoticstress(C)at24hptisplottedontheY-axis.TheVenndiagramshowsthenumberofBUGs(D);andBDGs(E)at18hpithatarealsoaffectedbyheat,
salinityandosmoticstressat24hpt.hpi/hpt,hourspostinoculation/treatment.
doi:10.1371/journal.pone.0125666.g002
ofdifferentiallyexpressedgenesrangedfrom2–3folds,thoughsomegenesshowed10-foldor
more(S2Table).ItisworthmentioningthatthenumberofgenesinvolvedinB.cinerea,cold,
salinityandosmoticstressresponsesseemstobegreaterthanthoseinvolvedindrought,heat
andoxidativestressresponses(Fig1Dand1E).ThismightbeduetothefactthatArabidopsis
isnaturallymoreadaptedtodrought,heatandoxidativestressthantootherenvironmental
stressconditions.
CommondifferentiallyexpressedgenesbyB.cinereaandmajorabiotic
stresses
TocomparenormalizedtranscriptionallevelsofgenesidentifiedasB.cinerea-andabiotic
stress-regulatedgenes,scatterplotswereconstructedonthecorrelatinggenesbetweenB.
cinerea[20]andheat,salinityorosmoticstress(Fig2A–2C).Similarpatternsofgeneexpres-
sionlevelswereillustratedbetweenArabidopsisplantsinfectedwithB.cinereaat18hpi,and
cold,droughtoroxidativestressat24hpt[20].Venndiagramsdisplayedthat37geneswere
commonlyupregulatedbyB.cinereainoculationandheattreatment;whereas87weredownre-
gulatedbythesamestresses,representing2.5%and7.6%ofthegenesthatwereupregulated
anddownregulatedbyB.cinerea,respectively(Table1).
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MicroarrayAnalysisofArabidopsis-StressedPlants
Thediagramalsodemonstratedthat284geneswereinducedbybothB.cinereaandsalinity
and215wererepressedbythesestresses(Fig2Dand2E),eachcorrespondingto19%ofeither
BUGsorBDGs(Table1).About40–50%oftheidentifiedB.cinerea-regulatedgeneswerealso
regulatedbyosmoticstress.Thelistoftheoverlappingup-anddown-regulatedgeneswithdis-
tinctresponsestoB.cinereaandabioticstresstreatmentisshowninS3Table.Tocomparethe
co-regulationbetweenB.cinereaandotherclassesofmajorabioticstressfromthosesubjected
here,theanalysiswasextendedtoincludeB.cinerea-regulatedgeneswithcold,droughtand
oxidativestressesthatwerepreviouslyidentified(Table1).Amongtheinducedgenes,251
weresharedinB.cinerea,salinityandosmoticstresstreatments,while18and14werecom-
monlyupregulatedbyB.cinerea/heat/osmoticstressandB.cinerea/heat/salinitytreatments,
respectively(Fig2D).Likewise,acommondownregulationofgeneswasobservedbetweenB.
cinereaandabioticstresstreatmentswherefiftyand39ofthesharedgenesshoweddownregu-
lationbyB.cinerea/heat/osmoticstressandB.cinerea/heat/salinitytreatments,respectively
(Fig2E),while13inducedgenesand29repressedwerecommonbetweenalltestedbioticand
abioticstresses(Fig2Dand2E).Whenwecomparedwithcold,droughtandoxidativestresses
data,wefoundthat15geneswerecommonlyresponsive;threegenesshowedcommoninduc-
tionwithBUGsand12genesshowedcommonrepressionswithBDGs(Table1).Takentogeth-
er,thesefindingssuggestanoverlapbetweenB.cinerea,salinityandosmoticstress.
Welookedcarefullyatthecommonup-anddown-regulatedmembersexpressedbyB.
cinerea,heat,salinityandosmoticstress;andwefoundthatsomegeneswerefrequentlyex-
pressedtocombinedtypes.Forexample,thecommonB.cinerea/heat/salinity/osmoticstress-
inducedAt5g22860andAt2g33380(RD20),andtherepressedAt5g25190(Table2)wereprevi-
ouslyidentifiedascommonrespondentstoB.cinerea,cold,droughtandoxidativestress[20].
