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Non-Host Defense Response in a Novel Arabidopsis- Xanthomonas citri subsp. citri Pathosystem
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Title: Non-Host Defense Response in a Novel Arabidopsis- Xanthomonas citri subsp. citri Pathosystem
Series Title: An C, Mou Z (2012) Non-Host Defense Response in a Novel Arabidopsis-Xanthomonas citri subsp. citri Pathosystem. PLoS ONE 7(1): e31130. doi:10.1371/ journal.pone.0031130
Physical Description: Journal Article
Creator: Mou, Zhonglin
An, Chuanfu
Publication Date: January 27, 2012
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Abstract: Citrus canker, caused by Xanthomonas citri subsp. citri (Xcc), is one of the most destructive diseases of citrus. Progress of breeding citrus canker-resistant varieties is modest due to limited resistant germplasm resources and lack of candidate genes for genetic manipulation. The objective of this study is to establish a novel heterologous pathosystem between Xcc and the well-established model plant Arabidopsis thaliana for defense mechanism dissection and resistance gene identification. Our results indicate that Xcc bacteria neither grow nor decline in Arabidopsis, but induce multiple defense responses including callose deposition, reactive oxygen species and salicylic aicd (SA) production, and defense gene expression, indicating that Xcc activates non-host resistance in Arabidopsis. Moreover, Xcc-induced defense gene expression is suppressed or attenuated in several well-characterized SA signaling mutants including eds1, pad4, eds5, sid2, and npr1. Interestingly, resistance to Xcc is compromised only in eds1, pad4, and eds5, but not in sid2 and npr1. However, combining sid2 and npr1 in the sid2npr1 double mutant compromises resistance to Xcc, suggesting genetic interactions likely exist between SID2 and NPR1 in the non-host resistance against Xcc in Arabidopsis. These results demonstrate that the SA signaling pathway plays a critical role in regulating non-host defense against Xcc in Arabidopsis and suggest that the SA signaling pathway genes may hold great potential for breeding citrus canker-resistant varieties through modern gene transfer technology.
Acquisition: Collected for University of Florida's Institutional Repository by the UFIR Self-Submittal tool. Submitted by Zhonglin Mou.
Publication Status: Published
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Non-HostDefenseResponseinaNovelArabidopsis-Xanthomonascitrisubsp.citriPathosystemChuanfuAn,ZhonglinMou *DepartmentofMicrobiologyandCellScience,UniversityofFlorida,Gainesville,Florida,UnitedStatesofAmericaAbstractCitruscanker,causedby Xanthomonascitri subsp. citri ( Xcc ),isoneofthemostdestructivediseasesofcitrus.Progressof breedingcitruscanker-resistantvarietiesismodestduetolimitedresistantgermplasmresourcesandlackofcandidate genesforgeneticmanipulation.Theobjectiveofthisstudyistoestablishanovelheterologouspathosystembetween Xcc andthewell-establishedmodelplant Arabidopsisthaliana fordefensemechanismdissectionandresistancegene identification.Ourresultsindicatethat Xcc bacterianeithergrownordeclineinArabidopsis,butinducemultipledefense responsesincludingcallosedeposition,reactiveoxygenspeciesandsalicylicaicd(SA)production,anddefensegene expression,indicatingthat Xcc activatesnon-hostresistanceinArabidopsis.Moreover, Xcc -induceddefensegeneexpression issuppressedorattenuatedinseveralwell-characterizedSAsignalingmutantsincluding eds1 pad4 eds5 sid2 ,and npr1 Interestingly,resistanceto Xcc iscompromisedonlyin eds1 pad4 ,and eds5 ,butnotin sid2 and npr1 .However,combining sid2 and npr1 inthe sid2npr1 doublemutantcompromisesresistanceto Xcc ,suggestinggeneticinteractionslikelyexist between SID2 and NPR1 inthenon-hostresistanceagainst Xcc inArabidopsis.TheseresultsdemonstratethattheSA signalingpathwayplaysacriticalroleinregulatingnon-hostdefenseagainst Xcc inArabidopsisandsuggestthattheSA signalingpathwaygenesmayholdgreatpotentialforbreedingcitruscanker-resistantvarietiesthroughmoderngene transfertechnology.Citation: AnC,MouZ(2012)Non-HostDefenseResponseinaNovelArabidopsisXanthomonascitri subsp. citri Pathosystem.PLoSONE7(1):e31130.doi:10.1371/ journal.pone.0031130 Editor: Ching-HongYang,UniversityofWisconsin-Milwaukee,UnitedStatesofAmerica Received September29,2011; Accepted January3,2012; Published January27,2012 Copyright: 2012An,Mou.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricted use,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited. Funding: ThisworkwassupportedbyagrantfromtheNationalScienceFoundation(IOS-0842716),aspecialgrantfromUnitedStatesDepartmentofAgriculture NationalInstituteofFoodandAgriculture,andagrantfromtheCitrusResearchandDevelopmentFoundationawardedtoZM.Thefundershadnoroleinst udy design,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript. CompetingInterests: Theauthorshavedeclaredthatnocompetinginterestsexist. *E-mail:zhlmou@ufl.eduIntroductionCitruscankerisadevastatingleaf,stem,andfruitspotting diseaseaffectingmanyimportantcitrusspeciessuchasgrapefruit ( Citrusparadisis Macf.),certainsweetoranges( C.sinensis (L.) Osbeck),Keylime( C.aurantifolia Swingle),andlemons( C.limon (L.)Burm.F.)