doi:10.1093/annbot/mcm148,availableonlineatwww.aob.oxfordjournals.org
ResistanceofRedClover(Trifoliumpratense)totheRootParasiticPlant
OrobancheminorisActivatedbySalicylatebutnotbyJasmonate
DAIKUSUMOTO1,YAAKOVGOLDWASSER2,XIAONANXIE1,3,KAORIYONEYAMA1,
YASUTOMOTAKEUCHI1andKOICHIYONEYAMA1,*
1WeedScienceCenter,UtsunomiyaUniversity,350Mine-machi,Utsunomiya321-8505,Japan,2R.H.SmithInstituteofPlantSciences&GeneticsinAgriculture,FacultyofAgriculture,Food&EnvironmentalSciences,TheHebrewUniversityofJerusalem,Rehovot76100,Israeland3UnitedGraduateSchoolofAgriculturalScience,TokyoUniversityofAgriculture
andTechnology,3-5-8Saiwai-cho,Fuchu-shi,Tokyo183-8509,Japan
Received:12February2007Returnedforrevision:21May2007Accepted:1June2007Publishedelectronically:27July2007
†BackgroundandAimsObligaterootholoparasitesofthegenusOrobancheattackdicotyledonouscropsandcauseseverelossesinmanypartsoftheworld.Chemicalinductionofplantdefencesystemssuchassystemicacquiredresistancewasproposedtobeanavailablestrategytocontroltherootparasite,butthedetailedmechanismsinvolvedhavenotbeenclarified.Theaimofthisstudywastoelucidatetheeffectsofsalicylicacid(SA),jasmonicacid(JA)andtheiranaloguesonresistanceofredclovertoOrobancheparasitism.
†MethodsRootsofredclovergrowninplasticchamberswereappliedwithSA,S-methylbenzo[1,2,3]thiadiazole-7-carbothioate(BTH),methyljasmonate(MeJA)andn-propyldihydrojasmonate(PDJ),andthenwereinoculatedwithO.minorseeds.Attachmentsoftheparasitewereobservedafter5weeks.
†KeyResultsSAandBTH,inducersofSA-mediateddefences,significantlyreducedthenumberofestablishedpara-sitesbymorethan75%.Bycontrast,MeJAandPDJ,inducersofJA-mediateddefences,didnotaffectparasitism.ThereductioninthenumberofestablishedparasitesbySAandBTHwasduetotheinhibitedelongationofO.minorradiclesandtheactivationofdefenceresponsesinthehostrootincludinglignificationoftheendodermis.
†ConclusionsTheseresultssuggestthatSA-inducedresistance,butnotJA-inducedresistance,iseffectiveininhibit-ingOrobancheparasitismandthattheresistanceisexpressedbythehostrootbothexternallyandinternally.
Keywords:Endodermis,haustoriuminducingsignal,inducedresistance,jasmonicacid,lignification,Orobancheminor,rootparasiticplant,salicylicacid,Trifoliumpratense(redclover).
INTRODUCTION
hempandtobacco(Gonsioretal.,2004)andO.cumana
RootparasiticplantsofthegenusOrobanchelackinsunflower(Mu
¨ller-Sto¨veretal.,2005).Foliarsprayofchlorophyllanddependontheirhostplantsfortheacqui-BTHwassitionofnutrientsandwater.Orobanchespp.infestagri-inpea(Pe
´alsoreportedtoreduceO.crenataattachments
rez-de-Luqueetal.,2004).AlthoughseveralculturallyimportantdicotyledonouscropsandcauseseverereportshavesuggestedtheeffectivenessofBTHapplicationyieldlossesoftheirproductioninmanypartsoftheworld,onthereductionofOrobancheattachments,physiologicalespeciallytheMediterraneanregion(ParkerandRiches,resistantmechanismstoOrobancheinfestationinducedby1993).Orobancheaegyptiaca,O.ramosa,O.crenata,BTHhavenotbeenclarified.
