52-2014.11.15-Gene苎麻根侵染线虫转录组测序分析

北京百迈客苎麻根侵染线虫转录组测序分析

Gene552(2014)67–74

ContentslistsavailableatScienceDirect

Gene

journalhomepage:/locate/gene

Genome-widetranscriptional

changesoframie(BoehmerianiveaL.Gaud)inresponsetoroot-lesionnematodeinfection

SiyuanZhu,ShouweiTang ,QingmingTang,ToumingLiu

InstituteofBastFiberCropsandCenterofSouthernEconomicCrops,ChineseAcademyofAgriculturalSciences,Changsha410205,China

articleinfoabstract

Ramie berextractedfromstembarkisoneofthemostimportantnatural bers.Theroot-lesionnematode(RLN)Pratylenchuscoffeaeisamajorramiepestandcauseslarge beryieldlossesinChinaannually.TheresponsemechanismoframietoRLNinfectionispoorlyunderstood.Inthisstudy,weidenti edgenesthatarepotentiallyinvolvedintheRLN-resistanceinramieusingIlluminapair-endsequencingintwoRLN-infectedplants(Inf1andInf2)andtwocontrolplants(CO1andCO2).Approximately56.3,51.7,43.4,and45.0millionsequencingreadsweregeneratedfromthelibrariesofCO1,CO2,Inf1,andInf2,respectively.Denovoassemblyforthese196millionreadsyielded50,486unigeneswithanaveragelengthof853.3bp.Atotalof24,820(49.2%)parisonofgeneexpressionlevelsbetweenCOandInframierevealed777differentiallyexpressedgenes(DEGs).Theexpressionlevelsof12DEGswerefurthercon rmedbyreal-timequantitativePCR(qRT-PCR).Pathwayenrichmentanalysisshowedthatthreepathways(phenylalaninemetabolism,caroten-oidbiosynthesis,andphenylpropanoidbiosynthesis)werestronglyin uencedbyRLNinfection.Aseriesofcan-didategenesandpathwaysthatmaycontributetothedefenseresponseagainstRLNinramiewillbehelpfulforfurtherimprovingresistancetoRLNinfection.

©2014PublishedbyElsevierB.V.

Articlehistory:

Received11July2014

Receivedinrevisedform27August2014Accepted5September2014

Availableonline8September2014Keyword:

Root-lesionnematodeRamie

IlluminasequencingTranscriptome

Differentiallyexpressedgenes

1.Introduction

Plantparasiticnematodesaremajoragriculturalpestsandcancauseupto20%lossesinawidevarietyofcropsworldwide.Annuallossesof125billionUSDhaveoccurredbecauseofnematodeinfestations(Chitwood,2003).Root-lesionnematode(RLN,PratylenchusFilipjev)isoneofthethreemostdevastatingplantparasiticnematodes.Itisdis-tributedworldwideandhasabroadhostrange,includingseveralim-portanttropicalandtemperatecropspecies.RLNisamigratory,endoparasiticnematode.Itdevelopsmainlyinthecorticalparenchymawhereitcausessevererootdamageandhinderstheabsorptionofwaterandnutrientsfromthesoil,resultingintheinhibitionofplantgrowth.Thereare97describedspeciesofRLN(Yuetal.,2012).

ToimprovethetoleranceofcropstoRLN,itisessentialtominedisease-resistancegenesandtoelucidatedefenseresponsemechanismsofcropstoRLNinfection.However,fewRLN-resistancegeneshavebeenidenti edandnonehavebeencharacterized(Yuetal.,2012).AmongtheRLN-resistancegenesreported,mostwerediscoveredusingthe

Abbreviations:RLN,root-lesionnematode;Inf,plantinfectedwithP.coffeae;CO,controlplants;DEG,differentiallyexpressedgene;qRT-PCR,real-timequantitativePCR;COG,TheClustersofOrthologousGroupsdatabase;GO,TheGeneOntologydatabase;KEGG,TheKyotoEncyclopediaofGenesandGenomesdatabase;RPKM,readsperkilobaseofexonmodelpermillionmappedreads;ROS,reactiveoxygenspecies. Correspondingauthors.

E-mailaddresses:zhusiy2015@(S.Zhu),cesc2012@(S.Tang),cstqm@(Q.Tang),liutouming@(T.Liu).geneticmappingmethodinwheat(Schmidtetal.,2005;Williamsetal.,2002;Zwartetal.,2005,2006,2010),barley(Sharmaetal.,2011),andbanana(Sundararaju,2010).TheseresistancegenescanbedirectlyusedtoimprovetheRLN-toleranceofplantsbymarker-assistedselectionincropbreeding.However,thesegenesarenothelpfulindeterminingdefensemechanismsofplantstoRLNinfectionbecausetheyhavenotbeenclonedandtheirmolecularfunctionsareunknown.

