Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mec...

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Title:
Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range
Physical Description:
Mixed Material
Language:
English
Creator:
Jalan, Neha
Kumar, Dibyendu
Andrade, Maxuel O.
Yu, Fahong
Jones, Jeffrey B.
Graham, James H.
White, Frank F.
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Bio-Med Central (BMC Genomics)
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Abstract:
Background: Citrus bacterial canker is a disease that has severe economic impact on citrus industries worldwide and is caused by a few species and pathotypes of Xanthomonas. X. citri subsp. citri strain 306 (XccA306) is a type A (Asiatic) strain with a wide host range, whereas its variant X. citri subsp. citri strain Aw12879 (Xcaw12879, Wellington strain) is restricted to Mexican lime. Results: To characterize the mechanism for the differences in host range of XccA and Xcaw, the genome of Xcaw12879 that was completed recently was compared with XccA306 genome. Effectors xopAF and avrGf1 are present in Xcaw12879, but were absent in XccA306. AvrGf1 was shown previously for Xcaw to cause hypersensitive response in Duncan grapefruit. Mutation analysis of xopAF indicates that the gene contributes to Xcaw growth in Mexican lime but does not contribute to the limited host range of Xcaw. RNA-Seq analysis was conducted to compare the expression profiles of Xcaw12879 and XccA306 in Nutrient Broth (NB) medium and XVM2 medium, which induces hrp gene expression. Two hundred ninety two and 281 genes showed differential expression in XVM2 compared to in NB for XccA306 and Xcaw12879, respectively. Twenty-five type 3 secretion system genes were up-regulated in XVM2 for both XccA and Xcaw. Among the 4,370 common genes of Xcaw12879 compared to XccA306, 603 genes in NB and 450 genes in XVM2 conditions were differentially regulated. Xcaw12879 showed higher protease activity than XccA306 whereas Xcaw12879 showed lower pectate lyase activity in comparison to XccA306. Conclusions: Comparative genomic analysis of XccA306 and Xcaw12879 identified strain specific genes. Our study indicated that AvrGf1 contributes to the host range limitation of Xcaw12879 whereas XopAF contributes to virulence. Transcriptome analyses of XccA306 and Xcaw12879 presented insights into the expression of the two closely related strains of X. citri subsp. citri. Virulence genes including genes encoding T3SS components and effectors are induced in XVM2 medium. Numerous genes with differential expression in Xcaw12879 and XccA306 were identified. This study provided the foundation to further characterize the mechanisms for virulence and host range of pathotypes of X. citri subsp. citri. Keywords: Xanthomonas citri, Wellington strain, Citrus canker, HR, Virulence, Transcriptome, RNA-Seq
General Note:
Jalan et al. BMC Genomics 2013, 14:551 http://www.biomedcentral.com/1471-2164/14/551
General Note:
doi:10.1186/1471-2164-14-551 Cite this article as: Jalan et al.: Comparative genomic and transcriptome analyses of pathotypes of Xanthomonas citri subsp. citri provide insights into mechanisms of bacterial virulence and host range. BMC Genomics 2013 14:551.

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University of Florida
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University of Florida
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RESEARCHARTICLEOpenAccessComparativegenomicandtranscriptomeanalyses ofpathotypesof Xanthomonascitri subsp. citri provideinsightsintomechanismsofbacterial virulenceandhostrangeNehaJalan1,DibyenduKumar2,MaxuelOAndrade1,FahongYu3,JeffreyBJones4,JamesHGraham5, FrankFWhite6,JooCSetubal7,8andNianWang1*AbstractBackground: Citrusbacterialcankerisadiseasethathassevereeconomicimpactoncitrusindustriesworldwide andiscausedbyafewspeciesandpathotypesof Xanthomonas.X.citri subsp. citri strain306(XccA306)isatypeA (Asiatic)strainwithawidehostrange,whereasitsvariant X.citri subsp. citri strainAw12879(Xcaw12879,Wellington strain)isrestrictedtoMexicanlime. Results: TocharacterizethemechanismforthedifferencesinhostrangeofXccAandXcaw,thegenomeof Xcaw12879thatwascompletedrecentlywascomparedwithXccA306genome.Effectors xopAF and avrGf1 are presentinXcaw12879,butwereabsentinXccA306.AvrGf1wasshownpreviouslyforXcawtocausehypersensitive responseinDuncangrapefruit.Mutationanalysisof xopAF indicatesthatthegenecontributestoXcawgrowthin MexicanlimebutdoesnotcontributetothelimitedhostrangeofXcaw.RNA-Seqanalysiswasconductedto comparetheexpressionprofilesofXcaw12879andXccA306inNutrientBroth(NB)mediumandXVM2medium, whichinduceshrpgeneexpression.Twohundredninetytwoand281genesshoweddifferentialexpressionin XVM2comparedtoinNBforXccA306andXcaw12879,respectively.Twenty-fivetype3secretionsystemgenes wereup-regulatedinXVM2forbothXccAandXcaw.Amongthe4,370commongenesofXcaw12879comparedto XccA306,603genesinNBand450genesinXVM2conditionsweredifferentiallyregulated.Xcaw12879showed higherproteaseactivitythanXccA306whereasXcaw12879showedlowerpectatelyaseactivityincomparisonto XccA306. Conclusions: ComparativegenomicanalysisofXccA306andXcaw12879identifiedstrainspecificgenes.Ourstudy indicatedthatAvrGf1contributestothehostrangelimitationofXcaw12879whereasXopAFcontributesto virulence.TranscriptomeanalysesofXccA306andXcaw12879presentedinsightsintotheexpressionofthetwo closelyrelatedstrainsof X.citri subsp .citri .VirulencegenesincludinggenesencodingT3SScomponentsand effectorsareinducedinXVM2medium.NumerousgeneswithdifferentialexpressioninXcaw12879andXccA306 wereidentified.Thisstudyprovidedthefoundationtofurthercharacterizethemechanismsforvirulenceandhost rangeofpathotypesof X.citri subsp .citri Keywords: Xanthomonascitri ,Wellingtonstrain,Citruscanker,HR,Virulence,Transcriptome,RNA-Seq *Correspondence: nianwang@ufl.edu1CitrusResearchandEducationCenter,DepartmentofMicrobiologyandCell Science,UniversityofFlorida,700ExperimentStationRoad,LakeAlfred,FL 33850,USA Fulllistofauthorinformationisavailableattheendofthearticle 2013Jalanetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Jalan etal.BMCGenomics 2013, 14 :551 http://www.biomedcentral.com/1471-2164/14/551

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BackgroundMembersofthegenus Xanthomonas arecapableof infectingatleast124monocotspeciesand268dicot speciesandprovideexcellentcasestudiesforunderstandingplant-microbeinteractions[1].Amongthe diseasescausedby Xanthomonas ,citruscankercaused by X.citri subsp .citri (Xcc)(syn. X.axonopodis pv. citri, X.campestris pv.citri, X.citri pv.citri)isan importantdiseasethathassevereeconomicimpact oncitrusindustriesworldwide.Asiatic(A)type strainsarethemostwidespreadand,hence,themost destructiveformofcitruscanker.Thestrainsinduce hyperplasicandhypertrophic(raised)lesionssurrounded byoilyorwater-soakedmarginsandayellowhaloon leaves,stems,andfruits.BesidesXcc,asecondspecies, X. fuscans subsp. aurantifolii (Xau),isalsoknowntocause citruscankerwithlimitedgeographicdistributionand limitedhostrange.TypeBstrainsofXauarerestrictedto SouthAmerica(Argentina ,UruguayandParaguay) andcausecankeronlemon( C.limon )andMexican lime( C.aurantifolia ).TypeBstrainscanalsobefoundon sweetorange( C.sinensis )andgrapefruit(Citrusx paradisi ) [2].TypeCstrainsofXauarerestrictedtoBrazilandcause cankeronlyonMexicanlime[3]. TwovariantsoftypeAstrainshavealsobeenidentified.ThevariantdesignatedA*wasfoundinSoutheast Asiainthe1990sinfectingMexicanlime[4,5].Asecond variant,designatedasthe “ Wellingtonstrain ” ,wasisolatedfromPalmBeachCountyinsouthernFlorida[4,6]. DNAhybridizationanalysisshowedthatXcawismore closelyrelatedtoXccAandXccA*strainsthantoXauB andXauCstrains[4].BothXcawandXccAhavesimilar symptomsandleafpopulationsonMexicanlime[7]. X. citri subsp. citri pathotypeAw(Xcaw)arepathogenicon Mexicanlimeandalemow( C.macrophyla )butnoton grapefruitandsweetorange.TheXcawstrainscausea hypersensitivereaction(HR)ingrapefruit[7].Thegene avrGf1 wasidentifiedinXcawstrain12879,andmutationof avrGf1 ofXcaw12879renderedthemutantvirulentongrapefruit,althoughthesymptomsweremuch reducedascomparedtosymptomsduetostrainsof XccA306[7].Acomprehensiveunderstandingofthe molecularmechanismsresponsibleforthedifferencesin virulenceandhostrangeofXcawandXccAislacking. Comparativegenomicanalysesofxanthomonadshave greatlyfacilitatedourunderstandingofthevirulencefactorsandhostrangedeterminantsofdifferentpathogens [8-10].Comparativegenomicanalysisof X.campestris pv.campestrisandXccA306hasbeenconductedpreviouslytounderstandthemechanismsofdifferenthost rangeandpathogenicprocessesofthetwo Xanthomonas species,whichhavedistincthostranges[8].Compared toXcc,whichinfectscitrusandcausescitruscanker, X. campestris pv campestrisaffectscruciferssuchas Brassica andcausesblackrot.Numerousspecies-specific geneshavebeenidentifiedwhichmightexplainthe differinghostspecificitiesandpathogenicprocessesof thetwopathogens.Comparativegenomicanalysisof XccA306and X.axonopodis pv citrumelowasalso conductedrecently[9]. X.axonopodis pv citrumeloF1 isanurseryinfectingstrainandshowslowvirulenceon citruscomparedtothatofXccA.Differencesingene contents,suchastypeIIIeffectors(e.g.,PthA),thetype IVsecretionsystem,andlipopolysaccharidesynthesis wereidentifiedandmaycontributetothedifferencesin bacterialvirulenceandhostrange[9].Furthermore,sequencingofXauBandXauCstrainsidentifieddifferent virulencefactorsaffectinghostrangeofcloselyrelated species[10]. Hereweconductedcomparativegenomicanalysisof Xcaw12879andthecloselyrelatedstrainXccA306using acompletegenomesequenceofXcaw12879tounderstandthedifferenceinvirulenceandhostrange.Recently, wehavecompletedthegenomesequencingofXcaw12879 [11].Wefurtherexaminedthetranscriptomesofboth XccA306andXcaw12879byRNA-Seqinnutrientrich conditionNutrientBroth(NB)andinXVM2,whichis knowntoinducehrpgeneexpression[12].Thecomparativegenomicandtranscript omeanalyseswillprovide thefoundationtofurthercharacterizethemechanisms forvirulenceandhostrangeofpathotypesof X.citri subsp .citri .ResultsMultilocussequencingtypinganalysisMultilocussequencetyping(MLST)basedphylogenetic analysiswasperformedforXcaw12879andother Xanthomonas spp.usingninehousekeepinggenes( uvrD, secA,carA,recA,groEL,dnaK,atpD,gyrB ,and infB )that arehighlyconservedinbacteria.Thenineprotein sequenceswerealignedandconcatenatedandthenused toconstructamaximum-likelihoodphylogenetictree (Figure1).TheresultsshowedthatXcaw12879isclosely relatedtoXccA306.Interestingly,thesetwocitrus cankerpathogensformacladewith X.citri pv. mangiferaeindicaeLMG941and X.axonopodis pv. punicaeLMG859,whichcausebacterialblackspotin mangoandbacterialleafblightinpomegranatesrespectively.BothstrainswereisolatedfromIndia[13,14],aputativeoriginofXccA.Hence,itispossiblethatthese pathogenshaveevolvedfromthesameancestorand evolvedtoadapttodifferenthosts.TheXccA306and Xcaw12879strainssharecloserelationshipwiththe othertwocitruscankercausingbacteriaXauBandXauC (Figure1).ThecloserelationshipbetweenXccA,Xcaw, XauBandXauCagreeswiththegenome-basedphylogenyofthegenus Xanthomonas [15].Jalan etal.BMCGenomics 2013, 14 :551 Page2of17 http://www.biomedcentral.com/1471-2164/14/551