O.cumanaandO.minorareeconomicallydamagingJasmonicacid(JA)isanotherdefence-inducerpromotingspecies.Severalcontrolstrategieshavebeenproposedandresistanceagainstinsects(McCornetal.,1997)andpatho-employed,butnonehasprovedefficaciousbecauseofthegens(Thommaetal.,1998).JAinducesproductionofpro-complexinteractionbetweenhostsandparasites.
teinaseinhibitors(FarmerandRyan,1990)andbasicSalicylicacid(SA)isachemicaldefence-inducerpro-pathogenesis-relatedproteinsinplants.Althoughtheregu-motingfungal,bacterialandviraldiseaseresistanceandlatorymechanismsandgeneexpressioninducedbyJAinducesproductionofacidicpathogenesis-relatedproteinsaredifferentfromthoseinducedbySA(ReymondandandhypersensitiveresponsesinmanyplantspeciesFarmer,1998;Thommaetal.,1998),theeffectofJAon(ReymondandFarmer,1998;SudhaandRavishankar,resistancetorootparasiticplantshasnotpreviouslybeen2002).S-methylbenzo[1,2,3]thiadiazole-7-carbothioate
demonstrated.Theaimofthepresentstudywastoelucidate(BTH)isasyntheticfunctionalanalogueofSA(Go
¨rlachtheeffectsandtheinhibitorymechanism(s)ofSAandJAetal.,1996;Bokshietal.,2003)anditsapplicationtoonresistancetoOrobancheparasitism.
plantshasbeenproposedtobeanapplicablestrategytocontrolOrobancheparasitism.Forexample,soakingofMATERIALSANDMETHODS
sunflowerseedsinBTHsolutionreducedthenumberofattachmentsofO.cumana(Sauerbornetal.,2002).BTHSourceofseeds
soildrenchingreducedtheattachmentsofO.ramosain
OrobancheminorSm.seedswerecollectedfrommatureplantsthatparasitizedredclover,grownintheWatarase*Forcorrespondence.E-mailyoneyama@cc.utsunomiya-u.ac.jp
basininJapanin2004.Seedsofredclover(Trifolium
#TheAuthor2007.PublishedbyOxfordUniversityPressonbehalfoftheAnnalsofBotanyCompany.Allrightsreserved.
ForPermissions,pleaseemail:journals.permissions@oxfordjournals.org
Downloaded from aob.oxfordjournals.org by guest on March 14, 2011538Kusumotoetal.—SA-InducedResistanceofRedClovertoOrobancheminor
pratenseL.‘Makimidori’),atypicalhostofO.minorandtheglass-fibrefilterpapersweretakenoutfromthe(ParkerandRiches,1993),werepurchasedfromSnowchambersonemonthaftercultivation.TheseedlingswereBrandSeedCo.,LtdinJapan.
handledcarefullysoasnottoseparatethemfromtheseed-bedsandtheglass-fibrefilterpapers.Theredcloverroots,Plantmaterialandgrowthconditions
theseedbedsandtheglass-fibre.filterpapersweresub-mergedinsolutionsof02mMSA,BTH,methyljasmonatePlasticchambers(14cminheightÂ10cminwidthÂ(MeJA)orn-propyldihydrojasmonate(PDJ),afunctional1.5cmindepth)witha1-cm-widerectangularholeintheanalogueofJA(Fujisawaetal.,1998),with0.1%Tweencentreofthetopwerefilledwithrockwool(NittoBoseki20for3handthenthoroughlyrinsedwithwatertoCo.Ltd,Tokyo,Japan).Aglass-fibrefilterpaper(14Âremovetheunabsorbedchemicals.Controlplantswere10cm;GC-90,ADVANTEC,Tokyo,Japan)wasplacedtreatedwithonly0.1%Tween20solutioninthesamebetweentherockwoolandthelidoftheplasticchamber,way.TheconditionedO.minorseedsweretransferredandasmallseedbedmadeofrockwoolwasputbetweenontotheglass-fibrefilterpaperssothattheywereintheplasticlidandtheglass-fibrefilterpaperneartheholecontactwiththeredcloverroots,15seedsper1cmof(Fig.1).Therockwoolandtheglass-fibrefilterpaperintheroot.Theglass-fibrefilterpaperswereuniformlymois-theplasticchamberweremoistenedwithtapwater.Seedstenedwith1mgL21GR24,asyntheticgerminationstimu-ofredcloverweresoakedinrunningtapwaterfor3dlant,toinduceseedgerminationsynchronously.Theredandthenoneseedeachwasplacedintheseedbedonthecloverseedlings,theseedbedsandtheglass-fibrefiltertop,atthecentreoftheplasticchamber.Theplasticpaperswerereturnedtotheplasticchambersandtheseed-chamberswerewrappedwithaluminiumfoilleavingthelingswereincubatedat238Cwitha16-hphotoperiodfor5toppartexposedandwereincubatedinagrowthchamberweeks.Fivereplicationswerepreparedforeachtreatment.