[1].Itiscausedbythebacterialpathogen Xanthomonascitri subsp. citri ( Xcc )[2].Althoughreducedquality andquantityoffreshandprocessedfruitshavebeencausinggreat economiclosstothecitrusindustry,noefficientwayhasbeen foundtocontrolthedisease.Currently,managementofcitrus cankerlargelyreliesonchemicalcontrolandagriculturalpractices [2].Becauseoftheeconomicandenvironmentalconcerns, developingresistantcultivarsperhapsisthebestlong-termsolution forthemanagement[3].However,limitedresistantscion germplasmresourcesandtheirinterferingwiththeexpressionof optimumtraitsrelatedtofruitqualityandproductionhamper developingcanker-resistantcitrusvarietiesthroughconventional breedingapproach,nottomentionitslabor-andtime-consuming characters[4].Incontrast,transgenicapproachcanquickly incorporateresistanceintocitruswithoutinterferingwiththe expressionofoptimumvarietaltraits.Nevertheless,itsaccomplishmentdependsontheunderstandingofthemolecular mechanismsofpathogenesisandtheavailabilityoftargetgenes formanipulation[3].Asahighlyheterozygous,polygenicspecies withlimitedgeneticresourcesandalongjuvenileperiod, functionalanalysisofcitrusgenesrelatedtoinnatedisease resistanceisimpaired,whichconsequentlyhindersthedevelopmentofcanker-resistantcitruscultivarsusingtransgenicapproach. Themodelplant Arabidopsisthaliana hasbeenshownasapromising alternativeforunderstandingplantdefensemechanisms[5–7]. Transferringmoleculartechnologiesincludinggenesinvolvedin innateimmunityfrommodelplanttocropsholdsgreatpotential forgeneticimprovement.Infact,severalstudieshavealready demonstrateditsfeasibilityinthedevelopmentofcitrusdisease resistantlines[3,8]. Innature,plantsareconstantlychallengedbyadiverserangeof microbes.However,foracertainplantspecies,onlyafewofthese microbesarepathogenic.Resistanceofanentireplantspecies againstallstrainsofapathogenthatisabletoinfectotherplant speciesisaphenomenonknownasnon-hostresistanceanddictates themostrobustformofplantimmunity[9].Despiteitsgreat potentialforprovidingcropplantswithdurableresistance,plant defensemechanismsunderlyingnon-hostresistancearenot sufficientlyunderstood[10].Accumulatingevidencehasindicated thatplantnon-hostresistanceiscomposedoflayersofdefense responses[10–13].Toestablishpathogenicity,pathogensneedto enterplanttissuetoobtainnutrientsandcounteracthostdefense. Phytopathogenicbacteriumlike Pseudomonassyringae entersthe internalplanttissuethroughopenstomataorwounds,whereas somefungalpathogensdirectlypenetrateplantcellwall. Preformedphysicalandchemicalbarriersarethoughttoconstitute PLoSONE|www.plosone.org1January2012|Volume7|Issue1|e31130

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theprimarytrancheofnon-hostdefensemechanisms[9].Several preformed(wax,cuticlelayer,cellwall)andinduciblebarriers, suchaspapilla/callose[12],aliphaticisothiocyanates[14], indoleglucosinolates[15],camalexin[16],andchloroplastgeneratedreactiveoxygenspecies(ROS)[17],playimportant rolesduringnon-hostinteractions.Twogenes AtGSNOR1 and F3OGT ,relatedto S -nitrosothiolandanthocyaninbiosynthesis, respectively,arethoughtimportanttonon-hostresistance [18,19].StudiesonArabidopsisagainstnon-adaptedphytopathogenicfungibarleypowderymildew( Blumeriagraminis f.sp. hordei ; Bgh )identifiedthreegenesinvolvedinlimiting Bgh penetration throughtwoseparatepathways.Oneinvolvesanexocytosis pathwaycontrolledbythePEN1syntaxinanditsworking partners[20,21]andtheotherrequiresthePEN2myrosinase andthePEN3ATP-bindingcassettetransporter[22,23]. Inhibitionoftheactinskeletalfunctionincombinationwith the eds1 mutationseverelycompromisesnon-hostresistancein Arabidopsisagainstwheatpowderymildew,whichsuggeststhat actincytoskeletonisalsoinvolvedinpreinvasionnon-host resistance[24].Comparativegeneexpressionprofilinganalyses revealedthesimilardefenseresponsesbetweennon-host resistanceandgene-for-generesistanceinArabidopsis[25,26]. Moreover,amongthenon-host Pseudomonas bacteria-regulated genes,approximately30%ofthemarealsoregulatedbyflg22, indicatingaroleofpathogen-associatedmolecularpattern (PAMP)signalinginnon-hostresistance[26].Species-or family-leveldifferenceinPAMPrecognitionalsosuggestsits associationwithnon-hostresistance[27–29].Meanwhile, pathogenmutantslackingafunctionalPAMPwereshownto gainatleastpartialvirulenceonnon-hostplants[30,31].These resultsindicatethatPAMPrecognitionisanotherimportant non-hostbarrier.Furthermore,somegeneticcomponents involvedingene-for-genehostresistancewereshowntofunction inpost-invasivedefense.Examplesof R genesfunctioningin non-hostresistancearefew[32,33].However,severalsignaling componentsinvolvedingene-for-generesistancehavebeen identifiedfromvariouspathosystems.Amongthemarethe EDS1-PAD4-SAG101complex[22,23],theHSP90-SGT1RAR1complex[34–37],ADS1[38],ARF1[39],EDR1[40], NDR1[41],HSP70/HSP90[42–44],andPAD3[45].In addition,aglycerolkinase-encodinggene NHO1 isrequiredfor Arabidopsisresistancetoheterologousbacterialpathogen P. syringae pv. phaseolicola and P.syringae pv. tabaci [46,47]. Recentgeneticandgenomicstudiesalsorevealedtheimportant