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ChromosomeorganizationandgenomeplasticityWhole-genomealignmentofXcaw12879tocloselyrelatedXccA306usingMAUVEinprogressivemoderevealednumerousinversionsandtranslocations (Figure2).Mostoftheseparatedblocksinthealignment areassociatedwithintegrasesand/orISelementsonat leastoneoftheirborders.TheISelementshavebeen knowntoaidhorizontalgenetransferandothergenome rearrangements[16]. Xcaw12879genomecontainstwoplasmidspXcaw19 andpXcaw58thataresignificantlydifferentfromthe plasmidsfoundinXccA306.PlasmidpXcaw19sequence hasnosimilaritywiththeplasmidsofXccA306,whereas pXcaw58isonlyabout35%similartopXAC64.Plasmid pXcaw58containsthe pthAw2 gene,ahomologof pthA4 ,whichiscapableofconferringtheabilitytocause canker-likesymptoms[17].However,theplasmid pXcaw58doesnotcontaintheVirliketypeIVsecretion systemgenesfoundonpXAC64.ThetypeIVsecretion systemhasbeenshowntocontributetovirulencein X. campestris pv.campestrisstrain8004[18]andabsence ofthesegenesfromtheplasmidcouldaffectvirulenceof Xcaw12879strain. Threeclusteredregularlyinterspacedshortpalindromicrepeats(CRISPRs)withshort(21 – 47bp)direct repeatsinterspacedwithunrelatedsimilarlysizednonrepetitivesequences(spacers)arefoundinXcaw12879 genome(Additionalfile1).TheCRISPR1andCRISPR2 repeatsarealsopresentinXccA306.CRISPR2and CRISPR3fromXcaw12879areidenticalexceptforaG atthebeginningofCRISPR2,indicatingthatitmightbe arecentduplication.CRISPRisabacterialimmunity systemthathelpsexcludeforeigngeneticelements. HoweverthevariabilityinXcaw12879andXccA306suggeststhatthestrainsmighthavehaddissimilarexposure toforeigngeneticmaterialassuggestedin X.oryzae [19]. TheTBLASTNanalysisofalltheproteinsfrom Xcaw12879andXccA306revealedvariousgeneclusters specifictoeachstrain.Ofthe4,760proteinsfrom Xcaw12879and4,603(176notannotatedpreviously[8]) proteinsfromXccA306,4,428proteinsarefoundtobe orthologoususingthecut-offe-value 10-10andalignments>60%sequenceidentity,>60%querygenelength. Amongthe4,428commonproteins,4,252wereannotatedinXccA306[8]whereas176arenotannotated. Xcaw12879has332proteinsthatareeithernonorthologoustoproteinsfromXccA306orunique, whereasXccA306has175suchproteins. The hrp and hrc genesencodingthetype3secretion system(T3SS)inXcaw12879arehomologoustothe hrp and hrc genesfoundinXccA306.Allthegenesare Xanthomonas campestris pv. campestris str. ATCC 33913 Xanthomonas campestris pv. campestris str. 8004 Xanthomonas campestris pv. campestris str. B100 XylellafastidiosaTemecula 1 Xylellafastidiosa M12 Xylellafastidiosa 9a5c Xanthomonas campestris pv. raphani 756C Xylellafastidiosa M23 Xanthomonas campestris pv. musacearumNCPPB 4381 Xanthomonas campestris pv. vasculorumNCPPB 702Xanthomonas gardneri ATCC 19865Xanthomonas vesicatoria ATCC 35937 Xanthomonas albilineans GPE PC73 Xanthomonas sacchari NCPPB 4393 Xanthomonasfuscans subsp. aurantifolli CXanthomonas fuscans subsp. aurantifolli BXanthomonas campestris pv.vesicatoria str. 85-10 Xanthomonas axonopodis pv. citrumelostr. FL1 Xanthomonas oryzae pv. oryzaeKACC10331 Xanthomonas oryzae pv. oryzaePXO99A Xanthomonas oryzae pv. oryzaeMAFF 311018 Xanthomonas citri subsp. citri str. Aw 12879 Xanthomonas citri subsp. citri str. 306 Xanthomonas perforans9 1-118 Xanthomonas axonopodis pv. punicaeLMG 859 Xanthomonas citri pv. mangiferaeindicaeLMG 941 Xanthomonas oryzae pv. oryzicolaBLS 25648 100 100 100 100 100 100 100 100 96 100 100 100 55 72 91 100 100 10010060 100 100 100 Figure1 Maximumlikelihoodphylogenetictreeofthegenomeof Xanthomonascitri subsp. citri Aw12879showingtherelationshipto other Xanthomonads andrelatedspecies. Thetreewasconstructedusingconcatenatedproteinsequencesofninehousekeepinggenes(UvrD, SecA,CarA,RecA,GroEL,DnaK,AtpD,GyrBandInfB)alignedusingClustalW.Phylogenictreefromconcatenatedsequenceswasconstructedin CLCGenomicsworkbenchv6.0usingtheMaximumlikelihoodmethod.Thepercentageofreplicatetreesinwhichtheassociatedtaxaclustered togetherinthebootstraptest(1000replicates)areshownnexttothebranches.Horizontalscalebar(0.11)atthebottomrepresentsnumberof amino-acidsubstitutionspersite. Jalan etal.BMCGenomics 2013, 14 :551 Page3of17 http://www.biomedcentral.com/1471-2164/14/551