Ramie(Boehmerianivea),commonlyknownasChinagrass,isoneofthemostimportantnatural bercrops.Ramie bersarestrippedfromthestembast,withmanyexcellentcharacteristicssuchassmoothandlong,andhaveexcellenttensilestrength,whichisthereasonwhyramieiswidelycultivatedinChina,India,andotherSoutheastAsianandPaci cRimcountriesforitshigh berquality.InChina,ramieisthesecondmostimportant bercrop,withitsgrowthacreageandquantityof berproductionsecondonlytocotton.Thegrowthoframiecanbeaffectedbybothbioticandabioticfactors,includingdrought,anthracnoseinfectionandRLN-infection(Liuetal.,2011;Zhuetal.,2012).Morphologicalandphysiologicalresponsesoframietodroughtstresshavebeenextensivelystudied(Liuetal.,2005,2013a).Changesinwhole-genomeexpressioninramieunderwaterde citcon-ditionshavebeencharacterizedbyIlluminatag-sequencing(Liuetal.,2013b).Usingsuppressionsubtractivehybridization,132genesin-volvedinanthracnoseresistancehavebeenidenti edinramie(Wangetal.,2012).

Pratylenchuscoffeaeisamajorroot-lesionnematodethatinfectsramie.P.coffeaeinfectioninramiecanleadtogreatyieldlossorplant

/10.1016/j.gene.2014.09.0140378-1119/©2014PublishedbyElsevierB.V.

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68S.Zhuetal./Gene552(2014)67–74

mortality.P.coffeaecausesdamagetotheroots,whichinhibitsthegrowthofplants.Italsocausesremarkablechangesinstemshape,resultinginasigni cantdecreaseinbast beryield(Zhuetal.,2012).ElucidatingthemechanismofinhibitionofstemelongationbyRLN-infectionofrootwillbehelpfulinimprovingresistanceoframietoRLNinfectionandincreasing beryieldintheRLN-infectedramie.How-ever,uptonow,themechanismofinhibitionofgrowthbyRLN-infectionispoorlyunderstood.Inthisstudy,thegenesassociatedwithdefensemechanismsintworamieplantsinfectedwithP.coffeae(Inf)andtwouninfectedplantswhichservedascontrols(CO)were rstidenti edbyIlluminatechnology.ThisstudywillbehelpfulforfurtherelucidatingthemoleculardefensemechanismsoframietoRLNinfec-tionandimprovingRLN-resistanceoframie.2.Materialsandmethods

2.1.Plantmaterial,RLNinfection,andRNAextraction

EliteramievarietyZhongzhu1wasusedinthisstudy.Zhongzhu1isanelitevarietywithhighyieldandgood berquality.IthashadthelargestgrowthareainChinaintherecentyears.CuttageseedlingsofZhongzhu1weretransplantedtopotsinMarch2013.InMay2013,tenpotted60-day-oldplantswereusedforexperiments.Fivepottedramiewereinfected(Inf)byinoculationwith200P.coffeaeRLNperplant.Theother vepottedplantswerenotinfectedandactedascon-trols(CO).Sevendaysafterinfection,thewholeplants,excludingroots,ofthetenpottedramiewereseparatelycollected.Thetissueswereimmediatelyfrozeninliquidnitrogenandstoredat 80°Cuntiluse.TwoCOtreatmentsandInftreatmentswereeachreplicatedtwice(CO1,CO2andInf1,Inf2)forIlluminasequencing.TheotherthreeCOandInfplantswereusedastriplicateforqRT-PCR.ThetissuescollectedforeachsamplewereextractedindividuallyforRNAusingTRIzolre-agent(TransgeneCompany,IllkirchGraffenstadenCedex,France)ac-cordingtothemanufacturer'sprotocol.TheRNAwasstoredat 80°C.2.2.cDNAlibraryconstructionandsequencing

IlluminasequencingwasperformedatBiomarkerTechnologiesCO.,LTD,Beijing,China(/).Brie y,RNAfromthefoursampleswereusedseparatelytoconstructthecDNAlibrarieswithfragmentlengthsof200bp(±25bp).Paired-endsequencingwasthenperformedusingtheIlluminasequencingplatform(HiSeq 2000)accordingtothemanufacturer'sinstructions(Illumina,SanDiego,CA).TherawsequencingdataofthefoursamplesaredepositedinNCBISequenceReadArchive(SRA,http://www.ncbi.nlm.nih.gov/Traces/sra)withaccessionnumbersSRR1021565,SRR1021566,SRR1021567,andSRR1021568,respectively.2.3.Data lteringanddenovoassembly