ThissuggeststhatalthoughsomegeneswerequitespecifictoB.cinerea,heat,salinityandos-
moticstress;othersshowedgeneralregulationtobioticandabioticstresses.Wealsoassesseda
selectednumberofcommonlydifferentiatedexpressedgenestoB.cinereainfectionusing
quantitativerealtime-PCR(qRT-PCR)tovalidatethemicroarrayanalysis.Relativegeneex-
pressionchangesmeasuredbyqRT-PCRinB.cinerea-infectedleavesat18hpiwerecompared
withArabidopsismicroarrays’data.Similartranscriptpatternsforthetestedgenes,ESE3,
BAG6,LCAT3andAt2g06890wereobservedinthetwoapproaches(qRT-PCRandmicroar-
rays)(Fig3).Webelievethattheoverlappinggenesarenotonlyfunctionalinsignal
Table1. RegulationofB.cinerea-regulatedgenesbydifferentstimuli.
Treatment Co-upregulatedgenes Co-downregulatedgenes
Noofgenes Percentagea Noofgenes Percentage
Coldb 373 24.9 377 33.1
Droughtb 92 6.1 77 6.8
Oxidativestressb 176 11.7 63 5.5
Heat 37 2.5 87 7.6
Salinity 284 19.0 215 18.9
Osmoticstress 618 41.2 546 47.8
Allstresses 3 0.2 12 1.1
ShownarepercentagesofBUGsandBDGs(atleasttwofold)thatwerealsoatleasttwofoldincreasedordecreasedbytheabioticstresslistedabove.
aPercentage=Noofup-ordown-regulatedgenesoftheabioticstress/NoofBUGs(1498genes)orBDGs(1138genes).BUGsandBDGswereobtained
from[20].
bResultswereobtainedfrom[20].
doi:10.1371/journal.pone.0125666.t001
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MicroarrayAnalysisofArabidopsis-StressedPlants
Table2. Changesinexpressionofup-/down-regulatedgenesencodingputativeproteinsduringB.cinereainfectionandheat,salinity,andosmot-
icstresstreatmentsinwild-typeArabidopsisplants.
Genelocus Genefamily Probeset B.cinereaa,b Abioticstressa
Heat Salinity Osmoticstress
At5g22860 serinecarboxypeptidaseS28 249860 6.511 2.222 3.116 12.929
At5g06190 Unknown 250722 2.241 2.133 3.335 3.757
At4g13800 permease-related 254683 2.487 2.425 3.214 12.075
At4g12910 SCPL20 254791 3.236 2.070 2.909 2.735
At2g33380 RD20 255795 5.153 2.360 5.936 26.651
At3g14067 subtilase 256997 2.271 2.166 2.684 6.830
At3g03310 LCAT3 259057 2.88 2.38 5.18 17.57
At3g05030 NHX2 259081 2.627 3.144 3.396 4.889
At1g70900 Unknown 262313 2.10 2.01 2.83 4.92
At2g42540 COR15A 263497 7.40 2.88 88.16 102.16
At2g06890 transposableelementgene 266214 2.43 2.40 2.18 2.44
At2g46240 BAG6 266590 2.631 2.023 56.992 3.703
At2g39250 SNZ 267010 2.413 2.432 4.054 11.476
At5g25190 ESE3 246932 -2.18 -3.85 -8.93 -5.73
At5g49450 BZIP1 248606 -2.94 -5.76 -2.47 -8.42
At5g48430 aspartylprotease/PepsinA30 248703 -2.08 -2.28 -4.65 -3.80
At5g41080 GDPD2 249337 -2.19 -11.50 -3.33 -8.52
At5g39580 Peroxidase 249459 -6.16 -9.85 -7.38 -11.29
At5g19120 aspartylprotease/PepsinA20 249923 -2.08 -5.61 -14.62 -27.66
At5g05440 PYL5/RCAR8 250777 -2.24 -8.22 -15.34 -11.26
At3g50560 SDR 252167 -5.21 -2.15 -6.98 -5.91
At3g50060 MYB77 252193 -3.01 -4.63 -5.27 -2.43
At3g46280 proteinkinase-related 252511 -10.92 -15.38 -5.26 -25.77
At4g21870 HSP26.5-P 254384 -2.18 -3.06 -9.16 -7.77
At4g12470 proteaseinhibitor(AZI1) 254818 -4.07 -13.71 -14.99 -14.45