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foundinsimilarorderwiththeexceptioningeneannotationbetween hrpF and hpaB .ThegenomeofXccA306 containstheannotatedgeneXAC0395betweenthetwo, whichisahypotheticalprotein.Theannotationin Xcaw12879inthesameregionisontheoppositestrand andcontains hpaI (XCAW_00803)and xopF1 (XC AW_00804/XCAW_00805)whichmaybenonfunctional duetoaframeshift.Thenucleotidesequencesinboth strainsarethesameandthedifferencesinannotation wereconfirmedbyBLASTsimilarityoftheannotated genesinXcaw12879tootherxanthomonads. TheT3SStranslocateseffectorproteinsintotheplant cells.Theeffectorscaneitheraidinnutrientacquisition andvirulenceoractasavirulencefactorsthattrigger hostimmuneresponse[20].ThetypeIIIeffector genesinXcaw12879werepredictedbyBLASTanalysis againsttheknownT3SSeffectordatabase[http://www. xanthomonas.org].Xcaw12879containsthirtyeffector genesofwhichtwenty-sixoverlapwithXccA306(except pthA1 pthA2 and pthA3 ).Nineteeneffectorsarepresent inallfoursequencedcitruscankercausingvariantscompared(XccA,Xcaw,XauB,andXauC)andthusrepresentthecoreeffectorsetfor Xanthomonas thatcause citruscanker.ItisnoteworthythatEscalonetal.[21]definea ‘ commonrepertoire ’ of26T3Seffectorgenes presentin55Xccstrainsfromseverallocationsaround theworld.TheydidnotusedatafromXauBandXauC incompilingthiscommonrepertoirewhichexplains why26T3Seffectorswereidentifiedpreviously[21] whereasweonlyidentified19commonT3Seffectors. Theeffectorgenes avrBs2,xopK,xopL,xopQ,xopR, xopX and xopZ arefoundinallothersequenced Xanthomonas genomesandhencethesesevengenes mightbeacoresetofeffectorsrequiredfor phytopathogenicityassuggestedbyMoreira etal. [10]. Twelveeffectorgenes( xopA,xopE1,xopE3,pthA4 or itsfunctionalhomologs ,xopI,xopV,xopAD,xopAI, xopAK,xopAP,hpaA ,and hrpW )arepresentinallfour citruscankercausingvariants(Xcaw,XccA,XauBand XauC).Ofthetwelveeffectorgenes, xopE3 and xopAI arepresentinXcaw12879albe itindifferentlocations ascomparedtothepotentialgenomicislandinthe otherthreestrainscausingcitruscanker.Howeverthey mayplayaroleincitruscankerassuggestedby Moreira etal. [10].Twoeffectorgenes avrGf1 and xopAF wereidentifiedinXcaw,XauBandXauCbut werenotpresentinXccA306genome(Table1). MultiplegenesclusteredintotengroupswereidentifiedinXcaw12879butnotinXccA306(Table2).Many genesoftheseclusterspresentinXcaw12879butnotin XccA306havehomologsinother Xanthomonas species. Alltheseregionscontaintransposase,integraseorphage relatedgenesindicatingpossibleacquisitionbyhorizontalgenetransfer.Aninterestingdifferencenotedin theabove-mentionedregionsisincluster5,whichencodesforlipopolysaccharide(LPS)biosyntheticpathway. Interestingly,theLPSclusterinXcaw12879containsregionsorthologoustobothXccA306and X.oryzae pv. oryzicolaBLS256asshowninFigure3,whichindicates thattherehasbeenhorizontalgenetransfer.Cluster4is almost100kblongandpartsofcluster4aresyntenic withregionsfrom X.campestris pv campestris8004,a blackspotpathogenofcabbage(Table2).AMUMmer comparisonbetweencluster4and X.campestrispv campestris8004showshighsynteny(Additionalfile2). Threetranscriptionalregulators(XCAW_01037,XC AW_01129,XCAW_01131)andonetwo-component system(TCS)sensorkinase(XCAW_01148)andits Figure2 MAUVEalignmentofthegenomesequencesof X.citri subsp. citri str.306and X.citri subsp. citri Aw12879. Conservedand highlyrelatedregionsarecoloredandlowidentityuniqueregionareinwhite(colorless).Thecoloredlinesindicatetranslocationsofthegenome sections.Samecoloredblocksonoppositesidesofthelineindicateinversion. Jalan etal.BMCGenomics 2013, 14 :551 Page4of17 http://www.biomedcentral.com/1471-2164/14/551

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Table1Effectorrepertoireof X.citri subsp. citri Aw12879(Xcaw12879), X.citri subsp .citri str.306(XccA306) ,X.fuscans subsp. aurantifolii str.ICPB11122(XauB)and X.fuscans subsp. aurantifolii str.ICPB10535(XauC)EffectorclassXcaw12879XccA306XauBXauCPfamdomainsReferences AvrBs2XCAW_00465XAC0076XAUB_16770XAUC_23650Glycerophosphoryldiester phosphodiesterase [ 22 ] PthA(AvrBs3,TAL)XCAW_b00018 (PthAw1) XACa0022 (PthA1) XAUB_40130XAUC_22430Transcriptionalactivator,nuclear localization [ 23 ] XACa0039 (PthA2) XAUC_24060 XCAW_b00026 (pthAw2) XACb0015 (PthA3) XAUB_28490XAUC_09900 XACb0065 (PthA4) XAUC_43080 XopA(Hpa1/ HpaG) XCAW_00826XAC0416XAUB_19280XAUC_43660-[ 24 ] XopE1(AvrXacE1)XCAW_00686XAC0286XAUB_37010XAUC_37580Putativetransglutaminase[ 25 ] XopE3(AvrXacE2)XCAW_03515XAC3224XAUB_14680XAUC_00040Putativetransglutaminase[ 26 ] XopF2XCAW_0138 XAC2785 XAUB_07540 XAUC_21000 -[ 27 ] XopIXCAW_03828XAC0754XAUB_39080XAUC_07100F-boxprotein[ 28 ] XopKXCAW_03372XAC3085XAUB_34090XAUC_12520-[ 29 ] XopLXCAW_03376XAC3090XAUB_34130XAUC_02900/ XAUC_12488 LRRprotein[ 30 ] XopQXCAW_04706XAC4333XAUB_10220XAUC_14670InosineuridinenucleosideNribohydrolase [ 27 ] XopRXCAW_00677XAC0277XAUB_36920XAUC_37490-[ 29 ] XopVXCAW_03980XAC0601XAUB_23140XAUC_21260-[ 29 ] XopXXCAW_00956XAC0543XAUB_14760XAUC_20690-[ 31 ] XopZ1XCAW_01815XAC2009XAUB_11532/ XAUB_13710 XAUC_25915-[ 26 ] XopADXCAW_00082XAC4213XAUB_02510XAUC_34870SKWPrepeatprotein[ 32 33 ] XopAIXCAW_01099XAC3230XAUB_26830XAUC_23780PutativeADP-ribosyltransferase[ 34 ] XopAKXCAW_04369XAC3666XAUB_02580XAUC_32490-[ 33 ] XopAPXCAW_03269XAC2990XAUB_13980XAUC_08760-[ 35 ] HpaAXCAW_00810XAC0400XAUB_19430XAUC_19990T3Scontrolprotein[ 36 ] HrpW(PopW)XCAW_03200XAC2922XAUB_19460XAUC_20020PectateLyase[ 37 ] XopAQXCAW_03514Notannotated* Notannotated**--[ 35 ] XopE2(AvrXacE3, AvrXccE1) XCAW_03520XACb0011XAUB_31660-Putativetransglutaminase[ 25 ] XopNXCAW_01387XAC2786XAUB_07520-ARM/HEATrepeat[ 38 ] XopPXCAW_01310XAC1208XAUB_06720--[ 27 ] XopAE(HpaF/ HpaG) XCAW_00801XAC0393XAUB_19500-LRRprotein[ 39 ] XopC2XCAW_01311 XAC1209/ XAC1210 Haloaciddehalogenase-likehydrolase[ 39 ] XopAF(AvrXv3)XCAW_b00003-XAUB_02310XAUC_00300-[ 40 ] XopAG(AvrGf1/ AvrGf2) XCAW_00608-XAUB_03570 XAUC_04910-[ 7 ] XopF1(Hpa4)XCAW_00804/ XCAW_00805 -XAUC_31730 [ 30 ] Jalan etal.BMCGenomics 2013, 14 :551 Page5of17 http://www.biomedcentral.com/1471-2164/14/551