roleofsalicylicacid(SA),jasmonicacid(JA),andethylene(ET)for maintenanceofnon-hostresistanceinspecificplant-microbe combinations[10].DegradationofSAinArabidopsissalicylate hydroxylase( NahG )transgenicplantsconferssusceptibilitytothe non-hostbacterium P.syringae pv. phaseolicola NPS3121[46].Nonhostresistanceagainstthecowpearustfungus Uromycesvignae requiresaccumulationofSAinArabidopsis[48].Non-host resistanceofArabidopsisto Alternariabrassicicola dependsonJA, as coi1 mutantissusceptibletofungalinfection[49].Moreover, tobaccoplantsimpairedinethyleneperceptionaresusceptibletoa varietyofsoil-bornespeciesinthegenusof Pythium [50].In anotherheterologouspathosystembetweenArabidopsisand Phakopsorapachyrhizi ,bothSAandJAsignalingpathwayare involved[6].TheJA/ETpathwayisalsoactivatedduringnonhostresistancetothehemibiotrophicpotatopathogen, Phytophthora infestans ,andthebiotroph, Blumeriagraminis f.sp. hordei in Arabidopsis[7].ArecentsurveyofapanelofArabidopsis mutants,involvedingene-for-generesistance,unveiledthatboth SAandJA/ETpathwaycontributetopost-invasiveresistance against Golovinomycescichoracearum UMSG1[51]. Inthisstudy,weestablishedanovelnon-hostpathosystem involvingArabidopsisandaneconomicallyimportantbacterial pathogen Xcc .Byexaminationofaseriesofpreviouslyidentified ArabidopsismutantscompromisedinSA,JA,andETdefense signaling,severalgeneticcomponentsoftheSAsignalingpathway werefoundtoplayprofoundroleinthe Xcc -induceddefensegene expressionandthenon-hostresistanceagainstXcc inArabidopsis. TheseresultssuggestthattheArabidopsisXcc pathosystemis highlyvaluableforidentifyingsignalingcomponentsthatpositively regulatenon-hostresistanceagainst Xcc .TheSAsignaling pathwaygenespinpointedinthisstudycouldpotentiallybe engineeredintocitrustocombatcankerdisease.Results Xcc isanon-hostpathogenofArabidopsisTotestwhetherthecitruscankerbacterialpathogen Xcc could causediseaseinArabidopsis,weinoculatedArabidopsisplants with Xcc bysyringeinfiltration,dip,andsprayinoculation methods.Leaftissueswerecollectedatdifferenttimepointsafter inoculationtodeterminethe inplanta growthof Xcc .Asshownin Figures1Ato1D,syringe-infiltrated Xcc didnotgrowatallduring thecourseofarelativelylong-term(15days)infectioninfour Arabidopsisecotypes,Columbia(Col-0),Landsberg erecta (L er ), Wassilewskija(Ws),andRLD.Similarresultwasobservedfrom experimentsusingdipandsprayinoculationmethods.Bacterial numberremainedalmostconstantduring9dayspost-inoculation (dpi)inbothCol-0andL er ecotypes(Figures1Eto1F).Wenoticed thatphysicalbarriersblockedalargeportionofbacteriacoatedon thesurfaceofleavesbydipandsprayinoculationmethods.The concentrationofthebacterialsuspensionsusedfordipandspray inoculationwas100foldhigherthanthatusedinsyringe infiltration,butbacterialgrowthwasatthesamelevel(dip)asor less(spray)thanthatobservedintheinoculationbysyringe infiltration(Figures1Eto1F).Interestingly,thenumbersof Xcc bacteriadidnotdeclineduringthecourseofinfection,indicating that Xcc isabletosurviveforalongperiodoftimeinArabidopsis. Thus, Xcc isanon-hostpathogenofArabidopsis.Furthermore,we didnotobserveanyvisiblesymptomsassociatedwiththeinfection, suggestingthat Xcc mayinducetypeInon-hostresistancein Arabidopsis[10].Xcc activatesamultilayereddefenseresponsein ArabidopsisTheinvolvementofROSinbothhostandnon-hostresponse hasbeenextensivelystudied[52–55].ToassaytheroleofROSin theArabidopsisXcc interaction,weexaminedROSaccumulation byDAB(3,3 9 -diaminobenzidinetetrahydrochloride)stainingand monitoredthedynamicexpressionof GST1 ,amarkergeneforthe engagementofROS-dependentdefense[56],in Xcc -infected Arabidopsisleaves.InbothCol-0andL er ecotypes,ROS accumulationwasdetectedat4hourspost-inoculation(hpi) (Figure2).Further, GST1 expressionpeakedat4hpi,andthen graduallydecreased(Figure3A).Together,theseresultsindicate thatROSmaybeanon-hostdefensecomponentinthe ArabidopsisXcc interaction.Moreover,PAMP-inducedearly responsegenesappearedtoparticipateinthenon-hostdefense responseintheArabidopsisXcc pathosystem.Threeflg22induciblegenes, FRK1 NHO1 ,and WRKY29 [31,57],were significantlyactivatedby Xcc infection(Figures3Bto3D). Expressionofthesegenesreachedthehighestlevelat4hpiand decreasedafterward,exceptthattheexpressionlevelsof FRK1 maintainedhighfrom4to12hpi(Figure3B).TotestwhetherSA isinvolvedinthenon-hostinteractionbetweenArabidopsisandANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org2January2012|Volume7|Issue1|e31130

