UniversityofNaturalResourcesandAppliedLifeSciences DepartmentofAppliedGeneticsandCellBiology

TheputativeRNAsilencingprotein ERL1isinvolvedinchloroplast ribosomalRNAprocessinginplants

Doctoratethesis submittedby DIJuttaMariaHelm March,2011

Declaration
IherebydeclarethatIhavewrittenthisthesisindependently.Allresultspresentedin theresultsectionhavebeenobtainedbymyownwork,probesandplasmidshave beensharedbyseverallabmembers.Relevantresultsobtainedbycoworkersare onlypresentedinthesupplementaryresultsectionwithpropercitations.Allintellec tualpropertyusedforthepreparationofthisworkhasbeencitedproperly. TheresultsobtainedfromtransgenicN.benthamianaplantsmisexpressingERL1in cludingcrossing,lightmicroscopyaswellasphotosyntheticanalysis,andrRNA cloningexperimentshavebeendescribedanddiscussedbyHeikoSchumacherin 2009.However,allexperimentalproceduresandplantmaintenancehavebeenexe cutedbyme.Thechloroplasticlocalization,analysisofchloroplastrelatedtranscripts andelectronmicroscopyexperimentshavebeenexecutedindependentlybyHeiko Schumacherandmetopreparereplicatesofthefindings. March2011,Vienna DIJuttaMariaHelm

Acknowledgements
Thisworkwouldnothavebeenpossiblewithoutthesupportofvariouspeople.Es peciallyIwouldliketothank DrKritonKalantidisforbeingavaluableteacherandstillleavingmeagreatfree domformyworkanddevelopment. DrMarieTheresHauserforgivingmetheopportunitytolearninherlabandfor allthesupportandgoodadvicefromfar. allmembersofthePlantMolecularBiologyLaboratoryinCreteformakingita placetoremember,Iwillkeepinmindthenicetimeswehad. themembersofthePlantDevelopmentalGeneticsLaboratoryinViennaforwel comingmesofriendlybeforeandaftermystayinGreece. SergiaTzortzakaki,EvaPapadogiorgaki,KosmasHaralampidisandmystudents Andreas,Evguenia,Giorgos,KalliaandRitsawhoallprovidedvaluablehelp. HeikoSchumacherforteachingmealessonforlife. allthepeoplewhohadbeenthereandwentthewayinCretealongwithmefora while,forsomeIamespeciallyproudtocallthemfriends. myfriendsbackathomeandaroundtheworldwhodidnotforgetmeevenfroma distance,forkeepingcontactbySkype,email,etc.andforspendingtheirholidays withme. myparentsforlettingmegoandforgivingmethefeelingthatIcanalwayscount onthemincaseofneed,despiteanylocaldistancebetweenus. mybrotherAndreasforconstantlysolvingallmycomputerproblemsinnotime. mysisterMartinaforalwaysbeingthere! HaraldZwillingforbeingHaraldZwilling!
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Abstract
DuringevolutioneukaryoteshaveacquiredasystemusingsmallRNAmolecules (siRNAs)asnegativeregulatorsofendogenousandexogenousRNAsequences calledRNAinterferenceorRNAsilencing.Thismechanismisalsousedinthede fenseagainstpathogensthathavethereforedevelopedseveralstrategiestocounter actitbyexpressionofviralsuppressorsofsilencing(VSRs).Aputativeendogenous suppressorofsilencingmightbethe35exonucleaseERI1(enhancedRNAi)andits homologuesinvariousspecies,whichspecificallybindanddegradesiRNAs.Re centlyanadditionalconservedroleofERI1homologueshasbeenidentified,where theycatalyzethefinalstepin5.8SrRNAprocessing. InthisworktheplanthomologuetermedERL1(ERI1LIKE1)isanalyzed.Thepro teinlocalizestothechloroplastandfailstoexhibitanyRNAsilencingsuppressor activity.ThisfindingisnotsurprisinginthiscontextsinceRNAsilencingisrestricted tothecytoplasm.AlsotheDrosophilamelanogasterhomologuedoesnotpossessthis functionsuggestingtwofunctionallydistinctgroupsofERI1homologues.Constitu tiveoverexpressionofERL1intransgenicNicotianabenthamianaplantsmanifestedin variegatedphenotypescharacterizedbyableachingoftheplants.Theresultcouldbe confirmedinArabidopsisthalianaplantsoverexpressingERL1.Theobservedpheno typesreachedfrompalegreen,yellow,mosaicgreenandwhitetocompletelossof chlorophyll.Theseverityofthebleachingcorrespondedtotheexpressionlevelsof ERL1.Thetransgenicplantlinesshowedmorphologicalandtranscriptionalaltera tionsreminiscentofreporteddefectsinchloroplasticribosomalRNAbiogenesis.In deeditcouldbeshownthat5SrRNAisdownregulatedaftertransientandconstitu tiveoverexpressionofERL1andelongatedbytwonucleotidesatits3endinafrac tionoftheanalyzedsamples.Aputativeribonucleasehasbeenproposedearlierto assistinchloroplasticrRNAprocessinginamutantbackgroundof RIBONUCLEOTIDEREDUCTASE1(RNR1)inArabidopsisthalianawhichmaybe constitutedbyERL1.Inadditionafractionof16SrRNAhasbeenelongatedbyone nucleotideinArabidopsisinsertionmutantssuggestingalsoafunctioninmaturation ofthischloroplasticrRNA.
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Zusammenfassung
ImLaufederEvolutionhabenEukaryonteneinSystemerworben,daskleineRNA Molekle(siRNAs)alsnegativeRegulatorenvonendogenenundexogenenRNA Sequenzenverwendet.DiesesogenannteRNAInterferenzoderRNASilencingwirkt auchalsImmunsystemgegenKrankheitserreger,deshalbhabendieseverschiedene Strategienentwickelt,umdiesemMechanismusdurchdieAusbildungvonviralen SilencingSuppressoren(VSR)entgegenzuwirken.Einmutmalichereukaryotischer endogenerSilencingSuppressorknntedie35ExonukleaseERI1sein,diespezi fischsiRNAsbindenundabbauenkann.AuerdemkatalysiertERI1denletzten SchrittbeiderReifungvon5.8SribosomalerRNA. IndieserArbeitwurdedasPflanzenHomologERL1(ERI1like1)analysiert.Das ProteinwirdinChloroplastengeschleustundbesitztkeineRNASilencing SuppressorAktivitt.DiesesErgebnisistinsofernnichtberraschend,daRNA SilencingaufdasZytoplasmabeschrnktist.DashomologeDrosophilaProteinzeigt ebenfallsdiesesVerhalten;wahrscheinlichexistierenzweiunterschiedlicheGruppen vonERI1HomologeninEukaryoten.berexpressionvonERL1intransgenenTa bakundArabidopsisPflanzenresultierteinvielfltigenPhnotypen,diedurchein BleichenderPflanzengekennzeichnetwaren:esreichtevonblassgrn,bergelb, grnundweigesprenkeltbiszukomplettemChlorophyllVerlust.DerSchwere graddesPhnotypsentsprachderberexpressionvonERL1.DietransgenenLinien zeigtenmorphologischeundtranskritptionelleVernderungen,dieanbereitsbe schriebeneMngelinderReifungribosomalerRNAsinChloroplastenerinnern.Es konntetatschlichgezeigtwerden,dassdie5SrRNALevelsnachderberexpression vonERL1verringertsindundam3EndeeinehufigeVerlngerungumzwei Nukleotidebesaen.ERL1knntediemutmalicheRibonukleasesein,diederRibo nukleotidreduktase1(RNR1)beiderVerarbeitungvonChloroplastenrRNAassis tiert.Auerdemzeigteauchdie16SrRNAinArabidopsisMutanten,beidenenERL1 unterdrcktwar,teilweiseVerlngerungenumeinNukleotid;eventuellbesitztERL1 aucheineFunktioninderReifungdieserrRNA.
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Abbreviations
% C ME m M Ci g J L 3 3hExo 5 7mG A A.thaliana/At A.tumefaciens aa ACS ADAR Ago/AGO APconjugate APS ARC Arg Asp ATP RLI2 attsites Aub BLAST bp BSA C C.elegans/Ce CBC cDNA Chp1 Percent DegreesCelsius Mercaptoethanol Micrometer(s) Micromolar Microcurie(s) Microgram(s) Microjoule(s) Microlitre(s) 3prime 3primehistoneexonuclease 5prime 7methylguanosine Adenosine ngstrm(s) Arabidopsisthaliana Agrobacteriumtumefaciens Aminoacid(s) Acetosyringone AdenosinedeaminasethatactsonRNA Argonauteprotein Alkalinephosphataseconjugate Ammoniumpersulfate ACCUMULATIONANDREPLICATIONOF CHLOROPLASTS Arginine Aspartate Adenosinetriphosphate RNASELINHIBITOR2 Attachmentsites Aubergine BasicLocalAlignmentSearchTool Basepair(s) Bovineserumalbumin Cytosine Caenorhabditiselegans Capbindingcomplex ComplementaryDNA ChromodomainproteininS.pombe
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Abbreviations
CHS Ci CLP CLSY1 cm cm CTAB Cterminus D D.rerio dATP DCL14 DCP2 DCR DCR1/2 dCTP ddC DDL DGCR8 dGTP DMSO DNA DnaQ DNase dNTP dpi DRB4 DRD1 DRM2 dsDNA dsRBD dsRNA DTT dTTP Duf E E.coli e.g. CHALCONESYNTHASE Curie(s) CASEINOLYTICPROTEASE CLASSY1 Centimeter(s) Squarecentimeter(s) Cetyltrimethylammoniumbromide Carboxyterminus Aspartate Daniorerio Deoxyadenosinetriphosphate DICERLIKE14 Decappingprotein2 Dicer DicerRelated1/2 Deoxycytosinetriphosphate Dideoxycytosine DAWDLE DiGeorgesyndromechromosomalregion Deoxyguanosinetriphosphate Dimethylsulfoxide Deoxyribonucleicacid DNApolymeraseIIIepsilonsubunit Deoxyribonuclease Deoxynucleosidetriphosphate Dayspostinfection/dayspostinfiltration DOUBLESTRANDEDRNABINDINGPROTEIN4 DEFECTIVEINRNADIRECTEDDNAMETHYLATION1 DOMAINSREARRANGEDMETHYLTRANSFERASE2 DoublestrandedDNA Doublestrandedribonucleicacidbindingdomain DoublestrandedRNA Dithiothreitol Deoxythymidinetriphosphate Domainofunknownfunction Glutamate Escherichiacoli exempligratia
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D.melanogaster/Dm Drosophilamelanogaster

Abbreviations
EDTA eIF4E ELSS ERI1/Eri1 ERL1 EST etal. EtBr EXOIII fmol For/F FoRTH FRY1 G g GFP GW182 h H H.sapiens H2O HCPro HEN1 HEPES HRP HYL1 i.e. IDN2 IGN IMBB IPTG K kb KD kDa KO KTN kV Ethylenediaminetetraaceticacid Eukaryoticinitiationfactor4E Extensivelocalsilencingspread EnhancedRNAi1 ERI1LIKE1 Expressedsequencetag andothers Ethidiumbromide Exonuclease(III)domain Femtomole Forward FoundationforResearch&TechnologyHellas 3(2),5BISPHOSPHATENUCLEOTIDASE/INOSITOL POLYPHOSPHATE1PHOSPHATASE Guanosine;Glycine Gramm(s);relativecentrifugalforce GreenFluorescentProtein Glycinetryptophanrepeatprotein182 Hour(s) Histidine Homosapiens Water Helpercomponentproteinase HUAENHANCER1 4(2hydroxyethyl)1piperazineethanesulfonicacid Horseradishperoxidase HYPONASTICLEAVES1 Idest INVOLVEDINDENOVOMETHYLATION2 Intergenicnoncodingtranscript InstituteofMolecularBiology&Biotechnology IsopropylS1thiogalactopyranoside Lysine Kilobase(s) Knockdown Kilodalton(s) Knockout KATANIN Kilovolt(s)
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Abbreviations
L LB let7 lin4/14 LOQS M M.musculus mA MES Met mg min miRNA miRNA* mL mM mm MMA MOPS mRNA MS N N.crassa N.tabacum/Nt NADP NAT natsiRNA NCBI NEP ng NiNTA nm NPC NRPD1a/b nt Nterminus O.sativa/Os OD600 Liter(s) Lysogenybroth/Luriabroth/LuriaBertanibroth LEThal7 AbnormalcellLINeage4/14 Loquacious Molar Musmusculus Milliampere(s) 2(NMorpholino)ethanesulfonicacid Methionine Milligram(s) Minute(s) MicroRNA MicroRNApassengerstrand Milliliter(s) Millimolar Millimeter(s) MS/MES/acetosyringone 3(NMorpholino)propanesulfonicacid MessengerRNA Murashige&Skoog Normal Neurosporacrassa Nicotianatabacum Nicotinamideadeninedinucleotidephosphate Naturalantisensetranscript NaturalantisensetranscriptderivedsiRNA NationalCenterforBiotechnologyInformation Nucleusencodedpolymerase Nanogram(s) NickelNitriloaceticacid Nanometer(s) Nuclearporecomplex NUCLEARRNAPOLYMERASED1A/B Nucleotide(s) Aminoterminus Oryzasativa Opticaldensityat600nm
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N.benthamiana/Nb Nicotianabenthamiana

Abbreviations
OH P.trichocarpa/Pt PAA PAGE PAP PAZ Pbody PCMP PCR PEP pH PIPES piRNA Piwi PLMVd pmol PNK Pol PolII/IV/V PPR PPV premiRNA PRG1 primiRNA PSRP1 PSTVd PTGS QDE2 QIP qPCR Qrich R2D2 RACE Ran rasiRNA RBCL RdDM RDR16/RdRP Rev/R Hydroxyl Populustrichocarpa Polyacrylamide Polyacrylamidegelelectrophoresis polyApolymerase Piwi/Argonaute/Zwille Processingbody Plantcombinatorialandmodularprotein Polymerasechainreaction Plastidencodedpolymerase pondusHydrogenii/potentiaHydrogenii PiperazineN,Nbis(2ethanesulfonicacid) PiwiinteractingRNA Pelementinducedwimpytestes Peachlatentmosaicviroid Picomole(s) Polynucleotidekinase Polymerase RNApolymeraseII/IV/V Pentatricopeptiderepeat Plumpoxvirus PrecursormiRNA Piwirelatedgene1 PrimarymiRNA PHLOEMSMALLRNABINDINGPROTEIN1 Potatospindletuberviroid Posttranscriptionalgenesilencing Quellingdeficient2 QDE2interactingprotein QuantitativePCR Glutaminerich TwodsRNAbindingdomains,associatedwithDCR2 RapidamplificationofcDNAends Rasrelatednuclearprotein RepeatassociatedshortinterferingRNA Ribulosebisphosphatecarboxylase,largechain RNAdirectedDNAmethylation RNADEPENDENTRNAPOLYMERASE16 Reverse
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Abbreviations
Rf rgsCaM RISC RITS RNA RNAi RNase RNasin RNR1 RPB1 rpm RPOB RRF rRNA RT RuBisCo s/sec S S.bicolor S.pombe S.purpuratus SAF SAP SDE3 SDN SDS SE SGS3 SINE siRNA siRNase SLBP SLSS snoRNA Snp snRNA snRNP SOB sp. Nuclearrestorer REGULATOROFGENESILENCINGCALMODULINLIKE RNAinducedsilencingcomplex RNAinducedtranscriptionalsilencing Ribonucleicacid RNAinterference Ribonuclease RNaseinhibitor RIBONUCLEOTIDEREDUCTASE1 RNApolymeraseIIlargesubunit Rotationsperminute RNAPOLYMERASESUBUNITBETA RNAdirectedRNApolymerasefamily RibosomalRNA Reversetranscription;Roomtemperature Ribulose1,5bisphosphatecarboxylase/oxygenase Second(s) Svedberg(sedimentationcoefficient) Sorghumbicolor Schizosaccharomycespombe Strongylocentrotuspurpuratus Scaffoldattachmentfactor SAFA/B,AcinusandPIAS SILENCINGDEFICIENT3 SMALLRNADEGRADINGNUCLEASE Sodiumdodecylsulfate SERRATE SUPPRESSOROFGENESILENCING3 Shortinterspacedelement SmallinterferingRNA Smallinterferingribonuclease Stemloopbindingprotein Shortrangelocalsilencingspread SmallnucleolarRNA Snipper SmallnuclearRNA Smallnuclearribonucleoprotein Superoptimalbroth Species
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Abbreviations
SPT5 SSC ssDNA ssRNA T TAE Taq TAS3 tasiRNA TBE TE TEM TEMED TIC/TOC Tm TNRC6A,B,C TRBP Tris tRNA TRV U UBA UTR UV V V.vinifera v/v VCS VSR W W w/v X.laevis XGal xray XRN14 Y Z.mays/Zm SuppressorofTyinsertion5 Sodiumchloride/sodiumcitratebuffer SinglestrandedDNA SinglestrandedRNA Thymine Tris/Acetate/EDTA Thermusaquaticus TRANSACTINGSIRNA3 TransactingsiRNA Tris/Borate/EDTA TrisEDTA Transmissionelectronmicroscopy Tetramethylethylenediamine Transloconattheinner/outerenvelopemembraneofchloro plasts Meltingtemperature Trinucleotiderepeatcontaining6A,B,C TransactivatingresponseRNAbindingprotein Tris(hydroxymethyl)aminomethan TransferRNA Tobaccorattlevirus Unit(s) Ubiquitinassociated Untranslatedregion Ultraviolet Volt(s) Vitisvinifera Volumepervolume VARICOSE Viralsuppressorofsilencing Watt(s) Tryptophan Weightpervolume Xenopuslaevis 5Bromo4chloro3indolylDgalactopyranoside Roentgenrays EXORIBONUCLEASE14 Tyrosine Zeamays
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TableofContent
1. Introduction..................................................................................................................... 25 1.1 RNAmoleculesandtheirlifebetweenDNAandprotein .............................. 25 1.2 RNAsilencingnewrolesfortheintermediate............................................... 27 1.2.1 siRNAmediatedgenesilencing...................................................................... 29 1.2.2 miRNAsmodulatetheexpressionofendogenoussequences.................... 32 1.2.3 ThepiRNApathwayprotectsthegermlinefromtransposonactivity...... 36 1.2.4 PlantspossessahighdiversityofsiRNAmolecules.................................... 38 1.2.4.1 CisactingsiRNAsmediatechromatinsilencinginplants ...................... 41 1.2.4.2 TransactingsiRNAs...................................................................................... 42 1.2.4.3 NaturalantisensesiRNAs ............................................................................ 44 1.2.5 PeculiaritiesofplantmiRNAs ......................................................................... 45 1.2.6 SpreadingofRNAsilencinginplantsresemblesanimmunesystem....... 46 1.2.7 ViralstrategiestosuppressRNAsilencinginplants................................... 49 1.2.8 RepressingtherepressorsendogenoussuppressorsofRNAsilencing.. 50 1.2.9 ERI1isanexampleforanendogenoussuppressorofRNAsilencing..... 51 1.2.10 ERI1LIKE1,theplanthomologueofERI1 ................................................ 56 1.3 Chloroplasts ........................................................................................................... 61 1.3.1 Photosynthesis ................................................................................................... 62 1.3.2 SpecificitiesofchloroplasticRNAs ................................................................. 64 1.4 Thesisobjectives .................................................................................................... 66 2. MaterialsandMethods .................................................................................................. 69 2.1 Materials ................................................................................................................. 69 2.1.1 Instruments ........................................................................................................ 69 2.1.2 Chemicals ........................................................................................................... 71 2.1.3 Consumables&kits .......................................................................................... 74 2.1.4 Solutions ............................................................................................................. 76 2.1.5 Others .................................................................................................................. 84 2.1.5.1 Enzymes.......................................................................................................... 84 2.1.5.2 Sizemarkers ................................................................................................... 85 2.1.5.3 Bacterialstrains.............................................................................................. 85 2.2 Methods .................................................................................................................. 85 2.2.1 Standardmolecularbiologymethods ............................................................ 85 2.2.1.1 Cultivation...................................................................................................... 85 2.2.1.2 Chemicallycompetentcells ......................................................................... 86 2.2.1.3 Transformation .............................................................................................. 86 2.2.1.4 Plasmidpreparation...................................................................................... 87 2.2.1.5 Agarosegel..................................................................................................... 88 2.2.1.6 Gelextraction ................................................................................................. 88 2.2.1.7 Digest .............................................................................................................. 88 2.2.1.8 Ligation ........................................................................................................... 88 2.2.2 Planttransformationtechniques ..................................................................... 88 2.2.2.1 Plantcultivation............................................................................................. 88 2.2.2.2 LeafDisctransformation.............................................................................. 89
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TableofContent
2.2.2.3 FloralDip........................................................................................................ 89 2.2.2.4 Agroinfiltration.............................................................................................. 89 2.2.3 Southernanalysis .............................................................................................. 90 2.2.3.1 DNAextraction.............................................................................................. 90 2.2.3.2 Southernanalysis .......................................................................................... 90 2.2.3.3 Capillaryblot ................................................................................................. 91 2.2.4 Northernanalysis .............................................................................................. 91 2.2.4.1 RNAextraction .............................................................................................. 91 2.2.4.2 Denaturingagarose/formaldehydegels..................................................... 91 2.2.4.3 PAGEnorthern .............................................................................................. 92 2.2.4.4 Semidryblot.................................................................................................. 92 2.2.5 Hybridization..................................................................................................... 92 2.2.5.1 Randomprimedlabelling ............................................................................ 92 2.2.5.2 Endlabelling .................................................................................................. 93 2.2.5.3 Hybridization,washesanddeveloping ..................................................... 93 2.2.6 Westernanalysis................................................................................................ 93 2.2.6.1 Proteinextraction .......................................................................................... 93 2.2.6.2 SDSPAGE ...................................................................................................... 93 2.2.6.3 Electroblot ..................................................................................................... 94 2.2.6.4 Westerndetection.......................................................................................... 94 2.2.7 rRNAcloning ..................................................................................................... 94 2.2.7.1 SelfLigation ................................................................................................... 94 2.2.7.2 LinkerLigation .............................................................................................. 94 2.2.7.3 ReverseTranscription(RT) .......................................................................... 95 2.2.7.4 PolymeraseChainReaction(PCR).............................................................. 95 2.2.8 RapidAmplificationofcDNAends(RACE)................................................. 96 2.2.9 QuantitativerealtimePCR(qPCR) ................................................................ 96 2.2.10 Protoplasts .......................................................................................................... 96 2.2.11 Preparationformicroscopicanalysis ............................................................. 97 2.2.12 Fluorescencemeasurement.............................................................................. 97 3. Results .............................................................................................................................. 99 3.1 Localization ............................................................................................................ 99 3.2 RACE..................................................................................................................... 101 3.3 TransgenicNicotianabenthamianaplants ...................................................... 103 3.3.1 KnockdownofERL1...................................................................................... 103 3.3.2 OverexpressionofERL1 ................................................................................. 104 3.3.2.1 Microscopy ................................................................................................... 107 3.3.2.2 EffectofERL1overexpressiononchloroplastmRNAs ......................... 110 3.3.2.3 EffectofERL1overexpressiononthephotosyntheticapparatus......... 112 3.4 TransgenicArabidopsisthalianaplants........................................................... 115 3.4.1 KnockdownofERL1...................................................................................... 115 3.4.2 OverexpressionofERL1 ................................................................................. 117 3.5 EffectofERL1onsilencing ................................................................................ 119
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TableofContent
Crosses .............................................................................................................. 119 LNA159 ............................................................................................................. 120 3.6 EffectofERL1onribosomalRNA .................................................................... 121 3.6.1 rRNAblots........................................................................................................ 121 3.6.2 Linkerligations ................................................................................................ 123 3.6.2.1 Effecton5.8SrRNA .................................................................................... 123 3.6.2.2 EffectonchloroplasticrRNAs ................................................................... 124 4. Discussion ...................................................................................................................... 129 4.1 ImplicationsofplantERL1inRNAsilencingprocesses ............................... 129 4.2 InvolvementsofERL1inchloroplastmetabolism.......................................... 131 4.3 SeverephenotypicalterationsafteroverexpressionofERL1suggestanin volvementinchloroplastdevelopment ........................................................................... 133 4.4 ERL1isinvolvedinchloroplasticribosomalRNAprocessing..................... 135 4.5 Conclusions .......................................................................................................... 140 5. References ...................................................................................................................... 143 6. Supplements .................................................................................................................. 171 6.1 Supplementarymethods .................................................................................... 171 6.1.1 Virus/viroidinfectionsinNicotianasp.plants ............................................. 171 6.1.2 Invitrotranscription........................................................................................ 171 6.1.3 PurificationofrecombinantERL1protein................................................... 171 6.1.4 InvitroassaysforrecombinantERL1protein............................................. 172 6.2 Supplementaryresults........................................................................................ 172 6.2.1 PSTVdderivedsiRNAsaresuppresseduponERL1overexpression ..... 173 6.2.2 ERL1failstoaffectRNAsilencinginAgrobacteriumcoinfiltrationassays 174 6.2.3 ExogenouslyinducedsilencingspreadmaybesuppressedafterERL1 overexpression .............................................................................................................. 176 6.2.4 ERL1overexpressingplantsarehypersensitivetowardsviralinfection 177 6.2.5 InvitroexperimentswithERL1..................................................................... 178 6.2.6 PreparationofaNbERL1suppressionconstructandanalysisofitseffects aftertransientandtransgenicexpression ................................................................. 179 6.3 Oligonucelotides.................................................................................................. 181 6.4 Vectormaps.......................................................................................................... 183 6.4.1 AtERL1GFP.................................................................................................... 183 6.4.2 AtleaderGFP .................................................................................................. 183 6.4.3 AtERL1over ................................................................................................... 184 6.4.4 NtERL1hp ...................................................................................................... 184 6.5 Sequences.............................................................................................................. 185 6.5.1 Newlyidentifiedsequences........................................................................... 185 6.5.2 Publishedsequencesusedforinsilicoanalysisandprimerdesign ......... 186 6.6 Curriculumvitae(March,2011) ........................................................................ 196
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3.5.1 3.5.2