Readsfromeachlibrarywereassembledseparately.Thetrimmingadaptersequenceswereremovedandlow-qualityreads(lessthan13bporreadswithunknownnucleotidesabove5%)were lteredwiththesoftwaredevelopedbytheBiomarkerTechnologyCompany.DenovoassemblywascarriedoutusingTrinity(Grabherretal.,2011).Trinityusesthreesoftwaremodules,Inchworm,Chrysalis,andButter y,sequentiallytoprocesslargevolumesofRNA-Seqreads.Inthe rststep,readsareassembledintocontigsbytheInchwormpro-gram.TheminimallyoverlappingcontigswereclusteredintosetsofconnectedcomponentsbytheChrysalisprogram,andthenthetran-scriptswereconstructedbytheButter yprogram(Grabherretal.,2011).Finally,thetranscriptswereclusteredbysimilarityofcorrectmatchlengthbeyondthe80%oflongertranscriptor90%ofshortertran-scriptusingthemultiplesequencealignmenttoolBLAT(Kent,2002).Theunigenewasde nedasthelongesttranscriptofeachcluster.

EMBOSSGetorfSoftwarewasusedtopredictitscodingregions(http://emboss.bioinformatics.nl/cgi-bin/emboss/getorf).2.4.Geneannotationandanalysis

Forannotationofunigenesusingvariousbioinformaticsapproaches,theunigeneswere rstsearchedagainsttheNr,theClustersofOrthologousGroups(COG)database,theSwiss-Protproteindatabase,andtheKyotoEncyclopediaofGenesandGenomes(KEGG)databaseusinglocalBLASTxwithanE-valuecutoffof10 5.WithNrannotation,theBlast2GOprogram()wasusedtogettheGeneOntology(GO)annotationaccordingtomolecularfunction,bi-ologicalprocess,andcellularcomponentontologies(Conesaetal.,2005).TheWEGOsoftware(/cgi-bin/wego/index.pl)wasthenusedtoperformGOfunctionalclassi cationofallunigenesandDEGstoviewthedistributionofgenefunctions.ThesequenceofallunigenesandDEGswerealsoalignedtotheCOGda-tabasetopredictandclassifypossiblefunctions.2.5.Digitalgeneexpressionanalysis

GeneexpressionlevelsweremeasuredintheRNA-Seqanalysisasreadsperkilobaseofexonmodelpermillionmappedreads(RPKM)(Mortazavietal.,2008).TheDEseqsoftwarewasusedtoidentifydiffer-entiallyexpressedgenesinpair-wisecomparisons(AndersandHuber,2010),andtheresultsofallstatisticaltestswerecorrectedformultipletestingwiththeBenjamini–Hochbergfalsediscoveryrate(FDRb0.01).Sequencesweredeemedtobesigni cantlydifferentiallyexpressediftheadjustedPvalueobtainedbythismethodwasb0.001andtherewasatleastatwofoldchange(N1orb 1inlog2ratiovaluewhichwerecalculatedbytheaverageRPKMvalueoftwoInflibrariesdividedbytheaverageRPKMvalueoftwoCOlibraries).TherawdatawasdepositedattheGeneExpressionOmnibuswithaccessionnumberGSE51900(http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE51900).

2.6.PathwayenrichmentanalysisofDEGs

PathwayenrichmentanalysisbasedonKEGG(KanehisaandGoto,2000)(KyotoEncyclopediaofGenesandGenomespathwaydatabase,http://www.genome.jp/kegg)wasusedtoidentifymarkedlyenrichedmetabolicpathwaysorsignaltransductionpathwaysindifferentiallyexpressedgenescomparedwiththewholegenomebackground.Theformulaforcalculationis:

M

N M mp¼X 11

i

ni¼0

N i;

n

where,NisthenumberofallgeneswithKEGGannotation,nisthenum-berofDEGsinN,Misthenumberofallgenesannotatedtospeci cpath-ways,andmisnumberofDEGsinM.

2.7.Real-timequantitativePCR(qRT-PCR)analysis

TheRNAofthreeCOandInfplantswereseparatelyusedforcDNAsynthesis.Foreachsample, rst-strandcDNAswerereverse-transcribedfromRNAstreatedwithDNaseI(Fermentas,Canada)byusingM-MuLVReverseTranscriptase(Fermentas,Canada)accordingtothemanufacturer'sinstructions.qRT-PCRwasperformedusinganoptical96-wellplatewithaniQ5multicolorrealtimePCRsystem(Bio-RAD,USA).Eachreactioncontained1.0μLofcDNAtem-platefromthereverse-transcribedreactionmentionedabove,10nMgene-speci cprimers,10μLofiTaq UniversalSYBRGreensupermix(Bio-RAD,USA)ina nalvolumeof20μL.Theramieactingene,which

北京百迈客苎麻根侵染线虫转录组测序分析

S.Zhuetal./Gene552(2014)67–7469

wasreportedtodisplayawellstabilityofexpressionintheCOandtheRLN-infectedramie(Liuetal.,2014),wasselectedastheendogenouscontrol.TheprimersequenceofDEGsandactingenewerelistedinTableS1.Thethermalcycleusedwasasfollows:95°Cfor30s,followedby40cyclesof95°Cfor10s,and55°Cfor25s.qRT-PCRwasper-formedintriplicateforeachsample.Relativeexpressionlevelswerede-terminedasdescribedpreviously(LivakandSchmittgen,2001).