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Xcc ,wemeasuredSAlevelsin Xcc -infectedleaftissues.Asshownin Figure4A,therewasaslightincreaseinfreeSAlevelsat8hpi, whichmaybecausedbytheinfiltration,asfreeSAlevelsalso increasedslightlyintheMgCl2-treatedleaftissues.However,total SAlevelsinthe Xcc -infectedleaftissuesincreasedsignificantly, reachingthehighestat24hpiandstayingattheplateautillthe endoftheexperiment(96hpi)(Figure4B).Wefurthertestedif Xcc infectioncouldtriggertheexpressionofSA-dependentpathogenesis-related( PR )genes[58].Comparedwiththemocktreatment (10mMMgCl2), Xcc inoculationinducedtheexpressionof PR1 PR2 ,and PR5 inCol-0plants(Figures5Ato5C).Inthisspecific interaction, PR2 and PR5 wereinducedearlier(4–8hpi)than PR1 (12hpi),whereas PR1 appearedtobeinducedtoahigherlevel than PR2 and PR5 .Theseresultsindicatethatthewell-definedSA signalingpathwayisactivatedduringtheArabidopsisXcc interaction. Figure1.XccdoesnotgrowinArabidopsis. ( A )Growthofsyringe-infiltrated Xcc inCol-0.( B )Growthofsyringe-infiltrated Xcc inL er .( C )Growth ofsyringe-infiltrated Xcc inWs.( D )Growthofsyringe-infiltrated Xcc inRLD.( E )Growthofdip-inoculated Xcc inCol-0andL er .( F )Growthofsprayinoculated Xcc inCol-0andL er .Four-week-oldplantswereinoculatedwith Xcc .BacterialsuspensionswithanOD600of0.002and0.02wereusedfor syringeinfiltrationanddip/sprayinoculation,respectively.The inplanta bacterialtitersweredeterminedonday0,3,6,9,12,and15post-inoculation forsyringeinfiltration,andonday3,6,and9post-inoculationfordipandsprayinoculation(cfu,colony-formingunits).Datarepresentthemeano f eightindependentsampleswithstandarddeviation.Theexperimentwasrepeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g001 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org3January2012|Volume7|Issue1|e31130

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Non-hostresistanceto Xcc iscompromisedinseveralSA signalingmutantsTorevealwhethertheSAsignalingpathwaycontributestothe non-hostresistanceagainst Xcc inArabidopsis,wequantifiedthe growthof Xcc inaseriesofsingleordoublemutantsrelatedtoSA signaling( npr1 eds1 eds5 sid2 pad4 ndr1 eds5npr1 ,and sid2npr1 ).A JAsignalingmutant( jar1 ),anETsignalingmutant( ein2 ),a camalexinmutant( pad3 ),andthenon-hostdefensemutant, nho1 whichwasidentifiedintheArabidopsisPseudomonassyringae pv. phaseolicola interaction[46,47],werealsoincludedinthe experiment.AsshowninFigure6, Xcc didnotgrowin npr1 sid2 pad3 ndr1 ein2 ,and jar1 ,buthadasignificantgrowth( 5 fold)in nho1 eds1 eds5 ,and pad4 .Interestingly,therewasa significantgrowthof Xcc inthedoublemutant sid2npr1 ,though Xcc didnotgrowineither npr1 or sid2 singlemutant.Thegrowthof Xcc in eds5npr1 wasalsohigherthaninthe eds5 singlemutant. Theseresultssuggestthat NPR1 maygeneticallyinteractwith SID2 and EDS5 inregulatingnon-hostresistanceagainst Xcc in Arabidopsis.Moreimportantly,allmutantsexcept nho1 with enhancedsusceptibilityto Xcc arerelatedtoSAsignaling, demonstratingthattheSAsignalingpathwayplaysanimportant roleinthenon-hostresistanceagainst Xcc inArabidopsis.Xcc -inducedcallosedepositionisnotalteredintheSA signalingmutantsTocharacterizeifcallosedepositioncontributestothe susceptibilityoftheSAsignalingmutantsto Xcc ,callosestaining wasperformedon Xcc -infectedArabidopsisleaves.Afterfixation, rehydration,andwashing,translucentleaveswerestainedwith anilineblueandexaminedbyepifluorescentillumination. Consistentwiththeobservationthatmutationsof sid2 ,encoding Figure2.XccinducesROSaccumulationinArabidopsis. Fourweek-oldArabidopsisplantsweresyringe-infiltratedwith Xcc (OD600=0.02)ormockcontrol(10mMMgCl2).At4hpi,infiltrated leaveswereexcisedandstainedwithDAB(3,3 9 -diaminobenzidine tetrahydrochloride).ThepresenceofROS(mainlyhydrogenperoxide) causedpolymerizationofDAB,yieldingareddish-browncolor.Tissue wasexaminedunderaLeicaMEIJIscope.Representativeimagesshown herecamefrom24leavesfrom12independentplants.Barsrepresent 1cmand1mminimagesmagnified0.7and4folds,respectively. doi:10.1371/journal.pone.0031130.g002 Figure3.XccactivatesbothROS-andflg22-inducibleearlyresponsegenesinArabidopsis. ( A )Expressionof GST1 .( B )Expressionof FRK1 ( C )Expressionof NHO1 .( D )Expressionof WRKY29 .Four-week-oldCol-0plantswereinoculatedwith Xcc (OD600=0.02)ormock-treatedwith10mM MgCl2.Leafsampleswerecollectedatdifferenttimepoints(0,4,8,12,and24hpi)fortotalRNAisolationandgeneexpressionanalysisusingRTqPCR.Expressionlevelswerenormalizedagainstconstitutivelyexpressed UBQ5 GST1 isamarkergenefortheengagementofROS-dependent defense. FRK1 NHO1 ,and WRKY29 areflg22-induciblegenes.Datarepresentthemeanofthreebiologicalreplicateswithstandarddeviation.The experimentwasrepeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g003 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org4January2012|Volume7|Issue1|e31130

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theSAbiosynthesisgene ICS1 [59],hadnoeffectoncallose production[60,61],callosedepositionwasinducedby Xcc infectioninalltheSAsignalingmutantssusceptibleto Xcc (Figure7),indicatingthattheSAsignalingpathwayisnotrequired for Xcc -inducedcallosedeposition.Moreover,noquantitative differencesincallosedepositionweredetectedamongthemutants andthewild-typeplants.Therefore,callosedepositiondidnot contributetotheobservedsusceptibilityoftheSAsignaling mutantsto Xcc .Xcc -inducedexpressionofearlydefense-responsegenes isdecreasedintheSAsignalingmutantsPAMPdetectionisanimportantcomponentofnon-host resistanceinplantsandservesasanearlywarningsystemfor thepresenceofpotentialpathogens[31,62].Similarly,oxidative burstisanotherquickdefenseresponseafterbothhostandnonhostpathogenrecognition[17].TodeterminewhetherPAMP-or ROS-dependentearlyresponsescontributetotheobserved susceptibilityoftheSAsignalingmutantsto Xcc ,wecompared theexpressionlevelsof GST1 FRK1 NHO1 ,and WRKY29 after Xcc infection[31,56,57].Expressionlevelsofthefourgeneswere generallylowerintheSAsignalingmutantsthaninthewild-type plants(Figure8).Thenon-hostdefensemutation nho1 onlyslightly loweredtheexpressionof GST1 NHO1 ,and WRKY29 at8hpi (Figures8I,8Jand8L).Although npr1 and sid2 didnotallow Xcc growth,expressionofthefourgeneswassimilarlyinhibitedin npr1 and sid2 asinothersusceptibleSAsignalingmutants. Figure4.XccinducesSAproductioninArabidopsis. ( A )FreeSA levels.