Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors

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Title:
Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors
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English
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Bocsanczy, Ana M.
Achenbach, Ute CM
Mangravita-Novo, Arianna
Chow, Marjorie
Norman, David J.
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BioMed Central (BMC Genomics)
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Abstract:
Background: Ralstonia solanacearum, the causal agent of bacterial wilt, is a genetically diverse bacterial plant pathogen present in tropical and subtropical regions of the world that infects more than 200 plant species, including economically important solanaceous crops. Most strains of R. solanacearum are only pathogenic at temperatures between 25 to 30°C with strains that can cause disease below 20°C considered a threat to agriculture in temperate areas. Identifying key molecular factors that distinguish strains virulent at cold temperatures from ones that are not is needed to develop effective management tools for this pathogen. We compared protein profiles of two strains virulent at low temperature and two strains not virulent at low temperature when incubated in the rhizosphere of tomato seedlings at 30 and 18°C using quantitative 2D DIGE gel methods. Spot intensities were quantified and compared, and differentially expressed proteins were sequenced and identified by mass spectrometry (MS/MS). Results: Four hundred and eighteen (418) differentially expressed protein spots sequenced produced 101 unique proteins. The identified proteins were classified in the Gene Ontology biological processes categories of metabolism, cell processes, stress response, transport, secretion, motility, and virulence. Identified virulence factors included catalase (KatE), exoglucanase A (ChbA), drug efflux pump, and twitching motility porin (PilQ). Other proteins identified included two components of a putative type VI secretion system. We confirmed differential expression of 13 candidate genes using real time PCR techniques. Global regulators HrpB and HrpG also had temperature dependent expression when quantified by real time PCR. Conclusions: The putative involvement of the identified proteins in virulence at low temperature is discussed. The discovery of a functional type VI secretion system provides a new potential virulence mechanism to explore. The global regulators HrpG and HrpB, and the protein expression profiles identified suggest that virulence at low temperatures can be partially explained by differences in regulation of virulence factors present in all the strains. Keywords: Bacterial wilt, Temperature, Virulent strains at low temperature, Type VI secretion system, Stress response
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Bocsanczy et al. BMC Genomics 2014, 15:280 http://www.biomedcentral.com/1471-2164/15/280; Pages 1-14
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doi:10.1186/1471-2164-15-280 Cite this article as: Bocsanczy et al.: Proteomic comparison of Ralstonia solanacearum strains reveals temperature dependent virulence factors. BMC Genomics 2014 15:280.

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© 2014 Bocsanczy et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
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RESEARCHARTICLEOpenAccessProteomiccomparisonof Ralstoniasolanacearum strainsrevealstemperaturedependentvirulence factorsAnaMBocsanczy1,UteCMAchenbach2,AriannaMangravita-Novo3,MarjorieChow4andDavidJNorman1*AbstractBackground: Ralstoniasolanacearum ,thecausalagentofbacterialwilt,isageneticallydiversebacterialplant pathogenpresentintropicalandsubtropicalregionsoftheworldthatinfectsmorethan200plantspecies, includingeconomicallyimportantsolanaceouscrops.Moststrainsof R.solanacearum areonlypathogenicat temperaturesbetween25to30Cwithstrainsthatcancausediseasebelow20Cconsideredathreattoagriculture intemperateareas.Identifyingkeymolecularfactorsthatdistinguishstrainsvirulentatcoldtemperaturesfromones thatarenotisneededtodevelopeffectivemanagementtoolsforthispathogen.Wecomparedproteinprofilesof twostrainsvirulentatlowtemperatureandtwostrainsnotvirulentatlowtemperaturewhenincubatedinthe rhizosphereoftomatoseedlingsat30and18Cusingquantitative2DDIGEgelmethods.Spotintensitieswere quantifiedandcompared,anddifferentiallyexpressedproteinsweresequencedandidentifiedbymass spectrometry(MS/MS). Results: Fourhundredandeighteen(418)differentiallyexpressedproteinspotssequencedproduced101unique proteins.TheidentifiedproteinswereclassifiedintheGeneOntologybiologicalprocessescategoriesof metabolism,cellprocesses,stressresponse,transport,secretion,motility,andvirulence.Identifiedvirulencefactors includedcatalase(KatE),exoglucanaseA(ChbA),drugeffluxpump,andtwitchingmotilityporin(PilQ).Other proteinsidentifiedincludedtwocomponentsofaputativetypeVIsecretionsystem.Weconfirmeddifferential expressionof13candidategenesusingrealtimePCRtechniques.GlobalregulatorsHrpBandHrpGalsohad temperaturedependentexpressionwhenquantifiedbyrealtimePCR. Conclusions: Theputativeinvolvementoftheidentifiedproteinsinvirulenceatlowtemperatureisdiscussed.The discoveryofafunctionaltypeVIsecretionsystemprovidesanewpotentialvirulencemechanismtoexplore.The globalregulatorsHrpGandHrpB,andtheproteinexpressionprofilesidentifiedsuggestthatvirulenceatlow temperaturescanbepartiallyexplainedbydifferencesinregulationofvirulencefactorspresentinallthestrains. Keywords: Bacterialwilt,Temperature,Virulentstrainsatlowtemperature,TypeVIsecretionsystem,StressresponseBackgroundSuddenchangesintemperaturecaninduceadaptive shockresponsesinbacteria,enablingthemtocolonize widelydiverseenvironments.Theseadaptiveresponses involvemajorchangesinthephysiologicalstateofthe bacterialcells[1,2]allowingthemtosurviveandfunctioninthenewenvironment.Smallerorslowerchanges intemperaturemaynotinduceshockresponsesbutcan modulateexpressionofparticularphysiologicalsystems suchastransportofnutrients,stressresponsesandvirulenceefficacyinpathogens.Thelattertypeofchangehas beenstudiedinanimalpathogensthatswitchexpressionof colonizationandinvasionfunctionswhenchanginghosts [3].Worksaddressingtemperature-dependentvirulence factorsinanimalpathogensandtheirregulationhavebeen publishedonlyrecently[4,5]. Virulencegeneexpressioninmostbacteriaismodulatedbydiverseparameters,includingpH,ionconcentration,growthphase,popu lationdensityandcontact withthehost.Temperaturemayactivateordeactivate *Correspondence: djn@ufl.edu1DepartmentofPlantPathology,UniversityofFlorida,IFAS,Mid-FloridaResearch andEducationCenter,2725BinionRd.,Apopka,FL32703,USA Fulllistofauthorinformationisavailableattheendofthearticle 2014Bocsanczyetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse, distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycredited.TheCreativeCommonsPublic DomainDedicationwaiver(http://creativecommons.org/publicdomain/zero/1.0/)appliestothedatamadeavailableinthis article,unlessotherwisestated.Bocsanczy etal.BMCGenomics 2014, 15 :280 http://www.biomedcentral.com/1471-2164/15/280