3.Results

3.1.Illuminapaired-endsequencinganddenovoassembly

RNAwasextractedindividuallyfromfourwholeplantbodies,ex-cludingroots(twocontrolplants,CO1andCO2,andtwoRLNinfectionplants,Inf1andInf2).RNAfromindividualplantswasusedtoconstructlibrariesofcDNAwithafragmentlengthof200bp.FragmentswerethensequencedusingIlluminapaired-endsequencingtechnology.Atotalof56.3,51.7,43.4,and45.0millionrawsequencingreadswithlengthsof90bpweregeneratedfromthelibrariesofCO1,CO2,Inf1,andInf2,respectively(Table1).Trinitysoftwarewasusedtoassemblethereadsequenceofeachsampledenovo.Sequencesnotextendedoneitherendwerede nedasunigene.Finally,36,934,34,505,32,918,and32,079unigeneswerefoundinCO1,CO2,Inf1,andInf2,respective-ly(Table1).

Allunigenesfromthefoursampleswerealignedbasedonnucleo-tidesequence.Theunigenesfromdifferentsamplesthatoverlappedmorethan35bpwereassembledfurther.Thedenovoassemblyyielded50,486non-redundantunigenes,withanaveragelengthof853.3bpandatotallengthof43.08Mb(Table1).Thelengthofassembledunigenesrangedfrom200to9911bp.Therewere15,640unigenes(31.0%)b300bp,13,578unigenes(26.9%)from301to500bp,7819unigenes(15.5%)from501to1000bp,7725unigenes(15.3%)from1001to2000bp,and5724unigenes(11.3%)N2000bp(Fig.1).

3.2.Functionalannotationandclassi cation

Forvalidationandannotationoftheassembledunigenes,sequencesimilaritysearcheswereconductedintheNr,COG,GO,KEGG,andtheSwiss-ProtproteindatabasewithanE-valuethresholdof10 5.There-sultsindicatedthat,outof50,486unigenes,24,463(48.9%),20,198(40.0%),21,302(42.2%),5137(10.2%),and8541(16.9%)unigenesshowedsigni cantsimilaritytoknownproteinsintheNr,SwissProt,GO,KEGG,andCOGdatabases,respectively(Fig.2).Together,24,820(49.2%)unigenesshowedsimilaritytoknownproteinsinthese veda-tabases(Fig.2).

TheGOdatabaseisaninternationalstandardizedgenefunctionalclassi cationsystem,andithasthreeontologies:molecularfunction,cellularcomponent,andbiologicalprocess.GOfunctionalclassi cationfortheseunigenesassembledshowedthat21,302unigenes(40.0%)withBLASTmatchestoknownproteinswereassignedtoGOclasseswith240,800functionalterms.Ofthese,assignmentstothebiologicalprocessontologymadeupthemajority(123,002;51.1%),followedbycellularcomponent(85,686;35.6%)andmolecularfunction(32,112;13.3%)ontologies(Fig.3).

Table1

SummaryoftranscriptomesassembledbyIlluminasequencing.SampleReadnumberUnigenenumberTotallengthMeanlength(million)assembled(Mb)(bp)CO156.336,93434.90944.8CO251.734,50533.76978.4Inf143.432,91832.34982.6Inf245.032,07931.58984.3Total

196.4

50,486

43.08

853.3

parisonofgeneexpressionlevelsbetweenCOramieandInframieTodeterminetheexpressionlevelsofthe50,486unigenesassem-bleddenovo,allsequencingreadswererealignedtotheunigenes.Thenumberofreadsalignedtoaunigeneappearinginthelibrarywasusedtoestimateitsexpressionlevelusingthenormalizedvalueofthegene'suniquelyalignedreadcountsperkilobaseofexonmodelpermil-lionreads(RPKM).DifferencesinreadfrequenciesbetweentheCOandInflibrarieswereusedtodeterminechangesingeneexpressioninre-sponsetoroot-lesionnematodeinfection.TheRPKMvaluesofallunigeneswerecomparedbetweenthetwoCOreplicatesandbetweenthetwoInfreplicates.Thereweresigni cantcorrelationsbetweenthetwoCOreplicatesandbetweenthetwoInfreplicates,withthecorrela-tioncoef cientsof0.95and0.99,respectively.Scatterdiagrams,inwhichthelogarithmicRPKMvaluesofeachgeneinthetworeplicatesofeachtreatmentwereassignedascoordinatevaluesoftwoaxes,showedthatalldatapointsweredistributedintheregionofthediago-nal(Fig.4).AlltheseresultssuggestedthattheabundancesoftheseunigenetranscriptsinthetwoCOreplicatelibrariesandinthetwoInfreplicatelibrariesweresimilar.