at238Cwitha16-hphotoperiod(photosyntheticphotonfluxdensitiesof52mmols21m22).Theredcloverseedsgerminatedwithin3dunderthesegrowthconditions.TheEstimationofO.minorpenetration
seedlingswerereplenishedwithtapwaterforthefirst5d,ThenumberofO.minorseedlingsattachingtoredcloverwithhalf-strengthTadano–Tanakamedium(Tadanoandrootsandtheirdevelopmentstageswererecorded5weeksTanaka,1980)forthenext6dandthenwithtapwaterafterinoculation.Developmentalstageswereclassifiedasuntiltheendoftheexperiments.
follows:S1,radicleattachingtohostrootsurface;S2,haus-toriumpenetratingintohostrootwithoutswellingofhostTreatmentwithchemicaldefence-inducersandroot;S3,hostrootswellingatpenetratedsite;S4,formationinoculationofO.minorseeds
oftubercle;andS5,tuberclewithroot-likestructures(Fig.2).
Orobancheminorseedsweresurface-sterilizedin70%ethanolfor2minandthenin1%sodiumhypochloritesolu-tionfor2min.SterilizedseedswereincubatedinasealedHistologicalstudy
PetridishlinedwithafilterpapermoistenedwithdistilledTheredcloverrootsitesofpenetrationbyO.minorwaterat238Cinthedarkfor7d(seedconditioning).seedlingsandtubercleswerefixedwith10%formaldehyde.TheplasticchamberspreparedasdescribedabovewereAfterdehydrationthroughagradedseriesofethanol(50,openedandtheredcloverseedlingswiththeseedbeds
75,95,100,100%;24h),sampleswereembeddedinLR
FIG.1.InducedresistanceofredcloverrootsagainstO.minorparasitismaftertreatmentwithwater(A),SA(B)orMeJA(C).Arrowsindicatetubercles
ofO.minor.
Downloaded from aob.oxfordjournals.org by guest on March 14, 2011Kusumotoetal.—SA-InducedResistanceofRedClovertoOrobancheminor539
andPDJwith1mgL21GR24.Seedgermination,lengthofradiclesandmorphologyofroottipswereobservedafterincubationfor7d.Viabilityoftheradicleswasdetectedbystainingwith0.4%trypanblue.FourPetridishespertreatmentwereprepared.
RESULTSANDDISCUSSION
InducedresistancetoO.minorinfectionbychemicaldefence-inducers
ThenumberofO.minorseedlingsthatsuccessfullyparasi-tizedredcloverrootswasreducedby86and77%bySAandBTHtreatments,respectively(Fig.1andTable1).Itwasfoundthatthisreductionwascausedbyacombinationoftwoseparateresistancemechanisms,namelytheinhib-itedelongationofO.minorradiclesandtheincreasedresist-anceofhostroots.