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responseregulator(XCAW_01150)arepresentin Xcaw12879,butareabsentinXccA306.PthAandhomologsAllthecitruscankercausingvariants(XccA,Xcaw, XccA*,XauB,andXauC)containPthAoritsfunctional homologs.Thus,PthAoritsfunctionalhomologsis likelythepathogenicitydeterminantofcitruscanker pathogenassuggestedinapreviousstudy[17]that linkedthestrainsof Xanthomonas withdifferenthost rangetogether.Al-Saadietal.[17]haveshownthatall thevariantscarryone pthA homologwith17.5repeats whichdeterminespathogenicityoncitrusandtriggers immunityinvariousotherplantspecies[42].The avrBs3 / pthA familyofeffectorsincludesvarious pth genesbutonlyPthA[42]isknowntoinducecanker. ThefunctionalhomologofthisgeneinXccAstrain306 is pthA4 ,whichalsohasthreeotherparalogsonitstwo plasmids(Table1).Wefoundtwohomologs pthAw1 and pthAw2 inXcaw12879genome,bothlocatedon plasmidpXcaw58.The pthAw2 geneis99%identicalto pthA4 fromXccAandalsoto pthAw sequencedfrom anotherWellingtonstrain0053thatisabletocomplementaknockoutmutantof pthA inXccAstrain3213 [17],indicatingthatPthAw2isthefunctionalhomolog of pthA inXcaw.PthAw2hasthesamerepeatnumber (17.5)asthefunctionalhomologsPthA4,PthBandPthC fromthethreerespectivecitruscankercausingstrains XccA,XauB,andXauC[10].Theotherhomolog PthAw1inXcawhas18.5tandemrepeats,whichis Table1Effectorrepertoireof X.citri subsp. citri Aw12879(Xcaw12879), X.citri subsp .citri str.306(XccA306) ,X.fuscans subsp. aurantifolii str.ICPB11122(XauB)and X.fuscans subsp. aurantifolii str.ICPB10535(XauC) (Continued)XopB--XAUB_09070/ XAUB_14842 XAUC_00260-[ 41 ] XopE4--XAUB_23330XAUC_31730Putativetransglutaminase[ 10 ] XopJ1--XAUB_20830XAUC_08850C55-familycysteineproteaseorSer/ Thracetyltransferase [ 27 ] Inactive/Pseudogene. *LocatedbetweenXAC3223andXAC3224from3,797,415bpto3,797,702bp. **LocatedbetweenXAUB_14670&XAUB_14680onNZ_ACPX01000163from23,262bpto23,549bp. Table2GeneclusterspresentinXcaw12879butabsentinXccA306Cluster number LocustagHomologsinothergenomesFunction 1XCAW_01029to XCAW_01069 hypotheticalproteins,RhsAfamilyprotein,transcriptionalregulator,integrase, adeninespecificDNAmethylase,typeIIIrestrictionenzyme:ressubunit,ATP dependentexoDNAse,thermonulease 2XCAW_01117to XCAW_01151 Somepresentin X campestris pv. campestrisATCC33913 transcriptionalregulator,phage-relatedtailproteins,TCSresponsesensorand regulator,chitinase,Znpeptidase,transcriptionalrepressor,protein-glutamate methylesterase 3XCAW_01571to XCAW_01582 Somepresentin X campestris pv. campestrisstr.8004 phagerelatedproteins,hypotheticalprotein 4XCAW_01620to XCAW_01726 Homologousto Acidovorax sp.JS42and X campestris pv.campestrisstr.8004 transposases,hypotheticalproteins,typeIIrestrictionenzyme:methylase subunit,phagerelatedregulatoryproteins,chromosomepartitioningrelated protein,solublelyticmureintransglycosylase,VirB6protein 5XCAW_04292to XCAW_04303 Homologousto X oryzae pv.oryzicola BLS256 lipopolysaccharidebiosynthesisgenes 6XCAW_04482to XCAW_04496 transposases,hypotheticalproteins,transcriptionalrepressor,polymeraseV subunit 7XCAW_04519to XCAW_04545 Homologousto X oryzae pv.oryzae PXO99A phagerelatedproteins,transcriptionalregulator,transposases,hypothetical proteins 8XCAW_a00001 to XCAW_a00017 Plasmidpartitionprotein,conjugaltransferprotein,hypotheticalproteins 9XCAW_b00002 to XACW_b00018 Transposases,plasmidstabilityproteins,avirulenceprotein,hypothetical proteins 10XCAW_b00048 to XACW_b00056 Plasmidmobilizationproteins,transposases,hypotheticalproteins Jalan etal.BMCGenomics 2013, 14 :551 Page6of17 http://www.biomedcentral.com/1471-2164/14/551

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differentfromPthAhomologsfoundinXccAthathave either15.5or16.5tandemrepeats.TheAvrBs3/PthA familyofeffectorsareknownastranscriptionactivatorlike(TAL)effectorssincetheyreprogramhostcellsby specificallybindingtothepromotersofplantgenesrecognizedbythecentraldomainoftandemrepeats[43]. ComparingtheDNAbindingTALeffectorcodesfor PthAfromXccAaspredictedbyBochetal.[44]to PthAwindicatethatthecodesforPthA4andPthAw2 arequitedivergent(Additionalfile3).Al-Saadietal.[17] predictedthatthewellconservedsequenceofthe17threpeatinfunctionalPthAmightbeimportantforpathogenicityoncitrus,andthissequenceispresentin PthAw2.TherestofPthAw2sequencehoweverpotentiallyencodesaDNAbindingcodethatisonlyabout 79%similartotheoneencodedbyPthA4ofXccA306 (Additionalfile3).Thismayresultinrecognitionofdifferenttargetgenesinhostplantordifferencesin strengthofinductionofplantgenesandthusaffectvirulenceofXcawandXccA.PathogenicityandgrowthassaysAllthreehostlimitedstrainsof Xanthomonas affecting citrus,Xcaw,XauBandXauChad avrGf1 and xopAF genesintheirgenomes.Thegene avrGf1 hasbeenpreviouslystudiedinXcawandisknowntoberesponsible forHRingrapefruit[7].Howeveritseffectonother varietiesofcitrussuchassweetorangeisunknown. Also,since xopAF istheotherputativeeffectorgene,its effectonhostlimitationwasfurthercharacterizedby pathogenicityandgrowthassaysofXcaw xopAF and Xcaw avrGf1 xopAF PathogenicityassaysindicatedthatXcaw12879didnot elicitanyreactiononValenciaorHamlinorangesatour testconditionswhilewildtypeXccA306causednecrotic raisedlesions,typicalofcitruscankerontheleavesata highbacterialinoculationconcentrationof108cfu/ml (Figure4).Xcaw12879showedaHRongrapefruitleaves thatwasabolishedbydeleting avrGf1 gene(Xcaw avrGf1),howeverthegrowthofthemutantwasvisibly reducedcomparedtoXccA306strain.Xcaw avrGf1 did notshowanysymptomsorreactiononeitherValencia orHamlin(Figure4). Tocheckwhethermutationof xopAF affects Xcaw12879growth inplanta ,thewild-typestrainsof XccA306andXcaw12879,Xcaw xopAF ,Xcaw xopAF 53: xopAF (complementarystrain),Xcaw avrGf1 and Xcaw xopAF avrGf1 mutantstrainswereinoculatedinto grapefruit,MexicanlimeandValencialeaves.Asshownin Figure5A,thepopulationofXcaw12879wasmuchlower comparedtoXccA306ingrapefruit.Thispopulationof Xcaw avrGf1 wasincreasedcomparedtothewildtype XooBLS256 Xcaw12879XccA306 Figure3 ComparisonoftheLPSgeneclustersof X.citri subsp. citri str.306, X.citri subsp. citri Aw12879and X.oryzae pv.oryzicolastr. BLS256. Conservedandhighlyrelatedgenes(over80%identity)arecoloredandsyntenicregionsbetweenthebacteriaareshadedingrey(over 50%identity). Jalan etal.BMCGenomics 2013, 14 :551 Page7of17 http://www.biomedcentral.com/1471-2164/14/551

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Xcaw12879andXcaw avrGf1 causedsymptomson grapefruit.However,thepopulationsofXcaw xopAF and Xcaw xopAF avrGf1 mutantswereoneordermagnitude lowerthanthatofXcaw12879andXcaw avrGf1 respectively,indicatingthatmutationof xopAF genedecreased thegrowthofXcaw12879 inplanta .Asimilartrendwas observedinMexicanlimewherethepopulationsof xopAF singleand xopAF,avrGf1 doublemutantswerelower comparedtoXcaw12879andXcaw avrGf1 respectively (Figure5B).ThegrowthofXcaw xopAF ingrapefruitand MexicanlimewasrestoredtosimilarlevelsasXcaw12879 bythecomplementation(Figure5).Nosignificantchanges wereobservedinValencialeavesasneitherXcaw12879 noranyofitsmutantsgrewwellinthesweetorangevarietyascomparedtoXccA306(Figure5C).TranscriptomeanalysesofXcaw12879andXccA306 undernutrientrich(NB)and hrp geneexpression inducing(XVM2)conditionsTodeterminethedifferentialgeneexpressionamongst thestrainsof X.citri subsp. citri ,wegrewXcaw12879 andXccA306undernutrientrichconditioninNutrient Broth(NB)andinXVM2[12].ThreebiologicalreplicatesofthestrainswereusedforRNA-Seq.Over45millionreadswereobtainedonaverageforeachsample. Aftertrimmingandmapping,approximately96%ofthe readsweremappedtothegenomes(datanotshown)indicatingthatRNA-Seqprovideshighqualityreadssuitablefor Xanthomonas transcriptomics.Ofallthereads, over6.5to14millionreadscouldbemappedfromeach sampletomRNAspecifically(Additionalfile4).This gaveanenrichmentofmRNAfrom11.3%upto28.5% foreachsample.Ithasbeensuggestedthat5 – 10million non-rRNAfragmentsenableprofilingofthevastmajorityoftranscriptionalactivityindiversespeciesincluding E.coli grownunderdiversecultureconditions[45].It wasalsofoundthatwhenRNA-Seqdatafrombiological replicatesisavailable,differentialexpressionofnumerousgenescanbedetectedwithhighstatisticalsignificanceevenwhenthenumberoffragmentspersampleis reducedto2 – 3million[45].ThusourRNA-Seqdatais likelysufficientforthetranscriptomeanalysisof XccA306andXcaw12879. Toquantifytheexpressionofeachgene,thereadsaligned toeachgenewerepooledandnormalizedforgenesizeby calculatingtheReadsPerKbperMillionreads(RPKM) values.Thevaluesforeachgenefromallthereplicateswere furtherquantilenormalizedtotestthemstatistically.The resultingvalueswerelog2transformedand t -testwas performedontheseexpressionvaluestocomparedifferentialgeneexpression(DGE)betweenXccA306and Xcaw12879underthesamegrowthconditionsorbetween thesamestrainsinNBorXVM2growthconditions.High correlationwasobservedbetweendifferentialexpression valuesofbiologicalreplicates(Additionalfile5),signifying thatthemethodwasreproducible.Principalcomponent analysisindicatesthatthebiologicalreplicatesofXccA formedaseparateclusterfromXcawinbothgrowth conditions(Additionalfile6). qRT-PCRwasusedtovalidatetheRNA-Seqdata. Eightgeneswerechosen(Additionalfile7)thatwere differentiallyexpressedinXcawascomparedtoXccA underbothNBandXVM2growthconditionstocomparedataobtainedfromthetwomethods.Theresulting Grapefruit Valencia Hamlin XccA306 Xcaw12879 Xcaw avrGf1 Figure4 Pathogenicityassay inplanta Inoculationbypressureinfiltrationof X.citri subsp. citri str.306, X.citri subsp. citri str.Aw12879and X. citri subsp. citri str.Aw avrGf1 mutantonyoungDuncangrapefruit,ValenciaandHamlinleaves.Thecultureconcentrationof108cfu/mlwasused forinoculationandleaveswerephotographedafter10daysofincubation.XccA306infectsallthreecitrusvarieties;Xcaw12879shows hypersensitivereactiononlyongrapefruit.Xcaw avrGf1 mutantshowsreducedsymptomsascomparedtoXccA306ongrapefruitandno symptomsonValenciaandHamlin. Jalan etal.BMCGenomics 2013, 14 :551 Page8of17 http://www.biomedcentral.com/1471-2164/14/551