TableofContent
ListofFigures: Figure1.1:Ghildiyal&Zamore,2009................................................................................. 28 Figure1.2:MacReaetal.,2006 ............................................................................................. 28 Figure1.3:Argonauteproteins............................................................................................ 29 Figure1.4:Okamuraetal.,2007........................................................................................... 31 Figure1.5:Carthew&Sontheimer,2009............................................................................ 35 Figure1.6:Zamore,2010 ...................................................................................................... 38 Figure1.7:Ghildiyal&Zamore,2009................................................................................. 40 Figure1.8:Matzkeetal.,2009 .............................................................................................. 39 Figure1.9:Allen&Howell,2010 ........................................................................................ 43 Figure1.10:Kalantidisetal.,2008 ....................................................................................... 47 Figure1.11:Cheng&Patel,2004......................................................................................... 51 Figure1.12:AlignmentofpublishedERI1homologs..................................................... 56 Figure1.13:AlignmentofERL1homologuesinvariousplantspecies......................... 57 Figure1.14:Winteretal.,2007 ............................................................................................. 57 Figure1.15:Winteretal.,2007 ............................................................................................. 58 Figure1.16:Campbelletal.,1996 ........................................................................................ 63 Figure1.17:Sternetal.,2010 ................................................................................................ 64 Figure3.1:LocalizationofplantERL1 ............................................................................. 100 Figure3.2:AlignmentsofNicotianasp.ERL1sequence................................................. 101 Figure3.3:AnalysisofpresumableERL1suppressorplants........................................ 101 Figure3.4:AnalysisofNicotianabenthamianaplantsoverexpressingERL1 ............... 104 Figure3.5:TEManalysisofphenotypesofNicotianabenthamianaplantsoverexpress ingERL1................................................................................................................................ 108 Figure3.6:PhenotypicanalysisbylightmicroscopyofNicotianabenthamianaplants overexpressingERL1 .......................................................................................................... 107 Figure3.7:Northernanalysisofselectedchloroplastrelatedgenes ........................... 109 Figure3.8:DeterminationofphotosyntheticparametersinNicotianabenthamiana plantsoverexpressingERL1 .............................................................................................. 111 Figure3.9:CharacterizationofselectedpubliclyavailableArabidopsisthalianaERL1 knockoutplants .................................................................................................................. 116 Figure3.10:AnalysisofArabidopsisthalianaplantsoverexpressingERL1.................. 118 Figure3.11:EffectofplantERL1ondifferentmoleculesoftheRNAsilencingappara tus .......................................................................................................................................... 117 Figure3.12:NorthernanalysisofchloroplasticribosomalRNAsandcytosolic5.8S rRNAfollowingoverexpressionofERL1 ........................................................................ 122 Figure3.13:Alignmentofcytosolic5.8SrRNAfromNicotianabenthamianaplants overexpressingERL1 .......................................................................................................... 123 Figure3.14:AlignmentofchloroplasticrRNAsofArabidopsisthalianaandNicotiana benthamianaplantsmisexpressingplantERL1 ................................................................ 126 Figure4.1:SecondarystructuresofchloroplasticrRNA(predictedbyRNAfold;Gru beretal.,2008). ..................................................................................................................... 135

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TableofContent
Figure6.1:ComparativeagroinfiltrationtimecourseinsystemicallyPSTVdinfected tobacco. ................................................................................................................................. 174 Figure6.2:AgrobacteriumcoinfiltrationassaysinN.benthamianaline16CtotestERL1 forRNAsilencingsuppressoractivity ............................................................................. 175 Figure6.3:SilencingoftheERL1phenotypeinducedbyagroinfiltration.................. 177 Figure6.4:ERL1overexpressorplantsarehypersensitivetowardsinfectionbyPPV ................................................................................................................................................ 178 Figure6.5:AnalysisofthesuppressionofERL1inNicotianabenthamiana. ................ 180 ListofTables: Table1.1:predictedlocalizationofplantERL1homologs .............................................. 58 Table3.1:SegregationofNicotianabenthamianaT2plantlinestransformedwitha hairpinconstructdesignedfordownregulationofERL1.............................................. 104 Table3.2:SummaryofsegregationandphenotypicpatternofNicotianabenthamiana T1plantlinesoverexpressingERL1 ................................................................................. 107 Table3.3:SummaryofcharacteristicsofArabidopsisthalianaERL1knockdownplant lines........................................................................................................................................ 117 Table3.4:SummaryofsequencealterationsinchloroplasticrRNAafterERL1misex pressioninNicotianabenthamiana(Nb)andArabidopsisthaliana(At)plants............... 125

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1. Introduction
SinceitsdiscoveryDNAhasbeenconsideredasaverystablemoleculewiththeabil itytoencodeforaninfinitenumberofproteins,whicharefactorswithanunlimited versatilityforcatalyticprocesses.TheintermediaryforminthisprocessisRNA,lack ingboththestabilityandtheflexibilityoftheabovementionedkeyplayers. Thisprocess,however,isatypicalchickenandeggparadox,sincenucleicacidsare requiredforproteinsynthesiswhileproteinsarenecessaryfornucleicacidproduc tion.OnehypothesistryingtoovercomethisparadoxconsidersRNAasthemolecule bothstoringgeneticinformationandcatalyzingchemicalreactionsinprimitivecells ofthissocalledRNAworld.DNAhasthendevelopedasamoresuitablemolecule forstoragesinceitsstabilityincreasedthemaximumsizeofthehereditarymolecules. Thismighthavehappenedinparallelwiththenecessityforstorageofincreased amountsofgeneticinformationafteraccumulationofadditionalproteincatalysts (Albertsetal.,2008).

1.1 RNAmoleculesandtheirlifebetweenDNAandprotein
mRNA(messengerRNA)isthefirstmoleculeinvolvedintheinformationflowfrom DNAtoprotein.AnRNApolymeraseguidesthetranscriptionofadefinedgenomic stretchbycatalyzingtheformationofphosphodiesterbondsbetweenribonucleo tides.Thepolymeraserecognizesacertainpromotersequencewhereitbindstothe DNAstrand.ItsynthesizesasinglestrandedmRNAmoleculein5to3direction andstopstheelongationwhenreachingtheterminatorsequence. Thisgeneralprocessismorecomplexineukaryotes.TheypossessthreeRNApoly meraseswithPolIIbeingresponsibleformostproteincodinggenesandPolIand PolIIItranscribinggenesforotherRNAmolecules.Thetranscriptioninitiationre quirestheactionofgeneraltranscriptionfactorsfacilitatingthebindingtothepro motersequenceandformingatranscriptioninitiationcomplex.Thesynthesized

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1.Introduction
mRNAstrandismodifiedatits5endbyadditionofacapof7methylguanosine (7mG)whichbindsaproteincomplexcalledcapbindingcomplex(CBC). Eukaryoticproteincodinggenestretchescontainexpressedsequences(exons)and noncodinginterveningsequences(introns).Theremovalofthelatterfromthepre cursormRNAstrandisperformedbythespliceosome.Theycontainanothertypeof RNA,thefivesmallnuclearRNAs(snRNAsU1,U2,U4,U5andU6)togetherwithat leastsevenproteinsformingthesmallnuclearribonucleoproteincomplex(snRNP). Theintronicsequenceiscircularizedintoastructurecalledlariat,excisedandthe endsoftheexonsarethenjoinedtogethertoacontinuouscodingsequence. ProteinfactorsaccompanyingthetranscribedmRNAmoleculerecognizethe3end ofthestrandandleadtoitscleavage.ThepolyApolymerase(PAP)thenaddsap proximately200adenosinenucleotides,thefinallengthofthispolyAtailisdeter minedbypolyAbindingproteins. AberrantRNAsaredegradedbythenuclearexosomeconsistingof3to5RNAex onucleases.OnlysplicedmaturemRNAwitha5capanda3polyAtailareex portedtothecytoplasmthroughthenuclearporecomplexes(NPCs). ThenucleotidesequenceofthemRNAistranslatedintoaminoacidsbycodonscon sistingofthreeconsecutivenucleotides.ThenecessaryRNAmoleculesforthisproc essaretRNAs(transferRNAs)whichareextensivelystructuredLshapedadaptor moleculeswithunusualbasesorganizedintothreesinglestrandedloops.Oneof themcontainstheanticodonwhichbindstothemRNAsequence.Therelevantamino acidisconnectedtothe3endofthetRNAmoleculebyaminoacyltRNAsyntheta ses.Thepolypeptidechainissynthesizedbythestepwiseadditionofaminoacidsto itsCterminalendthatisactivatedbythebindingtoatRNAmolecule(whichisthen calledpeptidyltRNA). Theabovedescribedprocesstakesplaceintheribosomes,acatalyticmachinerycon sistingofribosomalproteinsandribosomalRNAs(rRNA).Theyconstituteupto80 %ofthetotalRNAofcellsandareencodedinmultiplerRNAgeneswhichareoften arrangedintandemsandtranscribedbyPolI.
26

1.Introduction
LongrRNAprecursormoleculesareextensivelymodifiedatpositionsthatarespeci fiedbyguideRNAs,alsoknownassmallnucleolarRNAs(snoRNAs).Theyareoften encodedinandfurtherexcisedfromintronicsequences,especiallyfromribosomal proteins.ThenucleolusisadistinctstructureinthenucleuswhererRNAprocessing andribosomeassemblytakeplace.Thetworibosomalsubunitsarethenexportedto thecytoplasmwheretheyjointoformthematureribosome.Theprokaryotic70Sri bosomeconsistsofa50Ssubunit(with5Sand23SrRNA)anda30S(with16SrRNA). Theeukaryotic80Sribosomeconsistsofa60Ssubunit(with5S,5.8Sand23SrRNA) anda40Ssubunit(with18SrRNA).Thematureribosomecontainsfourbindingsites forRNA,oneformRNAandthreefortRNA:thelatterareboundattheAsite,the aminoacidsarethenconnectedtogetheratthePsiteandtheemptytRNAfinally getsreleasedattheEsite.ThemajorcatalyticreactionsarecarriedoutbytherRNA moleculeswhichcanbeconsideredasribozymes(Albertsetal.,2008).

1.2 RNAsilencingnewrolesfortheintermediate
FormanyyearsthecentraldogmaofmolecularbiologywaspostulatedbyFrancis Crick,statingthatthegeneticinformationonlyflowsfromnucleicacidstoproteins. TheimportanceofRNAhadbeenboostedsignificantlybythefinding,thatdouble strandedRNAisatriggerofgenesilencingandthereforeprovidinganegativefeed backmechanismwhichisindependentofproteinsynthesis(Fireetal.,1998). ThefirstobservationofanRNAsilencingmechanismwasreportedin1928when newlyemergingleavesofTRVinfectedNicotianatabacumplantswerefoundtobe freeofinfectionsymptoms(Wingard,1928).Manyotherimportantobservations whichfinallyleadtothediscoveryoftheRNAsilencingmechanismhadalsobeen madeinplants:afterexpressionofantisenseRNAthetobacconopalinesynthasewas inhibited(Rothsteinetal.,1987).Thisphenomenoncalledcosuppression,wherean endogenousgeneisdownregulatedafterstrongoverexpressionofthesamese quence,wasdiscoveredbytwogroupstryingtoincreasethepurplepigmentationof petunia.TheoverexpressionofCHALCONESYNTHASE(CHS),anenzymeofthe

27

1.Introduction
anthocyaninpathway,resultedinalargefractionofplantswithwhitepetals(Napoli etal.,1990;vanderKroletal.,1990). SeveralclassesofsmallRNAshavebeenidentifiedtoresultinvarioussilencingpro cedures.Commonfeaturesaretheinvolvementof1835nucleotidelongsmallRNAs whicharecomplementarytotargetRNAs.Bindingresultsintargetrepressioneither bytranslationalarrestorbycleavageofthetarget.Inaddition,chromatinremodeling canoccurinsomespecies.ThemajorplayersintheRNAsilencingmechanismare smallinterferingRNAs(siRNAs),microRNAs(miRNAs)andPiwiinteractingRNAs (piRNAs),thelatterbeingrestrictedtotheanimalkingdom.Moreover,membersof theArgonaute/Piwi(AGO)proteinfamiliesandtheRNaseIIItypeproteinDicer functioninallsilencingpathways.

Figure1.1:Ghildiyal&Zamore,2009 (A)ThesiRNApathwayischaracterizedbyadsRNAwhichisprocessedbyDicerintosiRNAs.They areincorporatedintotheRISCandleadtotargetcleavage.(B)InthemiRNAspathwayahairpin shapedprecursormoleculeisexportedintothecytoplasmandprocessedbyDicer.ThemiRNAis loadedintothemiRISCandleadstotranslationalrepressionofthetarget.(C)InthepiRNApathwaya singlestrandedprecursorRNAiscleavedbyaPIWIproteinintopiRNAswhichgetmethylatedatthe 3end.TheymayinitiatetheproductionofsecondarypiRNAs. 28

1.Introduction
1.2.1 siRNAmediatedgenesilencing
siRNAswereidentifiedtodirectendonucleolyticcleavageofthetargetRNAsin plants(Hamilton&Baulcombe,1999)andanimals(Zamoreetal.,2000;Hammondet al.,2000).TheyareproducedfromlongdsRNAmoleculesbytheenzymeDicer, whichisadsRNAspecificribonucleaseoftheRNaseIIIfamily(Bernsteinetal., 2001).ThefamilyisclassifiedintothreeclasseswithDicerbeingamemberofclassIII (Nicholsonetal.,1999).Itusuallyconsistsofsixdistinctdomains,somememberslack oneormoreofthem.ThePAZ[(Piwi/Argonaute/Zwille)(Ceruttietal.,2000)]domain functionsinbindingofsingleanddoublestrandedRNAandDNAwithaprefer enceforsinglestrandedRNAsmoleculesorsinglestranded3overhangs(Lingelet al.,2003).ItisdirectlyconnectedtotheendonucleolyticRNaseIIIdomainresponsible forRNAcleavage(Robertsonetal.,1968).AdditionaldoublestrandedRNAbinding domains(dsRBDs)mayfunctioninthebindingofdoublestrandedRNA.TheN terminusiscomprisedofaDExDhelicasedomain(Bernsteinetal.,2001)andado mainofunknownfunction(Duf283)thatmaybeinvolvedinstrandselection(Dlaki, 2006).CharacterizationoftheDicerhomologueofGiardiaintestinalisrevealedthata functionalenzymeonlyrequiresthecoreproteinconsistingofPAZandtwoRNase IIIdomainswhichdimerizeanduseatwometalionmechanismforRNAcleavage (MacReaetal.,2006). ThenumberofDicerproteinsvariesbetweenspecies.Vertebratesandnematodes possessasingleDicerprotein(Carmell&Hannon,2004);DrosophilahastwoDicer proteinswithDCR1beingresponsibleformiRNAproduction(seechapter1.2.2)and DCR2producingsiRNAs(Leeetal.,2004a).InArabidopsisfourdifferentDicerlike proteinshavebeenidentified[(Baulcombe,2004)(seechapter1.2.4)]. Theycleavetheirtargetapproximatelyevery21nucleotidesintoadoublestrandof 19nucleotidesandtwonucleotideoverhangsatthe3ends(Elbashiretal.,2001a),the strandspossessa5phosphateanda3hydroxyl(Elbashiretal.,2001b).Thelength oftheproducedsmallRNAisdeterminedbythedistancebetweenthePAZandthe RNaseIIIdomains(MacReaetal.,2006).
29

1.Introduction
(A) (B)

Figure1.2:MacReaetal.,2006 (A)DomainorganizationofhumanDicerwith Helicase,DUF283,PAZ,twoRNaseIIIandthe dsRBDdomainsandtheminimalDicerofGiardia intestinaliswiththePAZandthetwoRNaseIII domains.(B)Resolvedcrystalstructureofthe GiardiaDicerwiththeNterminalplatformdomain (blue),thePAZdomain(orange)connected(red)to theRNaseIIIadomain(yellow),whichhasabridge (grey)totheRNaseIIIbdomain(green).