FIG.2.FivedevelopmentalstagesofO.minorseedlingspenetratingintoSAtreatmentsignificantlyreducedthetotalnumberredcloverroot.S1,firstattachmentofO.minorradicletohostrootsurface.ofO.minorseedlingsattachingtotheredcloverrootS2,penetrationofthehaustoriumintothehostrootwithoutswellinghostroot.Theboundarylinebetweenthehaustoriumandthehostcortexwas(Table1).Asobservedmicroscopically,germinationofbrown.S3,hostrootswellingatthepenetratingsite.S4,tubercleformationO.minorseedswasnotdifferentamongthetreatmentsofO.minor.S5,formationofroot-likestructuresfromparasitetubercle.
(morethan80%).However,directedgrowthoftheparasiteradiclestowardtheSA-treatedredcloverrootswasinhib-whiteresinsoft(Sigma,StLouis,MO,USA).Sectionsited(Fig.3).Theinhibitedradicleshadpapillatecellson(5–10mm)werecutwitharotarymicrotome(RM2125RT;theirroottipsandsomeofthemexhibitedabifurcaterootLeicaMicrosystems,Tokyo,Japan)andstainedwithtipform(Fig.3).Thesemorphologiesseemedtobesafranin-fastgreendoublestaining(Johansen,1940).Withsimilartothatofattachmentorgansinducedafterroottipsthisstainingmethod,celluloseandsomecytoplasmareofOrobanchereachahostrootundernormalconditionsstainedblueorgreen,whilelignifiedcellwalls,phenolic(JoelandLosner-Goshen,1994).Whendirecteffectsofsubstancesandnucleiappearred.Lignifiedcellwallswerethechemicaldefence-inducersonGR24-elicitedseedger-alsodetectedwithphloroglucinol-HCl(Jensen,1962).
minationandseedlinggrowthofO.minorwereexamined,neitherthegerminationratenortheradicleelongationdetermined7dafterGR24treatmentwereaffectedbyDirecteffectsofchemicaldefence-inducersongerminationthesedefence-inducers.TheroottipsweresmoothandofO.minorseedsandelongationofradicles
conical,anddidnotdeveloptoattachmentorgansinallthetreatments.NoneoftheseedlingsexhibitedpronouncedGerminationtestswereconductedaccordingtoChaereductionofcellviabilitiesasdeterminedbystainingwithetal.(2004).AfterconditioningofO.minorseeds,theytrypanblue.Theseresultsindicatethatthereductionofweretransferredtothe0.2mMsolutionsofSA,BTH,MJ
theparasiteattachmentswasnotduetothedirecteffect
TABLE1.EffectsofSA,BTH,MJandPDJonthetotalnumberofO.minorseedlingsattachingtoredcloverrootandthe
numberofO.minorseedlingsineachdevelopingstageofparasitism
NumberofO.minorseedlingsortuberclesineachdevelopingstageofparasitismperplant
TotalnumberofRootlengthofO.minorPenetrationredclover
seedlings
throughhostRoot-likewhereO.minorattachingtoredAttachmentcortexbutnoSwellingofTuberclestructureEstablishmentseedswerecloverrootpertoroot
rootswellinghostrootformationformationofparasitismTreatmentplaced(cm)plant
surface(S1)(S2)
(S3)(S4)(S5)(S3þS4þS5)Water156.3+6.2a323.8+60.7a53.6+8.7a135.6+32.4a32.90.2+27.7a11.8+3.5a134.6+38.7aSA171.8+12.5a115.6+19.8b32.8+4.8a64.4+14.0a.6+8.6ab32+1.4c11.8+3.4b3.4+1.1ab18.4+5.2bBTH165.2+9.7a177.4+40.4ab43.4+9.7a103.2+26.5a8.0+5.1bc19.8+8.0b3.0+1.0b30.8+13.5bMJ157.5+15.1a298.4+60.9ab47.4+7.8a139.4+33.2a36.8+10.1a68.0+20.9ab6.8+1.8ab111.6+30.1abPDJ
155.2+4.7a235.8+15.3ab50.6+3.1a110.0+15.5a21.8+4.3abc46.0+4.4ab7.4+1.5ab75.2+6.3abFromanatomicaldata,theparasiticintrusivecellsinS2werepreventedfrompenetratingintothehostcentralcylinderbutthehaustoriuminS3–S5penetratedintothecentralcylinderandconnectedwiththehostvascularsystem.Therefore,thesumofS3–S5representsthenumberofO.minorseedlingsestablishingparasitism.Valuesarepresentedasmean+s.e.offivereplications.MultiplemeancomparisonswereperformedusingTurkey-Kramer’sHSDtest,anddifferentlettersindicatesignificancedifferencesbetweenmeansatP,0.05.