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transcriptionalratiofromqRT-PCRanalysiswaslog2transformedtocomparewiththeDGEvaluesobtained byRNA-Seq(Additionalfile8).Althoughthescaleof foldchangesbetweenthetwotechniquesisdifferent, highcorrelationcoefficientof0.87verifiesthatthegeneraltrendofgeneexpressionisconsistentforbothdata sets. WestudiedtheexpressionprofileofXccstrainsin XVM2ascomparedtoNB.Atthecut-offof fold change =3,FDR<0.05,292genesshoweddifferential expression(173up-regulatedand119down-regulatedin XVM2comparedtoNB)inXccA(Additionalfile9)and 281genes(129up-regulatedand152down-regulatedin XVM2comparedtoNB)forXcaw(Additionalfile10). TheentireT3SSclusterconsistingoftwenty-fivegenes exceptonegene(XAC0395)wasup-regulatedinXVM2 forbothXccAandXcawstrains(Additionalfiles9and 10).Amongalltheeffectors,sixteenwereinducedfor XccAwhereasnineteeneffectorswereoverexpressedfor XcawinXVM2comparedtoinNB.Asidentifiedinthis study,theeffectorgenes avrBs2,xopA,xopE1,xopE3, xopI,xopX,xopZ1,xopAD,xopAP,xopAQ,hpaA,xopN and xopP wereup-regulatedinXVM2inbothstrains, while pthA1 pthA2 avrXacE3 and xopK wereinduced onlyinXccAand xopL,xopR,xopAI,xopAK,xopAF and xopAG onlyinXcawstrain. The11-gene xps clusterencodesfortype2secretion system(T2SS)in Xanthomonas secretingvariousenzymesincludingpectatelyase,cellulase,andxylanase. The xps genesweredown-regulatedinXVM2ascomparedtoinNBforXcaw,with xpsE beingthemost significantlydown-regulated.ForXccA,the xps genes werenotdown-regulated.BesidestheT2SSgenes,at least22genesencodingT2SSsubstratesinXccAwere overexpressedinXVM2ascomparedtoonly12in Xcaw.Tothecontrary11genesforXcawand8for XccAweredown-regulatedinXVM2comparedtoin NB(Additionalfiles9and10). Ouranalysisshowedthatalltheflagellabiosynthesis genesencodedby flg and fli ,motilityby mot andchemotaxisby mcp che and tsr wererepressedinXVM2for XccAandXcawexcept cheY (XAC3284inXccAand Figure5 XopAFcontributestothegrowthofXcaw12879strain inplanta XccA306(A),Xcaw12879(Aw),Xcaw12879 xopAF (Aw xopAF), Xcaw12879 xopAF53: xopAF (Aw AF:53:xopAF,complementstrain),Xcaw12879 avrGf1 (Aw avrGf1),Xcaw12879 avrGf1 -34: avrGf1 (Aw Gf1:34: avrGf1,complementstrain),Xcaw12879 xopAF avrGf1 (Aw xopAF avrGf1)andXacw12879 xopAF avrGf1 -34: avrGf1 -53: xopAF (Aw Gf1 AF:34: avrGf1:53:xopAF,complementstrain)wereinoculatedatapproximately106cfu/mlconcentrationinto A .Duncangrapefruit, B .Mexicanlimeand C .Valenciasweetorangeleavesusinganeedlelesssyringe.Bacterialcellsfromtheinoculatedleaveswererecoveredatdifferenttime-points, dilutedandcountedtoplotthegrowthcurve.Thevaluesateachtimepointrepresentmeansofthreereplicates.MeansSDareplotted. Jalan etal.BMCGenomics 2013, 14 :551 Page9of17 http://www.biomedcentral.com/1471-2164/14/551

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XCAW_03412inXcaw)and tar (XCAW_03417, XCAW_04009andXCAW_02497).Thegenesencoding LPSweredown-regulatedinbothXcawandXccA, whereasthexanthangum(EPS)geneswereoverexpressed inbothexcept gumP inXccA.Afewgenesencodingouter membraneproteins,whichareinvolvedinadhesion,including ompW blc and hms wereup-regulatedinXVM2as comparedtoinNBforbothstrainswhile xadA and yapH wereinducedinXccAbutdow n-regulatedinXcaw.The TypeIVpiligenesencodedby pil and fim genesexcept pilB andfilamentoushaemagglutininrelatedgenes( fhaB XAC1816 )weredown-regulatedinbothXcawandXccA (Additionalfiles9and10). Inordertofurtherunderstandthemolecularmechanismsdeterminingthedifferencesinvirulenceandhost rangeofXcawandXccA,wecomparedtheexpression profileofcommongenesofXcawandXccA.When expressionoforthologousgenesinXcawwascomparedtoXccA,603genes(426overexpressedand177 down-regulated)inNB(Additionalfile11)and450 genes(319overexpressedand131down-regulated) genesinXVM2(Additionalfile12)conditionswere significantlydifferentiallyregulatedatcut-offvalueof foldchange =3andFDR<0.05.Oncomparingthe differentiallyexpressedg enesinbothconditions,126 genesweredifferentiallyregulatedinXcawascomparedtoXccA,irrespectiveofthegrowthconditions (Figure6).Ofthese87wereoverexpressedinXcaw and39geneswererepressedascomparedtoXccA (Additionalfile13).Ofthe87genesoverexpressedin Xcaw,35werevirulence-relatedgenesincluding hrpX hrpG phoP phoQ regulatorygenes,andT2SSsubstrate genes(XAC2537,XAC2763,XAC2999,XAC4004) (Additionalfile13).Ofthe39genesoverexpressedin XccA,21werevirulence-relatedgenesincluding cellulasegenes(XAC0028,XAC0029and engXCA ),reactiveoxygenspeciesscavengingenzymegenes,e.g., superoxidedismutasegene sodC2 ,andgenesencoding heatshockproteinGrpEandheatstressproteinMuc. SincethegeneexpressionofT2SSsubstrategeneswas different,wecomparedtheproteaseandpectatelyase activitiesofXccA306andXcaw12879.Xcaw12879showed higherproteaseactivityt hanXccA306(Figure7A). Xcaw12879showedlowerpecta telyaseactivitycompared toXccA306(Figure7B).DiscussionComparativeanalysisofXcaw12879andXccA306identifiedmultiplestrain-specificgenesthatmightcontribute tothedifferencesinvirulenceandhostrange.Among thegenespresentinXcaw12879,butabsentinXccA306, twoeffectorgenes xopAG(avrGf1) and xopAF were identifiedinXcaw,XauBandXauCbutwerenotpresent inXccA306genome(Table1).ThepresenceoftheseeffectorsinlimitedhostrangestrainscausingcitruscankerandnotinthebroaderhostrangeXccA306makes themprimecandidatesforeffectorsthatcouldaffect hostspecificity.Importantly,theroleof xopAG ( avrGf1 ) inlimitingthehostrangeofXcawhasbeenconfirmed previously[7].The xopAG genebelongstothe avrGf1 familyandhasbeenshowntotriggerHRingrapefruit [7].AvrGf1inXcawshowsonlyabout45%identitytoits homologXAUC_04910inXauCwhereasthehomolog XAUB_03570inXauBisinterruptedbyatransposon andmightbenon-functional,whichprobablycontributestothebroaderhostrangeofXauBcomparedto XcawandXauC[2,3].WhenthemutantXcaw xopAG wasinoculatedingrapefruititcausedtypicalcankerlike symptomsinsteadofHR,butthesymptomswerevisibly reduced[7].Also,Xcaw avrGf1 doesnotcausedisease Over-expressedUnder-ex p ressedWNB vs. ANB WXVM vs. AXVM 8739232 92 339138426 177 319 131 Figure6 Numberofdifferentiallyexpressedgeneswhencomparingexpressionofcommongenesin X.citri subsp. citri str.Aw12879 to X.citri subsp. citri str.306inNBandXVM2growthconditions. GeneexpressionoforthologousgenesbetweenXcaw(W)andXccA(A) wascomparedwhengrowninNutrientbroth(NB,nutrientrichmedium)andXVM2(XVM,hrpinducingmedium). Jalan etal.BMCGenomics 2013, 14 :551 Page10of17 http://www.biomedcentral.com/1471-2164/14/551