Thedoublestrandisseparateddependingontherelativethermodynamicstabilityof thetwoendsoftheduplex(Khvorovaetal.,2003;Schwarzetal.,2003).Thepassenger strandisdestroyed(Matrangaetal.,2005;Leuschneretal.,2006)andtheguidestrand isincorporatedintotheRNAinducedsilencingcomplex(RISC).Itischaracterized bytheguidestrandthatisboundtoanArgonauteproteinandauxiliaryproteinsde pendingonthespeciesandthetypeofsmallRNA(Hammondetal.,2000). Argonauteproteinscontainfourdomainswithpartlyunidentifiedfunctions(see Figure1.3a,b):anNterminaldomain,thePAZdomainwhichbindsssRNA(Lingel etal.,2003),themiddledomainwithresemblancetothesugarbindingdomainofthe lacrepressor(Friedmanetal.,1995)andthePiwi[(PElementinducedwimpytestis domain)(Lin&Spradling,1997)]withanRNaseHfoldthatfunctionsasaribonucle aseandconfersthecleavageofssRNA,whichisalsonamedslicing(Songetal.,2004). ThesilencingcomponentofArgonauteproteinscanbesubdividedintotwogroups: theAGOcladeconsistsofmembersthataresimilartoAGO1ofArabidopsisthaliana. TheybindsmallRNAsderivedfromdsRNAintheRISCandareexpressedubiqui tously.IncontrastthePiwicladeconsistsofthreeproteinswhichareprimarilyex
30

1.Introduction
pressedingonadaltissues(seeFigure1.3c):Piwi,theDrosophilaPelementinduced wimpytestesprotein(Lin&Spradling,1997),Aubergine(Aub)firstidentifiedbyits roleindorsoventralpatterning(Schpbach&Wieschaus,1991)andArgonaute3 (AGO3). TheincorporatedsiRNAbindstoitstargetsequenceandtherespectiveArgonaute proteincleavesthephosphodiesterbondofthetargetbetweenthetenthandeleventh nucleotideoftheboundguidestrand.Thecleavedtargetisthenreleasedofthema tureRISC(Elbashiretal.,2001b). HumanspossesseightArgonauteproteinsbutonlyAgo2showsSliceractivity(Liuet al.,2004;Meisteretal.,2004),DrosophilamelanogasterhasfiveArgonauteproteinsand allofthempossesstheabilitytoslice(Miyoshietal.,2005).Caenorhabditiseleganshas thelargestnumberofArgonauteproteinswith27differentmembers(Yigitetal., 2006).Arabidopsisthalianapossesses10Argonauteproteins[(Vaucheret,2008)(see chapter1.2.4)].
(A) (C)

(B)

Figure1.3:Argonauteproteins (A)DomainorganizationofhumanAGO2withtheNterminalPAZdomain,theMiddomaininclud ingthecapbindinglikeMCdomainandtheCterminalcleavagecompetentPIWIdomain(B)Crystal structureoftheArgonauteofPyrococcusfuriosusincludingthesiRNA(purple)andmRNA(turquoise) duplex.ActiveresiduesofthePIWIdomain(aDDHmotif)areshowninred.(Hutvagner&Simard, 2008)(C)MultiplesequencealignmentrevealedthreecladesofArgonauteproteins(Tolia&Joshua Tor,2007). 31

1.Introduction
1.2.2 miRNAsmodulatetheexpressionofendogenoussequences
ThefirstreportofamicroRNAgenewaslin4whichhadtheabilitytorepresscell proliferationinC.elegans(Chalfieetal.,1981).Althoughencodedbyagene,itwas latershowntobeonlytranscribedintoanoncodingRNAwithsomecomplementar itytothe3UTRofanothermRNAtranscript,lin14,whichconsequentlybecame downregulated(Leeetal.,1993).Firstconsideredasauniquephenomenonsome yearslaterlet7wasdiscoveredtousethesamemechanism(Reinhartetal.,2000). SubsequentlyitwasshownthatmiRNAsareanabundantclassofsmallRNAmole culesresponsibleformanyintracellularregulationprocesses(reviewedinCarthew& Sontheimer,2009). AnimalmiRNAsarehighlyconservedbetweenspecieswhichhasalsobeenusedfor theiridentificationinthepast(Ambrosetal.,2003).ManynewmiRNAshavebeen identifiedbydeepsequencingtechnologieswithnow15172entriesinmiRBasere lease16,Sept2010(GriffithsJonesetal.,2008). GenomicregionscodingformiRNAscanbelocatedinproteincodinggenesorin intergenicregions.Itwasshownthattheycontainstandardpromoterelements.Con sequentlytheyaretranscribedbyRNAPolymeraseII(PolII)(Leeetal.,2004b)into primarymiRNA(primiRNA)transcriptswhichareusuallyhighlystructured(Leeet al.,2002).Thefirstmaturationstepisalwayslocatedinthenucleusandexecutedby theRNaseIIIendonucleaseDrosha(Leeetal.,2003)assistedintheMicroprocessor complexbyadsRNAbindingprotein[Pashainflies(Denlietal.,2004)andDGCR8 inmammals(Gregoryetal.,2004;Hanetal.,2006)].ItresultsintheprecursormiRNA (premiRNA)whichiscomprisedofanimperfecthairpinstructure(Leeetal.,2002). InanimalsthemoleculeissubsequentlyexportedintothecytoplasmbyExportin5 andtheGTPaseRan(Yietal.,2003).Thesecondmaturationstepisexecutedbya Dicerenzyme(Grishoketal.,2001;Hutvgneretal.,2001;Kettingetal.,2001)again assistedbyadsRNAbindingprotein[infliesR2D2fordsRNA(Liuetal.,2003)and LOQSforstructuredloci(Frstemannetal.,2005;Saitoetal.,2005)andTRBPin mammals(Chendrimadaetal.,2005;Haaseetal.,2005)].ItgeneratesamiRNA/

32

1.Introduction
miRNA*duplexofapproximately21ntand2ntoverhangsatthe3end.Although resemblingsiRNAsinthisstep,theycanbedistinguishedbytheimperfectbinding betweenthetwostrands(Leeetal.,2002).TheduplexisloadedintotheRISCandthe miRNA*stranddegradedbytherespectiveAgoprotein(Filipowiczetal.,2008). 510%ofallmiRNAgeneswithlowexpressionarelocatedinintronswhichfoldinto shorthairpins.Thesesocalledmirtronsarefirstprocessedbythesplicingmachinery andthenlinearizedbythelariatdebranchase.Theyfurtherfoldintoahairpinsimilar topremiRNAswhichareprocessedasdescribedabove(Okamuraetal.,2007;Ruby etal.,2007). Recentlyadistinctbiogenesismechanismhasbeendiscoveredinmice(Cheloufiet al.,2010)andzebrafish(Cifuentesetal.,2010)formiR451whichpossessesanun usualsecondarystructurewithashortstemof17nucleotides.ThepremiR451is cleavedbyAgo2andtheresultingintermediatesarepolyuridylatedandfurther processedbyyettobedefinednucleasesintothematuremiRNA(Cheloufietal., 2010;Cifuentesetal.,2010).
Figure1.4:Okamuraetal.,2007 ThecanonicalmiRNApathwayconsistsofaPolII transcriptwhichfoldsintoahairpin.ThisprimiRNA isprocessedbytheDroshacomplexintothepre miRNAhairpin.Inthemirtronpathway,ashortin tronissplicedandbranchedintoahairpin.Thepre miRNAsareexportedtothecytoplasmbyEportin5 andcleavedbyDicer.

33

1.Introduction
AnimalmiRNAsbindtheirtargetsinadifferentmannerthansiRNAs:onlyaseed regionofapproximatelysixnucleotidesaroundtheusualcleavagesiterequiresper fectcomplementaritytothe3UTRsoftheirtargets,therestofthemiRNAspos sessesmismatchesandfrequentnonconventionalbasepairing(G:Uwobbles)with thetargetedmRNA.Theexactmechanismofsuppression,however,isstillunder investigation.TheimperfectbindingpreventstargetcleavagebyArgonautes.One modelproposescompetitionwiththecapbindingproteineIF4Eandsubsequentin hibitionofthetranslationinitiation.Anotherhypothesisaccountsforthefactthat manymiRNAregulatedmRNAsaredeadenylated.ItproposesRISCtostimulate deadenylationandsubsequentmRNAdecay.Athirdmodelusesthefindingthat RISChassomebindingaffinitytothe60Sribosomalsubunitandmaythereforepre venttheassemblyoftheribosome(reviewedinCarthew&Sontheimer,2009). ArgonauteproteinsinvolvedinthemiRNApathwayinteractwithGW182proteins (BehmAnsmantetal.,2006).Theycontainfrequentglycine(G)andtryptophan(W) repeats(Eystathioyetal.,2002)organizedinthreedistinctregions.TheNterminal GWrepeatregionisfollowedbyaubiquitinassociated(UBA)likedomainanda glutaminerich(Qrich)region.ThemiddleandaCterminalGWrepeatregionare separatedbyaRNArecognitionmotif(reviewedinDing&Han,2007;Eulalioetal., 2007).Whilethereexistthreeparaloguesinvertebrates(TNRC6A,BandC),fungi havenoGW182proteinsandDrosophilapossessesonlyoneorthologuemakingita goodmodelforstudyingtheirfunction(BehmAnsmantetal.,2006).TheC.elegans orthologuesAIN1andAIN2containonlytheNterminalGWrepeatregionbut functionalsoinmiRNAsilencing(Dingetal.,2005;Zhangetal.,2007).Bindingof ArgonautebyGWcontainingproteinshasbeenalsoidentifiedinplants(NRPD1b andSPT5liketranscriptionelongationfactor)(ElShamietal.,2007;BiesEtheveetal., 2009)andS.pombe[(Tas3)(Partridgeetal.,2007;Tilletal.,2007)]. GW182proteinsexhibitsomeintrinsicsilencingactivity(Eulalioetal.,2009a;Zip prichetal.,2009).InadditionlossofGW182suppressesmiRNAsilencing(Rehwinkel etal.,2005),butitactsdownstreamofmiRNAprocessingandloadingintotheRISC
34

1.Introduction
(Eulalioetal.,2009a;Miyoshietal.,2009).TheNterminalregionisrequiredforthe bindingtoAGO1(BehmAnsmantetal.,2006)andtogetherwiththeQrichregion responsibleforlocalizationtothePbodies[(processingbodies)(Eulalioetal., 2009b)].TheyarecytoplasmicgranuleswheretranslationallyrepressedmRNAscan concentrate.Allfactorsinvolvedin53exonucleolyticdecayofmRNA,including thedecappingenzymeDCP2andthemaincytoplasmic53exoribonucleaseXRN1 colocalizetothePbodiesinthecytoplasm.TheyarenotrequiredformiRNAsilenc ingbutcanbeformedasaconsequenceofit(reviewedinEulalioetal.,2007).Acon servedmotifofapproximately40residuesinsidethemiddleGWrepeatregionhas recentlybeenidentifiedasaPolyAbindingproteininteractingmotif(Fabianetal., 2009;Zekrietal.,2009),whichappearstobecriticalformiRNAmediatedsilencing (Huntzingeretal.,2010).

Figure1.5:Carthew&Sontheimer,2009 miRNAdependenttranslationalrepressionmaybemediatedbycompetitionwithcaporribosome binding,circularizationmaybeblocked,ortheribosomescoulddropoffaftertranslationinitiation. AlternativelydeadenylationandsubsequentmRNAdegradationmightbeinduced.

35

1.Introduction
miRNAsthemselvesareveryoftenundercontroloftheirtargetsinanegativefeed backloop.AnothercontrolpointistheregulationofmiRNAprocessing,wheremany factorshavebeenalreadyidentified,ortheregulationoftheeffectorproteinsofthe miRISC(reviewedinKroletal.,2010).Afirstreportofexonucleasesdegrading miRNAscomesfromplants(Ramachandran&Chen,2008)andahomologuehas alsobeenidentifiedinC.elegans(Chatterjee&Grosshans,2009). UndercertainconditionsthemiRNAloadedRISChasbeenshowntoactivatetrans lationbuttheexactreasonandmechanismisnotclear(Vasudevanetal.,2007;Henke etal.,2008;rometal.,2008).

1.2.3 ThepiRNApathwayprotectsthegermlinefromtransposonactivity
InDrosophilamelanogasteralargefamilyofsmallRNAswithalengthof2326ntwas identifiedtomaptorepetitiveheterochromaticregionsandtransposableelements, theywerecalledrepeatassociatedsmallinterferingRNAs(rasiRNAs)(Aravinetal., 2001)andcouldlateralsobeidentifiedinzebrafish(Chenetal.,2005). PiwianditsorthologueswereshowntobindrasiRNAsand,inmammals,smallRNA speciessimilartorasiRNAsbutnotderivedfromrepeatandtransposonsequences. ThenewlyidentifiedfamilyofsmallRNAswasfurthercalledPiwiinteractingRNAs [(piRNAs)(Malone&Hannon,2009)].Theyhave2Omethylated3ends,whichis depositedbyanorthologueoftheplantmethyltransferaseHEN1(Kirino&Mourela tos,2007;Oharaetal.,2007).Inaddition,theyhaveastrongbiasfora5uridineresi dueandaDicerindependentbiogenesispathwayresultinginalargersizecompared totheothersmallRNAs.Theyallarisefromchromosomalclustersandhaveasingle strandedRNAprecursor(reviewedinKlattenhoff&Theurkauff,2008).Thereisevi dencethatthe21URNAsofC.elegansarealsopiRNAssincetheycontaina5uridine (Rubyetal.,2006),theysuppresstransposonmobility(Dasetal.,2008)andtheyin teractwiththePiwirelatedgenePRG1(Wang&Reinke,2008). InDrosophilapiRNApopulationscanbematchedtotransposons,usuallyenriched forsequencesantisensetotransposons.TheseantisensepiRNAsareboundbyPiwi andAubwhereasthesenseorientatedfractioninteractswithAGO3.Thetwoclasses
36

1.Introduction
ofpiRNAshaveoverlapping5endsseparatedbytennucleotides(Brenneckeetal., 2007,Gunawardaneetal.2007),suggestingprocessingbyPiwiwhichhadbeen showntocleaveitstargettennucleotidesfromthe5endoftheguidestrand(Saitoet al.,2006).ThebiogenesisandamplificationofpiRNAsfollowsthesocalledping pongcycle.ItisinitiatedbyprimaryantisensepiRNAswhichtargetthecleavageof transposonmRNA.ThisresultsinsecondarysensepiRNAswhichareboundby AGO3anddirectthecleavageofantisensetransposonsequences(Brenneckeetal., 2007,Gunawardaneetal.2007).Partsofthiscyclehavealsobeendetectedinzebraf ish(Houwingetal.,2007)andmice,althoughthecyclethereseemstobeinitiatedby sensepiRNAsandisonlypresentinthemalegermline(Aravinetal.,2007).Thecycle sharessimilaritieswiththesilencingofheterochromaticregionsbyRNAdirected DNAmethylationinplantsandS.pombe(seechapter1.2.4.1).Inthelatterthetran scriptionofcentromericrepeatsleadstosiRNAswhichdirecttheAGOorthologueto cleavetargettranscriptsofthislocus.ThereactionactivatestheRNAdependent RNApolymerasecomplexwhichgeneratesfurthersiRNAs.FinallythesesiRNAs directthemodificationofhistones(Moazed,2009). AdditionalfactorshavebeenidentifiedtoberequiredforthepiRNApathway,muta tionsintheputativenucleasesZucchiniandSquashdisruptpiRNAproductionand releasetransposonsilencing(Paneetal.,2007).Thesamecanbeobservedafteraloss oftheputativehelicasesArmitage(Vaginetal.,2006)andSpindleE(Aravinetal., 2004).Theyareallpartofnuage,whichisagermlinespecificperinuclearstructure implicatedinRNAprocessing.ThissuggestsacompartmentalizationofpiRNAbio genesisandaction(Lim&Kai,2007;reviewedinKlattenhoff&Theurkauff,2008). C.elegans21URNAsarealsorequiredforfertility(Batistaetal.,2008)andthegerm linefunction(Wang&Reinke,2008). InDrosophilamainlyAubandAGO3associatedpiRNAstakepartinthepingpong cycle,PiwiassociatedpiRNAsmayonlycomprisetheprimarypiRNAsinthegerm linespecificcycle.However,anadditionalpathwayofpiRNAfunctionhasbeendis coveredrecentlyinsomaticovarianfolliclecells,dependingexclusivelyonPiwiand
37

1.Introduction
theflamencopiRNAcluster(Maloneetal.,2009a,b).Althoughinitiallymorefactors hadbeenidentified,recentfindingssuggestthefollowingproteinsbeingindispensa bleforthesomaticpiRNApathway:Zucchini,Armitageandtheputativehelicase/ tudordomainproteinYbarerequiredforsilencingofthegypsytransposoninthe somaticcells.Thelatterproteinsarebothlocalizedtocytoplasmicfoci.Piwiaccumu latesinthecytoplasmintheabsenceofZucchini,whichmaybeconfersshuttlingbe tweenthecytoplasmandthenucleus.InarmitagemutantsnopiRNAsaccumulate suggestingafunctionearlyinthepathway(Olivierietal.,2010;discussedinZamore, 2010).

Figure1.6:Zamore,2010 InsomaticcellspiRNAsaresynthesizedbyaPIWIdependentlinearpathwaywithoutamplification, whereasinthegermlinetheprimarypiRNAsaredependentonAubergineandgetamplifiedviathe socalledpingpongcycleandAgo3.

1.2.4 PlantspossessahighdiversityofsiRNAmolecules
PlantsdisplayanastonishingvarietyofsiRNAtypesandproteinswhichareneeded fortheirgeneration.ArabidopsisthalianahasfourDicerlikeandtenArgonautepro teinswithdistinctmolecularfunctions. EverymemberoftheDICERLIKEproteinfamilyinArabidopsishasdistinctfunctions althoughsomeredundancieshavealsobeenidentified:DCL1isthemainDicerin
38

1.Introduction
volvedinmiRNAprocessingwhichcannotbecompensatedbytheothermembers (Parketal.,2002;Reinhartetal.,2002;Pappetal.,2003).Inaddition,DCL1canprocess somenatsiRNAsfromendogenousinvertedrepeatsequences(Borsanietal.,2005; KatiyarAgarwaletal.,2006).DCL2alsoparticipatesinthelatterfunctionandgener ates22ntsiRNAsfromvirussequences(Xieetal.,2004).DCL3produces24nt siRNAswhicharemainlyinvolvedinchromatinsilencing(Xieetal.,2004).DCL4is themainplantDicerfortheproductionofviral21ntsiRNAs(Dunoyeretal.,2005; Bouchetal.,2006;Delerisetal.,2006).Moreover,itisinvolvedintasiRNAmetabo lism(Gasciollietal.,2005;Xieetal.,2005b)andintheproductionofmiR822and miR839(Rajagopalanetal.,2006).AfterprocessingbyDCLproteinsinplantsthere sultingsmallRNAsaremethylatedattheir3endsbytheSadenosyldependentme thyltransferaseHUAENHANCER1(HEN1)whichprotectsthemfromuridylation andfurtherdegradation(Lietal.,2005;Yangetal.,2006) PlantscontainahighnumberofArgonauteproteinswithtenmembersinArabidopsis thalianaand19membersinOryzasativa.Theformerwillbediscussedinmoredetails (reviewedinVaucheret,2008). AGO1isthefoundingproteinforthewholeArgonauteproteinfamilyanditspleio tropicdefectshavefirstbeendescribedin1998(Bohmertetal.,1998).Ithadlaterbeen identifiedtoactinRNAsilencing(Fagardetal.,2000).AGO1predominantlyactsin themiRNApathwaybutitcanalsobindseveralclassesofsiRNAs(Baumberger& Baulcombe,2005).ItisitselfregulatedbyafeedbackmechanismthroughmiR168 (Vaucheretetal.,2006).AGO10(originallytermedPINHEAD/ZWILLE)istheclosest paralogueofAGO1andpartlyshowsredundantfunctionindevelopment(Lynnet al.,1999).RecentstudiesimplicateAGO10asanegativeregulatorofAGO1(Mallory etal.,2010).AGO5isthethirdmemberofthisgroup.Itsexactfunctionisnotclear, butithasbeenshowntopreferentiallyinteractwitha5cytosine(Takedaetal.,2008). AnothercladeofArabidopsisAGOproteinscontainsAGO7whichisinvolvedinthe TAS3biogenesispathway[(Montgomeryetal.,2008a)(seechapter1.2.4.2)].AGO2

39

1.Introduction
andAGO3arehighlysimilarproteinswithcurrentlyunknownfunction.Theformer isprobablyregulatedbymiR403(Allenetal.,2005). ThethirdcladeiscomprisedofAGO4,themajorproteininvolvedintranscriptional genesilencing[(Zilbermanetal.,2003)(seechapter1.2.4.1)].AGO6hasapartialre dundantactivitywithAGO4(Zhengetal.,2007).Theroleoftheothertwoproteinsof thisclade,AGO8andAGO9,isstillunknown,buttheirsimilaritysuggestsaredun dantfunction.SincetheAGO8expressionisverylowithasbeensuggestedtobea pseudogene(Takedaetal.,2008). Ithasbeenshownthatthe5nucleotideofthesmallRNAspeciesactsasasorting signalintothedifferentAGOproteinsinplants:AGO2andAGO4preferentiallyin teractwithadenosine.AGO1prefersuridinewhichisthepredominant5nucleotide ofplantmiRNAs.FinallyAGO5bindssmallRNAswithaterminalcytosine(Mietal., 2008).