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formationhavenotbeenidentified,unlikesimilarparasiticplantsStrigaandTriphysaria(Yoder,2001).Recently,itwasnotedthatattachmentorgandifferentiationinOrobanchewastriggeredendogenouslyintheabsenceof
ahost(Gonza
´lez-Verdejoetal.,2005).Therefore,wepresumethatSA-treatedredcloverrootinterruptedradicleelongationviachemicalinteractionsalthoughrelationshipbetweentheexudatesfromSA-treatedrootsandthediffer-entiationofattachmentorgansremainsuncertain.
Asecondbarriertoparasitismoccurredaftercorticalinvasionbytheparasitebutbeforegrossmorphologicalchangeswereobservedinthehost.AnatomicalobservationsshowedthatthepenetrationofparasiticintrusivecellsinstageS2wasstoppedatthelignifiedendodermisofthehostrootanddidnotconnectwithhostvessels(Fig.4A,C).FIG.3.AbortionofO.minorseedlingsinducedbytheredcloverrootBycontrast,parasitesinstageS3–S5penetratedintothetreatedwithSA.Someoftheseedlingsshowedabifurcateroottipform
centralcylinderoftheredcloverrootandthehaustorial(arrows).
cellsoccupiedthecentralcylinderofthehostroots(Figs5and6).InthelaterstagesofS4andS5,thehaustoriaofthechemicalsonseedgerminationandradiclegrowth.expandedinthehostroot(Fig.6A)andaxiallyorientatedNormallyOrobancheradiclesstopelongationsoonafterxylemvesselsoftheparasiteoriginappearedtogethertheroottipsreachahost,andthenformattachmentwiththehostvessels(Fig.6B,C).Verticalparasiteorgans.However,signalsinducingattachmentorgan
vesselslinkedwiththeaxialparasitevesselswereobserved
FIG.4.TransversesectionsoftheredcloverrootinstageS2.Thesectionswerestainedbysafranin-fastgreen(A,B)andbyphloroglucinol-HCl(C).(A)Sectionofwater-treatedredcloverroot.PenetrationofO.minorintrusivecells(Pic)wasstoppedattheendodermis(En).Arrowindicateslignifiedthickwallsofhostvesselsinducedbytheparasitepenetration.(B)High-magnificationphotographofthecentralcylinderofSA-treatedredcloverroot.Fibrecells(F)accumulatedsafranin-positivesubstance(arrow)andhostvessel(Hv)wasoccludedbyastainedsubstance(arrowhead).(C)Cell-walllig-nification(arrowhead)oftheendodermis(En),pericycleandfibrecells(F)ofwater-treatedredcloverroot.Lignificationoccurredatthenearsideoftheendodermisfromtheparasiticintrusivecells(Pic)butnotattheoppositeside.Scalebar¼100mm(A),50mm(B,C).En,endodermis;F,fibre;Hc,host
cortex;Hv,hostvessel;P,parasite;Pic,parasiticintrusivecell.