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onsweetorange(ValenciaandHamlin)asshownin Figure4,indicatingthatthereareotherhostlimiting factorsintheXcaw12879genomeorothervirulencefactorsarerequiredforXccA306toinfectdifferenthosts. Anothercandidategene,whichmightcontributetohost specificity,is xopAF ,whichbelongsto avrXv3 familyand islocatedontheplasmidpXcaw58inXcaw12879.Homologsof xopAF ,XAUB_02310andXAUC_00300are foundinXauBandXauCbutnotinXccA306(Table1). Thus,weinitiallyhypothesizedthatXopAFmaycontributetorestrictinghostrangeofXcaw12879,XauB,and XauCtolimitedvarietiesofcitrusascomparedto XccA306.Additionally,an xopAF homolog avrXv3 from X.campestris pv.vesicatoriaisknowntoinduceHRin tomatolineHawaii7981andpepperplants[40].The sameworkalsoascertainedthatthegenewasplantinducibleandregulatedbythe hrp regulatorysystem.The Cterminalregionoftheproteinencodesforaputative transcriptionactivatordomainindicatingthatitmight interactwithplanthostgenes.Inthisstudywefound that xopAF mutantand xopAFavrGF1 doublemutant bothhavelowergrowth inplanta ascomparedto Xcaw12879and avrGF1 singlemutantrespectively (Figure5).Mutationof xopAF didnotmakeXcaw12879 strainpathogenicinsweetorangeValencia.Instead,mutationof xopAF slowedthegrowthofthepathogenin grapefruitandMexicanlime,whichwasrestoredby complementation,indicatingthatXopAFisimportant forbacterialgrowth inplanta .Inadditiontotheeffectorsdocumentedabove,othereffectorsthatdifferin theirpresenceare xopAQ xopE2 xopN xopP and xopAE ,presentinXcaw12879,XccA306andXauBbut notinXauCstrain.Also xopB xopE4 and xopJ1 are presentinbothXauBandXauCbutmissingfrom XccA306andXcaw12879.HowtheseeffectorscontributetovirulenceandhostrangeofXccA,Xcaw,XauB, andXauCrequiresfurtherinvestigation. OthergenecontentdifferencesbetweenXcaw12879 andXccA306includedifferencesinLPScluster(Figure3), phagerelatedgeneswithXcawcontainingXCAW_1134 toXCAW_1142,XACW_4520toXCAW_4227whereas XccAexclusivelyincludesXAC1063,XAC2628,andType IVsecretionsystemandaplant-likenatriureticpeptide (PNP)encodinggene(XAC2654).Interestingly,allthe genesinLPSclusterfromXcaw12879showhighsimilarity withLPSregionfromricepathogen X.oryzae pv.oryzicola BLS256,whereasonlyapproximat elyhalftheclusterissyntenictoXccA306LPScluster(Figure3).Thissuggeststhat HGThasprobablyresultedinahybridLPSclusterin Xcaw12879similarto X.oryzae pv.oryzicolaBLS256[46]. LPS,phagerelatedproteins,typeIVsecretionsystemand PNPhavebeenreportedtoplaycertainrolesinvirulence [18,47-51].HowtheycontributetothedifferenceofXcaw andXccAinvirulenceandhostrangeremainstobeinvestigatedexperimentally. Virulencerelatedgenesweredifferentiallyregulatedin XVM2ascomparedtoNBforbothXcaw12879and XccA306.InXccA306(Additionalfile9),fifty-nine virulencerelatedgeneswereinducedandthirty-eight geneswererepressedinXVM2comparedtoNB.In XccA306Xcaw12879A B Figure7 ProteaseandPectatelyaseactivityof X.citri subsp. citri str.306,and X.citri subsp. citri str.Aw12879.(A) Proteaseactivitywas testedbyinoculating1 lcultureon10%milkagarplatesat28Cfor6days.Zoneofclearancewasusedasthemeasureofproteaseactivity. (B) Pectatelyaseactivitywastestedbyinoculating1 lcultureonHildebrand ’ sagarmediumat28Cfor6days.Morepittingcanbeseenon mediumatpH8.5forXccAstraincomparedtoXcaw. Jalan etal.BMCGenomics 2013, 14 :551 Page11of17 http://www.biomedcentral.com/1471-2164/14/551

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Xcaw12879(Additionalfile10),fortyvirulencerelated geneswereinducedandtwenty-fourgeneswererepressedinXVM2comparedtoNB.Theinductionofthe virulencegenesinXVM2conditioncomparedtonutrientrichNBissupportedbyapreviousstudy[52].Inthe previousstudy,only279genesofXccApotentiallyassociatedwithpathogenicityandvirulenceweretestedand 31geneswereup-regulatedinXVM2,whileonly7genes wererepressed.Inourstudy,wefurtherexpandedthe previousstudybyincludingallgenesofXccAandprovidedacomprehensivepictureof Xanthomonas gene regulation. TheentireT3SSclusterconsistingoftwenty-fivegenes exceptone(XAC0395)wasup-regulatedinXVM2for bothXccAandXcawstrains.Thisisconsistentwithpreviousreportthat Xanthomonashrp geneswereinduced inXVM2[52,53].However,onlyeight hrp genesof XccAwerereportedtobeup-regulatedbyXVM2inthe previousstudy[52]comparedto24induced hrp genes identifiedinthisstudy.Amongalltheeffectors,16 wereinducedforXccAwhereas19effectorswere overexpressedforXcawinXVM2.Inthepreviousstudy [52],onlythreeeffectorgenes avrXacE1 avrXacE2 ,and Xac0076 ofXccAwereinducedinXVM2.Thus,our studyfurtherexpandedtheknowledgeofexpressionof the hrp andeffectorgenesinXVM2medium. Interestingly,both hrpX and hrpG geneswere overexpressedintheXcawcomparedtoXccA (Additionalfile13).Bothgeneshavebeenshowntobe criticalforvirulencein Xanthomonas spp.[54].The hrpX geneencodesanAraC-typetranscriptionalactivatorand hrpG geneencodesanOmpRfamilyregulator,whichareknowntoregulatemanyvirulence relatedgenesincludingT3SSeffectors,T2SSsubstrate, flagella,andchemotaxisgenes[55].Overexpressionof Xcaw hrpG in X.perforans elicitedHRingrapefruit andMexicanlimeleavesprobablybyinducing xopA andotheravirulencegenes[7].The xopA geneencodes harpinandwassuggestedtobeahost-limitingfactor byinducingHR.Itshomologues hpaG and hrpN are alsoknowntoinduceHR.However,the xopA genewas notoverexpressedsignifi cantlyinXcawcomparedto XccA(Additionalfile14).Thefoldchangeof xopA was morethan2,buttheFDRdidnotpassthecutoff value.Fiveothereffectorgenes xopL xopX xopAD hrpW ,and xopAQ wereoverexpressedinXcawin XVM2,whereasonlyoneeffectorgene xopAP wasinduced inXccAinNB(Additionalfile14).Overexpressionofthose effectorgenesinXcawmightcontributetothelimitedhost rangeofXcaw.Inaddition,the phoP phoQ twocomponent systemgeneswereoverexpressedinXcawcomparedto XccA(Additionalfile13).The phoP geneencodingaresponseregulatorispredictedtointeractwithvarioussignalsensorproteinsinadditiontoPhoQ.Itisknowntoactivate theresponseregulator hrpG in X.oryzae pv.oryzae,thus leadingtoactivationofvariousvirulenceandgrowthfactor genesdownstream[56].The phoQ geneontheotherhand isrequiredfortheactivityofAvrXA21in X.oryzae pv. oryzae,whichdetermineshost-variationofthestrain againstsomericelines[56].ThusinXcaw,overexpression of phoP-phoQ couldcontributetoact ivationofcertaineffectorgenesmentionedabove. T2SSisthemajorproteinsecretionsystem,whichsecretestoxinsandvariousdegradativeenzymestobreakdownthecellwallinplanthosts[20].T2SSandits substrateshavebeenshowntobeimportantforthevirulenceofXccA[57].The xps genesweredown-regulated inXVM2ascomparedtoinNBforXcawwith xpsE beingthemostsignificantlydown-regulated(Additional file10).XpsEisknowntobeakeycomponentofT2SS, thelossofwhichleadstolowervirulencein X.oryzae [4].ForXccA,the xps geneswerenotdown-regulated. Down-regulationof xps genesinXcawbutnotinXccA mightcontributetodifferencesinvirulenceondifferent hostsofXcawandXccA.InXccAatleast22genes encodingT2SSsubstrateswereoverexpressedascomparedtoonly12inXcaw.Onthecontrary11genesfor Xcawand8forXccAweredown-regulated.Thisis similartothepreviousstudywheregenesencoding T2SSsubstrateswerefoundeitherdown-regulatedor up-regulatedinXVM2[52].Specifically,fourT2SSsubstrateproteasegenes(XAC2537,XAC2763,XAC2999, andXAC4004)wereup-regulatedinXcawcompared toXccAinbothconditions(Additionalfile13). Consequently,Xcawshowedhigherproteaseactivity thanXccA(Figure7A).Incontrast,multiplecellulase genes(XAC0028,XAC0029,and engXCA )weredownregulatedinXcawcomparedtoXccA(Additionalfile 13).Pectatelyasegene pel (XAC03562)wasalsodownregulatedinXcawcomparedtoXccAinNBmedium (Additionalfile11).Consequently,Xcawshowedlower pectatelyaseactivityascomparedtoXccA(Figure7B). Thus,theproteaseandpectatelyaseactivitiesare consistentwiththedifferentialregulationofgenes encodingT2SSsubstratesinXcawandXccA. ComparedtoXcaw,multiplevirulencegeneswere overexpressedinXccAwhichmightcontributetoits adaptiontoabroadhostrange(Additionalfile13). Theseincludemanyreactiveoxygenspecies-scavenging enzymegenes,e.g., sodC2 and grpE ,whichindicatesthat XccAmightbemoreadaptedtostressfulconditionsdue tothehostdefenseresponsesofdifferenthosts. Attachmentof Xanthomonas toplantcellsurfacesis importantforpathogenicity[58,59].Multiplegenes involvedinadherencewereoverexpressedinXccAin NBmedium(Additionalfile11)includingfilamentous haemagglutiningene fhaB gum genes( gumB to gumK gumM ),chemotaxisgenes(XAC0611,XAC1666,Jalan etal.BMCGenomics 2013, 14 :551 Page12of17 http://www.biomedcentral.com/1471-2164/14/551