( B )TotalSA(SA + SAG)levels.Leavesofwild-typeCol-0plants wereinoculatedwith Xcc (OD600=0.02)ortreatedwith10mMMgCl2. Theinoculatedleaveswerecollectedatdifferenttimepoints(0,4,8,16, 24,48,72,and96hpi)forSAmeasurementbyHPLC.Datarepresentthe meanoffourindependentsampleswithstandarddeviation.The experimentwasrepeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g004 Figure5.XccinducesPRgeneexpressioninArabidopsis. ( A ) Expressionof PR1 .( B )Expressionof PR2 .( C )Expressionof PR5 .Fourweek-oldCol-0leaveswereinoculatedwith Xcc (OD600=0.02)ormocktreatedwith10mMMgCl2.Leafsampleswerecollectedatdifferent timepoints(0,4,8,12,and24hpi)fortotalRNAisolationandgene expressionanalysisusingRT-qPCR.Expressionlevelswerenormalized againstconstitutivelyexpressed UBQ5 .Datarepresentthemeanof threebiologicalreplicateswithstandarddeviation.Theexperimentwas repeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g005 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org5January2012|Volume7|Issue1|e31130

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Xcc -induced PR geneexpressionissuppressedintheSA signalingmutantsInArabidopsis, PR geneexpressionisnotonlytightlycorrelated withresistancetohostpathogens[58],butalsorelatedtonon-host resistance[5,46].Thefindingthat Xcc inducesSA-dependent defenseresponsepromptedustotesthow Xcc -induced PR gene expressionisregulatedintheSAsignalingmutantssusceptibleto Xcc .Geneexpressionanalysisrevealedthat Xcc -induced PR gene expressionwassignificantlysuppressedinthethreesusceptible singlemutants( eds1 eds5 ,and pad4 )andthetwosusceptibledouble mutants( eds5npr1 and sid2npr1 )(Figure9).Although Xcc -induced PR geneexpressionwasimpairedin npr1 and sid2 (Figures9Ato 9C),both npr1 and sid2 werenotmoresusceptibleto Xcc thanwild type. Xcc -induced PR geneexpressionin nho1 wasalsodecreased, buttoamuchlesserextentcomparedwiththatintheSAsignaling mutants(Figures9Gto9I).DiscussionResistanceofanentireplantspeciestoallisolatesofa microbialspeciesisreferredtoasnon-hostresistance[9].Itis thoughttocompriseavarietyofdistinctmechanismsinvolvedin layersofdiverseprocesses[11]. Morecomplicatedly,non-host resistancevariesamongdiffe rentpathosystems[63].Inthe presentstudy,weelectedtoemploy Arabidopsisthaliana asa modelforunderstandingnon-hostresistancetotheeconomicallyimportantbacterialpathogen Xcc ,whichcausescanker diseasetoseveralcitrusspecies.Onthechallengingroadto disease,presenceofpreformedbarriersisthefirstlineofplant defense,whichincludecellwall,antimicrobialenzymes,and secondarymetabolites[9,64, 65].Whenanon-hostpathogen managestoovercomeconstitutiv edefensivelayers,itbecomes subjecttotherecognitionattheplasmamembraneoftheplant cells.Elicitorsreleasedbyhos tornon-hostp athogenscan activatePAMPrecognitionwitht heinvolvementofleucine-rich repeat(LRR)-receptorkinasesandaMAPkinasecascade, whicheventuallyleadstobasal resistance[57,66].Inducible defenseresponsesinnon-hostpl antsalsoincludesynthesisand accumulationofROS,papillaryc allose,andphytoalexins,with orwithoutformationofthehypersensitiveresponse(HR)[67]. Thelastoptionoftheobstaclestothenon-hostpathogenisthe resistancemediatedbyindepe ndentlyandsimultaneously activatingpairsofpathogen avr andplant R genei.e.gene-forgeneresistance[13].Therefore ,similardefensemechanisms existbetweenhostandnon-hostinteractions[25,26].Here,we showedthat Xcc couldalsoactivatemultilayereddefense responsesinArabidopsis,whichincludeROSinduction (Figures2and3A),callosedeposition(Figure7),andPAMPandSA-induceddefensegeneex pression(Figures3and5).The involvementofROSinnon-hostresponsehasbeenfoundin variousplant-pathogensystems[52–55].However,ROS productiondoesnotalwaysleadtoHR.Severallinesof evidencefromvariousplantspeciessuggestthatthesourcesof ROSaredifferentduringnon-hostresponseandduringtheHR, butthesesourcesmayinteractwitheachother[68–70]. InteractionbetweenArabidopsisand Xcc inducesROSproduction(Figure2),butnotvisibleHR.Similarresponsewasfound intheinteractionbetweenArabidopsisandthesoybean pathogen Pseudomonassyringae pv. glycinea orthebeanpathogen P.syringae pv. phaseolicola [5].Nevertheless,severalspeciesof Figure6.GrowthofXccinseveralArabidopsisSA,JA,andETsinglingmutants. Leavesoffour-week-oldplantswereinoculatedwith Xcc (OD600=0.002).The inplanta bacterialtitersweredeterminedimmediately(day0)oronday5post-inoculation(cfu,colony-formingunits).Data representthemeanofeightindependentsampleswithstandarddeviation. Xcc grewsignificantlymorein eds5 pad4 nho1 eds5npr1 ,and sid2npr1 thaninthewild-typeCol-0plants(* P 0.01,0.001,0.001,0.001,and0.05,respectively).Similarly, Xcc grewsignificantlymorein eds1 thaninthewildtypeL er plants(* P 0.01).Theexperimentwasrepeatedthreetimeswithsimilarresults. doi:10.1371/journal.pone.0031130.g006 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org6January2012|Volume7|Issue1|e31130