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virulencegenes[6],althoughthemechanismsinvolved arelittleknown. Unlikemanyanimalpathogens,plantpathogenicbacteria typicallyonlyundergogradualtemperaturechangesrelated toseasonalchangeinenvironmentandarenotsubjected tosuddenchangesintemperatureswhenswitchinghosts. Whilemostplantpathogensofagivenspeciesareactive withinalimitedrangeoftemperature,somestrainsare virulentacrossawiderrangeduetoadaptiveandperhaps evolutionarypressures.Theriskofintroducingsuchstrains tonewcropzonescouldbeeconomicallydevastating. Thesepathogenicitydifferencesmayinvolvechangesin proteinregulationormaybeduetothepresenceof uniquegenesinvolvedinenablingvirulenceatlowtemperatures.Thisisthecaseofthebacterialwiltpathogen Ralstoniasolanacearum Bacterialwiltisasoil-bornebacterialplantdiseasewhich affectsmorethan200plantspeciesincludingsolanaceous cropssuchastomatoandpotato[7]. R.solanacearum itscausalagent,isaspeciescomplexwithstrainsclassifiedintofiveracesbasedontheirhostrange[8],and into6biovarsbasedonbiochemicalprofiles[9].Strains arefurtherclassifiedintophylotypesandsequevars basedonphylogeneticrelationshipsoftheinternaltranscribedspacerregionofthe16srRNAgenesequence andendoglucanase(egl)generespectively[10]. Asubgroupof R.solanacearum ,R3B2PhylotypeIIB, includescoldtolerantstrainswhichareabletoinfectpotato atlowtemperatures[11,12].Thissubgrouporiginatedin thehighlandsoftheAndesmo untainsandisadaptedto coolertemperaturesthantypicalstrainsof R.solanacearum [13].R3B2strainsaredesignatedas “ selectagent ” under theAgricultureBioterrorismProtectionActof2002[14] duetotheirthreattofoodsecurityintheU.S.Earlyreports [13]showedthatstrainsbelon gingtotheR3B2groupcould infectpotatoesattemperaturesaslowas16C.Recentresearchhassuggestedthatotherracesof R.solanacearum mayalsohavetheabilitytosurvivefreezingtemperaturesandthushavethepotentialtoestablishintemperateclimates[15,16]. R3B2arenotuniqueintheirabilitytoinfecthost plantsatlowtemperatures.Werecentlyreportedthat R1B1-sequevar4strainsare alsocapableofwiltingtomatoandpotatoplantsat18Cwhensoilinoculated [17].MoreoverpopulationsofR1B1strainsareindigenousintropicalandsubtropicalclimates,suchasin FloridaincontrasttoR3B2strains.Akeydifference, however,isthatwhileR1B1sequevar4cansurviveand multiplyaswellasR3B2atcooltemperatures,theyare notasvirulentonpotato18C,althougharesimilarly virulentontomato.Identif yingthefactorsinvolvedin R.solanacearum virulenceatlowtemperaturesmay providetoolsforeffectivecontrolofthepathogenat lowtemperatures. Transcriptomeandproteomeanalysishavebeenused toidentifymRNAandproteins,respectively,presentin specifictissues,underdiverseconditions.Bothmethods giveasnapshotofthestateofthecellatagiventime. Becauseproteinsarethekeyfunctionalmolecules,a characterizationoftheproteomeisconsideredtorepresentthebiologicalstateofacellmoreaccuratelythan themRNA.Currentproteomictechniquesinvolvethe useof2DDIGEwhichcandifferentiatethousandsof proteinsbymolecularweightandisoelectricpoint[18]. Theuseoffluorescentdyesandindependentprotein controlsin2DDIGEhaveimprovedthethroughputof 2Dgelsbyallowingmultipleproteinstobecompared simultaneously.Thistechniquehassignificantlyimproved gel-basedproteomicsanalysis,althoughoverallitrequires ahighquantity(atleast0.6g)ofproteinpersample. Inthisstudyweused2DDIGEgelandMS/MStechniquestocomparetheproteinprofilesat30Cand18C oftwostrainsof R.solanacearum (UW551andP673)that arevirulentatlowte mperaturewithtwostrains(GMI1000 andP597)thatarenotvirule ntatlowtemperature[17]. Wehypothesizedthateitherth edifferenceinregulationof virulencefactorspresentinall thestrainsordifferencesdue tonovelonespresentinvirulentstrainsatcooltemperaturesmustexplainthedifferenceinvirulenceatlowtemperatures.Theproteinsampleswereobtainedduringthe colonizationphaseoftheinfectionfromculturesincubated intherhizosphereoftomatoseedlingsmaintainedat30C and18C.Atotalof106cell-associatedandsecretedproteinsdifferentiallyexpressedinoneormorestrainswere identified,annotatedanddiscussed.ResultsComparativeproteinprofilesof Ralstoniasolanacearum strainsat30Cand18CComparisonat30Cand18Cofcell-associatedproteins fromstrainP597incubatedinrichmediainabsenceof tomatoplantsidentified61proteinspotsdifferentially expressedinapreliminarye xperiment.Fifteen(15)differentiallyexpressedspotswithabundantvolumewere selectedforsequencing,and9proteinswereidentified (Additionalfile1).Thelistincludesseveralproteinsrelatedtometabolismandtransport,aporinpartofthe typeIVtwitchingmotilityapparatus(PilQ),andacatalase (KatE),whichhavebeenpreviouslyrelatedtovirulenceof Ralstoniasolanacearum Cell-associatedcomparativeproteinprofilesattwo temperaturesobtainedfrombacterialculturesofstrains GMI1000,P597,P673andUW551incubatedincontact withtomatoseedlingsrhizosphererevealed872differentiallyexpressedproteinspots,determinedbystatistically differentialintensitiesforeachstrainsattwotemperatures. Further,secretedcomparativeproteinprofilesextracted fromculturesofstrainsGMI1000andUW551incontactBocsanczy etal.BMCGenomics 2014, 15 :280 Page2of14 http://www.biomedcentral.com/1471-2164/15/280

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withtomatoseedlingsrhizosphereidentified172spots differentiallyexpressed.Over all,atotalof1044differentially expressedproteinspotsforalltherhizospherecolonization experimentswereidentified.Outofthe1044spots,418 wereselectedforproteinsequencing.Theselectioncriteria aredescribedindetailinMethods. Atotalof101uniqueproteinswe reidentified(Additional file2).Detailedinformationabouttheproteinsidentified, includingnumberofpeptides,pr oteincoverage,statistical confidence,sizeandisoelectri cpointispresentedbystrain inAdditionalfile3. Thedistributionoftheidentifiedproteinsbyregulationshowsthat(Figure1)76outofthe101proteins wereexpresseddifferentiallyinindividualstrains.For example,22proteinswere differentiallyproducedat 30Ccomparedwith18ConlyinUW551.Theselectionofthespotswasfocusedonproteinsthatare down-regulatedat18Cinstrainsnotvirulentatlow temperature(P597,GMI1000)and/orup-regulatedin strainsvirulentatlowtemperatures(P673,UW551). InP597andGMI1000 67%ofthedifferentiallyexpressed proteinsaremoreabundantat30C.Incontrast,85%of theproteinsaremoreabundantat18Cinthestrains P673andUW551.FunctionaldistributionIdentifiedproteinswereannotated,classified,andlisted bybroadbiologicalcategoriesusingtheamiGOannotation database[19,20](Additionalfile2).Theunknownproteins represent23%oftheidentifiedproteins,andthemost abundant(Figure2).Thisgroupincludesproteinswith onlypredicteddomainsthatindicategeneralenzymatic functions,orinferredproteinstructure,suchastransmembraneproteins;however,n ospecificbiologicalprocess functionswereassigned.Overall,41%oftheidentifiedproteinsbelongtothemetabolismandcellprocessescategories (Figure2).Theabundanceof metabolismrelatedproteins indicatestemperaturedependentresponsesinaddition tocommonresponsestolimitednutritionalcompounds andexposuretoanoxidizingenvironmentfromplant exudates[21].Inthiscategory,aminoacid,energyproduction,andmacromoleculessubcategoriesarethemost numerous.Thestressresponsescategoryincludesproteinswithdetoxificationfunctionssuchascatalases, peroxidases,drugormulticoppereffluxpumps,chaperonesinvolvedinproteinproductionandfolding,and putativephasinsinvolvedinutilizationofpolymerreservegranules.Transportcategoryincludesmovement ofinorganicmolecules,suchasphosphate,transportof UW55130/18 GMI100030/18 P67330/18 P59730/187 2 1 71 1 3 5 2 3 225 421 1 2 2 1 2 22 4 5 5 42 15 7 1 6 5 3 7 3 Comparisons Upregulated Downregulated Opposite Total1 1 P597 & P673ALL 1 1 GMI & P673 & UW551 2 1 1 GMI & P673 & P597 GMI & 3 GMI & 18 UW551 0 P673P673 27 P597P597 GMI1000 2 GMI & P597 & UW551 3 1 2 GMI UW551 UW551 & 2 2 P597 & Figure1 Venndiagramshowingthenumberandrelationshipofproteinsdifferentiallyexpressedforthetemperaturecomparisons. Circlesrepresentthesetofproteinsdifferentiallyexpressedforthecomparisoninthelabel.Thenumberofproteinsdifferentiallyexpressedis indicatedineachsetorsubset.Tablesunderneaththecomparisonsindicatetheregulationofthedifferentiallyexpressedproteins. Bocsanczy etal.BMCGenomics 2014, 15 :280 Page3of14 http://www.biomedcentral.com/1471-2164/15/280