Figure1.7:Ghildiyal&Zamore,2009 (A)InplantscisactingsiRNAprecursormoleculesaretranscribedbyPolIVandadsRNAgenerated byRDR2.DCL3cleavesthe24ntcasiRNAswhichassociatewithAGO4.(B)InthetasiRNApathway aprecursormoleculeissubjecttomiRNAmediatedcleavage.RDR6generatesadsRNAwhichis processedbyDCL4intotasiRNAswhichassociatewithAGO1/7.(C)natsiRNAsderivefromoverlap pingtranscriptswhichareprocessedintoadsRNA.AdicermoleculethengeneratesthenatsiRNAs.

40

1.Introduction
1.2.4.1 CisactingsiRNAsmediatechromatinsilencinginplants RNAdirectedDNAmethylation(RdDM)isanepigeneticsiRNAmediatedmodifica tioninplants(reviewedinMatzkeetal.,2009;Law&Jacobsen,2010).Inplantsitis responsiblefor30%ofdenovomethylationofcytosinesinheterochromaticandsome euchromaticregionssuchastransposons(Cokusetal.,2008;Listeretal.,2008).This methylationisdepositedbyDOMAINSREARRANGEDMETHYLTRANSFERASE2 (DRM2).InadditionRdDMrequirestwoplantspecificPOLIIrelatedRNApoly merases,POLIVandPOLV(Pikaardetal.,2008,Wierzbickietal.,2008). Theirlargest(NRPD1andNRPE1)andsecondlargestsubunits(thesharedsubunit NRPD2/NRPE2)areuniquewiththelatterbeingalsorelatedtothelargestsubunitof POLII(RPB1)(Wierzbickietal.,2008;Reametal.,2009).Theyactincomplexes

Figure1.8:Matzkeetal.,2009 dsRNAisprocessedbyDCL3/HEN1into24ntsiRNAswhichareloadedontoAGO4.Transcription byPolVfacilitatesdenovomethylationatthesiRNAtargetedsite.PolIVtranscribesthemethylated DNAwhichisfurthercopiedbyRDR2intodsRNA. AfterprimaryRdDMPolIVtranscribesthemethylatedtemplateanddownstreamsequenceswhich resultinsecondaryRdDM. 41

1.Introduction
includingSNF2likechromatinremodelingfactors:POLIVtogetherwithCLASSY1 (CLSY1)isinvolvedintheinitiationofsiRNAbiogenesisbytranscribingalongsin glestrandedRNA.POLVtogetherwithDEFECTIVEINRNADIRECTEDDNA METHYLATION(DRD1)(Pikaardetal.,2008)identifiesandmaybealsotranscribes lowabundanceintergenicnoncodingtranscripts(IGN)(Wierzbickietal.,2008). RNADPENDENTRNAPOLYMERASE2(RDR2)producesdsRNAfromthesingle strandedPOLIVdependenttranscriptswhicharefurtherprocessedbyDCL3into 24ntheterochromaticsiRNAs(Mosheretal.,2008).TheseareboundbyAGO4[(or sometimesAGO6(Zhengetal.,2007)]whichcaninteractwithPOLVthroughacon servedGW/WGmotif(ElShamietal.,2007).TheIGNtranscriptsareprobablyrecog nizedbytheAGO4boundsiRNAs(Wierzbickietal.,2008).RecentlyPOLII dependentnoncodingtranscriptshavebeenidentifiedtoalsorecruitRdDMfactors (Zhengetal.,2009).INVOLVEDINDENOVOMETHYLATION2(IDN2)mayinter actwiththesiRNARNAduplexandrecruitRDM2(Ausinetal.,2009). TheactionofRDR2mayleadtotheproductionofsecondarysiRNAsandfurtherme thylationspreading(Daxingeretal.,2009).siRNAsalsoappeartoguideactivede methylation(Zhengetal.,2008).TheRdDMmechanismisconservedinS.pombeas wellwhereitleadstoheterochromatinization(reviewedinMoazed,2009). 1.2.4.2 TransactingsiRNAs TransactingsiRNAs(tasiRNAs)areaplantspecifictypeofsmallRNAsgenerated fromspecificTASloci.ThereexistfourTASfamilieswhichcanbefurthersubdivided intotwoclasses:oneconsistsofTAS1,TAS2andTAS4whichrequireonemiRNA bindingsitefortasiRNAbiogenesis.TheotheriscompromisedbytheTAS3family whichrequirestwomiRNAbindingsitesfortasiRNAbiogenesis(Allen&Howell, 2010). TheTAS1family,consistingofthreeloci,andTAS2werethefirstidentifiedTASloci (Peragineetal.,2004;Vazquezetal.,2004).TheydependonthecleavagebymiR173 (Yoshikawaetal.,2005),whereasTAS4dependsonthecleavagebymiR828(Ra jagopalanetal.,2006).TheinitialPOLIItranscriptsareboundbythemiRNAwith
42

1.Introduction
unusualmismatchesintheseedregion,substitutionofthesemismatchesabolishes theproductionoftasiRNAs(Montgomeryetal.,2008b;Felippes&Weigel,2009).The transcriptiscleavedbyAGO1(Vazquezetal.,2004)andboundbySUPPRESSOROF GENESILENCING3(SGS3)(Peragineetal.,2004;Vazquezetal.,2004).Itcaninteract withtheRNADEPENDENTRNAPOLYMERASE6(RDR6)(Kumakuraetal.,2009) whichsynthesizesthecomplementarystrandfromthe3endtowardsthe5cleavage site.TheresultingdsRNAisfurtherprocessedbyDCL4fromthemiRNAcleavage siteinto21ntlongsiRNAs(Peragineetal.,2004;Vazquezetal.,2004).Thedominant phasingpatterncanalsodriftforoneortwonucleotides,probablybytheredundant

Figure1.9:Allen&Howell,2010 InthefirstpathwaymiR173/828guidesthecleavageoftheTAStranscriptbyAGO1.Incontrastinthe secondpathwaymiR390bindstwicetotheTAS3transcriptandguidesitscleavagebyAGO7atthe3 site.ThecleavedtranscriptissynthesizedbyRDR6intoadsRNAwhichisprocessedbyDCL4intothe tasiRNAs. 43

1.Introduction
functionofotherDCLproteins(Howelletal.,2007).TheTAS3familyconsistsof threeloci(Howelletal.,2007),allcharacterizedbytwobindingsitesformiR390 whichflankthetasiRNAproducingsequence(Allenetal.,2008).Fromtheseonlythe 3siteiscleavedanddeterminestheresultingtasiRNAs.The5sitepossessescritical mismatchesanditisnotclearifitisalsocleaved.miR390wasshowntointeractwith AGO7.Whilethecleavageatthe3sitecanbeaccomplishedbyanothermiRNA AGOpair,thebindingofAGO7tothe5siteisindispensablefortasiRNAproduc tion(Montgomeryetal.,2008a). ThetasiRNAsaresortedintothecorrespondingArgonauteproteinsaccordingto their5nucleotide(Mietal.,2008).tasiRNAscanbeconsideredasanamplificationof aninitialsilencingsignal.Inadditiontheyarenoncellautonomousandcancreatea silencinggradientacrossneighboringcells(Chitwoodetal.,2009;Schwabetal.,2009). IdentifiedtargetsarePentatricopeptiderepeatproteins(Peragineetal.,2004; Vazquezetal.,2004),MYBtranscriptionfactors(Rajagopalanetal.,2006)andAuxin responsivefactors(Allenetal.,2005;Williamsetal.,2005;Adenotetal.,2006;Fahl grenetal.,2006;Garciaetal.,2006). 1.2.4.3 NaturalantisensesiRNAs NaturalantisensetranscriptderivedsiRNAs(natsiRNAs)havefirstbeendescribed asaresultofsaltstressinArabidopsis(Borsanietal.,2005).Thereexisttwodifferent typesofnaturalantisensetranscripts(NATs):cisNATshaveahighsequencecom plementaritysincetheyaretranscribedfromopposingstrandsofthesamelocus.In contrast,transNATsaretranscribedfromdifferentlociresultinginshortandimper fectcomplementarityofthedoublestrand(Jinetal.,2008). FactorsrequiredforthesynthesisoftheprimarynatsiRNAsareDCL2and/orDCL1, RDR6,SGS3andPOLIV(Borsanietal.,2005;KatiyarAgarwaletal.,2006;Zhang& Trudeau,2008).Theyguidethecleavageofthecomplementarytranscript.Although ratherrareunderphysiologicalconditions,theymaysubstantiallycontributetothe smallRNApopulationduringstressconditions,sinceNATpairscompromisemore than7%ofalltranscriptionalunitsinArabidopsisthaliana(Henzetal.,2007).
44

1.Introduction
1.2.5 PeculiaritiesofplantmiRNAs
ThefirstplantmiRNAshavebeenidentifiedin2002(Parketal.,2002;Reinhartetal., 2002).ExamplesofevolutionaryconservedmiRNAsbetweenanimalsandplantsare rare,thereforeitisbelievedthattheyevolvedindependentlyinthetwogenera(Ax tell&Bowman,2008).PlantmiRNAsconsequentlypossessseveralspecialfeatures comparedtoanimalmiRNAs(reviewedinVoinnet,2009). MostplantMIRgenesarelocatedinintergenicregions;thereexistmorethan100 familiesofrelatedmiRNAswhichareusuallyconservedbetweenangiosperms(Ax tell&Bowman,2008)andpartlyalsobacktomoss(Garcia,2008).Anothergroupof miRNAsareevolutionaryyounger,thereforelessconservedandwithahighlydi versetargetrange(Zhangetal.,2006).MIRgenesaretranscribedbyPOLIIintopri miRNAswhichgetcappedatthe3endandpolyadenylatedatthe5endlike mRNAtranscripts.Thestrandfoldsintoanimperfecthairpinwhichisprobablysta bilizedbytheRNAbindingproteinDAWDLE(DDL).ItalsobindsothermRNAs andmightbeinvolvedinsiRNAbiogenesistoo(Yuetal.,2008).TheprimiRNAsare furtherprocessedbyDCL1intomaturemiRNAsinatwostepprocess.Thefirsttran sitiontopremiRNAstakesplaceinnuclearprocessingcenters(Dbodies)where DCL1interactswiththedoublestrandedRNAbindingproteinHYPONASTIC LEAVES1(HYL1)andtheZincfingerproteinSERRATE(SE),thelatteralsohavinga roleinmRNAsplicing(Fang&Spector,2007;Kuriharaetal.,2006).Afterthesecond processingstepbyDCL1,thematuremiRNAduplexesarestabilizedbymethylation attheir3endswhichistransferredbyHEN1(Lietal.,2005;Yangetal.,2006).The miRNAsareexportedintothecytoplasmbythenuclearporecomplexHASTY,al thoughotherexportmechanismscannotbeexcluded(Parketal.,2005). ThefirstidentifiedmodeofactionofplantmiRNAsischaracterizedbyextensive complementaritytotheirtargetmRNAs.ThemiRNAsareloadedintotheArgonaute proteinAGO1oftheRISCwhichslicesthemRNAtargetsbetweenthetenthand eleventhnucleotide(Rhoadesetal.,2002).Lateramechanismoftranslationalrepres sionsimilartothemiRNAmechanisminanimalshasbeenidentifiedinmiRNA

45

1.Introduction
actiondeficient(mad)classIIImutants;itprobablyalsorequiresthefunctionof AGO1aswellasamicrotubuleseveringenzymeKATANIN(KTN)andthePbody componentVARICOSE(VCS)whichisrequiredformRNAdecapping(Brodersenet al.,2008).TheextentofmiRNAexpressionisregulatedondifferentlevels:MIRgenes havetheirownpromoters(Xieetal.,2005a)whichshowahighdegreeoftranscrip tionfactorbindingsites(Megrawetal.,2006).Tissuespecificdifferencesinprotein expressioncanaccountforalteredmiRNAaction.HighDCL3levelscompetewith DCL1formiRNAprocessing,resultingin24ntproductswhicharepredominantly sortedintoAGO4andthereforenotavailablefortheRISC(Vazquezetal.,2008). Abundanttranscriptionofshortinterspacedelements(SINE)RNAcancompetefor theessentialfactorHYL1(PouchPelissieretal.,2008).PlayersofthemiRNApath wayarefrequentlyitselftargetsofmiRNAregulation,suggestingafeedbackregula tion(Xieetal.,2003,Vaucheretetal.,2004).MoreoversinglestrandedmiRNAsare alsosubjecttodegradationbymembersoftheSMALLRNADEGRADING NUCLEASE(SDN)familyofexonucleases(Ramachandran&Chen,2008).

1.2.6 SpreadingofRNAsilencinginplantsresemblesanimmunesystem
InplantsRNAsilencingactsassortofanimmunesystemprovidingresistanceto viruses.Sincevirusesreplicaterapidlyandareabletospreadthroughoutthewhole plant,thehostneedsamechanismtransmittingtheinitialimmunityofRNAsilenc ingtootherleaves.ForstudyingthisphenomenonA.thalianaisusuallyreplacedby Nicotianasp.asamodelorganism,sincetheyhostawiderangeofvirusesandhavea lifecycleandsizemorefavorableforstudyingtheeffectsofviralinfections.Amajor disadvantage,however,isthemissinggenomicinformationofNicotianasp. TheinitiationofsilencingismediatedbytheoverexpressionofexogenousRNA whichistransformedintodsRNAbytheactionofRNAdependentRNApoly merases[(RDRs)(Wassenegger&Krczal,2006)].Theiractivityisstimulatedbythe presenceofaberrantRNAwithmissing5capstructuresor3polyadenylation(Herr etal.,2006;Luo&Chen,2007).RDR6andRDR1arethemajorplayersinantiviralsi lencingmechanisms(Schwachetal.,2005;DiazPendonetal.,2007;Quetal.,2008)
46

1.Introduction

Figure1.10:Kalantidisetal.,2008 ExamplesofsilencingspreadofaGFPtransgene:(A)notsilenced(B)spontaneousshortrangelocal silencing(C)inducedshortrangelocalsilencing(D)fullysilenced(E)systemicsilencing(F)extensive localspread(left)andsystemicsilencing(right).

togetherwiththecofactorsSILENCINGDEFICIENT3(SDE3),aputativeRNAheli case(Dalmayetal.,2001)andSUPPRESSOROFGENESILENCING3(SGS3),a coiledcoileddomainprotein(Mourrainetal.,2000;Kumakuraetal.,2009).Silencing ofDNAvirusesandtheRNATobaccorattlevirus(TRV)additionallyrequiresRDR2 (Donaireetal.,2008). Exogenousfactorssuchastemperature(Szittyaetal.,2003)andlight(Kotakisetal., 2010)caninfluencetheefficiencyofsilencingonset.Endogenoussequencescannot serveassubstratesforRDRs(Himberetal.,2003;Schwachetal.,2005;Bleysetal., 2005). ThespreadingofRNAsilencingcanbedifferentiatedintothreedifferentstages, shortrangelocalspread(SLSS),extensivelocalspread(ELSS)andsystemicsilencing. TheshortrangelocalsilencingspreadisnotlimitedtoexogenousRNAandcanalso affectendogenoussequences.Afterinitiationofsilencinginacellthesignalistrans
47

1.Introduction
ferredto1015cellssurroundingtheinitialsourceofsilencingwithoutamplification (Himberetal.,2003).Theexactnatureofthesignalcouldnotbediscoveredyet,butit probablyreliesonpassivediffusionthroughtheplasmodesmata(Voinnetetal.,1998; Himberetal.,2003;Kalantidisetal.,2006)withamobilitycomparabletosolublepro teinsof2754kDa(Kobayashi&Zambryski,2007).Severalfactorshavebeenshown tobeindispensablefortheshortrangespread:lossofDCL4abolishesthemechanism suggestinganinvolvementof21ntsiRNAswhicharetheproductsofDCL4action (Dunoyeretal.,2005).SeveralotherproteinsofthevariousRNAsilencingmecha nismshavebeenshowntobeaprerequisitefortheshortrangespreadincluding HEN1,DRB4,AGO1,thePOLIVsubunitNRPD1a,RDR2anditspresumableinter actingproteinCLSY1(Hiragurietal.,2005;Adenotetal.,2006;Yangetal.,2006; Dunoyeretal.,2007;Nakazawaetal.,2007;Smithetal.,2007).Thismultitudeofin volvedproteinssuggestsanintensivecrosstalkofthedistinctRNAsilencing mechanisms. Extensivelocalspreadofsilencingischaracterizedbythefactthatthesignalexceeds thelimitof1015cellsbutdoesnotspreadtothewholeplant.Itisaccompaniedby amplificationoftheinitialsignalasaresultofanRDR6dependentmechanism (Himberetal.,2003;Schwachetal.,2005).OtherrequiredfactorsappeartobeDCL4 andtheputativeRNAhelicaseSDE3(Dalmayetal.,2001).Itisunclearwhichfactors definetheonsetofextensivelocalspread;itwasproposedthatacertainthreshold hastobeexceededtotriggerastrongerreaction.Itisrestrictedtosinktissueswhich receivethesignalfromthesourcetissuesoftheleaveswhichhadinitiallybeenchal lengedwiththeexogenousRNA(Kalantidisetal.,2006). Theleastunderstoodmechanismissystemicspreadwhichisprobablyaccompanied bythetransportofthesilencingsignalthroughthephloem(Voinnet&Baulcombe, 1997;Fagard&Vaucheret,2000;Mlotshwaetal.,2002;Tournieretal.,2006).Itsaction mightbeexecutedbyavirusspecificsiRNARISC(Lakatosetal.,2006).Thesignal transmittingthesilencingspreadofviralsequencesisbelievedtobeanRNAmole cule(Jorgensenetal.,1998),butithasbeenprovennottobeofthesizeofsiRNAs
48

1.Introduction
(Malloryetal.,2003).Anotherproteinnecessaryforbindingandfacilitatingthe movementofRNAmoleculesbetweencellshasbeendiscoveredincucurbits(Yooet al.,2004).However,sofarnohomologuesofthisPHLOEMSMALLRNABINDING PROTEIN1(PSRP1)havebeenidentifiedinArabidopsisthalianaorNicotianasp.