ComparisonsoftheaverageRPKMvaluesofthetworeplicatesforeachgenebetweenCOandInframiewereperformedtoscreenthedif-ferentiallyexpressedgenes(DEGs)involvedintheRLN-defensere-sponse.Atotalof777DEGs(592genesup-regulatedand185genesdown-regulated)withatleastatwo-folddifferencebetweenCOramieandInframiewereidenti ed(FDRb0.01)(Fig.5,TableS1).AmongtheseDEGs,16(12genesup-regulatedand4genesdown-regulated)genesshowedmorethanatwenty-folddifferencebetweenCOramieandInframie(Fig.5,TableS2).Therewere40transcriptionfactors,whichweredistributedinto21families,regulatedbyRLNinfection(Table2,TableS3).

3.4.ValidationoftheexpressiondifferenceofDEGs

Inordertovalidatetheexpressionpro lingbyIlluminasequencing,theexpressionlevelsoftwelvegenes,includingtwocatalasegenes,twoNACtranscriptionfactors,threeperoxidasegenesand vedisease-resistancerelatedgenes,werefurtheranalyzedbyqRT-PCR.TheqRT-PCRresultshowedthatsevengeneswereup-regulatedexpressionand vegeneswasdown-regulatedexpression(Table3)(Pb0.05).ThetrendofexpressionchangesoftheseselectedgenesbasedonqRT-PCRwassimilarwiththosedetectedbyIllumina-sequencingmethod.However,thechangefoldsofthesegeneexpressionlevelsrespondingtoRLN-infectiondetectedbyqRT-PCRhadsomedifferencewiththosedetectedbyIlluminasequencing(Table3).

3.5.Functionalclassi cationofDEGsandthepathwaysenrichedbyDEGsTheCOGdatabasewasusedtoclassifytheorthologousgeneprod-ucts.EveryproteinintheCOGdatabaseisassumedtohaveevolvedfromanancestorprotein.Thewholedatabaseisbuiltoncodingproteinswithcompletegenomes,aswellasonsystemevolutionrelationshipsofbacteria,algae,andeukaryotes.AllDEGswerealignedusingtheCOGda-tabasetoclassifypotentialfunctions.Intotal,214DEGswereassignedtothe23COGclassi cationswith335functionalterms(Fig.6).TheGOfunctionalclassi cationofDEGswasperformedand694DEGswereclassi edinto51GOcategorieswith7641functionalterms(Fig.3).InmostGOcategories,theratioofDEGsagainstbackgroundunigenesrangedfrom2%to4%.Threefunctionalcategories,metallochaperoneac-tivity,antioxidantactivityandcellproliferation,contain6.67%,7.87%,and8.08%oftheDEGsintheirtotalunigenes,respectively,whichsug-geststhatthesethreefunctionalcategorieswereenrichedbyDEGs.

Thein uenceofRLNinfectiononbiologicalpathwayswasevaluatedbyenrichmentanalysisofDEGs.Atotalof61pathwaysinramiewerepossiblyaffectedbyRLNinfection(TableS4).Threepathways,phenylal-aninemetabolism,carotenoidbiosynthesis,andphenylpropanoid

北京百迈客苎麻根侵染线虫转录组测序分析

70S.Zhuetal./Gene552(2014)67–74

Fig.1.Lengthdistributionofassembled

unigenes.

Fig.2.Unigenenumbersannotatedinthe vepublicdatabases

searched.

Fig.3.Geneontologyclassi cationsofassembledunigenesandDEGs.Theresultsaresummarizedinthreemaincategories:biologicalprocess,cellularcomponent,andmolecularfunction.