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XAC1891,XAC1893,XAC1894,XAC1895,XAC1896, XAC1897,XAC1899,XAC1900,XAC1902), mcp genes(XAC1996,XAC2448,XAC2866,XAC3132), cheA (XAC2865), cheR (XAC1890), cheR (XAC2869), cheY (XAC1904)and cheD (XAC1889).Multipletransporter genes,whichareknowntoplaycriticalrolesinbacteriato acquirenutrientsfromtheintercellularenvironment,were overexpressedinXccAinXVM2ascomparedtoXcaw,e.g., thepotassiumtransportergenes kdpB kdpC and kdpD and theironsiderophoretransportergene fhuA (XAC2185)and XAC2830(Additionalfile12).Altogether,theymightcontributetothevirulenceonbroadhostofXccAascompared toXcaw.ConclusionsInconclusion,comparativegenomicanalysisofXcaw12879, XauB,XauC,andXccA306providesinsightsintothevirulencemechanismof X.citri subsp .citri. Ourstudyindicated thatAvrGf1mainlycontributestothehostrangelimitation ofXcaw12879whereasXopAFcontributestovirulence.In addition,wecomparedthege neexpressionprofilesof XccA306andXcaw12879inNBandXVM2.Ourdata demonstratedthatvirulenceg enesincludinggenesencodingT3SSanditseffectorsareinducedinXVM2medium. NumerousgeneswithdifferentialexpressioninXcaw12879 andXccA306wereidentified.Thisstudylaysthefoundationtofurthercharacterizet hemechanismsforvirulence andhostrangeofstrainsof X.citri subsp .citri andother bacterialpathogens.MethodsPhylogeneticandcomparativeanalysisThededucedproteinsequencesofninehousekeeping genes( uvrD,secA,carA,recA,groEL,dnaK,atpD,gyrB and infB )from13completelysequencedand10draft Xanthomonas spp.,andthree Xylellafastidiosa strains (out-groupspecies)wereusedtoconstructthephylogenetictree.Aminoacidsequenceswerealignedusing ClustalW2.1[60].Aphylogenetictreewasconstructed fromtheconcatenatedsequencesusingCLCGenomics Workbenchv6.0(CLCBio,Aarhus,Denmark)bythe maximumlikelihoodmethod.Comparativeanalysesof XccA306andXcaw12879wasconductedby,atwo-way BLASTofthenucleotidesequencestoidentifyunique genesineachstrainusingthestandaloneblast+software(ncbi-blast-2.2.4).Thegeneswereconsidered orthologousifreciprocalTBLASTNhitswerefoundbetweentwogeneswithe-valuelessthanorequalto10-10andalignmentsexceeding60%sequenceidentityand 60%querygenelength.Agenewasconsideredsingleton oruniquetoeachstrainifithadnohitsorwithan e -valueslessthanorequalto10-5[61,62].The CRISPRfinderserver[63]wasusedtoidentifyCRISPRs. Onlyconfirmedstructuresarereportedhere.Alignment betweenwholechromosomeswasdoneusingthescript PromerfromtheMUMmerpackage[64].Promerdoes alignmentsbetweentranslatednucleotidesequences.PreparationofRNAsamplesfortranscriptomeanalysisRNAsamplepreparationandcDNAlibrarygeneration wereperformedaccordingtoproceduresoutlinedby Filiatraultetal.[65]withsomemodifications.RNAsampleswereextractedfromXccA306andXcaw12879 growntoOD600of0.4inXVM2mediumandNB mediumat28Conshakerat200rpm.Thestarting OD600foreachculturewas0.03.Threebiological replicatesofeachstrainineachmediumwereusedfor RNAextraction.WhentheOD560reached0.4foreach condition,RNAwasstabilizedimmediatelybymixing10 mloftheculturewithtwovolumesofRNAprotect bacterialreagent(Qiagen,Valencia,CA).Thecellswere centrifugedat5000gat4Candcellpelletswere treatedwithlysozymeandRNAextractionswere performedusingRiboPurebacteriakit(Ambion,Austin, TX)permanufacturers ” instructions.GenomicDNA wasremovedbytreatmentwithTURBODNA-freekit (Ambion,Austin,TX).TotalRNAsampleswerequantifiedusingspectrophotometry(NanodropND-1000, NanoDropTech.Inc.).RNAqualitywasassessedusing theAgilent2100bioanalyzer(AgilentTechnologies,Palo Alto,CA).MRNAenrichmentandlibraryconstructionmRNAwasenrichedfromtotalRNAusingMicrobExpress kit(Ambion)toremovethe23Sand16SribosomalRNAs (rRNAs).RemovalofrRNAswasassessedusinganAgilent Bioanalyzer.DoublestrandedcDNAsynthesiswas performedfollowingtheIlluminamRNASequencingsamplepreparationguide(Cat.No.RS-930-1001)inaccordancewiththemanufacturer ’ sstandardprotocol.Enriched mRNAwasfragmentedviaincubationfor5minat94C withtheIllumina-suppliedfragmentationbuffer.Thefirst strandofcDNAwassynthesizedbyreversetranscription usingrandomoligoprimers.Second-strandsynthesiswas conductedbyincubationwithRNAseHandDNApolymeraseI.TheresultingdsDNAfragmentswerefurther end-repaired,andA-nucleotideoverhangswereadded. AftertheligationofIlluminaadaptors,thesampleswere runonadenaturinggelandthebandcorrelatingto200 (25)basepairsonthedenaturedDNAladderwasselected.TheselectedDNAconstructswereamplifiedby PCRusingtheprimersprovidedintheIlluminalibrarykit. Theamplifiedconstructswerepurifiedandthelibrarywas validatedusingAgilent2100bioanalyzer.IlluminasequencingandalignmentPaired-end,75-cyclesequencingofthelibrarieswas performedusinganIlluminaGAIIxatYaleCenterforJalan etal.BMCGenomics 2013, 14 :551 Page13of17 http://www.biomedcentral.com/1471-2164/14/551

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GenomicAnalysis.TherawsequencingreadswerefurtheranalyzedusingCLCGenomicsWorkbenchv6.0 (CLCBio,Aarhus,Denmark).Thereadsweretrimmed usingthequalityscorelimitof0.08andmaximumlimit of2ambiguousnucleotides.Thetrimmedreadswere mappedtothegenomeandtheprotein-codinggenes ofXccA306(GenBankaccessionno.NC_003919, NC_003921.3andNC_003922.1)andXcaw12879,with theparametersallowingmappingofreadstothe genomewithupto2mismatches.Thereadsmapped torRNAandthereadsnotuniquelymappedwereremovedfromfurtheranalysis.Theexpressionlevels wereevaluatedbyRPKMmethodasdescribedby Mortazavietal.[66].DifferentialgeneexpressionanalysisThedifferentialgeneexpressionofthepooledsamples fromeachconditionwasanalyzedusingCLCGenomics Workbenchv6.0(CLCBio,Aarhus,Denmark).RPKM valueswerenormalizedusingquantilenormalization andfurtherlog2transformedforstatisticalanalysis.Box plots,hierarchicalclusteringofsamplesandprincipal componentanalysisweredonetoexaminedataquality andcomparability.A t -testwasperformedonlog2transformeddatatoidentifythegeneswithsignificant changesinexpressionbetweenthetwogrowthconditionsandbetweenthetwostrains.The p-values were adjustedforthefalsediscoveryrate(FDR)usingthe BenjaminiandHochbergmethod[67].Quantitativereversetranscription-PCR(qRT-PCR)ToverifytheRNA-Seqresult,qRT-PCRassayswere carriedoutusingthesamesetsofRNAforRNA-Seq analysis.GenespecificprimerslistedinAdditionalfile7 weredesignedtogeneratesequencesof100 – 250bp inlengthfromtheXccA306genome.qRT-PCRwas performedforall3biologicalreplicatesofXccA306 andXcaw12879growninNBandXVM2ona7500 fastreal-timePCRsystem(AppliedBiosystems)using QuantiTect ™ SYBRGreenRT-PCRkit(Qiagen)following themanufacturers ’ instructions.16SrRNAwasusedasan endogenouscontrol.Thefoldchangeofgeneexpression wascalculatedbyusingtheformula2C T[68].Thefold changewasfurtherlog2transformedtocomparewiththe RNA-Seqdata.Generationofthe xopAF mutantand xopAF,avrGf1 doublemutantToconstructthe xopAF deletionmutant,the1096-bp fragmentcontainingentire xopAF genewasamplified usinggenomicDNAofXcaw12879astemplateand primersxopAFF1andxopAFR.ThisresultedinF1, containinga Bam HIrestrictionsitewithinthe xopAF gene.A422bpfragmentcontaining337bpof xopAF geneanditsdownstreamregionwasamplifiedfurther fromF1usingprimersxopAFF2Bam HIandxopAFR (Additionalfile7),resultinginF2.BothF1andF2were digestedwith Bam HIandfragmentsF3(414bp)andF4 (500bp)weregelpurified.Thefragmentswereligated andclonedintopGEM-Teasyvector,resultinginthe constructnamedpGEMxopAF thatwasconfirmedby PCRandsequencing.FrompGEMxopAF ,an Apa IPst Ifragmentcontaining xopAF genewith192bpinternaldeletionwastransferredinto Apa IPst Idigested suicidevectorpNTPS138,resultinginpNTPSxopAF TheconstructpNTPSxopAF wastransformedinto E. coli DH5 PIR.Theconstructwaspurifiedfrom E.coli andsubsequentlytransferredintoXcaw12879and Xcaw12879 avrGf1 generatedinapreviousstudy[7]by electroporation.TransformantswereselectedonNA mediumsupplementedwith50 g/ lkanamycin.PositivecolonieswerereplicatedonbothNAplates supplementedwith5%(w/v)sucroseandkanamycin, andonlyNAandkanamycin.ThesucrosesensitivecolonieswereselectedfromNApluskanamycinplateand growninNBmediumovernightat28C.Theculture wasthendilution-platedonNAcontaining5%sucrose toselectforresolutionoftheconstructbyasecond cross-overevent.Theresultingdeletionmutantof xopAF anddoublemutantof xopAF and avrGf1 was confirmedbyPCR(datanotshown).Thecomplete xopAF and avrGf1 geneswerecomplementedinthe singleanddoublemutantsusingpUFR053andpUFR034 respectively.Theresultingcomplementstrainswere Xcaw12879 xopAF -53: xopAF ,Xcaw12879 avrGf1 -34: avrGf1 andXcaw12879 avrGf xopAF -34: avrGf1 -53: xopAF wereusedinthisstudy.PathogenicityassayPathogenicityassayswereconductedinaquarantine greenhousefacilityatCitrusResearchandEducation Center,LakeAlfred,FL.XccA306,Xcaw12879,and Xcaw avrGf1 strainsweregrownwithshakingovernight at28CinNB,centrifugeddownandsuspendedinsteriletapwaterandtheconcentrationswereadjustedto 108cfu/ml.Thebacterialsolutionswereinfiltratedinto fullyexpanded,immatureleavesofDuncangrapefruit, ValenciasweetorangeandHamlinsweetorange,with needlelesssyringes[54].Thetestwasrepeatedthree timeswithsimilarresults.Diseasesymptomswere photographed10dayspostinoculation.Growthassay inplantaXccA306,Xcaw12879,Xcaw xopAF ,Xcaw xopAF -53: xopAF, Xcaw avrGf1 andXcaw xopAF avrGf1 strains weregrownwithshakingovernightat28CinNB, centrifugeddownandsuspe ndedinsteriletapwaterJalan etal.BMCGenomics 2013, 14 :551 Page14of17 http://www.biomedcentral.com/1471-2164/14/551