1.2.7 ViralstrategiestosuppressRNAsilencinginplants
VirusesalsopossessstrategiestocounteracttheRNAsilencingmechanismsusedfor theirclearancefromtheplantgenomes.Theyencodeforproteinsactingasviralsup pressorsofsilencing(VSRs)inaverydiversemanner.Morethan35VSRfamilies couldbefoundinallplantvirustypes,theycanbeclassifiedintothreedifferent categories:classIVSRssuppressthelocalsilencingoftheviralRNAs,classIIVSRs suppressthelocalsilencingspreadandclassIIIVSRsarethelargestclasssuppress ingthesystemicspreadingofsilencing(reviewedinDazPendn&Ding,2008). TheclassIsuppressorpotyviralhelpercomponentproteinase(HCPro)isamulti functionalproteinwhoseperformancepartlydependsonitssilencingsuppression activity(Kasschau&Carrington,2001).Itinterfereswithmethylation(Ebhardtetal., 2005)andpreventsRISCassembly(Meraietal.,2006;Yuetal.,2006;Shibolethetal., 2007).ThepoleoviralP0isalsoamemberofclassIVSRsandappearstomediatethe degradationofAGO1whichabolishesintracellularRNAsilencingprocesses(Pfeffer etal.,2002;Pazhouhandehetal.,2006;Baumbergeretal.,2007;Bortolamioletal., 2007).Thetobamoviralproteinp126containsmethyltransferaseandhelicasedo mainsandispresentinacomplexwithp183(Komodaetal.,2007).Itbindsduplex siRNAsandinterfereswithmethylationbyHEN1(Blevinsetal.,2006;Csorbaetal., 2007;Vogleretal.,2007)whichhasalsobeenshownforp21(Yuetal.,2006).Several othervirusencodedproteinshavebeenshowntopossessviralRNAsilencingsup pressoractivitywithyetunknownmechanisms,suchasthetranscriptionfactor AL2/AC2/C2(Trinksetal.,2005;Yangetal.,2007),thetranslationalenhancerP6 (Loveetal.,2007)andthep23proteinwhichcontrolsviralRNAaccumulation(Sat yanaryanaetal.,2002;Luetal.,2004).

49

1.Introduction
ManyviralmovementproteinshavebeenshowntobemembersofclassIIVSRs.The potexviralp25isanRNAhelicasethatinterfereswiththeplasmodesmata(Bayneet al.,2005).Thetymoviralp69appearstotargetplantsilencingupstreamofRDR dependentdsRNAsynthesis(Chenetal.,2004).p50inhibitssystemicspreadofsi lencing(Yaegeshietal.,2007),whilep25targetsdownstreamofdsRNAsynthesis (Voinnetetal.,2000;Bayneetal.,2005;Moissiardetal.,2007). MostviralsuppressorsofsilencingbelongtoclassIII.Thetomoviralp19bindsshort dsRNAmoleculeswithhighaffinity.ThesequesteringofsiRNAduplexesmaypre ventRISCfunctionality(Silhavyetal.,2002;Vargasonetal.,2003;Yeetal.,2003;Laka tosetal.,2004;Omarovetal.,2007).Therelatedp14alsobindsdsRNAmolecules (Haveldaetal.,2003,Meraietal.,2005;Pantaleoetal.,2007).Thecucumoviral2bpro teinmayhaveadualrolesinceitweaklysuppressesintracellularsilencinginaddi tiontoitspotentinhibitionofRNAsilencingspreadwhichallowslongdistancevi rusmovement(Guo&Ding,2002).ItblockstheproductionofRDR1dependentsec ondaryviRNAs(Caoetal.,2005;Yaegeshietal.,2007).Someviralcoatproteinsalso exhibitVSRfunction,thecarmoviralp38proteinisabletoreplacep19(Qu&Morris, 2002),itcanselectivelyinhibitDCL4andalsosuppress22ntsiRNAswhichare DCL2products(Meraietal.,2006).

1.2.8 RepressingtherepressorsendogenoussuppressorsofRNAsilencing
ThepowerofthesmallRNAmoleculesandthepresenceofVSRswhichspecifically sequestersmallRNAssuggestthattherealsoexistendogenoussuppressorsofRNA silencing.AfirstreportwastheCa2+sensorproteinREGULATOROFGENE SILENCINGCALMODULINLIKE(rgsCAM)whichbecameupregulateduponHC Proexpression.EctopicoverexpressionofrgsCAMmimicsthesymptomsofHC Procontainingviruses(Anandalakshmietal.,2000).RNaseLinhibitor2(RLI2)has beenfoundtobeupregulatedwhentransgenicplantsaresubjecttoRNAinterference (Brazetal.,2004).Laterithasbeenshownthatuponsimultaneousoverexpressionit reducestheamountsofsiRNAs(Sarmientoetal.,2006).Thecytoplasmicexonuclease XRN4hasbeenproposedasanendogenoussuppressorofsilencingsinceinthemu
50

1.Introduction
tantbackgroundxrn4RDRdependentsilencingwasincreased(Gazzanietal.,2004). Inaddition,overaccumulationofmiRNAcleavageproductscouldbedetected (Souretetal.,2004).Similarresultshavebeenobtainedforthenuclearexonucleases XRN2andXRN3(Gyetal.,2007).Inthesamescreenthe3,(2),5bisphosphatenu cleotidase/inositolpolyphosphate1phosphataseFIERY1(FRY1)hasbeenidentified asasuppressorofvirusandtransgeneinducedposttranscriptionalgenesilencing (PTGS)(Gyetal.,2007). InC.elegansthelossoftheputativeRNAdirectedRNApolymeraseRRF3leadto hypersensitivitytoRNAi(Simmeretal.,2002).Fornoneoftheabovedescribedpro teinsaspecificeffectonsiRNAshasbeenprovenandsecondaryeffectscannotbe excluded. FormiRNAsithasrecentlybeenshownthattheyarespecificallydownregulatedbya familyofexoribonucleasescalledSMALLRNADEGRADINGNUCELASE(SDN)in plants(Ramachandran&Chen,2008)andC.elegans(Chatterjee&Grosshans,2009)

1.2.9 ERI1isanexampleforanendogenoussuppressorofRNAsilencing
InCaenorhabditiseleganstheneuronalcellsarerefractorytoRNAinterference.Ina geneticscreeneri1nullmutantswereidentifiedtopossessenhancedsensitivityto dsRNAsthroughoutthewholeorganism.Themutantswereviableandshoweda weakphenotypeexceptforsterilityduetoadefectinspermfunction.ERI1ismainly localizedinthecytoplasmofdevelopingsomaticgonadsandinasubsetofneurons. Itisanevolutionaryconservedproteinwithnucleicacidbindingproperties(con ferredbyaSAP/SAFboxdomain)andaDEDDhlike35exonucleasedomain.It partlydegradeddoublestrandedsiRNAswith2nt3overhangsinvitro.Inaddition eri1mutantsaccumulatedmoresiRNAsafteringestionoflongdsRNAsorinjection ofsiRNAs(Kennedyetal.,2004).Thefirstidentificationofanendogenousinhibitor ofsilencinginC.eleganswastheproteinRRF3whichissimilartoRNAdependent RNApolymerasesRdRPs).rrf3mutants(Sijenetal.,2001;Simmeretal.,2002)exhib itedasimilarphenotypetoeri1mutants,includingtheenhancedRNAiphenotype (Timmons,2004).BothproteinswerefoundinascreenforDCR1interactingpro
51

1.Introduction
teins.Inadditiontwonewlyidentifiedgeneseri3anderi5wereinteractingwith DCR1.TheyalsoshowedanenhancedRNAiphenotypeandpromotedtheDCR 1/ERI1interaction.OnlythelongisoformofERL1(ERI1b)couldbedetectedin DCR1immunoprecipitatessuggestingdistinctmolecularfunctionsofthetwoiso forms(Duchaineetal.,2005). AhomologueofERI1hadbeendescribedearlierbyDominskietal.,2003.Theyiden tifiedaproteinbindingthehighlyconservedstemloopstructureofmetazoanhis tonemRNAs.3hExocontainsaSAP(Kippetal.,2000)anda35exonucleasedo main.Itbindsthe3terminalACCCAofthestemloopanddegradesit,unlessthe histonemRNAisprotectedbythestemloopbindingproteinSLBP(Dominskietal., 2003).TheconservedstemloopsequenceofhistonemRNAsisnecessaryforitsselec tivedegradationandconfersthesamereactionwhenintroducedtoothermRNAsat their3ends.The3hExoGFPproteinpredominatelyaccumulatedinthecytoplasm andthenucleoli.DeletionoftheSAPdomainabolishedbindingof3hExotothe stemloopRNAbuttheresidualexonucleaseexhibitedenzymaticactivity.There placementofArg105intheSAPdomaineliminatedbindingtothestemloop.Asp234, Asp298andMet235areindispensiblefortheenzymaticactivityof3hExo.Mutationof thelatteraminoacidleadstoglobalstructuralchangesunabletobindthestemloop (Yangetal.,2006).3hExorequiresaterminalhydroxylgroupandcannotprocess RNAsterminatingwithaphosphategroup(Dominskietal.,2005).Itcanremovethe 2ntoverhangsandthefirstnucleotideofthedoublestrandedregionofthesiRNAsin vitro(Yangetal.,2006). Thecrystallographicstructureoftheexonucleasedomainof3hExoboundtorAMP, areactionproductoftheenzyme,hasbeenresolvedataresolutionof1.6inthe presenceofMg2+.Itiscomposedofasixstranded,twistedsheetwhichisbracketed byninehelices(Cheng&Patel,2004).ThestructureissimilartoDnaQlike35 exonucleaseswhichusuallybindtoDNAandproducehydrolyticproductsreleasing anucleotide5monophosphateandleavinga3hydroxylonthepenultimatenucleo tide(Viswanathan&Lovett,1999).Theactivesiteiscomprisedoftheconserved
52

1.Introduction
acidicDEDDmotifwhichbindstwomagnesiumions.Thesetwotogetherwiththe conservedHistidineareindirectcontactwiththemonophosphateofrAMP. TheexonucleasedomainappearstolackabindingpocketaccommodatingRNAs longerthandinucleotides.ProbablytheSAP(SAFbox,AcinusandPIAS) DNA/RNAbindingdomainpositionsthe3overhangswithintheactivesite(Cheng &Patel,2004).

Figure1.11:Cheng&Patel,2004 ThepositioningoftherAMPsubstrateinthe3hExoreactioncenterrequirestwoMagnesiumions andisconferredbytwohydrogenbonds.

Thehomologueof3hExoinDrosophilamelanogasterisnamedSnipper(Snp),ahighly activeandpromiscuous35exonuclease.Ithasabroadsubstratespecificityandde gradesinvitrosinglestrandedanddoublestrandedDNAandRNAwiththere quirementofaminimal25nt3flank.Itdoesnotrequirea2OHforsubstraterec ognition,catalysisorproductrelease.3hExoandERI1share38%sequenceidentity and60%sequencesimilarity,Snpshares31%sequenceidentitywithERI1andhas acharacteristicDEDDhmotif(compareFigure1.12).Snpisamoreefficientnuclease than3hExotowardhistonestemloopRNAssinceitcanalsocleavethedouble strandedstemportion.OnereasonmaybetheabsenceoftheSAPdomainwhich

53

1.Introduction
mightbindthestemintheotherhomologuesandtherebyprotectsitfromdegrada tion.SnpcancleaveDNAsubstratesandisalsoabletodegradethestemportionof theDNAhairpin.Theminimumlengthofthe3flankforassociationinmobilityshift assayswasfoundtobeatleasttwonucleotides.HomozygousSnpmutantsshowed noincreasedRNAifunction,italsodoesnotplayamajorroleintheclearanceof apoptoticDNAinDrosophila(Kupscoetal.,2006). InNeurosporacrassaRNAiisessentialforthedsRNAortransgene(quelling)induced genesilencing,itscomponentsareQDE2,anArgonauteproteinassociatedwith siRNAsandtwoDicerproteins.ThehypotheticalproteinQIPcopurifiedwithQDE 2andcontainsa35exonucleasedomainbelongingtotheDEDDhsuperfamily.In QIPKOssiRNAlevelsweresignificantlyhigherthaninwildtype.Genesilencing wasimpairedintheabsenceofQIP,suggestingthatQIPisessentialforfunctional RNAi.ItisrequiredforefficientprocessingofsiRNAduplexes,butactingdown streamofQDE2.siRNAduplexesfromqipKOswerelessstableandsinglestranded at57C,unlikesiRNAduplexesfromqdeKOs,suggestingthatinqipKOsthe siRNAshadalreadybeenpreprocessed.QIPprobablyfunctionsintheRISCactiva tionprocessbyremovingthenickedpassengerstrandfromthesiRNAduplexes (Maitietal.,2007). InfissionyeastheterochromatinassemblyrequirestheRNAimachineryandisiniti atedbysiRNAs.Theyarederivedfromheterochromaticregionsandprocessedby theRNAinducedtranscriptionalsilencing(RITS)complexwhichcontainsAgo1, Chp1andTas3.DeletionofEri1,thesingleS.pombeorthologue,causesanincreasein siRNAsassociatedwiththeRITScomplexandenhancesheterochromaticsilencing.It containsconservedSAPandDEDDhexonucleasedomainsandshowsmorethan30 %identitytoC.elegansERI1.ItdegradesdsRNAwith2ntoverhangsandtheRNA moietyofRNADNAhybrids.InanelectrophoreticmobilityshiftassaytheSAP domainefficientlybounddsRNAandRNADNAhybridsbutnotssRNA,dsDNAor ssDNA.LossofEri1whichpredominatelylocalizestothecytoplasmdidnotaffect normalcellulargrowthbuttheoverexpressionofEri1causedaseveregrowthdefect.
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1.Introduction
TheamountofcentromericsiRNAswasconsiderablygreaterthaninwildtypecells (Iidaetal.,2006).NewlygeneratedsiRNAscanalsorecruitheterochromatinproteins andinitiatedenovosilencingintrans,butthisintranssilencingisstronglyinhibited byEri1(Bhleretal.2006). InvivosubstratesofERI1inCaenorhabditiselegansandSchizosaccharomycespombe havelongbeenpoorlyunderstooduntilthediscoverythat5.8SrRNAineri1null mutantwormsislongerthaninwildtype.Atleastoneadditionalnucleotideatthe3 endcouldbedetectedinallmutantworms;asubstantialfractioncontainedtwoto four.ThesamewasfoundforS.pombeeri1KOswherethe5.8SrRNAhadtwoto eightadditional3nucleotides,suggestingacommonancestorforthisfunctionin animalsandfungi.BothERI1isoformsrescuedthe5.8SrRNAlengthinvivo,but onlyERI1bwasfunctionalinRNAirescuewhileitalsofailedtorescuetherRNA processinginvitro.MostrRNAprocessingoccursinthenucleolus.Inthematureri bosomethe3endof5.8Sispairedwiththe5endofthe2528SrRNA,reminiscent ofthehistonemRNAstemloopandsiRNAstructures.MutationsinH317andD321 completelydisruptedthefunctionofC.elegansERI1(Gabel&Ruvkun,2008). SuppressionofthemouseorthologueofERI1increasedtheeffectofRNAi.After introductionofhighamountsofexogenoussiRNAs,mouseERI1andADAR1 (adenosinedeaminasesactingonRNAwhichconvertadenosineintoinosine)tran scriptlevelsareincreased,maybeleadingtotheobservablereboundaftertheinitial RNAiinducedtargetsuppression.(Hongetal.,2005). InmiceERI1isubiquitouslyexpressed,withmaximainspleen,thymusandtestis.It ispresentinthecytoplasm,nucleusandslightlyenrichedinthenucleolus.Thebirth weightofEri1KOmiceisreducedandthisremainedsignificantinthe10%surviv ingadultmice.Growthdefectswerealsoobservedforcellsculturedinvitro.ERI1 wasfoundtobindindependentlytoeachribosomesubunit(40Sand60S).Under stringentlysisconditionsonly5.8SrRNAwasabletocoimmunoprecipitatewith endogenousERI1.InEri1KOmicethe3endsof5.8SrRNAwerevariablewith1or 2nt3extensions.PointmutationsshowedthatthecatalyticallyinactiveD130andE132
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1.Introduction
mutantsstillbound5.8SrRNA,whereaslinkerregionmutantsK107andK108showed impairedbinding.TheSAPandlinkerdomainshavesupportivefunctioninrRNA bindingbutarenotcrucialfor5.8SrRNAinteraction.WildtypeERI1wasableto converttheabnormal5.8SrRNAofpurifiedribosomesinvitro.Thenaturallyoccur ring5.8S28Sduplexissufficient,butefficientprocessinginvolvesinteractionwith otherfeaturesoftheribosome(Anseletal.,2008).
C. elegans H. sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus C. elegans H. sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus C. elegans H. sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus C. elegans H. sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus C. elegans H. sapiens M. musculus D. rerio X. laevis D. melanogaster S. pombe Consensus (1) (1) (1) (1) (1) (1) (1) (1) (99) (83) (79) (69) (80) (20) (27) (101) (196) (176) (172) (164) (175) (113) (92) (201) (277) (256) (252) (244) (255) (205) (192) (301) (376) (331) (327) (319) (330) (274) (262) (401) 1 100 MSADEPSPEDEKYLESLRDLLKISQEFDASNAKQNDEPEKTAVEVESAETRTDESEKSIDIPREQQLLPSERVEPLKSMVEPEYVKKVIR--QMDTMTAE -------MEDPQSKEPAGEAVALALLESPRPEGGEEPPR--PSPEETQQCKFDGQET-----KGSKFITS----SASDFSDPVYKEIAITNGCINRMSKE -------MEDERGRE---RGGDAAQQKTPRPECEESRP---LSVEKKQRCRLDGKET-----DGSKFISS----NGSDFSDPVYKEIAMTNGCINRMSKE -------METKEKSR------------KPPNKTPQSEG-----DQEDQPCPDTSCEK-----NEDQEPSSP---KQGEFSDPVYKEIALANGAINRMNRE -------MEEQKENRP-LDTEDSVVEEDLCKKLSRNLD----LVGVKQRCRFDGQED-----NGTSTVSS----NTSDFSDPVYKEIAIANGCVNRMTKD ---------------------------------------------------------------------------------MALIKLARQLGLIDTIYVD --------------------------------------------------------------------------MESPVQILVWPFPCDEMNQKTPSTVE MED Q CR D E ISS SDFSDPVYKEIAI NG INRMTKE 101 * * 200 QLKQALMKIKVSTGGNKKTLRKRVAQYYRKENALLNRKMEPNADKTARFFDYLIAIDFECTCVEIIY---DYPHEIIELPAVLIDVREMKIISEFRTYVR ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESNFADS---YYDYICIIDFEATCEEGNPP--EFVHEIIEFPVVLLNTHTLEIEDTFQQYVR ELRAKLSEFKLETRGVKDVLKKRLKNYYKKQ--KLMLKESSAGDS---YYDYICIIDFEATCEEGNPA--EFLHEIIEFPVVLLNTHTLEIEDTFQQYVR ELRAKCTELKLDTRGVNDVLRKRLKSYYKKQKLMHSPAAEGNSDM---YFDYICVVDFEATCEENNPP--DYLHEIIEFPMVLIDTHTLEIVDSFQEYVK ELKAKLVEHKLDTRGVKDVLRKRLKNYYKKQKLTHALHKDSNTDC---YYDYICVIDFEATCEAGNSL--DYPHEIIEFPIVLLNTHTLEIEDVFQCYVR GARPDPNNDPEESFNEDEVTEANSVPAKSKK-------SRKSKRLAMQPYSYVIAVDFEATCWEKQAPPEWREAEIIEFPAVLVNLKTGKIEAEFHQYIL EIRIALQELGLSTNG-----------------------NK---------R-YLLIVDVEATCEEGCGF--SFENEIIELPCLLFDLIEKSIIDEFHSYVR ELRAKL E KLETRGVKDVLRKRLKNYYKKQ D YYDYICIIDFEATCEEGN DF HEIIEFPVVLLNTHTLEIED FQ YVR 201 * * 300 PVRNPKLSEFCMQFTKIAQETVDAAPYFREALQRLYTWMRKFN-------------------LGQKNSRFAFVTDGPHDMWKFMQFQCLLSNIRMPHMFR PEINTQLSDFCISLTGITQDQVDRADTFPQVLKKVIDWMKLKE-------------------LGTK-YKYSLLTDGSWDMSKFLNIQCQLSRLKYPPFAK PEVNAQLSEFCIGLTGITQDQVDRADAFPQVLKKVIEWMKSKE-------------------LGTK-YKYCILTDGSWDMSKFLSIQCRLSRLKHPAFAK PVLHPQLSEFCVKLTGITQEMVDEAKTFHQVLKRAISWLQEKE-------------------LGTK-YKYMFLTDGSWDMGKFLHTQCKLSRIRYPQFAR PEINPQLSEFCVNLTGITQDTVDKSDTFPNVLRSVVEWMREKE-------------------LGSK-YKYAILTDGSWDMSKFLNMQCRISRLKYPRFAK PFESPRLSAYCTELTGIQQKTVDSGMPLRTAIVMFNEWLRNEMRARNLTLPKMN--------KSNILGNCAFVTWTDWDFGICLAKECSRKGIRKPAYFN PSMNPTLSDYCKSLTGIQQCTVDKAPIFSDVLEELFIFLRKHSNILVPSVDEIEIIEPLKSVPRTQPKNWAWACDGPWDMASFLAKQFKYDKMPIPDWIK P INPQLSEFCI LTGITQDTVDKA F QVLKKVIEWMR KE LGTK YKYAFLTDGSWDMSKFL QCKLSRIKYP FAK 301 * 400 -SFINIKKTFKEKFNGLIKGNGKSGIENMLERLDLSFVGNKHSGLDDATNIAAIAIQMMKLKIELRINQKCSYKENQRSAARKDEERELEDAANVDLTSV -KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHCGLDDSKNIARIAVRMLQDGCELRINEKMHAGQ---------------------LMSV -KWINIRKSYGNFYKVPRS---QTKLTIMLEKLGMDYDGRPHSGLDDSKNIARIAVRMLQDGCELRINEKILGGQ---------------------LMSV -KWINIRKSYGNFYKVPRT---QTKLICMLENLGMEYDGRPHCGLDDSRNIARIAIHMLKDGCQLRVNECLHSGE---------------------PRSV -KWINIRKSYGNFYKVPRT---QTKLTTMLEKLGMTYNGRLHSGLDDSKNIARIAAHMLQDGCELRVNERMHAGQ---------------------LMTV -QWIDVRAIYRSWYKYRPCN-----FTDALSHVGLAFEGKAHSGIDDAKNLGALMCKMVRDGALFSITKDLTPYQ------------------------GPFVDIRSFYKDVYRVPRT-----NINGMLEHWGLQFEGSEHRGIDDARNLSRIVKKMCSENVEFECNRWWMEYE------------------------KWINIRKSYGNFYKVPRT QTKLT MLEKLGM YDGR HSGLDDSKNIARIAIKML DGCELRINEKL GQ L SV 401 473 DISRRDFQLWMRRLPLKLSSVTRREFINEEYLDCDSCDDLTDDKVKHLHSCDIYEIFDEKTSASFTDSKCLIC SSSLPIEGTPPPQMPHFRK-----------------------------------------------------SSSLPVEGAPAPQMPHSRK-----------------------------------------------------PISAPIEGAPAPQPPKKRD-----------------------------------------------------SSSLPFEGAPVPQNPHLKN----------------------------------------------------------------QLNPRFVL--------------------------------------------------------K-NGWIPNRSYPPYFAS----------------------------------------------------S P EG P PQ P R

Figure1.12:AlignmentofpublishedERI1homologues Thesequencesshareatotalidentityof7%andasimilarityof54%.WhiletheNandCterminalre gionsvary,ahigherlevelofconservationispresentintheexonucleasedomain.Theconserved DEDDHmotifisindicatedwithasterisks(*).TheN.crassaQIPproteinhasbeenexcludeddueto highersequencevariability.