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FIG.5.TransversesectionsoftheredcloverrootinstageS3(A,B)andS4(C,D).Sectionswerestainedbysafranin-fastgreen(A,C,D)andbyphloroglucinol-HCl(B).(A)SectionofSA-treatedredcloverroot.Parasitehaustorium(Pha)intrudedintothehostcentralcylinderandcompressedthehostendodermalcells(En).(B)High-magnificationphotographofthecentralcylinderofthesamerootin(A).Lignifiedcells,i.e.hostvessels(arrow-head)andlignifiedfibres(arrow),remainedinthecentralcylinder.(C)SectionofBTH-treatedredcloverrootandparasitetubercle(Pt).(D)High-magnificationphotographofthehostxylemin(C).Mostofhostvesselsaccumulatedastainedsubstance(arrowhead).Scalebar¼100mm
(A,C),50mm(B,D).En,endodermis;Hc,hostcortex;Hx,hostxylem;P,parasite;Pha,parasitehaustorium,Pt,parasitetubercle.
FIG.6.LongitudinalsectionsoftheredcloverrootandO.minortubercleinstageS5stainedbysafranin-fastgreen.(A)Low-magnificationphotographofthehostrootandtheparasitetubercle(Pt).Parasitehaustorium(Pha)completelyfusedwiththehostcellsinthecentralcylinder(Hcc).(B)Linkageofthehostandparasitevessels.Parasiticvesselsintheverticaldirection(Vpv)continuouslyconnectedwiththeaxialparasiticvessels(Apv),whichwereformedalongthehostvessels(Hv).(C)Axialparasitevessels(Apv)lyingadjacenttothehostvessels(Hv).Vesselelementsoftheparasiteareshorterandwiderandhavemuchlargerpitsthanhostvesselelements.Scalebar¼500mm(A),50mm(B,C).Apv,axialparasiticvessel;Hcc,hostcentralcylinder;
Hv,hostvessel;Pha,parasitehaustorium;Pt,parasitetubercle;Vpv,verticalparasiticvessel.
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TABLE2.EffectsofSA,BTH,MJandPDJonpercentageoffourstages(S2,S3,S4andS5)inthetotalnumberofO.minorseedlingswhosehaustoriumpenetratedintored
cloverroot(S2þS3þS4þS5)
Penetrationintocentralcylinderthrough
PenetrationhostendodermisthroughhostcortexRootlikebutnorootSwellingofTuberclestructureswellinghostrootformationformation(S2)
(S3)(S4)(S5)Water50.12.1+1.33.0+6.2a4.5+0.9aSA.5+8.4a766+4.5b.9ab3.4+15c15.BTH77.0+6.6b4.7+2.1bc.3+3.2ab4.7+1.3a149+3.6b3.5+2.MJ56.0+4..0a25.7+3.1ab3.PDJ
.3ab14.4+1.9a58.5+41ab11.5+1.6ab.9+16a26.1+4.3ab39+0.7aFromanatomicaldata,theparasiticintrusivecellsinS2werepreventedfrompenetratingintothehostcentralcylinderbutthehaustoriuminS3–S5penetratedintothecentralcylinderandconnectedwiththehostvascularsystem.Valuesarepresentedasmean+s.e.offivereplications.MultiplemeancomparisonswereperformedusingTurkey-Kramer’sHSDtest,anddifferentlettersindicatesignificancedifferencesbetweenmeansatP,0.05.
inthemiddleofthetubercle(Fig.6B).TheseanatomicaldatasuggestthatO.minorseedlingsinS2failedinsucces-sivepenetrationintohostvascularsystemsduetohostresistanceandthoseinS3–S5successfullyconnectedandinfectedthehostroot.
Thepercentageofindividualparasitismstagesafterpara-siticcellsinvadedintoredcloverroot(S2,S3,S4andS5)wascalculatedtoclarifyfurthertheeffectsofthechemicaldefence-inducersoninternalresistanceoftheredcloverroot(Table2).SAandBTHinducedanincreaseintheper-centageoftheparasiticcellspenetratingthroughthecortexbutnotintothecentralcylinder(S2)anddecreasedtheper-centageofthestageswherethehaustoriasucceededinpenetratingintothecentralcylinder(S3andS4).Bycon-trast,MeJAandPDJhadlittleinfluenceontheseprocesses.TheseresultssuggestthatSAandBTHactivatedefenceresponsesintheredcloverrootandthuspreventthesuc-cessfulinvasionoftheparasite,whereasMeJAandPDJhavelittleeffectsontheresistancetotheOrobanchepenetration.