At4g01250 WRKY22 255568 -2.15 -5.63 -3.75 -4.13
At4g01720 WRKY47 255596 -2.58 -2.52 -3.12 -4.49
At3g14770 nodulinMtN3 256548 -3.54 -2.60 -2.56 -3.25
At3g15950 TSA1-LIKE(NAI2) 257798 -23.49 -2.54 -2.69 -3.33
At3g16460 jacalinlectin 259327 -16.43 -2.29 -4.22 -7.73
At1g28010 ABCB14/MDR12/PGP14 259579 -2.80 -2.89 -3.29 -3.49
At1g21910 DREB26 260856 -5.69 -14.79 -22.89 -3.68
At1g19610 PDF1.4/LCR78 261135 -4.85 -5.36 -5.36 -7.44
At1g21830 Unknown 262488 -2.72 -2.92 -3.11 -3.56
At1g14890 Invertase/pectinesteraseinhibitor 262844 -2.82 -2.05 -2.37 -3.59
At1g23870 TPS9 263019 -3.45 -3.50 -2.54 -4.46
At1g54740 Unknown 264238 -2.60 -3.62 -3.10 -3.75
At1g76930 EXT4 264960 -2.30 -7.08 -3.18 -4.63
At1g24530 transducin/WD-40repeat 265028 -4.69 -6.35 -5.48 -4.05
At2g20670 Unknown 265387 -4.33 -15.19 -3.60 -17.86
At2g26980 CIPK3 266313 -3.18 -2.10 -2.75 -3.84
(Continued)
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 9/21
MicroarrayAnalysisofArabidopsis-StressedPlants
Table2. (Continued)
Genelocus Genefamily Probeset B.cinereaa,b Abioticstressa
Heat Salinity Osmoticstress
At2g40000 HSPRO2 267357 -2.16 -4.50 -2.63 -8.24
aFoldchangeinexpressionforeachgenewascalculatedbydividingtheexpressionlevelofaB.cinerea-infectedorabioticstress-treatedsamplebythe
expressionlevelofanon-infectedornon-treatedsample,respectively.AtwofolddifferenceinexpressionlevelbetweenB.cinerea-inoculatedandnon-
inoculatedorabioticstress-treatedandnon-treatedsampleswassetasthethresholdforconsideringagenetobeB.cinerea-orabioticstressup-/down-
regulatedgene(P<0.05).
bB.cinereaup-/down-regulatedgenesdatawereobtainedfrom[20].
-,downregulation.
doi:10.1371/journal.pone.0125666.t002
transductionpathways,mediatedbyphytohormones,butalsoinbioticandabioticstresspath-
waysthatsharemanyoverlappingstepsinnon-enzymaticfreeradical-catalyzedpathway.
PhenotypicanalysisofT-DNAinsertionmutantsofoverlappinggenesto
B.cinereainfection
Todeterminethefunctionoftheoverlappinggenesinresponsestobioticandabioticstress
treatments(Table1),weisolatedmutantsinselectedregulatedgenesencodingputativeregula-
toryproteins.T-DNAinsertionlinesforthesegeneswereidentifiedfromtheSyngentaArabi-
dopsisInsertionCollection(SAIL),theSalkInstitute(SALK)T-DNAcollectionandthePlant
Fig3.ComparisonofvaluesobtainedfordifferentialexpressionusingqRT-PCRandmicroarrays.
RelativeexpressionlevelsobtainedthroughqRT-PCRwerecomparedwithmicroarrayexpressionlevels
(NASCArrays)forselectedcommonB.cinerea-andabioticstress-upregulatedor-downregulatedgenesafter
infectionwithB.cinereaat18hpi.ExpressionofB.cinerea-inducedor-repressedgeneswasquantitated
relativetocontrolconditions(noinfection),andcorrectedforexpressionofthecontrolβ-actingene.
MicroarrayexpressiondatawereobtainedfromTables1and2.ErrorbarsforqRT-PCRvaluesarethe
standarddeviations(n(cid:2)3).hpi,hourspostinoculation;AtActin2,ArabidopsisActin2gene.
doi:10.1371/journal.pone.0125666.g003
PLOSONE|DOI:10.1371/journal.pone.0125666 May1,2015 10/21