1.2.10 ERI1LIKE1,theplanthomologueofERI1
SofarnoplanthomologueofERI1hasbeencharacterized.BLASTsearch (http://blast.ncbi.nlm.nih.gov/Blast.cgi;Altschuletal.,1997)identifiedasinglehomo logueinArabidopsisthalianaencodedonthethirdchromosomeatthelocus At3g15140.Itcodesfora337aminoacidproteinwith50%similaritytotheC.elegans
56

1.Introduction
protein.AccordingtoplantnomenclatureconventionstheproteinistermedERI1 LIKE1(ERL1).Thegeneiscurrentlyannotatedtoresultinonetranscriptassembled ofsixexons,butasecondsplicevariantlackingtheNterminalregioncannotbeex cluded(http://www.ncbi.nlm.nih.gov/IEB/Research/Acembly/;ThierryMieg& ThierryMieg,1997).TheproteincontainsthecharacteristicconservedDEDDhex onucleasedomainwhereitalsoshareshighersimilaritywiththeC.elegansERI1,but itlackstheNterminalSAPdomainliketheDrosophilamelanogasterERI1homologue Snipper(Kupscoetal.,2006).
A. thaliana N. benthamiana V. vinifera O. sativa S. bicolor Z. mays P. trichocarpa Consensus (1) (1) (1) (1) (1) (1) (1) (1) 1 100 MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCN--------------SSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSEN-----ARWRP ------SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRK-ISISASRSTTEESTSSLIQPTPSR------TRWKP ---------MAFYRVSPFRYGSLS-S---LIPYVS-------------SP-----SSLSPPVRT-FTLSASISTPHPSPPSLLTASPKAS-----DRWRP ---------MALARVSPPAFSSPFLIHSLLRPFSSPSSVLRPR---------VTRVPHHRGFAIAAALSQASPLPSADGDGAVMEAPPRPS--SRRPWKP ---------------------------------------------------------------------------SAASSATVRASGSVG------------------MALARVSPSSLANLIPPLLQSFFRPFSSDFPIR-------------NSRRRSSPVAAAFSLTSQSAHAAREGLVMEAPRPSS---RYPWKP ----MSFPRIPLSRVPSYLHNSNN--CFHLLHPPFIPVSKTP------------SLPTYQTARTYTDFNSQTQTQPPLSLPSLIPSPPVNNPNATHRWKP MALARVSPF LI S SASS T AS VM SP RWKP 101 * * 200 MCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKY TCLYFTQGKCTKMDDPMHIDKFNHNCSLELMQNAAGLKNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGKY MCLYYTQGKCTKMDDPTHLETFNHNCSRELQVNAANFQHLQSQHLDFFLVLDLEGKIEILEFPVLMINAKTMDVVDLFHRFVRPSEMSEQRINEYIEGKY TCLYYTQGKCTMLNDTLHLEKFNHNLPTDLPVNYSAADKVKSQKLDYFLVLDLEGKVEILEFPVVMIDAQSMEFVDSFHRFVHPTAMSEQRIREYIEGKY ----------CHMNDAMHLEKFGHNLKMDLPVNASATDKFKPQKLEYFLILDLEGRVEILEFPVVMIDAQSTEFIDSFHRFVRPTAMSEQRTTEYIEGKY TCLYYTQGKCTMMNDAMHLEKFSHNLKMDLPVNASATDKSKPQKLEYLLILDLEGRVEILEFPVMMIDAQNREFVDSFHRFVRPTAMSEQRTTEYIEGKY MCLYHTHGKCTKIDDPVHVERFNHDCSRDFQVSAADFERKRPQDFDFFLVFDLEGKVEILEFPVLIIDAKTMGVVDLFHRFVRPTAMSEERVNEYIYNKY CLYYTQGKCTKMDDPMHLEKFNHNCS DL VNAAA DK K Q LDYFLVLDLEGKVEILEFPVLMIDAKTMEVVD FHRFVRPTAMSEQRINEYIEGKY 201 * * 300 GELGVDRVWHDTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWINLKDVYLNFYG--REARGMVSMM GKLGVDRVWHDTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQCKVAGTKLPPYFMEWINLKDVFLNFYK--RRAKGMLSMM GKLGVDRVWHDTSIPFKEVIQQFEAWLTQHHLWTKEMGGRLDQAAFVTCGNWDLKTKVPQQCKVSKMKLPPYFMEWINLKDVYLNFYK--RRATGMMTMM GKFGVDRVWHDTAIPFMEVLQEFEDWIEHHKFWKKEQGGALNSAAFITCGNWDLKTKVPEQCRVSKIKLPSYFMEWINLKDIYLNFYN--RRATGMMTMM GKFGVDRVWHDTAVPFKEVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKT-----------------------------------KATGMMTMM GKFGVDRVWHDTATPFKQVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKVPEQCKVSKINLPTYFMEWINLKDIYLNFYN--RRATGMMTMM GKFGVDRVWHDTALPFNEVLQQFESWLTQHNLWEKTRGGRLNRAAFVTCGNWDVKTQVPHQCSVSKLKLPPYFMEWINLKDVYQNFYNPRNEARGMRTMM GKFGVDRVWHDTAIPFKEVLQEFE WLGNHNLWKKE GG LNRAAFVTCGNWDLKTKVP QC VS I LPPYFMEWINLKDVYLNFY RRA GMMTMM 301 * * 358 RQCGIKLMGSHHLGIDDTKNITRVVQRMLSEGAVLKLTARRSKSNMRNVEFLFKNRIK RELQMPLLGSHHLGIDDAKNIARVLQHMLSDGALVQITARRNPHSPEKVEYLFEDRIV KELQIPLLGSHHLGIDDTKNIARVLQRMLADGALLQITARRNADSPENVEFLFKNRIR RELQMPIVGSHHLGIDDAKNIARVVQRMLADGAVMQITAKRQS-ATGDVKFLFKNRIR RELQLPIIGNHHLGIDDSKNIARVVQRMIADGAVIEITAKRQ-STTGNVKFLFKDRIR RELQLPIVGNHHLGIDDSKNIARVVQRMLADGAVIQITAKRQ-SATGDVRFLFKDRIR SQLKIPMVGSHHLGLDDTKNIARVLLRMLADGAVLPITARRKPESPGSVNFLYKNRIRELQIPLLGSHHLGIDDTKNIARVLQRMLADGAVL ITARRN S V FLFK RIR

A. thaliana (82) N. benthamiana (88) V. vinifera (64) O. sativa (81) S. bicolor (16) Z. mays (76) P. trichocarpa (83) Consensus (101) A. thaliana N. benthamiana V. vinifera O. sativa S. bicolor Z. mays P. trichocarpa Consensus A. thaliana N. benthamiana V. vinifera O. sativa S. bicolor Z. mays P. trichocarpa Consensus (182) (188) (164) (181) (106) (176) (183) (201) (280) (286) (262) (248) (171) (274) (283) (301)

Figure1.13:AlignmentofERL1homologuesinvariousplantspecies TheplanthomologuesshowaveryhighdegreeofconservationintheCterminalexonucleasedo main;allaminoacidsoftheDEDDhmotifarepresent,whichisindicatedwithasterisks(*).Anexcep tionistheS.bicolorhomologuewhichpossessesadeletioninsidetheexonucleasedomainandalsoa shortenedNterminus.

HighlyconservedplanthomologuesofERL1couldbeidentifiedinthefullyse quencedplantgenomesandtheESTcollectionsofmanyotherplantspecies.Oryza sativaprobablycontainsseveralERL1homologues.TheSorghumbicolorsequence containsaprominentdeletionof33aminoacidsinsidetheexonucleasedomain.In additionthesequenceisnotannotatedcompletelyattheNterminussinceitlacksa

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1.Introduction
Methionine.Thesameistrueforthepoplarsequencebutanupstreamstartcodon couldbeidentifiedpossiblyleadingtothefulllengthprotein(compareFigure1.13). Subcellularlocalizationpredictionalgorithmsrevealedapotentialchloroplastsignal peptideoftheindividualplantERL1homologues.AnexceptionistheSorghumse quencebutitcannotbeexcludedtobetargetedtothechloroplastaswellbecauseof itsincompleteannotation(compareTable1.1).
Table1.1:PredictedlocalizationofplantERL1homologues FivepredictionalgorithmscalculateachloroplasticlocalizationofplantERL1homologuesin60%of allcases,29%predictamitochondriallocalization,11%predictotherornosubcellularlocalization. Caption:mito=mitochondrial,chloro=chloroplastic,nucl=nuclear,none=noneoftheabove

iPSORT BaCelLo (Bannaiet (Pierleoni al.,2002) etal.,2006) mito chloro mito mito mito mito mito chloro chloro chloro chloro nucl chloro chloro

PCLR (Scheinet al.,2001) chloro chloro chloro chloro none chloro chloro

Predotar (Smallet al.,2004) chloro none mito chloro none mito mito

WoLF PSORT

(Hortonet al.,2007) chloro chloro chloro chloro chloro chloro mito

A.thaliana P.trichocarpa O.sativa Z.mays S.bicolor V.vinifera N.benthamiana

ERL1expressionlevelshavebeenobtainedfrompubliclyavailablemicroarraydata ofArabidopsisthaliana(http://www.bar.utoronto.ca/;Winteretal.,2007).Thegeneis foundtobeubiquitouslytranscribedatverylowlevels(forcomparisonALPHA TUBULINisexpressedat40timeshighervalues).ThehighestERL1expressioncould bedetectedintheshootapex,inthereproductiveorganspollenandstigmataaswell asinimbibedseeds.Infullydevelopedtissuestheexpressiondropsbyafactoroften (compareFigure1.14&Figure1.15). ExpressiondataisalsoavailableforP.trichocarpaandM.truncatula;theabsoluteex pressionlevelsarehigherthaninArabidopsis,butcomparableinrelationtoALPHA TUBULIN.InMedicagothehighestexpressionisalsoobservedinthevegetativetis sue,whereasinPopulusitisdetectedintheseedlings(compareFigure1.14).
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1.Introduction

Figure1.14:Winteretal.,2007 ThedevelopmentalmapofA.thalianashowsincreasedERL1expressionintheshootapexandim bibedseeds.InP.trichocarpaERL1ismainlytranscribedinetiolatedandlightgrownseedlings. 59

1.Introduction

Figure1.15:Winteretal.,2007 AmoredetailedexpressionmapofA.thalianashowsERL1expressionmaximainthe shootapicalmeristem,ovariesandduringearlypollendevelopment. 60

1.Introduction
1.3 Chloroplasts
Chloroplastsdevelopfromproplastidswhicharesmallprecursorplastidswitha doublemembranepresentinallimmatureplantcells.Theyareinheritedfromthe ancestorplantsintothezygote.Allofthemcontainthesamegenomicinformation buttheyareabletodifferentiateintodifferentplastidtypes.Plastidshavevarious functionsrangingfromphotosynthesisforenergysupply,storageofenergy containingmoleculestometabolictaskssuchassynthesisofnucleotides,certain aminoacidsandfattyacids(Albertsetal.,2008).Themetabolicfunctionscanalsobe performedbyproplastids,althoughtheydependonthesupplywithenergyandpre cursormetabolitesbytheplantcell(Brutigam&Weber,2009). Etioplastsaredevelopedwhenaplantisgrowninthedark.Theycontaininner membranestructureswithayellowchlorophyllprecursor,whichisabletoconvertto normalchlorophyllinthecaseoflightexposure.Leucoplastsareplastidswhich, unlikechloroplasts,canalsobefoundinepidermalplantcells.Theycanserveas storageplastidsforstarch(amyloplasts)orlipids(elaioplasts).Anotherplastidtype arechromoplastswhichstorethepigmentsofflowerpetalsorfruits. Themostprominentplastids,however,arethechloroplasts.Evolutionaryevidence suggeststheirorigininphotosyntheticbacteriawhichhadbeenendocytosedbythe eukaryoticcell.Theypossesstheirowngenomecodingforapproximately120genes andproteinsynthesismachinery.Duringevolutiongenetransferfromthechloro plastintothenucleustookplace,thereforemostoftheirproteinsarenowencodedin thenucleusandimportedbyaspecialimportcomplex. AsaruleofthumbthechloroplasticribosomalandtransferRNAsandphotosyn theticproteinsarestillencodedinthechloroplasts,butthelargerenzymecomplexes andribosomalproteinsusuallycontainalsonuclearproteins(Albertsetal.,2008). TheseareimportedintothechloroplaststhroughtheTOC(transloconattheouter envelopemembraneofchloroplasts)andTIC(transloconattheinnerenvelopemem braneofchloroplasts)complexes(Inaba,2010).ThereplicationofchloroplasticDNA isindependentfromtheSphaseofthecells,butitisregulatedinawaythatthe
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1.Introduction
numberofchloroplastshasdoubledbeforecelldivisiontoensureaconstantamount ofplastidsinthedaughtercells(Albertsetal.,2008).Thedivisionofchloroplastsis controlledbyARC(ACCUMULATIONANDREPLICATIONOFCHLOROPLASTS) genes(Marrisonetal.,1999). Inthedevelopmentfromproplastidstheirinnermembranefoldsandgetsconstricted leadingtothethylakoidmembranesinsidethechloroplasts(Vothknecht&Westhoff, 2001).Theyformaninternalcompartmentcalledthethylakoidspace,themembranes themselvesareorganizedinstackswhicharecalledgranaandcontainthephotosyn theticsystemsofthechloroplasts(Albertsetal.,2008).

1.3.1 Photosynthesis
Photosynthesisconsistsoftwodifferentreactiontypes:thelightreactions,where sunlightistransformedintoelectricenergyandtheconsecutivesynthesisreactionsof theCalvincycle,wherecarbondioxideisfixedintosugarmolecules. Thethylakoidmembranescontaindifferentpigmentswhichareabletoabsorblight. Themostprominentischlorophyllaconsistingofalightabsorbingporphyrinring andahydrophobicphytolgroupanchoringthemoleculeinthemembranes.Ithas absorptionmaximaat430nmand662nm,togetherwithchlorophyllbandcaroti noidsthewavelengthwhichcanbeusedforphotosynthesisisbroadened,moreover thelatterhaveafunctioninprotectingchlorophyllfromexcessivelightenergy. Photosynthesisisperformedintwodistinctphotosystems.Botharecomprisedofa lightharvestingcomplexformedbyseveralhundredpigmentmoleculeswhichchan neltheabsorbedlightenergytoareactioncenter. InphotosystemIIthechlorophyllamoleculesofthereactioncentertransfertheir highenergyelectronsontoaprimaryelectronacceptor.Toreturnbacktoitsinitial stateitgetsreducedbyelectronsoriginatingfromwaterwhichhadbeenphotolyzed. TheresultingprotonscanbeusedforATPsynthesisandinadditionoxygenisre leased.Theelectronsresidingintheprimaryelectronacceptorarefurthertrans portedviaseveralmoleculestophotosystemI,wheretheabsorbedlightenergyhas alsoresultedinoxidizedchlorophyllamoleculeswhichnowgetreduced.Thepri
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1.Introduction
maryelectronacceptorofphotosystemIleadstothereductionofNADP+toNADPH. Thisreactionmechanismiscallednoncyclicelectrontransportresultinginequimo laramountsofATPandNADPH. AlternativelyphotosystemIcanalsoproduceextraATPbythecyclicelectrontrans portwheretheprimaryelectronreceptorreducesthereactioncenterofthephotosys tem.TheabsorbedenergythenleadstothechemiosmoticproductionofATP. InthefirstphaseofthesubsequentCalvincyclecarbondioxideisattachedontoribu lose1,5bisphosphatebytheenzymeribulose1,5bisphosphatecarboxylase.Thein stableproductdissociatesandgetsfurtherphosphorylatedandreducedtoglyceral dehyde3phosphatewhichcanbeusedfortheproductionofsucroseandstarch.Ina laststepthecarbondioxideacceptorisregenerated(Campbell,1996).