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aminoacids,andsugarderivatives,probablylinkedto acquisitionofnutrientsinstressfulenvironments.The smallercategoriesinterms ofnumbersalthoughcontainingimportantproteinsforvirulence,weremotility, secretion,andvirulence. Theclassificationofproteinsbycomparativeabundance profileat30Cand18C(Table1)helpedtoidentify patternsofregulation.Ofinterestinthesearchforcandidatevirulencefactorsatlowtemperaturearegroups2,4 and8(Table1;Additionalfile2)whichcombineproteins moreabundantat30Cinstrainsnotvirulentatlow temperaturesand/ormoreabundantat18Cinstrains virulentatlowtemperatures. Weselectedacoreof22proteinsthatbelongtogroups 2,4,8,and/orhavebeenrelatedtovirulenceinprevious works(Table2).Theseproteinsarestrongcandidatesas factorsthatmayaffectvirulenceof R.solanacearum at lowtemperaturesdirectlyorindirectly.mRNArelativeexpressionof13candidatecoolvirulence factorsandglobalregulators hrpG and hrpB correlate withproteinexpressionprofilesInordertoconfirmtheproteomicsresults,therelative expressionof13genesat30and18Cwasquantifiedby real-timereversetranscriptionqRT_PCRanalysis.Therelativeexpressionofeachgenetoareferencegene(16sRNA) Figure2 Distributionofallsequenceddifferentiallyexpressedproteinsbybiologicalprocesscategories. Piediagramindicatesthe percentageofproteinsidentifiedinthelabeledcategory.CategoriesaredefinedperamiGOdatabaseintheGeneOntologywebpage (http://www.geneontology.org/). Table1TemperaturedependentproteinsgroupingcriteriaGroup number Groupingcriteria 1Proteinsthataremoreabundantat30Cinalltypeofstrains 2 Proteinsthataremoreabundantat30Cinatleastastrain that isnot virulentatlowtemperature 3 Proteinsthataremoreabundantat30Cinatleastone strainthat is virulentatlowtemperature 4 Proteinsthataremoreabundantat30Cinatleastastrain that isnot virulentatlowtemperatureandaremore abundantat18Cinatleastonestrainthat is virulentat lowtemperature 5 Proteinsthataremoreabundantat30Cinatleastastrain that is virulentatlowtemperatureandaremoreabundant at18Cinatleastonestrainthat isnot virulentatlow temperature 6Proteinsthataremoreabundantat18Cinalltypeofstrains 7 Proteinsthataremoreabundantat18Cinatleastone strainthat isnot virulentatlowtemperature 8 Proteinsthataremoreabundantat18Cinatleastone strainthat is virulentatlowtemperature 9NopatternCriteriausedtogroupproteinsbytheirtemperatureregulationbasedonthe virulenceofstrainsatlowtemperature.Bocsanczy etal.BMCGenomics 2014, 15 :280 Page4of14 http://www.biomedcentral.com/1471-2164/15/280

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ateachtemperaturewascalculated,andpresentedas theratioat30Cversus18C(Figure3).Expressionof 12candidategenestestedwasup-regulatedatthe highertemperatureforstrainP597.Theonlygenethat wasdown-regulatedat30Cwas phaP1 whiletheexpectedresultwasup-regulation.InP673,resultswere variable;however,ratiosw erelowcomparedwiththe ratiosinthenotvirulentstrainsGMI1000andP597, andmostofthegenestestedweredown-regulated.In UW551allgenestestedweredown-regulatedat30C. TheprofileinFigure3correlateswiththeexpected patternofexpressioninferredfromtheproteomics dataforthetargetgenestested:inP597andGMI1000 genesareup-regulatedat30CandinP673andUW551 genesmaintainsimilarexpressionatbothtemperaturesor areup-regulatedat18C,respectively. Relativeexpressionofthe hrpG and hrpB globalvirulence regulatorswasalsotestedwithqRT_PCR(Figure3).The relativeexpressionat30CinP597andGMI1000isupregulated,whileinP673andUW551itisdown-regulated, suggestingacorrelationbetweenexpressionofregulators HrpGandHrpBandvirulenceatlowtemperature.FunctionalannotationandcomparativetemperatureprofileInthissectionproteinsdifferentiallyexpressedandtheir profilesaredescribedbybiologicalprocesscategory.Metabolism/cellprocessesCellprocessesclassincludedpr oteinsrelatedtotranslation, transcription,andchaperoning(Additionalfile2).Mostof theproteinsinthiscategorybelongtogroups2and8 (Table1).Theirprofileofregulationsuggestsamoreactive Table2AselectedsubsetofcandidatevirulenceproteinswhoseexpressionistemperaturedependentBiologicalprocess category ProteinnamePutativefunction Proteinaccession GMI1000 Genetagin GMI1000 Profileof regulationgroup MetabolismMdh Malatedehydrogenase.Glyoxylatecycle17546717Rsc19984 LeuC Isopropylmalateisomeraselargesubunit.Amino acidbiosynthesisleucinepathway 17546709Rsc19904 StressresponseClpB ATP-dependentprotease.ClpBarechaperones toproteinstaggedfordestruction. 17546054Rsc13352 PpO PolyphenolOxidase.Contributestoresistance tophenoliccompounds 17545056Rsc03372 GroES 10kDAchaperonin.Foldingandassemblyofproteins 17545360Rsc06414 GroEL 60kDAsubunitchaperonin.Foldingand assemblyofproteins 17545361Rsc06424 PhaP1 Phasin_2domain.Usuallyassociateswith PHBgranulesinbacteria. 17546324Rsc16054 HtpG Putativeheat-shock90.Molecularchaperone.17545709Rsc09902 Dps DNAprotectionstarvationprotein.17547406Rsc26872 KatE/katB CatalaseIhydroperoxidaseHpiIoxidoreductase.17549800Rsp15811 KatG Heme-dependentperoxidase.17545494-495Rsc0775-07764 Transport/motility/ secretion SecB Putativetranslocase.Secdependentpathway17545075Rsc03564 PilQ Porin,partoftypeIVtwitchingmotilitysystem17547690Rsc29712 Rsp0744 AssociatedwithbacterialtypeVIsecretion apparatussecretion 17548965Rsp07442 Hcp TypeVIsecretionsystemtranslocator,Hcp1family17548966Rsp07454 UnknownRsc1727 HypotheticalproteinwithanEmrB/QacAfamily multidrugresistancetransmembraneprotein 17546446Rsc17272 Rsp0167 HomologtoRaxSTPAMPeffectorin Xanthomonas oryzae pv. oryzae 17548388Rsp01672 VirulenceEgl Endoglucanase.Degradationofpolysaccharides17548383Rsp01623 Rsp0275 Putativechitinase17548496Rsp02751 ChbA Putativeexoglucanase.CellobiohydrolaseAdomain17548804Rsp05832 HrcC SecretinoftheconservedfamilyoftypeIII apparatussystem 17549095Rsp08748 WecC(EpsD) Enzymepartoftheexopolysaccharide operon(EpsD). 17549237Rsp10168 Bocsanczy etal.BMCGenomics 2014, 15 :280 Page5of14 http://www.biomedcentral.com/1471-2164/15/280