北京百迈客苎麻根侵染线虫转录组测序分析

S.Zhuetal./Gene552(2014)67–7471

Fig.4.ThecorrelationofRPKMvaluesofallunigenesbetweenthetworeplicatesofCOramieandbetweenthetworeplicatesofInframie.ThelogarithmicRPKMvaluesofeachgeneinthetworeplicateswereassignedascoordinatevaluesoftwo

axes.

biosynthesis,werestronglyin uencedbyRLNinfection(Pb0.05)(Table4).4.Discussion

4.1.Denovoassemblyof50,486non-redundantgenesinramie

Sequencingandanalysisofexpressedsequencetags(ESTs)areapri-marytoolingenediscoveryandgenomicsequenceannotationinplants.ESTscanalsobeusedforotherfunctionalgenomicprojects,includinggeneexpressionpro ling,microarrays,molecularmarkers,andphysical

mapping.Forthepasttenyears,alargenumberofcDNAlibrarieshavebeenconstructedandsequencedforrice(Kikuchietal.,2003),maize(Alexandrovetal.,2009;Soderlundetal.,2009),wheat(Zhangetal.,2004),andothercropstoobtainESTinformation.However,thecostlyandtime-consumingtraditionalsequencingmethodshaverestrictedlarge-scaleESTsequencingofnon-modelplantsandminorcrops.Aslessexpensiveandlesstime-consumingnextgenerationsequencing(NGS)techniqueshavebeendeveloped,large-scaleESTsequencingofnon-modelplantsandminorcropshavebecomefeasible.Todate,thesequencingofanumberofminorcropshasbeencompletedandhun-dredsofthousandsofESTshavebeenidenti edinthesespecies(Barakatetal.,2009;Dengetal.,2012;Gargetal.,2011;Sunetal.,2010;Wangetal.,2010).

Althoughramieisoneofthemostimportant bercrops,fewofitsESTshavebeensequencedanddepositedintheGenBankdatabase.Toobtainlarge-scaleESTs,Liuetal. rstcharacterizedthetranscriptomeoframieusingIlluminasequencingtechnology(Liuetal.,2013c).How-ever,onlyapproximately53millionreadswereusedtoassemblethetranscriptome,whichgenerated43,990ESTswithatotallengthof36.26Mb(Liuetal.,2013c).Inthisstudy,196.4millionreads(3.7-foldmorethanthatofLiuetal.)weresequencedand50,486unigeneswithatotallengthof43.08Mbwereobtained,resultinginthediscoveryofatleast6496newgenes.Inaddition,thetotalaveragesequencesizegeneratedinthisstudywasmuchlarger(853.3bpversus824bpinLiuetal.).Therefore,thetranscriptomeassembledinthecurrentstudyismorecompletethanthatofLiuetal.

4.2.Identi cationof777genesexpressedinresponsetoRLNinfectioninramie

Apreviousstudyfoundthatthelengthanddiameteroframiestem,whicharedeterminingfactorsof beryield,hadasigni cantdecreasewhenramieisinfectedbyRLN(Zhuetal.,2012).However,theexactmechanismofinhibitioninovergroundvegetativegrowthbyRLN-infectionispoorlyunderstood.Inthepresentstudy,thewholeplant,ex-cludingroots,wereusedtoanalyzethetranscriptome.Obviously,therearemanygenesexpressedinrootswhichhaveregulatedthedefensere-sponsetoRLNinfection.However,becausetheinfectedrootwaspara-sitizedbyalargenumberofRLNswhichcannotberemoved,iftheinfectedrootissampled,thetranscriptomeofbothramieroot

and

Fig.5.GenesexpresseddifferentiallyinCOramieandInframie.Theparameters“FDR≤0.01”and“log2ratio≥1”wereusedasthresholdstodeterminethesigni canceofthedifferenceingeneexpression.GreendotsrepresenttheDEGsandreddotsindicatetranscriptsthatdidnotchangesigni cantlybetweenthetwotreatments.Thexaxisrepre-sentstheaveragelogarithmicRPKMvaluesofeachgeneinCOandInframie,andtheyaxisrepresentsthelogarithmicfoldchange(FC)valuesofeachgene.