Figure1.16:Campbell,1996 Graphicrepresentationofthephotosyntheticreactionsinthechloroplast.Lightenergyisabsorbedin photosystemIIandleadstotheproductionofATPandO2.Theenergygetstransportedviathecyto chromecomplexandPhotosystemItotheNADP+reductasefortheproductionofNADPH.Thereac tionproductsfunctioninthesubsequentCalvincyclewhichleadstothesynthesisofsucrose. 63

1.Introduction
1.3.2 SpecificitiesofchloroplasticRNAs
Thechloroplastgenomeoflandplantsencodescomponentsrequiredforchloroplast proteinsynthesis(rRNA,tRNAs,ribosomalproteinsandRNApolymerasesubunits) andforphotosynthesis. ThechloroplasticrRNAgenesarehighlyconservedbetweenplantspeciesandor ganizedintheribosomalRNA(rrn)operonwhichistranscribedintoapolycistronic molecule.Inlandplantsthetranscriptstartswiththe16SrRNA,followedbytwo

Figure1.17:Sternetal.,2010 Chloroplasticgenesexpressioninvolvesthetranscriptionofapolycistronicprecursorwhichgets processedatits5and3ends.Afterintercistroniccleavagetheresultingtranscriptsareeditedby PPRproteinsandsplicedintofunctionalRNAs. 64

1.Introduction
tRNAs,23SrRNA,4.5SrRNAandfinally5SrRNAandanothertRNA(Harrisetal., 1994).Thefirstprocessingoftheprimaryprecursorinvolvestheexcisionofthe tRNAs.Additionalendonucleolyticcleavagegeneratesthe16Sand5SrRNA.The resultingdicistronic23S4.5SrRNAintermediaterequires3maturationbeforethe finalprocessingintothemonocistronicrRNAstakesplacealreadyintheribosome. AlsotheotherrRNAmoleculesneedfurtherendoandexonucleolyticprocessing untilthematurerRNAsareready(Ksseletal.,1985;Bellaouietal.,2003;Bisanzetal., 2003;Bollenbachetal.,2005;Nishimuraetal.,2010). IncontrasttotheprokaryoticRNAmetabolism,chloroplasticRNAspossessintrons andpassthroughmanyRNAeditingsteps.Mostofthemarealsoinfluencedbyheli calrepeatproteins(reviewedinSternetal.,2010). Plantspossessalargegenefamilywithmorethan450membersinArabidopsiswhich encodeforproteinswithpentatricopeptiderepeats(PPRs)Itisadegenerate35 aminoacidmotifwhichisoftenarrangedintandemof227repeatsperpeptide (Small&Peeters,2000;Lurinetal.,2004).Unliketetratricopeptideproteinsfrom whichtheirnamewasderivedof,pentatricopeptideproteinsareabletobindRNA andDNAinasequencespecificmanner(Sahaetal.,2007). Theyaremostlytargetedtoorganelles[(chloroplastsandmitochondria)(Small& Peeters,2000;Lurinetal.,2004)]andcanbegroupedintotwosubfamilies:classical PPRproteinswithonlytheabovedescribedmotifsandplantcombinatorialand modularproteins(PCMPs)containingalsoshort(31aminoacids)andlong(3536 aminoacids)motifs[(Lurinetal.,2004)(compareFigure1.18a)].Anotherconserved featureisaCterminalEmotif,EandE+orE,E+andDYWstretchwhichispresent inalargefractionofPPRproteins(Lurinetal.,2004).PPRgenesareusuallyshort sincemostofthemdonotcontainintronsandtheygenerallyhavealowexpression rate(Lurinetal.,2004). Althoughtherearereportsforfunctionsinmitochondriainotherspecies,inplants theyaremainlyparticipatinginchloroplastRNAmetabolisminprocessessuchas RNAtrimming,stabilization,translationandediting(compareFigure1.18b).They
65

1.Introduction
areinvolvedinchloroplasttranslationalcontrol,embryogenesisandorgandevelop mentaswellastherestorationofmalefertilitythroughmodificationorsilencingof cytotoxicmitochondrialtranscripts(Sahaetal.,2007).Mitochondriaencodedcyto plasmicmalesterilitycanbeovercomebynuclearrestorer(Rf)geneswhichareusu allyPPRproteins(SchmitzLinneweber&Small,2008).Rfhomologousgeneshave beenshowntogeneratesiRNAsandmightbeundergenesilencingregulationthem selves(Howelletal.,2007).
(A) (B)

Figure1.18:PPRproteins (A)DomainorganizationofPPRandPCMPproteins(Lurinetal.,2004).(B)FunctionsofPPRpro teinsin(1)Translation,(2)RNAediting,(3)RNAsplicingand(4)RNAstability(SchmitzLinneweber &Small,2008).

1.4 Thesisobjectives
Theinitialaimofthisworkwasthecharacterizationoftheplanthomologueofthe3 5exonucleaseERI1(enhancedRNAi)inthetwomodelplantsArabidopsisthaliana andNicotianabenthamiana.TheproteinisfurthercalledERI1LIKE1,ERL1.Sofar, noscientificpublicationdescribesthisplantprotein.Duetothecompletegenomic sequenceinformation,invitrocharacterizationoftheArabidopsishomologueispossi bleandhasbeenpresentedinchapter1.2.10.Incontrast,onlypartialsequencein formationwasavailablefortheNicotianahomolog,thereforeitshouldalsobeidenti fiedinthecourseofthiswork.Theinsilicopredictedchloroplasticlocalizationofthe Arabidopsisproteinshouldbeproveninvivoaswell.

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1.Introduction
SincethenematodeproteinissuggestedtosuppressRNAsilencinginCaenorhabditis elegans,theeffectofERL1inplantRNAsilencingprocessesshouldbeinvestigated.In thecourseoftheworkcytosolic5.8SrRNAhadbeenidentifiedasanewsubstrateof ERI1anditshomologues.Hence,theeffectofERI1onplantribosomalRNAmole culesshouldhavebeenexaminedinmoredetail.Interestingly,thechloroplastisbe lievedtobefreeofRNAsilencing,butitcontainsfourchloroplasticribosomalRNA species.TheeffectofplantERL1ontheseandthehomologous5.8SrRNAshouldbe exploredinmoredetails. Summarizedthefollowingquestionshouldbeaddressed: InvivocharacterizationofERL1inArabidopsisthaliana. InvivocharacterizationofERL1inNicotianabenthamiana. InvolvementofERL1inplantRNAsilencingprocesses. InvolvementofERL1inribosomalRNAprocessing.

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2. MaterialsandMethods

2.1 Materials
Theusedmaterialsofthisworkarepresentedbelowtogetherwiththeirstandard supplier;alternativesuppliersarepossibleinsomecases.

2.1.1 Instruments
Agarosegelelectrophoresis BlottingDevice A.Kabbalos,Greece OwlB2EasyCast,ThermoScientific,USA SD20SemiDryMidi,CleaverScientific, UK MiniTransBlotElectrophoreticTransfer Cell,BioRad,Germany Centrifuges Confocalmicroscope Developer DigitalCamera ElectronMicroscope Fluorescencemeasurement Geldocumentationsystem Hybridizationbottles Hybridizationoven Icemachine Eppendorf5415D,Eppendorf,Germany Eppendorf5810R,Eppendorf,Germany Biofuge15R,Heraeus,Germany BiofugeStratos,Heraeus,Germany Kubota5800,Kubota,Japan LeicaTCSSP,Leica,Germany Curix60,AGFA,Belgium Coolpix990,Nikon,Japan Coolpix5600,Nikon,Japan JEM100C,JEOLLtd.,Japan HandyPEA,HansatechInstruments,UK UVT28MP,Herolab,Germany Hybaid,ThermoScientific,USA ShelLab,USA ShelLabModel1004,USA BremaIcemakers,Italy
69

2.MaterialsandMethods
Incubator B5060,HeraeusInstruments,Germany FormaIncubatedConsoleOrbitalShakers, ThermoScientific,USA LaminarFlowHood ESIFlufranceAriane18UV,JouanGmbH, Germany Biocyt180NFX44201,JouanGmbH,Ger many Magneticstirrer/heater Microlitrepipettes Microscope(optical/fluorescent) Microwaveoven PAAgelelectrophoresis Powersupply IKAMAGRET,IKAWerke,Germany Pipetman,Gilson,USA EclipseE800,Nikon,Japan Localelectronicsupplier MiniProtean3,BioRad,Germany ,Greece PowerPacBasic,BioRad,Germany PharmaciaECPS3000/150,GEHealthcare, UK MicrocomputerElectrophoresis,Renner GmbH,Germany qPCRmachine Scintillationcounter RG3000ARotorGene,Qiagen,Germany ScintillationSystemLS1701,Beckman, USA Spectrophotometer NanoDropND1000,ThermoScientific, USA Thermocycler Lambda2,PerkinElmer,USA DNAEnginePTC200Gradient,BioRad, Germany UVcrosslinker UVlamp,handheld Vortexmixer Stratalinker1800,Agilent,USA BlakRayB100AP/R,UVP,USA VortexGenie2,ScientificIndustries,USA
70

2.MaterialsandMethods
Waterbath Xraycassettes LaudaA100,LaudaDr.R.Wobser GmbH&CO.KG,Germany C.B.S.ScientificCo,USA

2.1.2 Chemicals
[32P]dATP[C10H16N5O12P3] [32P]dCTP[C9H16N3NaO13P3] [32P]ATP[C10H16N5O13P3] Aceticacid[CH3COOH] Acetosyringone(ACS)[C10H12O4] Acrylamide[C3H5NO] Agaragar[(C12H18O9)x] Agarose[(C12H18O9)x] Agarose,lowmelting[(C12H18O9)x] Ammoniumnitrate[NH4NO3] Ammoniumpersulfate[(NH4)2S2O8] Ammoniumthiocyanate[NH4SCN] Ampicillin[C16H19N3O4S] 6Benzylaminopurine(BAP)[C12H11N5] Boricacid[B(OH)3] PerkinElmer,USA PerkinElmer,USA PerkinElmer,USA Merck,Germany SigmaAldrich,USA Merck,Germany SigmaAldrich,USA Lonza,Switzerland SigmaAldrich,USA SigmaAldrich,USA SigmaAldrich,USA Merck,Germany BristolMyersSquibb,USA Duchefa,Netherlands Merck,Germany

5Bromo4chloro3indolylDgalacto MelfordLaboratories,England pyranoside(XGal)[C14H15BrClNO6] Bromophenolblue[C19H9Br4O5SNa] Bovineserumalbumine(BSA) Calciumchloride[CaCl2] Cefotaxime[C16H17N5O7S2] Cetyltrimethylammoniumbromide (CTAB)[C19H42BrN] Chloroform[CHCl3]
71

SigmaAldrich,USA Merck,Germany SigmaAldrich,USA Localpharmaceuticalsupplier Merck,Germany

Merck,Germany

2.MaterialsandMethods
CoomassieBrillantblueG250 [C47H48N3O7S2Na] Deoxyribonucleotide(dNTP)set Dimethylformamide(DMF)[C3H7NO] Dimethylsulfoxide(DMSO)[C2H6OS] Dithiothreitol(DTT)[C4H10O2S2] DNAoligos Ethanol,absolute[C2H6O] Ethidiumbromide(EtBr)[C21H20BrN3] Merck,Germany MBI,Fermentas,Lithuania Bioron,Germany SigmaAldrich,USA SigmaAldrich,USA Merck,Germany IMBBMicrochemistry,Greece Metabion,Germany Merck,Germany SigmaAldrich,USA

Ethylenediaminetetraaceticacid(EDTA) Merck,Germany [C10H16N2O8] Formaldehyde[HCOH] Formamide[CH3NO] Glucose[C6H12O6] Glycerol[C3H5(OH)3] Glycine[C2H5NO2] Guanidiniumthiocyanate[C2H6N4S] Hydrochloricacid,fuming[HCl] 4(2hydroxyethyl)1piperazineethane sulfonicacid(HEPES)[C8H18N2O4S] Kanamycin[C18H36N4O11] Magnesiumchloride[MgCl2] Magnesiumsulfate[MgSO4] Manganesechloride[MnCl2] Mercaptoethanol[C2H6OS] Methanol[CH3OH]
72

Merck,Germany Merck,Germany Merck,Germany BiomolGmbH,Germany BiomolGmbH,Germany Merck,Germany Merck,Germany Merck,Germany SigmaAldrich,USA Merck,Germany Merck,Germany Fluka,Switzerland Fluka,Switzerland Merck,Germany

2.MaterialsandMethods
N,Nmethylenebisacrylamid(BIS) [C7H10N2O2] 2(NMorpholino)ethanesulfonicacid (MES)[C4H8ONC2H4SO3HH2O] Merck,Germany SigmaAldrich,USA

3(NMorpholino)propanesulfonicacid Merck,Germany (MOPS)[C7H15NO4S] Murashige&Skoogmacroelements Murashige&Skoogmicroelements Murashige&Skoogvitamins Naphthaleneaceticacid(NAA) [C12H10O2] Phenolcrystals[C6H5OH] PiperazineN,Nbis(2ethanesulfonic acid)(PIPES)[C8H18N2O6S2] Polysorbate20(Tween20)[C58H114O26] Polyvinylpyrrolidone(PVP40) [(C6H9NO)n] Potassiumacetate[CH3COOK] Potassiumchloride[KCl] Potassiumhydroxide[KOH] Potassiumnitrate[KNO3] Merck,Germany Merck,Germany Merck,Germany SigmaAldrich,USA Sigma.Aldrich,USA SigmaAldrich,USA Fluka,Schwitzerland Merck,Germany Duchefa,Netherlands Duchefa,Netherlands Duchefa,Netherlands Duchefa,Netherlands

Potassiumphosphate,dibasic[K2HPO4] Merck,Germany Potassiumphosphate,monobasic [KH2PO4] 2Propanol(isopropanol)[C3H8O] Rifampicin[C43H58N4O12] SilwetL77 Skimmedmilkpowder Sodiumacetate[CH3COONa] Merck,Germany Merck,Germany Duchefa,Netherlands OSISpecialities,USA Localmarket Merck,Germany
73

2.MaterialsandMethods
Sodiumcarbonate[Na2CO3] Sodiumchloride[NaCl] Sodiumcitrate[Na3C6H5O7] Sodiumdodecylsulfate(SDS) [C12H25NaO4S] Sodiumhydroxide[NaOH] Sodiumhypochlorite[NaClO] Sodiumphosphate,dibasic[Na2HPO4] Sodiumphosphate,monobasic [NaH2PO4] Sodiumthiosulfate[Na2S2O3] Sorbitol[C6H14O6] Spectinomycin[C14H24N2O7] Sucrose[C12H22O11] Tetramethylethylenediamine(TEMED) [C6H16N2] Tris(hydroxymethyl)aminomethan (TRIS)[C4H11NO3] TritonX100[C14H22O(C2H4O)n] Tryptone Urea[(NH2)2CO] Water,nanopure XylenecyanolFF[C25H27N2O6S2Na] Yeastextract Merck,Germany SigmaAldrich,USA Merck,Germany Millipore SigmaAldrich,USA SigmaAldrich,USA BiomolGmbH,Germany Merck,Germany SigmaAldrich,USA SigmaAldrich,USA SigmaAldrich,USA Merck,Germany SigmaAldrich,USA SigmaAldrich,USA Merck,Germany Merck,Germany Merck,Germany Merck,Germany Merck,Germany Merck,Germany

2.1.3 Consumables&kits
antiRabbitAPconjugate Blottingpaper Promega,USA Whatman3MM,UK

FermentasMaximaSYBRGreenqPCR ThermoFischerScientific,USA MasterMix(2X)

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2.MaterialsandMethods
GatewayLRClonaseIIEnzymeMix, Invitrogen,USA HRPSuperSignalWestPicoChemilumi ThermoFischerScientific,USA nescentsubstrate IllustraMicroSpinS200SpinColumnsGEHealthcare,UK Laboratoryfilm Membrane PechineyParafilmTMM,Pechiney,USA ProtranTMNitrocellulose0.22m, Whatman,UK NucleoBondXtraMidiKit NucleoSpinExtractII Pasteurglasspipettes Petridishes10cm pGEMTEasyVectorSystem Pipettetips Nylon0.45m,NytranN,Whatman,UK MachereyNagel,Germany MachereyNagel,Germany Volac,Poulten&GrafLtd.,UK Sarstedt,Germany Promega,USA Sarstedt,Germany&GreinerBioOne, Germany PlatinumTaqDNApol.HighFidelity Polypropylenetubes,15/50mL RadPrimeDNALabelingSystem Reactiontubes,0.2/0.5/1.5/2mL Gloves Invitrogen,USA Sarstedt,Germany Invitrogen,USA Sarstedt,Germany DigitilNpowderfree,Hartmann,Ger many Scalpelblades Syringe1mL Syringefilters TaKaRa3FullRACECoreSet TOPOTACloningKit XrayfilmsFujiSuperRX Paragon,UK HMDHealthcareLtd.,Horsham,UK PallCorporation,USA TaKaRa,Japan Invitrogen,USA Fujifilm,Japan

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2.MaterialsandMethods
2.1.4 Solutions
30%(w/v)acrylamidemix(29:1) 40%(w/v)acrylamidemix(38:2) BindingBuffer Blockingbuffer Churchhybridizationbuffer Coomassiedestainingsolution Coomassiestainingsolution 40%(v/v)methanol 10%(v/v)aceticacid 50%(v/v)water 0.1%(w/v)Coomassie 15%(v/v)ethanol 10%(v/v)aceticacid 75%(v/v)water 0.5MphosphatebufferpH7.2 1%(w/v)BSA 1mMEDTA 7%(w/v)SDS 1xPBS 1%(w/v)skimmedmilkpowder 0.05%(v/v)Tween20 1xPBS 2%(w/v)skimmedmilkpowder 0.05%(v/v)Tween20 38%(w/v)acrylamide 2%(w/v)N,Nmethylenebisacrylamide 29%(w/v)acrylamide 1%(w/v)N,Nmethylenebisacrylamide

76

2.MaterialsandMethods
CTABbuffer2x DNAloadingdye6x dNTPmix Fixingbuffer HighSaltSolution2x LB
77

2%(w/v)CTAB 100mMTrisHClpH8.0 20mMEDTApH8.0 1.4MNaCl 1%(w/v)PVP40(Mw=40000g/mol)

10mMTrispH7.6 60mMEDTA 60%(v/v)glycerol 0.03%(w/v)bromophenolblue

10mMdATP 10mMdCTP 10mMdGTP 10mMdTTP

2%(v/v)glutaraldehyde 2%(w/v)paraformaldehyde 0.1Mcacodylatebuffer

0.8Msodiumcitrate 1.2MNaCl

1%(w/v)tryptone 0.5%(w/v)yeastextract 1%(w/v)NaCl adjustpHto7.07.4withNaOH,autoclave

2.MaterialsandMethods
LBagar LBRif LBRifagar MMA MS MSagar LaemmliBuffer4x LDMBase MS 1xMurashige&Skoogvitamins 3%(w/v)sucrose
78

LB 1.5%(w/v)agaragar

LB 120g/mLRifampicin

LBRif 1.5%(w/v)agaragar

MS 10mMMESpH5.7 200Macetosyringone

1xMurashige&Skoogmacroelements 1xMurashige&Skoogmicroelements adjustpHto5.7withKOH,autoclave

MS 1%(w/v)agaragar

250mMTrisHClpH6.8 8%(w/v)SDS 40%(v/v)glycerol 20%(v/v)mercaptoethanol 0.01%(w/v)Bromophenolblue

2.MaterialsandMethods
LDMI LDMII LDMIII Lowmeltingagarose1%(w/v) MOPS10x,pH7.0
79

0.8%(w/v)agar adjustpHto5.7withKOH,autoclave

LDMBase 0.8mg/LBAP 0.1mg/LNAA adjustpHto5.7withKOH,autoclave LDMI 250g/mLCefotaxime 200g/LKanamycin adjustpHto5.7withKOH,autoclave addtheantibioticsaftercoolingto55C

LDMBase 250g/mLCefotaxime 200g/LKanamycin adjustpHto5.7withKOH,autoclave addtheantibioticsaftercoolingto55C

1%(w/v)agarose,lowmelting autoclave,temperto30Cpriortouse

0.4MMOPS 0.1Msodiumacetate 10mMEDTA adjustpHto7.0withNaOHpellets

2.MaterialsandMethods
MOPSrunningbuffer PAGEnortherngel PBS10x Proteinextractionbuffer Proteinrunningbuffer5x Protoplastextractionbuffer MS 0.4Msucrose 2mMCaCl2
80

1xMOPS 0.74%(v/v)formaldehyde

815%(w/v)acrylamidemix(38:2) 8Murea 1xTBE 0.0375%(w/v)APS 0.075(v/v)TEMED

1.4MNaCl 27mMKCl 100mMNa2HPO4 18mMKH2PO4 adjustpHto7.4,autoclave

20mMHEPESpH7.9 150mMNaCl 0.5%(v/v)Tween20 5%(v/v)glycerol 1%(v/v)proteaseinhibitors 0.2%(w/v)PMSF

125mMTris 96mMglycine 0.5%(w/v)SDS

2.MaterialsandMethods
RadPrimeBuffer2.5x Resolvinggel RNAextractionbuffer RNAloadingdye5x Sequencingdye2x 98%(v/v)formamide 10mMEDTApH8.0
81

25mMMESpH5.7 1%(w/v)cellulose 0.5%(w/v)macerozyme

125mMTrisHClpH6.8 12.5mMMgCl2 25mMmercaptoethanol

375mMTrisHClpH8.8 1015%(w/v)acrylamidemix(29:1) 0.1%(w/v)SDS 0.01%(v/v)TEMED 0.1%(w/v)APS

38%(v/v)phenol 0.8Mguanidinethiocyanate 0.4Mammoniumthiocyanate 0.1MsodiumacetatepH5.0 5%(v/v)glycerol

4xMOPS 31%(v/v)formamide 0.27%(v/v)formaldehyde 4mMEDTApH8.0 20%(v/v)glycerol 0.03%(w/v)bromophenolblue

2.MaterialsandMethods
SOBmedium SolutionI SolutionII SolutionIII SouthernDenaturation SouthernDepurination SouthernNeutralization
82

0.03%(w/v)bromophenolblue 0.03%(w/v)xylenecyanolFF

2%(w/v)tryptone 0.5%(w/v)yeastextract 10mMNaCl 2.5mMKCl 1mMMgCl2 adjustpHto7.0andautoclave

50mMglucose 10mMEDTA 25mMTrispH8.0

0.2MNaOH 1%(w/v)SDS

3MKCH3COO 11%(v/v)glacialaceticacid

1.5MNaCl 0.5MNaOH

0.2MHCl

1.5MNaCl 1MTrispH7.5

2.MaterialsandMethods
SSC20x Stackinggel Sterilizationsolution Sucrosedippingsolution TAE50x TB TBE10x 0.9MTris 0.9Mboricacid 20mMEDTApH8.0
83

3MNaCl 0.3Msodiumcitrate adjustpH7.0withHClandautoclave

125mMTrisHClpH6.8 4%(w/v)acrylamidemix(29:1) 0.1%(w/v)SDS 0.01%(v/v)TEMED 0.1%(w/v)APS

0.5%(w/v)sodiumhypochlorite 0.05%(v/v)Tween20

5%(w/v)sucrose 0.03%(v/v)SilwetL77

2MTris 5.71%(v/v)glacialaceticacid 50mMEDTApH8.0

10mMPIPESpH6.7 15mMCaCl2 250mMKCL 55mMMnCl2 freshlyprepared,sterilizedbyfiltration

2.MaterialsandMethods
TE1x,pH8.0 TransferBuffer1x WashI WashII WashIII WashingBuffer 1xPBS 0.05%(w/v)Tween20 0.5xSSC 0.1%(w/v)SDS 1xSSC 0.1%(w/v)SDS 2xSSC 0.1%(w/v)SDS 25mMTris 150mMglycine 20%(v/v)methanol 10mMTrispH8.0 1mMEDTApH8.0

2.1.5 Others
2.1.5.1 Enzymes Cellulase DNaseI Macerozyme Proteaseinhibitorcocktails(P9599) ProteinaseK Duchefa,Netherlands Roche,Switzerland Duchefa,Netherlands SigmaAldrich,USA Invitrogen,USA

Restrictionendonucleases:BamHI,EcoRI, inotech,Greece&NewEnglandBiolabs, M HindIII,KpnI,NotI,PstI,XbaI,XHoI Reversetranscriptase USA HTBiotechnologyLtd.,UK

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2.MaterialsandMethods
RNaseA RNaseH RNaseinhibitor(RNasin) TaqDNApolymerase T4DNALigase T4Polynucleotidekinase(PNK) T4RNALigase KlenowFragment 2.1.5.2 Sizemarkers 1kbDNALadder 100bpDNALadder LambdaDNA,PstIdigested PrestainedSDSPAGEstandard, broadrange(1610318) 2.1.5.3 Bacterialstrains AgrobacteriumtumefaciensC58C1 EscherichiacoliDH5 IMBB,Heraklion,Greece Stratagene,Agilent,USA NewEnglandBiolabs,USA Invitrogen,USA NewEnglandBiolabs,USA Invitrogen,USA Minotech,Greece BioRad,Germany Qiagen,Germany Invitrogen,USA HTBiotechnologyLtd.,UK Minotech,Greece Promega,USA NewEnglandBiolabs,USA NewEnglandBiolabs,USA Minotech,Greece

2.2 Methods
Thefollowingmethodswereusedtoobtaintheresultspresentedinchapter3.

2.2.1 Standardmolecularbiologymethods
2.2.1.1 Cultivation UnlessstatedotherwisebacteriaweregrowninLBmedium,ifnecessary,antibiotics wereaddedasselectivemarkerstoafinalconcentrationof100g/mL.