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pathogensfromgenus Xanthomonas causetypeII(withHR)nonhostresistanceindifferenthetergologousinteractions[10].The depositionofalinear-1,3-gluca npolymer,callose,inresponse topathogenattacking/woundi ngstressesisabasicdefense mechanismthatenablestheplant toarrestpathogenproliferationbyreinforcingthecellwall[71–74].Duringthenon-host interactionbetwe enArabidopsisand Xcc ,wefoundthatcallose isstronglyinduced(Figure7).H owever,asageneralresponseto bio/abioticstresses,callose itselfisjustonecomponentof multilayerednon-hostdefensemechanismandneedscoordinationwithothers[12,75].Nodifferenceincallsoedepositionwas observedbetween Xcc susceptibleSAsignalingmutantsand wild-typeplants.Itisthusuncl earwhethercallosedeposition contributestothenon-hostresistanceto Xcc inArabidopsis. Previousstudieshaveestablish edthatPMAP-triggereddefense responseplaysanimportantrole innon-hostresistance[31,67]. Threeflg22-induciblegenes, FRK1 NHO1 ,and WRKY29 ,were agroupofearlyresponsegenesinducedby Xcc (Figures3Bto 3D),suggestinganimportantroleofflagellin-inducedinnate immunityinthispathosystem.Anothercomponentofthe multilayereddefensebarriersofthenon-hostArabidopsisplant to Xcc istheSA-mediateddefenseresponsewiththeactivationof PR genes(Figures4and5).InfectionofArabidopsisplantswith P.syringae pv. phaseolicola NPS3121inducesSAaccumulationand non-hostresistance[76].Incontrast,removalofSAbya NahG transgeneconferssusceptibilitytothesamenon-hostpathogen Figure7.Xcc-inducedcallosedepositionisnotchangedintheSAsignalingmutants. Four-week-oldArabidopsisplantswereinoculated with Xcc (OD600=0.2)ormock-treatedwith10mMMgCl2.At9and15hpi,inoculatedleaveswereexcisedandstainedwithanilineblue. FluorescencewasobservedusinganOlympusBH-2epifluorescentmicroscope.Nosignificantdifferencesweredetectedamongwildtype(Col-0and L er )andthemutantplants.Representativeimagesshownherecamefrom24leavesfrom12independentplants.Barsrepresent100mm. doi:10.1371/journal.pone.0031130.g007 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org7January2012|Volume7|Issue1|e31130

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[46].Inaddition,inductionof PR genesuponinfectionof differentnon-hostpathogenshasbeenfoundinmultipleplant species[5,12,41,46,77].Therefore,itseemsclearthatSA signalingisinvolvedinnon-hostresistance.However,thereis anobservationsuggestingthatcatechol(degradationproductof SA),insteadofSA,isresponsibleforthelossofnon-host resistanceinArabidopsis NahG plantsduetotheresistant phenotypeofseveralmutantswithdefectsinSAsignaling[76]. Figure8.ExpressionofearlyresponsegenesintheXccsusceptiblemutants. ( A to D )Expressionof GST1 FRK1 NHO1 ,and WRKY29 in npr1 eds5 sid2 eds5npr1 ,and sid2npr1 .( E to H )Expressionof GST1 FRK1 NHO1 ,and WRKY29 in eds1 .( I to L )Expressionof GST1 FRK1 NHO1 ,and WRKY29 in nho1 and pad4 .Four-week-oldplantswereinoculatedwith Xcc (OD600=0.02).Leaftissueswerecollectedatdifferenttimepoints(0,4,8,and 12hpi)fortotalRNAisolationandgeneexpressionanalysisusingRT-qPCR.Expressionlevelswerenormalizedagainstconstitutivelyexpressed UBQ5 Datarepresentthemeanofthreebiologicalreplicateswithstandarddeviation.Mutant eds1 isinL er geneticbackground,whereasothers( nho1 eds5 pad4 sid2 npr1 eds5npr1 ,and sid2npr1 )areinCol-0geneticbackground.Theexperimentwasrepeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g008 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org8January2012|Volume7|Issue1|e31130

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Thisdivergencemaybeexplain edbyvariationsofpathogen growthexperimentsanddifferentcriteriafornon-hostresistance/susceptibilityamonglaboratories. TodetermineiftheknownSA-dependentand/orJA/ETdependentsignalingpathwayareinvolvedinnon-hostresistance ofArabidopsisagainst Xcc ,agroupofArabidopsismutantsthatare impairedinSAorJA/ETsignalingwereemployedforbacterial growthexamination.InadditiontoEDS1andPAD4,two componentsoftheEDS1-PAD4-SAG101signalingcomplex, whichhavebeenrevealedtofunctioninSA-mediatednon-host resistanceinmultiplepathosystems[22,23],non-hostresistance against Xcc wasabolishedintheabsenceofafunctional EDS5 in Arabidopsis(Figure6).Unlikepreviousreports,wedidnotobserve Xcc growthinmutant jar1 [48], ein2 [78], npr1 [48], pad3 [45], ndr1 [41],and sid2 [48,51],whichprovidedanotherlineofevidence thatthemechanismsofnon-hostresistancevaryamongpathosystems[25].Interestingly,mutationof npr1 or sid2 alonedoesnot confersusceptibilityto Xcc ;however,combiningbothmutations togethercompromisesnon-hostresistanceto Xcc inthedouble mutant sid2npr1 (Figure6).Thisresultreveledanundefined interactionbetween NPR1 and SID2 inthenon-hostresistance against Xcc .Similarly, NPR1 mayalsointeractwith EDS5 inthe ArabidopsisXcc pathosystem,sincethedoublemutant eds5npr1 is moresusceptibleto Xcc than eds5 (Figure6).Thus, NPR1 ,amaster regulatorofmultipleimmuneresponses,mayalsoplayan importantroleinnon-hostresistance via eitherdirect[48]or indirectways(asshownhere). Inthe Xcc susceptibleSAsignalingmutants,inductionofboth earlyresponsegenes(ROS-andflg22-inducible)and PR genesis inhibitedinresponseto Xcc infection(Figures8and9).However, decreasedexpressionofthesedefensereadoutsincertainmutants suchas npr1 and sid2 doesnotconstitutesusceptibilityto Xcc (Figure6).Furthermore,nodifferenceintheexpressionofthe defensegeneswasfoundbetween sid2 or npr1 andthesusceptible doublemutant sid2npr1 (Figures8and9).Similarly,although eds5npr1 ismoresusceptibleto Xcc than eds5 and npr1 ,inductionof Figure9.ExpressionofPRgenesintheXccsusceptiblemutants. ( A to C )Expressionof PR1 PR2 ,and PR5 in npr1 eds5 sid2 eds5npr1 ,and sid2npr1 .