Histologicalobservations
Ahistologicalstudyoftheinfectionsitewasperformedtoclarifythemechanismbywhichthehostrootpreventedpenetrationoftheparasite.TheintrusivecellsofO.minorinstageS2penetratedthroughtheepidermisandthecortexoftheredcloverrootbutfurtherpenetrationwasstoppedattheendodermis(Fig.4A).Theboundarywallsbetweenthehaustoriaandthehostcortexwerebrowninnon-stainedsections.Insomecases,browndropletsaccu-mulatedinhostfibresclosetotheparasiticintrusivecells.Thesedropletswerestainedbysafranin(Fig.4B)andphloroglucinol-HCl(Fig.4C),suggestingthattheycon-tainedlignin-likelow-molecular-weightsubstances.Thecellwallsofthefibresthataccumulatedbrowndroplets
werelignified(Fig.4C).Thickvesselwallswereformedneartheparasiticintrusivecellsandstaineddenseredbysafraninandphloroglucinol-HCl(Fig.4A),indicatingthatthewallswerehighlylignified.Somehostvesselswereoccludedbyastainedsubstancerevealedbysafranin-fastgreen(Fig.4B).Thephloroglucinol-HClstainingrevealedlignificationofthecellwallsoftheendodermisandthepericyclethatcontactedtheparasiticintrusivecells,whenthepenetrationstoppedattheendodermis(Fig.4C).Inthemoredevelopedstages,S3–S5,theintrusivecellspene-tratedthroughthehostendodermisintothecentralcylinderandhaustoriaoccupiedtheinside(Figs5and6).Hostcellsinthecentralcylinderweredestroyedbytheparasiteexceptforhostvesselsandlignifiedfibrecells.Mostofthehostvesselseitherhadlignifiedthickwalls(Fig.5B)orwereoccludedbyastainedsubstance(Fig.5D)suchasthoseobservedinS2.Noanatomicaldifferenceselicitedbythechemicaltreatmentswereobservedaslongastheobservedsiteswereinthesamestageofparasitism.
BeforediscussingtheeffectsofSAandBTHontheresistanceoftheredcloverrootstoOrobanche,itisusefultosummarizethethreeaspectsofhostrootresistanceduringtheearlystagesofthepenetration.Corticalresistanceisthefirstresistancethatthepenetratinghaustoriaencounter.Ithasoftenbeenrepresentedasanapoplasticsecretionofbrownsubstances(Goldwasseretal.,2000;Rubialesetal.,2003).However,somereportsindicatedparasitecells(Pe
´thatthissecretionoriginatedfromthe
rez-de-Luqueetal.,2005).Inmorerecentstudies,suberization(Echevarrı
´a-Zomen˜2006)andprotein´oetal.,a-Zomen˜oetal.,2006;Pe
´cross-linking(Echevarrı
rez-de-Luqueetal.,2006a)ofthecorticalcellwallshavebeensuggestedtobeinvolvedintheresist-ance.Thehaustoriathenfaceendodermalresistanceafterpassingthroughthecortex.Agenotypeofvetchwithendodermalresistance(Goldwasseretal.,2000)exhibitedhigheraccumulationoflignininresponsetoparasiteinfectionthanasusceptiblegenotype(Goldwasseretal.,1999).AnatomicalstudiesrevealedthatlignificationwasinducedintheendodermalandpericyclecellsincontactwithPe
´theparasiticintrusivecells(Maitietal.,1984;rez-de-Luqueetal.,2005,2006b).Thelignifiedcellsarepresumedtorestrictthepenetrationoftheparasitecellsintothehostcentralcylinderasaphysicalbarrier.Iftheparasitepenetratesthroughtheendodermisbeforethelignificationisinducedoritovercomesthelignifiedbarrier,vesselocclusionplaystheroleinresistance.Afterpenetratingintothecentralcylinder,theparasiteconnectstohostvascularsystemsandformsatubercle.However,massivevesselocclusionbysecretionofsubstancescon-tainingcarbohydrates2001;Pe