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cellstateat30Cforthestrainsnotvirulentatlow temperature,andat18Cforthevirulentones. Inthemetabolismcategorya pproximatelyonethirdof theproteinswererelatedtoaminoacidbiosynthesis.The leucine-isoleucinebiosynthet icpathwayinparticular,was representedby4differentproteins(LeuC,IlvC,IlvD,ThrC) whoseprofileofexpressionbelongtogroups2,4,and8 (Additionalfile2).LeuCisthelargeunitoftheisopropylmalateisomerase,akeyenzymeintheL-leucinebiosynthesisfromvaline.ThepatternofexpressionofLeuCwas confirmedbyqRT_PCR(Table2,Figure3).Enzymesthat belongtocysteineandglutamatewerealsomoreabundant at30CinP597. Energyproductionisanotherimportantsubcategoryin metabolism.ProteinsAceE,GltA,NuoF,andMqo,related totheTCAcyclehaveavariablepatternofabundanceratio; howeverproteinsGapA,Mdh,andAceArelatedtoglycolysisbelongtogroups2,4and8(Additionalfile2). Metabolismofmacromoleculessubcategoryrepresented 9%ofthedifferentiallyexpressedproteins,theirabundance profilewasvariable,andnocorrelationwasobservedwith virulenceatlowtemperature.StressresponseStressresponseproteinsrepresented14%ofthedifferentiallyexpressedproteins(Figu re2).Proteinsinthiscategory relatetobiosynthesisofcarbo nandenergyreserves,protein foldinginstressfulenvironments,andproteinsinvolvedin detoxification.TwoproteinsPhaP2,PhaP1whichcontain aphasindomainweredifferentiallyexpressed.Phasindomainsareassociatedwithsynthesisordegradationofpoly -Hydroxybutyrate(PHB)granules,formedasanalternate -60-40-20020406080HrpG HrpB ChbA Hcp Rsp0744 PilQ KatG KatE AhpC1 Dps HtpG Ppo ClpB Phap1 LeuCRatio 30/1830C 18CUW551 P673 P597 GMI 1000Virulent at low temperature Not virulent at low temperature Figure3 Relativeexpressionof15genesforGMI1000,P673,P597,andUW551attwotemperatures. Positivebarsrepresenthigher expressionat30Candnegativebarsrepresenthigherexpressionat18C.Quantitativereversetranscription-polymerasechainreactionwas performedonequalamountsoftotalRNApersampleextractedfrombacterialsuspensionsgrowninco-culturewith invitro tomatoseedlings. 16sRNAwasusedasreferencefortherelativeexpressionofeachgene.Barsrepresentstandarderrorofthreebiologicalreplicates. Bocsanczy etal.BMCGenomics 2014, 15 :280 Page6of14 http://www.biomedcentral.com/1471-2164/15/280

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carbonreservefororganismsfacingstarvationinharshenvironmentalconditions[22,23].In Rhodospirilliumrubrum PhaPproteinisanactivator ofthePHBdegradationpathway[24].Inparticular,PhaP1wasdifferentiallyexpressedin allthestrainsanditsabundanceprofile(Table2)correlated withvirulenceatlowtemperature.TheRT-PCRconfirmed thepatternofexpressionofallstrainsexceptforP597 wherethegenewasdown-regulated(Figure3),although withasmallratio. GroEl/GroEs,formacomplexthatfunctionsaschaperone infoldingandrefoldingofproteins,usuallyinducedunder adverseenvironmentalconditions[25].Heat-shockprotein HtpGandproteaseClpBwereshowntofacilitate denovo foldingofproteinsduringhightemperatureconditions in E.coli [26].Althoughthetemperaturechangesin R.solanacearum arenotsudden,theseproteinsmayaid instabilizingproteinsduringlowtemperatureconditions (Table2). Differentiallyexpresseddetoxificationproteinswhoseexpressioncorrelatewithvirulenceatlowtemperature(groups 2,4,8)includedPpo,KatG,Dps(Table2,Additionalfile2), Rsc0754,andRsp1530(Additionalfile2).Ppoisapolyphenoloxidasewhichhasbeenshowntohavestrongtyrosinase activitysuggestingapossibleroleincounteractingphenolic compoundsproducedbythehostplants[27].Dpscontributestooxidativestresstoleranceandvirulenceontomato plants[28].KatGandRsc0754arepredictedperoxidases [21],whileRsp1530,haslaccaseactivity,similarinfunctiontothetyrosinasePpo[27]anditwasup-regulated byhydrogenperoxideincultureand inplanta [21]. CatalaseKatEwasmoreabundantat30CinGMI1000, P597andP673(Table2,Figure3).KatEwasalsoupregulatedat30CwhenstrainP597wasgrowninrichmedia (Additionalfile1).AhomologofKatEin Agrobacterium tumefaciens (KatA)hasbeenshowntocontributetoitssurvivalinadverseenvironmenta lconditionsasinthepresence ofhydrogenperoxide[29].In R.solanacearum ,KatEprobablycontributestosurvivalinthepresenceofphenolic compoundsproducedbytheplanthosts.Theresponse totheoxidativeenvironmentistemperaturedependent; however,itdoesnotcorrelatewithvirulenceofthestrains atlowtemperature. Conversely,AhpC1predictedperoxidasewasmore abundantat18CinGMI1000,P673andUW551 (Additionalfile2,Figure3).Theratiosofabundancein thesecomparisonsweresmallerthanforthecaseofKatE.TransportThepatternofexpressionof9p roteinsrelatedtotransport ofinorganiccompoundswereup-regulatedinallthestrains comparedat30C(group1).Thoseproteinsincluded putativealkalinephosphat asesandseveralABC-type transporters(Additionalfile2).Theirpatternofregulation suggestsmoreionactivityat30Cirrespectivethevirulence ofthestrainatlowtemperatures.Fourproteinsrelated tocarbohydratetransportandtworelatedtoaminoacid transportweredifferentiallyexpressed.Theirpatternof regulationvaried,andtherewasnoobviousco-relation withvirulenceatlowtemperatures.Motility/secretionInthesubcategorymotilityonlyPilQwasidentifiedas differentiallyexpressed(Table2).PilQisaporin,integralpartofthestructuralapparatusresponsibleforthe twitchingmovementof R.solanacearum [30].Twitching motilityhasbeenassociatedwithattachmentandvirulence of R.solanacearum ontheirhost[31],anditsexpressionis dependentonthequorumsensingregulatorypathway throughPehS/Pehrtwocomponentssystem[31].PilQwas up-regulatedat30CinP597(Additionalfile1,Table2)in richmediaandinco-culturewithtomatorhizosphere.In theRT_PCRexperimentPilQwasup-regulatedat30C inGMI1000andP597,andup-regulatedorunchanged at18CinUW551andP673(Figure3).Thisprofileofexpressioncorrelateswithvirulenceatlowtemperatures. Twitchingmotilitywasalsotemperaturedependent,independentofthepresenceofplantsinpreviousstudies[17]. Rsp0744andHcp(Rsp0745)wereidentifiedaspartofa recentlydescribedtypeVIsecretionsystem[32].Theirregulationprofilecorrelateswithv irulenceatlowtemperature (groups2,4)(Additionalfile2,Figure3).Rsp0744isa homologtoatypeVIsecretionapparatusproteinknown asVipB,orVCA0108in Vibriocholerae ,whichisthought tobepartofthecoresecretionapparatus[33].Rsp0745is ahomologtoHcpofseveralgram-negativebacteria[34]. ThisisaproteinsecretedbytheputativetypeVIsecretion system[32,33]andthoughttobeatranslocator[32,35]. TypeVIsecretionsystemshaveonlyrecentlybeen describedinanimalandplantpathogens[32,33,36,37]. Thissystemislinkedtovirulencein Vibriocholerae and Pseudomonasaeruginosa .SincethenthetypeVIsecretion systemhasalsobeenidentified insilico inmanygram negativebacterialgenomesincluding R.solanacearum [34].Similarlytoothersecretionsystemsitisalsowidespreadamonggram-negativebacteria[38].Interestingly, aclustercalled imp withhomologytothelaterdescribed typeVIsecretionwasshowntohavearoleintemperaturedependentproteinsecretionin R.leguminosarum [39],congruentwithourresults.Thepresenceandpatternofabundanceoftheseproteins,plustheinvolvementinvirulenceof typeVIsecretionsystemsinotherbacterialpathogens,make theidentifiedtypeVIsecretionastrongcandidatetovirulencefactoratlowtemperaturesin R.solanacearum .UnknownsAmongthetwentythreeunknownproteinsthatwere differentiallyexpressed,a cell-associatedhypothetical Rsp0167(Table2)wasconsideredofinterestbecauseisBocsanczy etal.BMCGenomics 2014, 15 :280 Page7of14 http://www.biomedcentral.com/1471-2164/15/280