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Table2

SummaryofDEGsannotatedastranscriptionfactor.Genefamily

DEGnumberTotal

ARFB3bHLHbZIPC2H2C3HCOLE2FERFGATAHD-ZIPHSFMYBNACSBPTALETCPWhirlyWRKYYABBYZF-HD

1210111111211521221112

Up-regulated129101011210100011011

S.Zhuetal./Gene552(2014)67–74

Down-regulated001010100001421210101

genestoRLNinfectionwillbeidenti edinramie.Finally,atotalof777DEGsinvolvedinovergroundvegetativegrowthwerefoundtoberegulatedbyRLNinfection.Moreover,twelvegenes(twoNACtran-scriptionfactors, veantioxidantgenes,and vedisease-resistancere-latedgenes)werechosentovalidatetheirdifferentialexpressionbyqRT-PCR,andallthese12genescommonlyagreedwiththeresultofIlluminasequencing,i.e.thetrendsofexpressionchangesofthese12genesdetectedbyqRT-PCRaresamewiththosebyIlluminasequencing.However,thefoldsofexpressiondifferenceforthese12genesintheInfandCOramiedetectedbyqRT-PCRaredifferentwiththosedetectedbyIlluminasequencing.Actually,thesimilarcasehadbeenobservedinpreviousstudies(Liuetal.,2013b;Wuetal.,2010).TheqRT-PCRisbasedonthePCRmethodsandcyclethreshold(Ct)valuetoidentifytheDEGs,whereasthetechnologyofIlluminasequencinginidentifyingDEGsbycomparingtheabundanceofsequencereadappearinginCOandInflibraries,whicharetwocompletelydifferentstrategies.Proba-bly,thesensitivedegreeofIlluminasequencingmethodinidentifyingDEGshassomedifferencewiththatofqRT-PCR.Althoughthescalesofexpressiondifferencedetectedbytwomethodsaredifferent,thetrendsofexpressionchangesofthese12genesdetectedbyqRT-PCRcommon-lyagreedtotheresultofIlluminasequencing,whichsuggestedthattherewasapotentiallowrateoffalseDEGs.Theexpressionpro lingcharacterizedbythisstudywillaccumulatetheunderstandingoframiedisease-resistancetoRLN.

RLNwillbesequenced.Moreover,amajorobjectiveinthisstudywastounderstandtheexpressionresponseofgenesinvolvingintheovergroundvegetativegrowth,includingstemelongation,toRLNinfec-tion.TheinfectionofRLNcancauseseveredamageoframierootandhinderstheabsorptionofwaterandnutrientsfromthesoil,thusleadingtothewaterde citandnutritionalde ciencyinramie,andresultingintheinhibitionofplantgrowth.Inotherwords,manygenes,whichareexpressedintheovergroundtissuesoframie,andareinvolvedinplantgrowthanddevelopment,tolerancetowaterde citandnutrition-alde ciency,mightberegulatedexpressionbyRLN-infection.Identi -cationofthesegenesexpressedintheovergroundtissueswillbehelpfulforunderstandingthepotentialmechanismofgrowthinhibitionofRLN-infectedramie.Therefore,thewholeplantwithoutrootswaschosenforIlluminasequencing.

Inaddition,twoindividuals(Inf1andInf2)usedforIlluminase-quencingwereinfectedbyRLNwithinsevendays.Generally,whentheplantsufferingfrombiotic/abioticstresses,somestress-signaltrans-ductionrelatedgenesandtranscriptionregulatorswillshowafastre-sponsetostresssignalinseveralhours.Thereafter,thesetranscriptionregulatorsactivatetheexpressionofgenesinvolvinginstresstolerance,and nallymakingtheplantadapttheenvironmentstresses.Hence,theactivationofexpressionoftolerancegenesmightrequirearelativelylongtime,andstableexpressionofthesetolerancegenespossiblyap-pearsinseveraldaysafterstresstreatment.Inthisstudy,amajoraimistoidentifythetolerance-relatedgenestoRLNinfectioninramie.Therefore,theexperimenttreatedbyRLN-infectionwasperformedwithinsevendaysinthisstudy,whichcanensurethatmoretolerance

Table3

TheDEGsvalidatedbyqRT-PCR.Unigene

T1_Unigene_BMK.20119T2_Unigene_BMK.19209T4_Unigene_BMK.22483T1_Unigene_BMK.2274T2_Unigene_BMK.19179T3_Unigene_BMK.18796T3_Unigene_BMK.13840T3_Unigene_BMK.791T3_Unigene_BMK.15403T1_Unigene_BMK.2026T4_Unigene_BMK.16944T4_Unigene_BMK.14594

qRT-PCR 5.66 8.87 4.80 5.41 3.9510.424.6529.5115.892.6519.343.08

4.3.AntioxidantgenesinresponsetoRLN-infection

About7.87%ofgeneswereclassi edintheantioxidantactivitycate-goryoftheGOfunction,whichisfarhigherthanotherfunctionalcatego-ries.AlthoughRLNinfectionoccursintheroots,superoxidedismutase(SOD)declinesandmalondialdehyde(MDA)concentrationsincreaseintheleaves(Zhuetal.,2012).SODcaneliminatereactiveoxygenspe-cies(ROS)causedbystressfactorsandprotectplantcellsbyscavengingsuperoxideradicals,H2O2,andothersuperoxidecompounds(LiuandZhang,1994;SchreiberandNeubauer,1990).MDAisthelastproductoftheperoxidationofmembraneliposomesanditsconcentrationisameasureofthedegreeofperoxidationofmembrane-boundliposomes(JiangandHuang,2001).ThedecreaseinSODactivitiesandtheincreaseinMDAconcentrationsinleafsuggestthatleafcellsaredamagedwhentherootisinfectedbyRLNinramie(Zhuetal.,2012).Inthisstudy,twocatalasegenesandthreeperoxidasegenesinvolvedinROS-scavengingwerefoundtobearegulatedexpressionbyRLN-infectionbyIlluminasequencing.Moreover,theirdifferentialexpressionofthese vegeneswasfurthervalidatedbyqRT-PCR.However,amongthese vegenes,threegeneswerefoundtobedown-regulatedexpression,whichseemtobeinconsistentwiththeincreaseofantioxidantenzymeactivity.Infact,intheplantcell,ROSnotonlycandirectlycauselipidperoxidationandmembranedamage,butalsoisimportantsignalsmediatingdefensegeneactivation(Torresetal.,2006).Therefore,a neadjustmentforROSisessentialtodiseasedefense.Thedown-regulatedexpressionfor