( D to F )Expressionof PR1 PR2 ,and PR5 in eds1 .( G to I )Expressionof PR1 PR2 ,and PR5 in nho1 and pad4 .Theexperimentwasperformedas inFigure7.Expressionwasnormalizedagainstconstitutivelyexpressed UBQ5 .Datarepresentthemeanofthreebiologicalreplicateswithstandard deviation.Mutant eds1 isinL er geneticbackground,whereasothers( nho1 eds5 pad4 sid2 npr1 eds5npr1 ,and sid2npr1 )areinCol-0genetic background. Xcc -inducedexpressionof PR1 PR2 ,and PR5 wasdramaticallyinhibitedinallthetestedmutantsexcept nho1 .Theexperimentwas repeatedtwicewithsimilarresults. doi:10.1371/journal.pone.0031130.g009 ANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org9January2012|Volume7|Issue1|e31130

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thedefensegenesiscomparableinallthreemutants(Figures8and 9).Clearly,non-hostresistanceagainst Xcc isdeterminedbythe interactionofmultipledefensemechanisms.Althoughsome mutationscouldsuppresscertain Xcc -induceddefensereadouts, whethertheycouldpromote Xcc growthdependsontheirposition inthecomplexdefensenetwork[5].Weidentified EDS1 EDS5 PAD4 ,and NHO1 ascrucialcomponentsintheinteractionof Arabidopsisand Xcc .Mutationsinanyofthesegenesleadto Xcc growthinArabidopsis.Incontrast,othergenes,like NPR1 and SID2 ,maygeneticallyinteractwitheachotherinnon-host resistanceagainst Xcc Inthisstudy,wecharacterizedanovelnon-hostpathosystem involvingArabidopsisandthecitruscanker-causingbacterial pathogen Xcc .Usinggeneticandmolecularanalysis,weobtained anoverviewofthemultilayereddefenseresponsesassociatedwith thenon-hostresistanceagainst Xcc inArabidopsis.Thepathosystemdescribedherenotonlyofferedanexcellenttoolforimproving ourunderstandingofnon-hostdefenseresponsebutalsoshedlight ondevelopingdisease-resistantcitrusvarietiesbytransferring defenseknowledgefrommodelplants.Thefeasibilityofthis strategyhasbeenprovedbyarecentstudyshowingthat overexpressionoftheArabidopsis NPR1 geneincitrusincreases resistancetocitruscanker[3].Usingnon-hostresistanceforcrop improvementhasattractedmuchattentionbecausethisformof immunityisdurableandcanprovideprotectionagainstallisolates ofapathogenspecies[63].Anexcellentexampleisthatanon-host wheatstriperustresistancegene Yr9 fromryeplayedavery importantroleincontrollingwheatstriperustworldwideforalong time[79,80].GenesrevealedtoplayimportantroleinthenonhostinteractionbetweenArabidopsisand Xcc holdgreatpotential forbreedingcanker-resistantcitrusvarietiesthroughmoderngene transfertechnology.MaterialsandMethods Plantmaterials,growth,andpathogeninfectionThewild-typeplantsusedwere Arabidopsisthaliana (L.)Heynh. Columbia(Col-0),Landsberg erecta (L er ),Wassilewskija(Ws),and RLDecotypes,andthemutantallelesusedwere npr1-1 [81], eds1-2 [82], eds5-1 [83], sid2-1 [84], pad3-1 [85], pad4-1 [86], ndr1-1 [87], ein2-2 [88],and jar1-1 [89].Twodoublemutants, eds5npr1 and sid2npr1 ,weregeneratedbycrossing npr1-1 with eds5-1 and sid2-1 respectively.The nho1 mutantseedswereobtainedfromthe ArabidopsisBiologicalResourceCenter(ABRC)(SALK_067205) [46,47],andhomozygousT-DNAinsertionplantswereidentified byPCR.AllmutantsareinCol-0geneticbackgroundexcept eds12 ,whichisinL er background.PlantsweregrowninMetromix MVPsoil(Bellevue,WA)undera16hrlight/8hrdark photoperiodat 22 u C.Four-week-oldplantswereinoculated with Xcc strain306bysyringeinfiltration[90],diporspray inoculation[91].Fordipandsprayinoculation,plantswerekept athighhumiditybyaplasticdomefortwodays.Afterinoculation, eightleaveswerecollectedfromdifferentplantsateachtimepoint foreachgenotypetodetermine inplanta growthof Xcc .BacterialcultureThecitruscankercausativebacterium Xcc strain306was obtainedfromDr.JamesGraham(CitrusResearchandEducation Center,UniversityofFlorida)[92].Thebacteriawerestreaked fromaglycerolstockontoNutrientBroth(NB)-agarplate containing20mg/mlrifampin.Afterculturedat30 u Cfortwo days,asinglecolonywaspickedupandculturedovernightin 3mLliquidNB/rifampinat30 u Cwitharotationalspeedof 220rpm.Forsyringeinfiltrationinoculation,the3mLovernight culturewasdirectlyused.Fordipandsprayinoculation,the3mL overnightwasaddedto500mLliquidmediumandfurther culturedovernight.Bacterialcellswerespundownandthepellet wasresuspendedin10mMMgCl2todesiredOD600valuesfor differentexperiments:0.002forsyringeinfiltrationinoculation, 0.02forgeneexpression,SAquantification,andROSstaining, and0.2fordipandsprayinoculationandcallosestaining.ROSandcallosestainingFour-week-oldplantsweresyringe-infiltratedwithasuspension