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ahomologofraxST,acomponentofatypeIsecretion cassetteAvrXa21in Xanthomonasoryzae pv. oryzae [40].Inthislatterorganismthecassettecontainsfour genes: raxA raxB raxC and raxST ,whichencodea sulfotransferase-likeproteinandarenecessaryforrecognitionofXa21,areceptor-likekinaseinrice.Thisputative pathogen-associatedmolecularpattern(PAMP)effector couldbeinvolvedinvirulencein R.solanacearum .Rsp0167 wasdown-regulatedat18CinGMI1000. AnotherproteinofinterestisRsc1727(Table2).Thisisa hypotheticalproteinwithanE mrB/QacAfamilymultidrug resistancedomain.Thissubfamilyofdrugeffluxproteins arerelatedtodrug/antibioticresistancetransporters.Transportersmembersofthissubfamilyincludemultidrugresistancelocusin Escherichiacoli and Vibriocholerae [41,42]. Althoughtheexperimentaldesignfavoredidentification ofproteinsthatarepresentinallthestrains,twodifferentiallyexpressedproteinsabsentinGMI1000wereidentified:RRSL_00447andRSPO_m01224(Additionalfile2). Thefirsthasamethyltransferasedomainandispresent inUW551,P597andP673.Itwasup-regulatedat30Cin P597.Thesecondisannotateda sahypotheticalproteinand ispresentinP597,P673,andinsequenced R.solanacearum strainPo82[43].Itwasup-regulatedat18CinP673.VirulenceSecretedvirulencefactorsidentifiedincludedaputativecellobiohydrolaseencodedbythe chbA gene,an endoglucanase(Egl)andaputativechitinase(Rsp0275) (Table2).Theirfunctionisrelatedtoattackanddegradationofthehost ’ scellularwalls.Ithasbeensuggestedthat ChbAattacksthehemicellulosefractionofplantcellwalls [44];however, invitro enzymeactivityonregularexoglucanasesubstratessuchasmethyl-umbelliferyl-cellobioside (MUC)hasneverbeendemonstrated[44].Liu etal. [45] demonstratedthataGMI1000straindefectiveinChbA productionislessvirulentwhensoilinoculated.Inthesame way,anEglmutantimpairedinendoglucanaseactivityis alsolessvirulent[45].TheirdatasuggestthatChbAandEgl contributeequallytovirulenceinanadditivefashion[45].It wasexpectedthatChbAandEglwouldbeco-regulated. TheproteomicsandRT_Pcrresultsdonotsupportthis assumption.Inourresults,ChbAwasmoreabundantat 30CinGMI1000,andEglwasmoreabundantat30Cin UW551(Additionalfile2);however,ChbAwasmoreabundantat18CinUW551intheRT_PCRresults(Figure3). Thecell-associatedproteinsHrcCandWesCwere moreabundantat18CinUW551(group8inTable2), supportingaroleinvirulenceatlowtemperature. HrcCisanintegralpartofthetypeIIIsecretionapparatuswhichisadeterminantvirulencefactorin R.solanacearum [46],andWesC(EpsD)ispartofthe exopolysaccharideoperon,involvedinaggressiveness duringhostcolonization[47].DiscussionInpreviousworkwedeterminedthatthereisabarrierto virulenceatlowtemperatureatthestageofcolonization ofthehostrhizosphere[17].Thereforeinthisstudywe focusedourcomparativeanalysisonthatstageofthe disease.Wedesignedan invitro systemthatmimics thenaturalrhizosphereconditionsandatthesame timefacilitatetheretrievalof R.solanacearum proteins fromasterileenvironment.Bacteriainco-culturewith tomatoseedlingsexploitplantexudatesinorderto multiplyandapproachrootsasinthenaturalrhizosphere [48,49].Weused2D-DIGEgels toidentifyproteinsthat weredifferentiallyexpressedin4strainsof R.solanacearum thatareeithervirulentornotvirulentatlowtemperatures. Ourexperimentsprovidedin formationabouttheidentity ofproteinsexpresseddifferentiallyandtheirpatternof regulationattwotemperatures.Followingwediscuss thebiologicalsignificanceofourdata. Overall,theabundanceofproteinsrelatedtocellprocessesandmetabolismatlowtemperaturesuggeststhat themetabolicstateofvirulentstrainsisnotreducedatlow temperatureasitisfornotvirulentstrains.Forexample,the leucine-isoleucinebiosynthet icpathwaycouldbeimportant forsurvival,growth,orcompetitionof R.solanacearum in therhizosphereenvironment,andstrainUW551which increasesitsexpressionat18Cmayhaveacompetitive advantageforgrowthandcolonizationoftherootsat lowtemperatureovertheotherstrains.Theglycolysis processmayalsocontributetofitnessofvirulentstrains atlowtemperatureintherhizosphere. Thepresenceofstressresponseproteinswasexpected because R.solanacearum encountersanoxidativeenvironmentintherhizosphereofthehostplant[21];however, theseproteinswerealsotemperaturedependentand thestrainsrespondeddifferentlytothisenvironment atlowtemperatures.Thedifferentbehaviorsbetween KatE,AhpC1,andotherpredictedperoxidasessuggest thatthedifferentcatalases/peroxidasesmayhavespecific functionsandmaybeactivated/de-activatedbydifferent regulators.Thehighervolumesofheat-shockprotein HtpGandproteaseClpBinstrainsthatarevirulentatlow temperatures,suggestahigherlevelofcellularprotection atlowtemperaturesinvirulentstrains. ThepresenceofanHcphomologinourresultsamong thesecretedproteinsindicatedafunctionaltypeVIsecretionsystem.SecretionofHcpisusedtoidentifyfunctional typeVIsecretionsystemsinbacterialspecies[35,50]since presenceofHcpinthesecretomeofbacterialspeciesis requiredforthefunctionofthissecretionsystem[35].An importantobservationisthattheregulationofthissystem istemperaturedependentsimilartothe imp clusterin Rhizobiumleguminosarum [39].Thepatternofexpression ofthetypeVIrelatedproteinssuggeststhatthissystem mayplayaroleinvirulenceatlowtemperature.Bocsanczy etal.BMCGenomics 2014, 15 :280 Page8of14 http://www.biomedcentral.com/1471-2164/15/280