Illuminasequencing 2.58 2.12 2.46 23.93 3.265.843.013.092.022.667.837.37

Functionannotated

CatalaseCatalasePeroxidase

NACdomain-containingprotein43-likeNACdomain-containingprotein8-likeDesiccation-relatedproteinPCC13-62-likePeroxidase3

GDSLesterase/lipaseAt2g04570-like

Diseaseresistanceresponseprotein206-likePeroxidase12-like

GDSLesterase/lipaseAt5g55050-likePathogenesisrelatedprotein1isoform1

北京百迈客苎麻根侵染线虫转录组测序分析

S.Zhuetal./Gene552(2014)67–7473

Fig.6.HistogramsoftheClustersofOrthologousGroups(COG)classi cationofDEGs.

antioxidantenzymeencodedgeneshadbeenreportedinthepreviousstudy(Doreyetal.,1998).4.5.In uenceofRLNinfectiononpathwaysassociatedwithphenylpropanoidmetabolisminramie

Phenylpropanoidscontributetoallplantresponsestobioticandabi-oticstimuli(Vogt,2010).Theyarenotonlyindicatorsofplantstressre-sponsestovariationsinlightandmineraltreatments,butarealsokeymediatorsofplantresistancetopests(Cameraetal.,2004).Alargenumberofplantgeneswithinthephenylpropanoidpathwaytakepartinthedefenseresponseagainstpests(Porthetal.,2011).Inthisstudy,threepathwaysshowedsigni cantenrichmentofDEGsandwere,therefore,determinedtobein uencedbyinfection.SixandsevenDEGsinthephenylpropanoidbiosynthesispathwayandthephenylala-ninemetabolismpathwaywereenriched,respectively.Phenylalanineisanimportantprecursorforthebiosynthesisofphenylpropanoid(Vogt,2010).Therefore,twooutofthreepathwayswithenrichedDEGsarein-volvedinphenylpropanoidmetabolism.Itislikelythatthechangesintheexpressionofgenesinphenylpropanoidmetabolismpathwaysleadtoachangeinphenylpropanoidproduction,whichcanincreasere-sistancetoRLNinramie.

Supplementarydatatothisarticlecanbefoundonlineat/10.1016/j.gene.2014.09.014.

4.4.TranscriptionfactorsinresponsetoRLN-infection

Recently,expandingtranscriptomedatahaveuncoveredaglobalpictureofbioticstressresponsivegenesinplant,anddozensoftran-scriptionfactors(TFs)arefoundtobeinvolvedintheplantdefensere-sponsetopathogenorpestattack(Lietal.,2012;Santosetal.,2012;Wuetal.,2010).MostoftheseTFsfallintoseverallargeTFfamilies,suchasAP2/ERF,bZIP,NAC,MYB,MYC,andWRKY(Mooreetal.,2012;Puraniketal.,2012;vanVerketal.,2011).TheexpressionofTFsregulatestheexpressionofdownstreamtargetgeneswhichareinvolvedinthedefenseresponseandresistancetodisease.Inthisstudy,atotalof40transcriptionfactorswereregulatedexpressionbyRLNinfection.Amongthem,twoNACTFs(T1_Unigene_BMK.2274andT2_Unigene_BMK.19179)weredown-regulatedexpressioninInframie,andthedown-regulationofexpressionwasfurthervalidatedbyqRT-PCR.Inthepreviousstudy,atotalof32ramieNACTFshadbeenidenti ed(Liuetal.,2014).Expressionanalysisforthese32NACTFsre-vealedthat4genes(BnNAC16,BnNAC20,BnNAC29,andBnNAC30)wereregulatedexpressionbyRLNinfection(Liuetal.,2014).Interestingly,theT1_Unigene_BMK.2274andT2_Unigene_BMK.19179ofthisstudyaretheBnNAC16andBnNAC20,respectively.There-fore,thedown-regulatedexpressionofT1_Unigene_BMK.2274andT2_Unigene_BMK.19179inInframieinthisstudyisconsistentwiththeresultsofthepreviousstudy(Liuetal.,2014).

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