of Xcc bacteriaormockcontrol(10mMMgCl2).Twenty-four leavesfrom12plantswereusedforbothstainingpurposes.DAB (3,3 9 -diaminobenzidinetetrahydrochloride)stainingforROS (mainlyhydrogenperoxide)wasreportedelsewhere[93].Leaf sampleswereexcisedat4hpiforDABstaininganddestainedleaf sampleswereexaminedforreddish-browncolorationundera LeicaMEIJIscope(Wetzlar,Germany).Forcallosestaining,leaf sampleswerecollectedat9and15hpi,fixedin3:1ethanol-toglacialaceticacidunderbriefvacuumandthenonashakerwith severalchangesoffixativeuntilleavesappearedslightlytranslucent.Thentheleafsampleswererehydratedsequentiallyin70% and50%ethanolsolutioneachforovertwohours.Afterwashing twicewithwater,theleafsampleswereleftinwaterovernightona shaker.Theleafsampleswerethenincubatedin150mM K2HPO4(pH9.5)solutioncontaining0.01%anilinebluefor overfourhours[94].Theleafsamplesweremountedonslides with50%glycerolanddetectedwithanOlympusBH-2 epifluorescentmicroscope(Shinjuku,Tokyo,Japan)underUV illuminationwithbroadbandDAPfilterset(excitationfilter 390nm,dichroicmirror420nm,emissionfilter460nm).RNAextractionandreal-timequantitativePCRanalysisWeusedsyringeinfiltrationasbacterialinoculationmethodfor geneexpressionanalyses.RNAextractionfollowedtheprotocol describedpreviously[95].Briefly,100mgleaftissuesinfectedwith Xcc weregroundtofinepowdersinliquidnitrogenwithaSpex SamplePrep2000Geno/Grinder(OPSDiagnostics,Lebanon,NJ) andextractedwith80 u Cpre-warmedwater-saturatedphenoland RAPDbuffer(100mMLiCl,100mMTrispH8.0,100mM EDTA,and1%SDS).Theaqueousphasewasextractedwith chloroform,andtheresultingaqueousphasewasprecipitatedwith ethanolat 2 80 u Cforonehour.RNAwaspelletedby centrifugation,washedoncewith80%ethanol,driedonice,and suspendedin40mlDEPC-treatedwater.RNAqualitywas checkedwithformaldehyde-agarosegelelectrophoresis,and RNAconcentrationwasmeasuredwithaNanoDrop2000 spectrometer(ThermoScientific,Wilmington,DE).Forreverse transcription,totalRNAwastreatedwithDNaseI(Ambion, Austin,TX)at37 u Cfor30minutes.Afterinactivationofthe DNase,2mgRNAwasreversetranscribedbyM-MLVReverse Transcriptasefirst-strandsynthesissystem(Promga,Madison,WI). TheresultingcDNAproductswerediluted20foldswithwater, and2.5mlofthedilutedcDNAproductswereusedfor quantitativereal-timePCRanalysisinanMx3005PqPCRsystem (AgilentTechnologies,SantaClara,CA).AllqPCRreactionswere performedinduplicateusingtheSYBRGreenprotocol(Applied Biosystems,FosterCity,CA)witha12.5mlreactionvolumeanda 0.4mMprimerconcentration.Theamplificationconditionwas 95 u Cfor10minfollowedby40cyclesof94 u Cfor30sec,55 u C for1min,and72 u Cfor1min.PCRspecificitywascheckedby dissociationanalysisaftertherunwascompleted.RelativemRNA abundancetothereferencegene UBQ5 wascalculatedaccording tothedeltaCtmethod.Primersforamplificationof UBQ5 PR1 PR2 ,and PR5 werereportedelsewhere[96].PrimersequencesofANovelArabidopsisXcc Non-HostPathosystem PLoSONE|www.plosone.org10January2012|Volume7|Issue1|e31130

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theothergenesarelistedbelow: GST1 ( qGST1F :5 9 -GTTCCAGCCTTTGAAGATGG-3 9 ; qGST1R :5 9 -TCCTTGCCAGTTGAGAGAAG-3 9 ), FRK1 ( qFRK1F1 :5 9 -TGAGTCAGGTCGTTATGGAG-3 9 ; qFRK1R1 :5 9 -ATTCACTACCTTGCTCGAGG-3 9 ), NHO1 ( qNHO1F :5 9 -CCACAGCTAACAACCTTCTG-3 9 ; qNHO1R :5 9 -AGAGAATCTGTTGTCGGACG-3 9 ),and WRKY29 ( qWRKY29F :5 9 -AGAGAATCTGTTGTCGGACG-3 9 ; qWRKY29R :5 9 -ACACCCTTTTGAGCTACTGC-3 9 ).SalicylicacidquantificationLeaftissuessyringe-infiltratedwith Xcc ormockcontrol(10mM MgCl2)werecollectedattheindicatedtimepoints.Measurement ofbothfreeandtotalSAwasperformedbyHPLCmethodas reported[97].StatisticalanalysisDataanalysistool t -TESTinExcelofMicrosoftOffice2007for Macintoshwasusedforallstatisticalanalyses.AcknowledgmentsWethankDr.JamesGrahamforprovingthecitruscankerbacterial pathogen Xanthomonascitri subsp. citri strain306,Dr.DavidOppenheimer forsharingArabidopsisRLDecotypeseeds,Dr.SixueChenforaccessto HPLCequipment.WealsothankYezhangDingandXudongZhangfor technicalassistance.AuthorContributionsConceivedanddesignedtheexperiments:CAZM.Performedthe experiments:CA.Analyzedthedata:CAZM.Wrotethepaper:CAZM.References1.GottwaldTR,GrahamJH,CiveroloEL,BarrettHC,HearnCJ(1993) Differentialhostrangereactionofcitrusandcitrusrelativestocitruscankerand citrusbacterialspotdeterminedbyleafmesophyllsusceptibility.PlantDis77: 1004–1009. 2.GrahamJH,LeiteRP(2004)Lackofcontrolofcitruscankerbyinduced systemicresistancecompounds.PlantDis88:745–750. 3.ZhangX,FrancisMI,DawsonWO,GrahamJH,Orbovic V,etal.(2010)OverexpressionoftheArabidopsis NPR1 geneincitrusincreasesresistancetocitrus canker.EurJPlantPathol128:91–100. 4.ViloriaZ,DrouilardDL,GrahmJH,GrosserJW(2004)Screeningtriploid hybridsof‘Lakeland’Limequatforresistancetocitruscanker.PlantDis88: 1056–1060. 5.MishinaTE,ZeierJ(2007)Bacterialnon-hostresistance:interactionof Arabidopsis withnon-adapted Pseudomonassyringae strains.PhysiolPlant131: 448–461. 6.LoehrerM,LangenbachC,GoellnerK,ConrathU,SchaffrathU(2008) Characterizationofnonhostresistanceof Arabidopsis totheAsiansoybeanrust. 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