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Thedifferenceinpatternofregulationofthevirulence proteinsidentifiedsuggestsdifferentregulationpathways. ThiswaspartiallydeterminedforChbA,Egl,EpsandTek [51].EglandTekaredirectlyr egulatedbytheglobalregulatorPhcA[52],whileChbA,andEpsareregulatedindirectly throughaVsrA/VsrDtwocomponentsystem[53]. GlobalvirulenceregulatorsHrpGandHrpBexpression wasfoundtobetemperaturedependentandcorrelated withvirulenceatlowtemperature(Figure3).Ourresults supportthehypothesisthatvirulenceatlowtemperaturesisexplainedprimarilybydifferencesintemperature dependentregulationofproteinspresentinallthestrains. R.solanacearum hasacomplicatedregulationnetwork thatitisstilllargelyunknown.Theremayyetberegulationsubsystemsthatcontrolspecificvirulenceandsurvivalfactorsatlowtemperatures.Thefuturedirectionis toinvestigatetheknownglobalvirulenceregulatorsand perhapsidentifynewones. Proteomicsisapowerfulmethodtogiveapictureof thestateofanorganismanddiscoverproteinsthatmay contributetospecificfunctions;howeverthepresenceof veryabundantproteinsobscuredtheidentificationof proteinsproducedinsmallerquantities.Forexample, wenotedtheabsenceoftypeIIIeffectorsinourresults.WeidentifiedaputativeporinHrcCassociated withthetypeIIIapparatus,whichsuggestthattypeIII effectorscannotbediscardedaspotentialtemperature dependentproteins.AnindicationthatthetypeIIIsecretionmaybetemperaturedependentwastheprofile ofthetypeIIIglobalregulatorHrpBinourqRT_PCR expressionexperiment. Wesequenced40%ofthedifferentiallyexpressedspots, reducingourcapacitytoidentifyalltheproteinspotsthat weredifferentiallyexpressedinthecomparisons.Wehypothesizedthataproteinimportantforvirulenceshould beexpressedinlessquantityatlowtemperaturefornot virulentstrains,andinmorequantityforvirulentonesat lowtemperatures;thereforemostproteinspotsthatfitthe criteriawereselectedforsequencing.Wecombinedexperimentstoobtainamorecomp letepicturewithverygood results.Veryfewoftheproteinswerecontradictoryacross experimentsandtherestwereindependentorcongruent, providinguswithanextraqualitycontrolstep.ConclusionsThecomparativeproteinanalysispresentedinthiswork identifiedlikelycandidatesfortemperaturedependent virulencefactors.Toourknowledge,thisisthefirstreportoftemperaturedependentproteinexpressionina plantpathogen.Thisalsoisthefirstreportofaninferred functionalsystemin Ralstoniasolanacearum .Thissecretionsystemwaspreviouslyidentifiedbybioinformatic methodsbutnotpreviouslyconfirmed.Ourresultssupportthehypothesisthatvirulenceatlowtemperatureis mainlyduetoregulationdiffe rencesbetweenstrainsthat arevirulentandstrainsthatarenotvirulentatlowtemperatures.Thisworkprovidesalistofcandidatevirulencefactorsatlowtemperatureswhose expressionistemperature dependentandaworkinghypothesisforthestudyofthe virulencemechanismatlowtemperatures.MethodsExperimentaldesignWecomparedcell-associatedproteinsinseparateexperiments:1)P597(R1B1,Sequevar18),obtainedfrom bacterialpopulationsgrown invitro in20mlofrich mediaat30Cand18Cwithoutplantsinoneexperiment. 2)UW551(R3B2,Sequevar1),P673(R1B1,Sequevar4), GMI1000(R1B3,Sequevar38),andP597,obtained frombacterialpopulationsincubatedincontactwith therhizosphereofsterilet omatoseedlings(seedetails innextsection),inthreeexperimentswithdifferent combinationsofstrains(Figure4a). WealsocomparedsecretedproteinsofUW551and GMI1000extractedfromtheliquidmediumwhenbacterial populationswereincubatedincontactwiththerhizosphere ofsteriletomatoseedlingsino neexperiment(Figure4a). Eachcomparativeexperimentincludedthreebiological replicatespertreatmentperstrain(30Cand18C)that wereloadedin n comparativegels,where n isthenumberofstrainscomparedineachexperimentbythe numberofbiologicalreplicates.Eachcomparativegel wasloadedwithtwosamplesandaninternalcontrol (see2DDIGEproteingelssection)indifferentcombinations.Figure4bexemplifiestheexperimentaldesignforthe comparisonoftwostrains:westartedwith6samplesat 30Cand6at18C.Thesampleswereloadedin6gelsin differentcombinationsdyedwithCya3andCya5,including comparisonsofthesamestrainattwotemperaturesand differentstrainsinthesamegel.The6gelsproduced 18imagesincludingtheinternalcontrolsandsamples (Figure4b).Allimageswerematchedandnormalized. Amastergelwascreatedwithinformationfromallgels. Temperatureproteinprofileswerecomparedusingt-tests. Asubsetofapprox.40%ofthedifferentiallyexpressed proteinswasselectedforMS/MSanalysis.Thefollowing criteriawereusedfortheselection:1-Sufficientproteinspotvolumeforsequencing;2-Moreabundantat 30Cthanat18Cforthestrainsnotvirulentat18C (P597andGMI1000);3-Moreabundantat18Cthan at30Cforthestrainsvirulentat18C(P673,UW551). Sinceonespotcouldpotentiallyrepresentmultipleproteins,thebestfitproteinforeachspotwasdeterminedby selectingthebestpercentageofconfidence( 99%)for thepeptideassignationandbestcoverageoftheprotein usingtheproteinsoftwareScaffold(ProteomeSoftware Inc.,Portland,OR).Proteinswithconflictingresultswere eliminated.Bocsanczy etal.BMCGenomics 2014, 15 :280 Page9of14 http://www.biomedcentral.com/1471-2164/15/280

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Invitro tomatoseedlingsandinoculationwith R.solanacearumTomatocultivarWalter( Solanumlycopersicon ‘ Walter ’ ) plantsweregrownaspreviouslydescribed[17].Seedswere germinatedonwateragarplates(BactoAgarat16.0g/liter, pH7.0).Seedlingsweretransferredtotesttubescontaining 20mlofMSMOliquidmedium(Murashige-Skoogplus organics,SigmaM6899)supplementedwithsucroseat 30g/liter,pH5.8.Plantsweregrown invitro about 10weeks.Toestablishbacteria-plantrootco-cultures invitro R.solanacearum populationswereculturedin casaminoacids-peptone-glu cose(CPG)liquidmedium (5.0g/literglucose,1.0g/litercasaminoacids,10.0g/liter peptone)toanexponentialphase(OD600=0.8).Eachtest tubecontainingfivetomatoplantletswasinoculatedwith 200 lofthecellsuspensionforafinalconcentration ofapprox.8106cells/ml.Co-cultureswereincubated for5daysat18Candfor2daysat30C,bothonarotary shaker(150rpm).Bacterialsamplecollection,proteinextraction,and CyDyelabelingBacterialsuspensioninMSMOmediawascollectedfrom the invitro tomatoseedlingstubesinsterileflasks,andthen filteredwitha20 mSteriflipfilter(SCNY00020Millipore Corp.)toeliminateplantdebris.Theresultingsuspension Figure4 Experimentaldesignflowchart.a .Conditionsofbacterialpopulationsandtypeofproteinsextractedbycomparisons. b .Exampleof experimentaldesignforthecomparisonoftwostrains.ProteinsamplestobecomparedwerestainedwitheitherCy3orCy5fluorescentdyes. Threebiologicalrepsofeachsampletobecomparedwerecombinedinseveralgelstoprovidestatisticalpowerforcomparisons.Thepoolof gelsisnormalizedinordertocomparespotsfromdifferentgels.Spotswereidentifiedandlocalizedacrossgelsandtheirabundancecompared statistically.Alistofdifferentiallyexpressedspotswasanalyzedandasubsetofthetotalnumberofspotsofinterestwereexcisedandsequenced byMSanalysis. Bocsanczy etal.BMCGenomics 2014, 15 :280 Page10of14 http://www.biomedcentral.com/1471-2164/15/280