´andpolyphenols(Labrousseetal.,
rez-de-Luqueetal.,2005,2006b)and/ortylosis(Maitietal.,1984)isassumedtodiminishwaterandnutri-entsupplytotheparasiteandcausetuberclenecrosis.Thesethreeresistantmechanismsdependonthegenotypeofhostplants(Labrousseetal.,2001)andsingleormultiplemechanismsprobablycontributetotheresistancetotheparasitepenetrationinaresistantgenotype.Bycontrast,nooronlyweakresistantresponsesappearedinasuscep-tiblegenotype.Inthepresentstudy,thenon-treatedred
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cloverrootexhibitedtheendodermalresistanceinhalfofthepenetrationsites(seestageS2inTable2).SAandBTHtreatmentincreasedthepercentageofpenetrationsitesinstageS2,suggestingthatthesechemicalsenhancedendodermalresistance,presumablythroughtheactivationoflignification(Table2,Fig.4C).Ontheotherhand,noclearcorticalresistanceappearedintherootstreatedwiththesechemicals.Althoughvesselresistancewasshownaslignifiedthickwallsandocclusionbysubstances,SAandBTHtreatmentsdidnotaffectthefrequencyoflignificationofcellwallsandvesselocclusion,northegrowthornecro-sisofthetubercles.Therefore,itislikelythatthemainformofresistanceenhancedbySAandBTHisendodermalresistance.TheeffectsofSAonthesynthesisofligninarenotfullyunderstood(ReymondandFarmer,1998),butsomereportssuggestedthatSAactivatedlignificationinresponsetopathogeninfection(Mauch-ManiandSlusarenko,1996;HeandWolyn,2005).
PossiblestrategyofOrobancheparasitisminhostroots
ThepresentresultsshowedthatSA-dependentdefenceresponseseffectivelyinhibitedOrobancheparasitismonahostplantbutJA-dependentdefenceresponseshadlittleeffect.SAandJAinducedifferentdefencegeneexpressionviaindependentsignallingpathways(ReymondandFarmer,1998).RecentreportsdescribedthedefencegeneexpressionofhostplantsinfestedbyOrobanchespp.BasedonexaminationofmRNAaccumulationofdefencegenesinAradopsisthalianainfestedbyO.ramosa,expressionofseveralgenesmediatedbyaJA-dependentpathwaywereactivatedbyO.ramosainfestationwhereasgenesmediatedbyaSA-dependentpathwaywerenot(VieiraDosSantosetal.,2003a,b).Similarly,byusingtransgenictobaccocontainingapromoter:GUSfusion,PR-1a,anindicatorgeneoftheSA-dependentpathwayinduction,wasnotexpressedbyO.aegyptiacainfesta-tion(Griffittsetal.,2004).Thus,itislikelythathostplantsinduceaJA-dependentpathwayratherthanaSA-dependentpathwayinresponsetoOrobancheinfesta-tion.ActivationoftheJA-dependentpathwayinducedbyOrobancheinfestationareassumedtobeinsufficientfortheinhibitionofOrobancheparasitismbecausetheexogen-ousapplicationofJAanaloguesdidnotpromoteinhibition.Meanwhile,itmayhavebeenunnecessaryforOrobanchetoacquiretheabilitytoovercometheinducedresistancebySAduringevolutionbecauseSA-mediateddefenceisanunusualresponseinthenormalhost–parasiteinteraction.ItisassumedthattheeffectivenessofSAtreatmentoninhi-bitionoftheOrobancheparasitismiscausedbythediscor-dancebetweenSA-andOrobanche-inducibleresponses.
ACKNOWLEDGEMENTS
ApartofthisstudywassupportedbyGrants-in-AidforScientificResearch(15830079,1820810)fromtheJapaneseSocietyforPromotionofScience(JSPS),andagrantforEminentResearchatUtsunomiyaUniversity.
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