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andtheconcentratio nswereadjustedto106cfu/ml. Thebacterialsolutionswereinfiltratedintofully expanded,immatureleavesofDuncangrapefruit, MexicanlimeandValenciasweetorangewith needlelesssyringes[54].Toevaluatethegrowthof variousXccstrainsandmutantsintheseplants2inoculatedleaveswerecollectedfromeachplantat0,2,4, 7,10,14and21days.1cm2leafdisksfrominoculated leaveswerecutwithacorkborerandthengroundin1 mlsterilewater.Thesewereseriallydilutedandplated onNAplates.Thebacterialcolonieswerecounted after3-dayincubationat28C.Thetestwasrepeated threetimesindependently.PectatelyaseandproteinaseassayXccA306andXcaw12879weregrownonnutrientagar at28C,thensuspendedinsteriledeionizedwatertothe O.D.of0.3at560nm.Hildebrand ’ smediumA,BandC wereusedtotestforpectolyticactivity[69].Inshortthe mediumcontainedbromothymolbluedye,calcium chloride,2%sodiumpolypectateand0.4%agar.ThepH wasadjustedto4.5,7.0and8.5forthemediumA,B andC.One lofthecultureswereinoculatedonto theplatesandincubatedat28Cfor6daysbefore confirmingpittingduetopectatelyaseproduction.10% skimmilkagarwasusedtotestthebacterialprotease activity.Theculturesweregrownandsuspendedin sterilewaterasexplainedabove.One lofthecultures wereinoculatedontotheskim-milkplatesandcultured at28Cfor6daystoobserveproteaseactivity.AvailabilityofsupportingdataThegenomesequencesof Xanthomonascitri subsp. citri strainAw12879areavailableatGenBankundertheaccessionnumbersCP003778,CP003779andCP003780. TheRNA-Seqdatafromthisstudyareavailableinthe NCBI ’ sGeneExpressionOmnibusdatabaseunderthe accessionnumberGSE41519.AdditionalfilesAdditionalfile1: Clusteredregularlyinterspacedshortpalindromic repeats(CRISPRs)in X.citri subsp. citri str. Aw12879genome predictedusingCRISPRfinder. Additionalfile2: Dot-Plotcomparisonofuniquecluster4from Xcaw12879andgenomeof X.campestris pv.campestrisstrain8004 doneusingMUMer. Reddotsrepresentundisturbedsegment conservationwhereasbluedotsindicateinversion. Additionalfile3: PredictionandcomparisonoftheTALeffector codesencodedby pthA genesof X.citri subsp. citri str. 306,and X. citri subsp. citri str.Aw.PanelA:PredictionofTALeffectorcodesof PthAw1andPthAw2.PanelB:TheknownTALeffectorcodesofPthA genesfromXccA.PanelC:ComparisonoftheTALeffectorcodesof PthAw2andPthA4,homologsinXcaw12879andXcc306respectively. Additionalfile4: SummaryofRNA-SeqdataofXcaw12879and XccA306inNBandXVM2. Additionalfile5: CorrelationbetweenbiologicalreplicatesforRNASeq. Additionalfile6: PrincipalcomponentanalysisofDEGof X.citri subsp. citri str. 306(A),and X.citri subsp. citri str.Aw12879(W)under NBandXVM2conditions. Additionalfile7: Primersusedinthisstudy. Additionalfile8: RNA-seqvalidationbyqRT-PCR. Comparisonof geneexpressionbyqRT-PCRandRNA-seq.Thelog2-foldchangeofeach genewasderivedfromcomparisonofeitherWNBvsANBorWXVM2vs AXVM2.The16SrRNAgenewasusedasanendogenouscontrolinqRTPCR.Valuesoflog2foldchangearemeansofthreebiologicalreplicates. Errorbarsindicatestandarddeviation.Bluebarsrepresentvaluesfrom RNA-seqandyellowbarsarevaluesfromqRT-PCR. Additionalfile9: Differentiallyexpressedgenesof X.citri subsp citri str. 306(A)inXVM2mediumascomparedtoNB. Additionalfile10: Differentiallyexpressedgenesof X.citri subsp citri str. Aw12879(W)inXVM2mediumascomparedtoNB. Additionalfile11: Differentiallyexpressedgenesbetween X.citri subsp. citri str. 306(A)and X.citri subsp. citri str.Aw12879(W)inNB medium. Additionalfile12: Differentiallyexpressedgenesbetween X.citri subsp. citri str. 306(A)and X.citri subsp. citri str.Aw12879(W)inXVM2 medium. Additionalfile13: Shareddifferentiallyexpressedgenesbetween X.citri subsp. citri str. 306(A)and X.citri subsp. citri str.Aw12879(W)in bothNBmediumandXVM2. Additionalfile14: Differentialexpressionofeffectorgenes between X.citri subsp. citri str. 306(A)and X.citri subsp. citri str. Aw12879(W)inbothNBmediumandXVM2medium.FDRvaluesarein parenthesis.Theeffectorgenesthatpasscut-offvalueof0.05aremarked ingreen. Competinginterests Theauthorsdeclarethattheyhavenocompetingfinancialinterests. Authors ’ contributions Conceivedanddesignedtheexperiments:NWandNJ,Performedthe experiments:NJandMOA,Analyzedthedata:NJ,NW,DK,FY,JBJ,FFW,JCS andJHG,Wrotethepaper:NJ,NW,JBJ,FFW,JCSandJHG.Allauthorsread andapprovedthefinalmanuscript. Acknowledgements ThisworkhasbeensupportedbyFloridaCitrusResearchandDevelopment FoundationandUnitedStatesDepartmentofAgriculture-CooperativeState ResearchEducationandExtensionServicesSpecialCitrusCankerGrant Project73402.Supportwasalsoprovided,inpart,byAFRIgrant2012-67013 -19384fromUSDANIFA(FW,JJ,NW,WF). Authordetails1CitrusResearchandEducationCenter,DepartmentofMicrobiologyandCell Science,UniversityofFlorida,700ExperimentStationRoad,LakeAlfred,FL 33850,USA.2WaksmanGenomicsCoreFacility,RutgersUniversityBusch Campus,Piscataway,NJ08854,USA.3ICBR,UniversityofFlorida,Gainesville, FL32611,USA.4DepartmentofPlantPathology,UniversityofFlorida, Gainesville,FL32611,USA.5DepartmentofSoilandWaterScience,Citrus ResearchandEducationCenter,UniversityofFlorida,700ExperimentStation Road,LakeAlfred,FL33850,USA.6DepartmentofPlantPathology,Kansas StateUniversity,4024ThrockmortonHall,Manhattan,KS66506,USA.7DepartamentodeBioqumica,InstitutodeQumica,UniversidadedeSo Paulo,SoPaulo,SP05508-000,Brazil.8VirginiaBioinformaticsInstitute, VirginiaPolytechnicInstituteandStateUniversity,Blacksburg,VA24060-0477, USA. Received:10May2013Accepted:6August2013 Published:14August2013Jalan etal.BMCGenomics 2013, 14 :551 Page15of17 http://www.biomedcentral.com/1471-2164/14/551

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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 Jalan etal.BMCGenomics 2013, 14 :551 Page17of17 http://www.biomedcentral.com/1471-2164/14/551