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wascentrifugedfor10minutesat8000gtocollectthe bacterialpellet.Thispelletwasresuspendedin500 lof DIGEbuffer(8Murea,2Mthiourea,4%CHAPS,0.2% SDS,and10mMTrisHClpH8.5),fastfrozenandstored at Š 80Cforfurtherprocessing.Thesupernatantwasfilteredagainwitha0.22umSteriflipfilter(SEIM179M6 MilliporeCorp.)toeliminate anyresidualbacteriaandconcentratedat4CwithAmico nUltra-1530K(Millipore UCF903024)filter.Concentrated bacterialsecretedprotein wasextractedandcleanedfromsaltsandothercontaminantswithacidphenolaccordingtoNissinenetal.[54]. Thecontaminant-freeproteinpelletwasdissolvedin 250 lofDIGEbuffer.Proteinsamplesolutionswere furtherclarifiedbyultracentrifugationat40kgat 15Cfor30min.Proteinconcentrationsweremeasured withtheEZQProteinAssaykit(Invitrogen)afteradjustingtheproteinsolutiontopH8.5. Tolabeltheproteinsamples,1 lofCyDye(400pmole) dilutedinDMFwasaddedto100 gofproteinandincubatedoniceinthedarkforexactly30min.Tostopthereaction,1 lof10mMlysinewasaddedandincubatedfor 10moreminutes.2DDIGEproteingelsForeachgel,100 gofCy2labeledinternalreference, 100 gofCy3labeledcontrolsample,and100 gofCy5 labeledexperimentalsampleweremixedbeforerehydration ofIPGstrip(GEhealthcare).Dyeswappingwasused andsamplemixturesforthegelswerefrozenat Š 80C untilreadyfor2DGE. BeforeIPGstriprehydration,DTTconcentrationof eachsamplemixturewasadjustedto100mM,IPGbuffer pH3to11to0.5%,andfinalvolumeto500 lwithlabelingbuffer.AsmallamountofOrangeGwasalsoaddedas trackingdye.AnonlinearIPGstrip(24cmpH3to11) wasrehydratedO/Nwith500 lofIEFsamplemixturein thedarkatroomtemperature. FirstDimensionIEFrunwascarriedoutinanIPGphor 3unit(GEHealthcare)underalayerofmineraloilwith gelsurfaceat19Cinthedark.Electrodeswereplacedon eachendoverpiecesoffilterpaperdampenedwithMilli Qwater.Theinitialvoltagewassetat500Vfor1500Vhr (voltagehour).Voltagethenwasrampedupto1000V in6000Vhr,rampedupto8000Vin13.5kVhr,then rampedupto10000Vin16.5Vhr,andfinallywasfocused at10000Vfor80kVhruntilitreachedasteadystateof around27 A. AftercompletionofIEF,proteinsineachstripwere firstreducedin15mlofasolutioncomposedof50mM Tris – HClpH6.8,6MUrea,30%(v/v)glycerol,2%(w/v) SDS,and100mMDTT,for20mininthedarkatroom temperature,thenwerealkylatedin15mlof50mM Tris – HClpH6.8,6MUrea,30%(v/v)glycerol,2%SDS, and2.5%idoacetamidefor20minatroomtemperaturein thedark.Afterreductionandalkylation,thestripwas transferredandwasmountedona8to16%precastTris Glycinepolyacrylamidegelwhichwascastbetweentwo lowfluorescentglassplatesunderalayerofwarm0.5% agarosemadeinSDSelectrophoresisrunningbuffer.Electrophoresiswasfirstrunat12Cand10mA/gelforone hour,andthenO/Nat12mA/gelwithalimitof150V untilthedyefrontreachedthebottomoftheplate. Immediatelyaftergelelectrophoresis,gelcassettes wererinsedwithdeionizedwateranddriedwithlintfreetowelingbeforebeingscannedwithaTyphoon 9400VariableModeImager(GEHealthcare).Theexcitation/emissionwavelengthsforCy2,Cy3andCy5were 488/520,532/580and633/670nmrespectively.Three images(internalreference,controlandexperimental)were acquiredforeachgel.Examplesofgelsandimagesfora comparisonofP597andGMI1000cell-associatedproteins arepresentedinAdditionalfile4toillustratetheprocess. Thedigitalimageinformationacquiredwasthenanalyzed withDeCyder2Dversion7.0, automatedimageanalysis softwarebyGEHealthcare.Allspotspresentedinallimages ofallgelswereco-detected,matchedandnormalized withDIAModule(DifferentialIn-GelAnalysis).InformationfromreplicategelswasanalyzedwithBVAModule (BiologicalVariationAnalysis).InBVA,amastergelwas createdwithinformationfromallgels,withmatchesof multipleimagesfromdifferentgelstoprovidestatistical datafordifferentialproteinexpressionlevelsbetween controlandexperimentalgrou ps.Proteinspotsofinterest (POI)wereselectedbysettingthefolddifferencethreshold to1.5folds.Anyproteinspotfromtheexperimentalgroup (18C)expressedunderorover1.5foldswhencompared withspotsfromthecontrolgroup(30C)wasselectedas POI.Aproteinspotpicklistwasmadeafterfilteringthe spotinformationbasedonmatchingquality,appearance inallgels,andstatisticalconfidencewhenStudent ’ s T-test p valueisequaltoorlessthan0.05.Excisionand Digestionofproteinspotswasdoneasfollows:witha setofpaperreferencecirclesattachedtoeachsideof theglassplate,theordinanceinformationforeachPOI wastranslatedandtransferredtoanautomaticspot picker(ProPicbyGenomicSolutions)throughthepick list.Spotsthenwereexcisedbythepickerandtransferredtoa96-wellcollectingplate.Proteinspotswere washedanddestained,proteinsinthespotwerefirstreducedbyDTTandalkylatedby40mMIodoacetamide beforeO/Ntrypsindigestionat37C.MS/MSsequencingTheenzymaticallydigestedsampleswereinjectedonto acapillarytrap(LCPackingsPepMap)anddesaltedfor 5minata3 l/minflowrateof0.1%v/vaceticacid. ThesampleswereloadedontoanLCPackingC18Pep MapnanoflowHPLCcolumn.TheelutiongradientoftheBocsanczy etal.BMCGenomics 2014, 15 :280 Page11of14 http://www.biomedcentral.com/1471-2164/15/280

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HPLCcolumnstartedat3%solventBand97%solvent A,andfinishedat60%solventAand40%solventBfor 30minforproteinidentification.SolventAconsisted of0.1%v/vaceticacid,3%v/vACN,and96.9%v/vH2O. SolventBconsistedof0.1%v/vaceticacid,96.9%v/v ACN,and3%v/vH2O.LC-MS/MSanalysiswascarried outonahybridquadrupole-TOFmassspectrometer (QSTARelite,AppliedBiosy stems,Framingham,MA).The focusingpotentialandionsprayvoltagewassetto225V and2400V,respectively.Theinformation-dependentacquisition(IDA)modeofoperationwasemployedinwhich asurveyscanfromm/z400 – 1800wasacquiredfollowed bycollisioninduceddissociation(CID)ofthefourmost intenseions.SurveyandMS/MSspectraforeachIDA cyclewereaccumulatedfor1and3s,respectively. Fortheproteinsearchalgorithm,tandemmassspectra wereextractedbyABIAnalystversion2.0.AllMS/MS sampleswereanalyzedusingMascot(MatrixScience, London,UK;version2.2.2).Mascotwassetuptosearch NCBIwithtaxonomyBacteriadatabaseassumingthe digestionenzymetrypsin.Mascotwassearchedwitha fragmentionmasstoleranceof0.50Daandaparent iontoleranceof0.50Da.Iodoacetamidederivativesof Cys,deamidationofAsnandGln,oxidationofMet,are specifiedinMascotasvariablemodifications.Scaffold (ProteomeSoftwareInc.,Portland,OR)wasusedtovalidate MS/MSbasedpeptideandproteinidentifications.Peptide identificationswereacceptediftheycouldbeestablishedat greaterthan95.0%probabilityasspecifiedbythePeptide Prophetalgorithm[55].Proteinidentificationswere acceptediftheycouldbeestablishedatgreaterthan 99.0%probabilityandcontainedatleast3identifiedunique peptides.Proteinprobabilitie swereassignedbytheProtein Prophetalgorithm[56].TotalRNAextractionandcDNAsynthesisCellsamplesfrombacterialco-cultureswithtomato rootswereextractedasfollows:thesuspensionwasfilteredwitha20 mSteriflipfilter(MilliporeCorp.)to eliminateallplantdebris.Filtratewascentrifugedat 8000gandpelletwascollectedandsuspendedin RNABacteriaProtectReagent(QiagenCat.76506) followingthemanufacturer ’ sinstructions.Prepared sampleswerestoredat Š 70Cforfurtherprocessing. TotalRNAwasextractedfromthestoredsamplesas perJahnetal.[57].QuantityofextractedRNAwas measuredusingtheNanodrop2000Cspectrophotometer.Qualitywasassessedondenaturingelectrophoresisgels.PurifiedRNAwasreverse-transcribedto cDNAusingtheSuperScriptViloTMcDNASynthesis Kit(LifeTechnologiesCat.11754 – 050),accordingto manufacturer ’ sinstructions.Qualityandquantityof cDNAwereassessedbyspectrophotometry(Nanodrop Technologies,Inc.).RealtimeqRT-PCRrelativequantificationandprimerdesignPrimersweredesignedfora188-bpfragmentofthe 16sRNAsequenceandfragmentsrangingfrom100bpto 250bpofthetargetgenes(Additionalfile5).Preliminary experimentsweremadewiththreedilutionsofeach cDNAsamplestofindthebestdilutionfortherelative quantification.RelativeqRT-PCRwasperformedusingthe RocheLightcycler480withtheSYBRSelectMasterMix Kit(LifeTechnologiesCat.4472908)accordingtothe manufacturer ’ sinstructions.Samplesof250ngwereused fortheamplificationusingtheprogram:forprimerswith Tm>=60C(UDGactivation50Cfor2min,Amplitaq activation95Cfor2min,and40cyclesofdenaturationat 95Cfor15secandanneal/extendat60Cfor1min). Meltingcurvesofthesampleswereassessedtoevaluatecontamination.Thetargetgene/16sRNAratioof amplificationat18Cwasnormalizedagainsttheratio ofeachstrainat30Cforther elativequantification analysis.AvailabilityofsupportingdataThedatasetssupportingtheresultsofthisarticleare includedwithinthearticleanditsadditionalfiles.The proteinandpeptidedatasetssupportingtheresultsare presentedinAdditionalfile6.AdditionalfilesAdditionalfile1: Proteinsproduceddifferentiallyat30and18C instrainP597whenincubatedinabsenceofplants. Listof proteinsproduceddifferentiallyat30and18CinstrainP597of R. solanacearum whenincubatedinrichmedia,preliminaryexperiment. Theinformationforeachproteinincludesname,putativefunction, broadbiologicalprocesscategory,equivalentproteinaccession numberandgenetaginGMI1000andUW551,predictedcellular localization,andregulation. Additionalfile2: Proteinsproduceddifferentiallyat30and18Cin strainsGM1000,P597,P673,andUW551of R.solanacearum in co-culturewithtomatoseedlings. Listofproteinsproduced differentiallyat30and18CinstrainsGM1000,P597,P673,andUW551of R.solanacearum whenincubatedincontactwith invitro tomatoplants roots.Theinformationforeachproteinincludesname,putative function,broadbiologicalprocesscategory,equivalentprotein accessionnumberandgenetaginGMI1000andUW551,predicted cellularlocalization,andregulation. Additionalfile3: Bestfitproteindetailedidentificationinformation bystrain. Theinformationforeachspotsequencedincludesregulation at30C,ratioofexpressioncomparedwith18C,isoelectricpoint,from thegels;bestfitproteindescription,accessionnumber,proteinsizein Da,numberofpeptides,andproteincoveragefromScaffold. Additionalfile4: Gelimagesforcomparativeexperimentoftwo strainstoillustratetheprocessofgelcomparisons. Includes comparativegelsandimageswithspotsnumberedandnormalizedfor GMI1000andP673comparativegelsofcell-associatedproteins. Additionalfile5: ListofprimersdesignedfortheqRT-PCRrelative quantification. IncludestheprimerpairsequencesusedforqRT-PCRand theestimatedlengthoftheproducts. Additionalfile6: PeptidereportfromScaffold. Excelworkbook containing5worksheetswiththecompletepeptidereportforthe proteinsbyexperimentexportedfromScaffoldviewerprogram.Bocsanczy etal.BMCGenomics 2014, 15 :280 Page12of14 http://www.biomedcentral.com/1471-2164/15/280

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Abbreviations R3B2: Race3Biovar2;R1B1:Race1Biovar1;2D-DIGE:Two-dimensional differentialgelelectrophoresis;MS/MS:Tandemmassspectrometry; PCR:Polymerasechainreaction;qRT_PCR:Quantitativereversetranscription polymerasechainreaction;mRNA:Messengerribonucleicacid;PHB:Poly -Hydroxybutyrate;TCA:Tricarboxylicacidcycle;MUC:Methyl-umbelliferylcellobioside;CHAPS:3-[(3-cholamidopropyl)dimethylammonio]-1propanesulfonate;IPG:ImmobilizedpHgradient;DTT:Dithiothreitol; IEF:Isoelectricfocusing;SDS:Sodiumdodecylsulphate. Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions AMBdesignedtheexperiments,performedtheinoculations,bacterialand secretedproteinspreliminaryextraction,primerdesign,RNAextraction, qRT-PCRexperiments,dataanalysisandinterpretation,andwrotethe manuscript.UCMAandAMNconceivedtheco-cultureintomatomethod, designedandperformedthepreliminaryexperiments.MCperformedthe 2D-DIGEproteincomparisonsanddeterminationofthespotsdifferentially expressed.DJNconceivedofthestudy,participatedinallstepsoftheproject ascoordinatorandcriticallyreviewedthemanuscript.Allauthorsreadand approvedthefinalmanuscript. Acknowledgements WeacknowledgetheUnitedStatesDepartmentofAgricultureFloriculture andNurseryResearchInitiativeandtheUniversityofFloridaInstituteofFood andAgriculturalSciencesforprovidingthefundsforthisresearch.Thanksto Dr.StevenArthursandBarbHennyforthecriticalreviewofthewritingand editingofthemanuscript.ThankstoCarolynDiazforthesequencingand preliminaryidentificationoftheproteinsbasedonthepeptideprofiles. Authordetails1DepartmentofPlantPathology,UniversityofFlorida,IFAS,Mid-FloridaResearch andEducationCenter,2725BinionRd.,Apopka,FL32703,USA.2Development LeadNorth-EastEurope,SyngentaAgroGmbH,AmTechnologiepark1-563477, Maintal,Germany.3BurnhamInstituteforMedicalResearchatLakeNona,6400 SangerRoad,Orlando,FL32827,USA.4ICBRProteomicsCore,Universityof Florida,Gainesville,FL32610,USA. Received:6November2013Accepted:9April2014 Published:12April2014 References1.HurmeR,RhenM: Temperaturesensinginbacterialgeneregulation-what itallboilsdownto. MolMicrobiol 1998, 30 (1):1 – 6. 2.RamosJL,GallegosMT,MarquesS,Ramos-GonzalezMI,Espinosa-UrgelM, SeguraA: Responsesofgram-negativebacteriatocertainenvironmental stressors. CurrOpinMicrobiol 2001, 4 (2):166 – 171. 3.KonkelME,TillyK: Temperature-regulatedexpressionofbacterial virulencegenes. 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JMicrobiolMeth 2008, 75 (2):318 – 324.doi:10.1186/1471-2164-15-280 Citethisarticleas: Bocsanczy etal. : Proteomiccomparisonof Ralstonia solanacearum strainsrevealstemperaturedependentvirulencefactors. BMCGenomics 2014 15 :280. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Bocsanczy etal.BMCGenomics 2014, 15 :280 Page14of14 http://www.biomedcentral.com/1471-2164/15/280


ERROR CAUGHT WHILE SAVING NEW DIGITAL RESOURCE
6/13/2014 7:50:06 AM

Error while executing stored procedure 'SobekCM_Save_Item'.
at SobekCM.Resource_Object.Database.SobekCM_Database.exception_caught(String StoredProcedureName, Exception Exception)
at SobekCM.Resource_Object.Database.SobekCM_Database.Save_New_Digital_Resource(SobekCM_Item ThisPackage, Boolean TextFlag, Boolean OnlineSubmit, String Username, String Usernotes, Int32 Userid)
at SobekCM.Library.MySobekViewer.New_Group_And_Item_MySobekViewer.complete_item_submission(SobekCM_Item Item_To_Complete, Custom_Tracer Tracer) in c:\GitRepository\SobekCM\SobekCM-Web-Application\SobekCM_Library\MySobekViewer\New_Group_And_Item_MySobekViewer.cs:line 839