85

2.MaterialsandMethods
Liquidculturesweregrowninavesselatleastthreetimestheculturevolumeonan orbitalshaker,nutrientplatescontained1.5%(w/v)agaragar. 2.2.1.2 Chemicallycompetentcells EscherichiacoliDH5cells ChemicallycompetentcellswerepreparedwithanadaptedprotocolfromInoueet al.,1990. 250mLSOBmediumina2LErlenmeyerflaskwereinoculatedwithasinglecolony ofrecentlystreakedDH5cellsandincubatedat18Cwithvigorousshakingat250 rpm.WhenanOD600of0.60.8hadbeenreachedtheflaskwasleftonicefor10min andthecellsweresubsequentlyharvestedbycentrifugationat2500xgfor10minat 4C.Thepelletwasgentlyresuspendedin80mLicecoldTB,leftonicefor10min andharvestedasdescribedabove.Thepelletwasresuspendedinanother20mLice coldTBandDMSOwasaddedtoafinalconcentrationof7%(v/v).Afteranother incubationonicefor10minthecellswerealiquotedandquickfrozeninliquidni trogen.Thecellscouldbestoredat80Cforseveralmonths. AgrobacteriumtumefaciensC58C1cells ForthepreparationofcompetentAgrobacteriumtumefacienscells5mlLBRifwere inoculatedwithasinglecolonyofrecentlystreakedC58C1cellsandincubatedover nightat28Cwithvigorousshakingat250rpm.2mLofthestarterculturewere usedtoinoculate50mLLBRifandthecellsweregrownasaboveuntilanOD600of 0.60.8hadbeenreached.Theflaskwasleftonicefor10minandthecellsweresub sequentlyharvestedbycentrifugationat2500xgfor10minat4C.Thepelletwas resuspendedgentlyin1mLicecold20mMCaCl2solutionandaliquoted.Thecells werethenquickfrozeninliquidnitrogenandstoredat80C. 2.2.1.3 Transformation EscherichiacoliDH5cells 50100LofchemicallycompetentDH5cellswerethawedoniceandmixedwith thedesiredamountofDNA.Themixturewasleftonicefor20min,heatshockedat
86

2.MaterialsandMethods
42Cfor45secandquickchilledonicefor1minute.250mLLBwereaddedandthe cellsincubatedfor4560minat37Cwithvigorousshakingat250rpm.Thecells werethenstreakedonselectiveLBagarplatesandincubatedovernightat37C. AgrobacteriumtumefaciensC58C1cells AgrobacteriacellsweretransformedwithanadaptedprotocolfromHfgen& Willmitzer,1988. 100LofchemicallycompetentC58C1cellswerethawedoniceandmixedwiththe desiredamountofDNA.Themixturewasleftonicefor10min,quickfrozeninliq uidnitrogenfor1minandafterwardsincubatedat37Cfor10min.250mLLBwere addedandthecellsincubatedfor4hat28Cwithvigorousshakingat250rpm.The cellswerethenstreakedonLBRifagarplatescontainingtheselectiveantibioticand incubatedfor2daysat28C. 2.2.1.4 Plasmidpreparation PlasmidDNAwasextractedbyalkaliclysisasdescribedinSambrook&Russel, 2001. Bacterialcellsofa5mLLBovernightculturewerepelletedbycentrifugationat maximumspeedandresuspendedin100LSolutionI.200LSolutionIIwere addedandmixedgently.After5minincubationonice150LSolutionIIIwere added,mixedwellandleftoniceforanother10min.Thecelldebriswaspelletedby centrifugationandthesupernatantprecipitatedwith0.7volumesofisopropanol.The pelletwaswashedwith70%(v/v)ethanol,airdriedandresuspendedinwater.Ifa higherpuritywasdesired,thesupernatantwasextractedpriortoprecipitationwith anequalvolumeofphenolandthenchloroformorinasinglestepwithaphe nol/chloroform1:1mixture. LargescaleplasmidpreparationswereperformedwithaNucleoBondXtraMidiKit accordingtothemanufacturersprotocol.

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2.MaterialsandMethods
2.2.1.5 Agarosegel 0.72.0%(w/v)agaroseweremeltedin1xTAEand0.01%(w/v)ethidiumbromide addedbeforecasting.Thegelswererunin1xTAEat20120Vdependingonthesize ofthegeltankandtheapplication.NucleicacidswerevisualizedunderUVlight. 2.2.1.6 Gelextraction DNAfragmentsseparatedonagarosegelsin1xTAEwerecutunderUVlightand extractedwithNucleoSpinExtractIIaccordingtothemanufacturersprotocol. 2.2.1.7 Digest Forsitespecificcleavage110gofDNAwereincubatedat37Cfor12hourswith 110Uoftherespectiverestrictionenzymeinitscorresponding1xbuffer. 2.2.1.8 Ligation VectorandinsertDNAsequenceswerecutbyrestrictionenzymes,separatedon0.7 2.0%(w/v)agarosegelsin1xTAEandextractedoutofthemasdescribedabove.The extractedsamplesweremixedinamolarrationof1:3ofvectortoinsertandincu batedwith1UT4DNAligasein1xligasebufferat16Covernight.

2.2.2 Planttransformationtechniques
2.2.2.1 Plantcultivation Forselectionpurposesseedsweresterilizedfor3mininsterilizationsolutionand subsequentlywashedthreetimeswithsterilewater.Afteradditionof250L1% (w/v)lowmeltingagarose,seedswerespreadonMSagarplatescontainingkanamy cinatafinalconcentrationof50g/mLforArabidopsisthalianaseedsor200g/mL forNicotianabenthamianaseeds. Approximately2weekspostgerminationgreenseedlingsweretransferredontopot tingsoilandcoveredwithplasticbags.Thebagswereopenedgraduallytoacclima tizetheseedlingstogreenhousehumiditylevels.Nicotianasp.plantswerefurtherre pottedtofreshsoil,containingpeatmoss,perliteandfertilizer.

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2.MaterialsandMethods
2.2.2.2 LeafDisctransformation TransgenicNicotianabenthamianaplantswerecreatedbyleafdisctransformationus inganadaptedprotocolfromHorschetal.,1985. Youngleavesweresterilizedfor10mininsterilizationsolutionandconsecutively washedthreetimeswithsterilewater.AgrobacteriaweregrowninLBRifcontaining theappropriateselectiveantibiotictoanOD600ofapproximately0.7.Theywerehar vestedbycentrifugationat2500xgfor10minat4Candresuspendedin20mLMS medium.About60leafdiscswerecutandsoakedintheAgrobacteriasuspensionfor 20min.TheleafdiscswererinsedinfreshMSandtransferredtoLDMIplateswith thestomatafacingtowardstheair.After2daystheleafdiscsweretransferredto LDMIIplatesandchangedrepeatedlytofreshplatesonceaweekuntilshootforma tionstarted.ShootsfromindependentcalliweretransferredtoLDMIIIandafter rootinginthepresenceof200g/mLkanamycinplantletswereputinsoiland movedtogreenhouseconditions. 2.2.2.3 FloralDip TransgenicArabidopsisthalianaplantswerecreatedbyfloraldiptransformationbased onClough&Bent,1998. 5mLLBRifandtheappropriateselectiveantibioticwereinoculatedwithasingle colonyofrecentlystreakedAgrobacteriaandincubatedat30Cwithvigorousshak ing.Theovernightculturewasfurtherusedtoinoculate300mLLBmediumina2L Erlenmeyerflaskandincubatedagainat30Cwithshakingat180rpm.Thecells wereharvestedbycentrifugationat2500xg.Thepelletwasresuspendedinsucrose dippingsolutionandtheOD600wasadjustedto0.8.Theshootsoftheplants,which hadbeenpreparedpreviouslybyremovingallripesiliques,weredippedintothe solutionfor2minwithslightshaking.Theplantswerestoredhorizontallyinthe darkfor2daysandthenstoredundernormalgrowthconditionsuntilmaturity. 2.2.2.4 Agroinfiltration ThemethodisbasedonSchoebetal.,1997.
89

2.MaterialsandMethods
5mLLBRifandtheappropriateantibioticwereinoculatedfromaglycerolstock.The cellswereincubatedat28Cwithvigorousshakingof250rpmuntilanOD600ofap proximately0.7wasreached.Theagrobacteriawereharvestedbycentrifugationat 2500xgfor10minat4C.Thepelletwasresuspendedin5mLofMMAandincu batedinashakerat28Cforatleast2hours.Thebacteriawerepelletedbycentrifu gationasaboveandresuspendedin2mLof10mMMgCl2.Afterrepeatingthis washingsteptwice,theOD600wasadjustedtoapproximately0.25.Thesolutionwas infiltratedwithasyringeintotheleafthroughapreviouslymadesmallpuncture. Thevolumescouldbeadjustedaccordingtotheamountofnecessaryleaves.

2.2.3 Southernanalysis
2.2.3.1 DNAextraction DNAwasextractedwithanadaptedprotocolbasedonRogersandBendichs(1985) CTABbasedprotocol. Approximately20mgoffinelygroundplanttissuewereresuspendedin200L2x CTABbufferbyvigorousvortexingandincubatedat65Cforatleast5min.After additionofanequalvolumeofchloroform,thesampleswereagainvortexedvigor ouslyandafterwardscentrifugedatmaximumspeed.Theclearsupernatantwas transferredtoanewtubeandprecipitatedwith0.7volumesofisopropanol.Theex tractedDNAwastreatedwithRNaseAandusedforPCR.ForSouthernAnalysisthe amountswereupscaledaccordingly. 2.2.3.2 Southernanalysis 2g(Arabidopsisthaliana)or20g(Nicotianabenthamiana)ofgenomicDNAweredi gestedwith1030Urestrictionenzymeinitsappropriate1xbuffer.Thereactions wereincubatedat37Cforapproximately3hours.After2hoursasmallaliquotwas separatedonageltotestifthedigestionhadbeenalreadycompleted.Priortoload ingthesamplevolumewasdecreasedbyisopropanolprecipitation.Thesamples(in 1xDNAloadingbuffer)wereloadedona0.7%(w/v)agarosegelandrunatap proximately20Vovernight.Thegelwasdepurinateduntilthebromophenolblueof
90

2.MaterialsandMethods
theloadingbufferchangedtoyellow,thendenaturedfor30minandfinallyneutral izedwiththecorrespondingsolutionsfromchapter2.1.4.Thevolumesoftheliquids shouldbeatleasttwotimesthegelvolumetoensurecompletecovering. 2.2.3.3 Capillaryblot Thegel,agelsizedmembraneandfourwhatmanpaperswereequilibratedin10x SSC.TheblotwasassembledbyaWhatmanpapersubmergedin10xSSCactingasa bridge,twogelsizeWhatmans,thegel,themembrane,twomoreWhatmanpapers anda10cmthicktissuepaperstackontopofthesandwich.Thenucleicacidswere blottedontothemembranebyapplyingconstantpressurefromtopovernight.The nucleicacidswerecrosslinkedonthemembranebyapplyingUVradiationof100x 1200J/cm2.Thedriedmembranewasstoredatroomtemperatureuntildetection.

2.2.4 Northernanalysis
2.2.4.1 RNAextraction TotalRNAfromplantswasextractedusinganadaptedprotocolfromChomczynski &Sacchi,1987. Approximately100mgoffinelygroundplanttissuewereresuspendedin1mLRNA extractionbufferbyvigorousvortexingandincubatedatroomtemperatureforat least5min.Afterpelletingandremovingcelldebris,200Lchloroformwereadded totheclearedsupernatantandthesampleswereagainvortexedandincubatedfor15 minatroomtemperature.Afterseparationofthetwophasesbycentrifugation,the upperphasewasusedforamodifiedisopropanolprecipitation,byadding1xhigh saltmixturetotheisopropanol.TheRNApelletwaswashedwith70%(v/v)ethanol, airdriedandresuspendedinwater. 2.2.4.2 Denaturingagarose/formaldehydegels FordetectionofRNAmoleculeslongerthan200bases1020goftotalRNAwere separatedon1.2%(w/v)agarosegelsin1xMOPScontaining0.7%(v/v)formalde hydeand0.01%(w/v)ethidiumbromide.Thegelswereprerunat100Vin1xMOPS
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runningbuffer.TheRNAwasboiledin1xRNAloadingdyeandquickchilledbefore loading.Thesampleswererunat100Vforatleast3hours.Thegelswererinsedin waterandusedforcapillaryblottingasdescribedabove. 2.2.4.3 PAGEnorthern FordetectionofRNAmoleculesshorterthan200bases2050goftotalRNAwere separatedon815%(w/v)denaturingpolyacrylamide(PAGEnorthern)gels.After polymerizationthegelswereprerunin1xTBEuntiltheyreachedatemperatureof 50C.TheRNAwasboiledin1xsequencingdyeandquickchilledbeforeloading. Thewellswererinsedwith1xTBEtoremovereleasedureapriortoloading.Gels wererunat1025Wattforkeepingaconstantgeltemperatureof50C.Thegelswere stainedinethidiumbromideandequilibratedin1xTBEuntilthetransfer. 2.2.4.4 Semidryblot TheRNAwasblottedwithaSD20SemiDrydevice,theblotconsistedoffivegelsize Whatmanpapersoakedin1xTBE,followedbytheequilibratedgelfacingthecath ode,thenthemembranefacingtheanodeandanotherlayeroffiveWhatmanpapers. Aconstantamperageof3mApercm2membraneareawasappliedfor30minand theRNAwassubsequentlycrosslinkedonthemembranebyUVradiationof100x 1200J/cm2.Thedriedmembranewasstoredatroomtemperatureuntildetection.

2.2.5 Hybridization
2.2.5.1 Randomprimedlabeling 100ngofpurifiedDNAtemplateweredenaturedbyboilingfor3minandsubse quentquickchilling.Thetypicalreactionvolumewas50L,consistingof1xRad PrimeBuffer,3grandomprimers,10mMdTTP,10mMdGTP,20Ci32PdATP and20Ci32PdCTP,aswellas20UKlenowFragment.Afteranincubationof60 minat37Cthelabeledprobewaspurifiedfromunincorporatednucleotideswitha MicroSpinS200columnaccordingtothemanufacturersprotocol.Priortousethe probewasdenaturedbyboilingfor3minandsubsequentquickchilling.
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2.2.5.2 Endlabeling 8pmolDNAoligowereendlabeledbyincubationwith20UT4PNKcontaining40 Ciof32PATPin1xPNKreactionbufferat37C.After45minthelabeledprobe waspurifiedfromunincorporatednucleotideswithaMicroSpinS200columnac cordingtothemanufacturersprotocol.Priortousetheprobewasdenaturedbyboil ingfor3minandsubsequentquickchilling. 2.2.5.3 Hybridization,washesanddeveloping ThemembraneswereprehybridizedforatleastonehourinChurchhybridization buffer.Thehybridizationtemperaturedependedonthelengthofprobeandsample andwasusuallysetto42CforsmallRNAs<50ntand65CforlongRNAsand DNA.Theboiledprobewasaddedandthemembranehybridizedovernight.The membranewasrinsedatroomtemperaturewithWashIandthenwashedathybridi zationtemperatureaccordingtothefollowingprotocol:twowashesfor15minwith excessWashI,twowashesfor10minwithexcessWashIIandinthecaseofrandom primedprobestwoadditionalwashesfor5minwithexcessWashIII.Finallythe membranewasrinsedin2xSSC,sealedinplasticbagsundsubsequentlyexposedon Xrayfilms.Signalscouldbedetectedbydevelopingthefilmafter110dayexposure at80Cdependingontheamountofthetargetednucleicacid.

2.2.6 Westernanalysis
2.2.6.1 Proteinextraction 100mgfinelygroundleafpowderwasmixedwithatleastfourvolumesofprotein extractionbufferandvortexedthoroughly.After30minincubationonicethesam pleswerecentrifugedatfullspeedfor15minat4C.Theclearsupernatantwas transferredtoanewtubeandstoredat20C. 2.2.6.2 SDSPAGE SDSPolyacrylamidegelsof1015%(w/v)consistingof7.5mLresolvinggeland2.5 mLstackinggelwerepreparedwithaBioRadcastingdevice.Thesampleswere
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boiledin1xLaemmlibuffer,loadedandrunat70140mAin1xproteinrunning buffer.ThegelswerestainedwithCoomassiestainingsolutionanddestainedinde stainingsolutionuntiltheproteinbandswerevisible. 2.2.6.3 Electroblot Thegelaswellastwosponges,fourgelsizewhatmanpapersandthenylonmem branewereequilibratedin1xtransferbuffer.Theblotwasassembledbytwo Whatmanpapersfollowedbythegelfacingthecathodeandthemembranefacing theanodeandanothertwoWhatmanpapersandonespongeoneachside.Thepro teinwasblottedbyapplying90mAconstantamperageovernightat4C. 2.2.6.4 Westerndetection Themembranewasblockedinblockingbufferforatleastonehourandwashedthree timesfor5minwithwashingbuffer.Themembranewasincubatedwithprimary antibody,comprisedofserumfromERL1injectedrabbits(Vlatakis,2010)diluted 1:20000inbindingbufferforonehour.Afterthreewashesthemembranewasincu batedwithsecondaryantibodyantiRabbitAPconjugatediluted1:2500inbinding bufferforanotherhour.Afterthreefinalwashesthemembranewasincubatedwith HRPsubstrateandexposedonXrayfilmuntilproteinbandsappeared.

2.2.7 rRNAcloning
2.2.7.1 SelfLigation Theprecise5and3endsof5.8SrRNA,5SrRNAand4.5SrRNAweredetermined byamodifiedcircularRTPCRprotocolasdescribedbefore(Bollenbachetal.,2005). 5gtotalRNAwereselfligatedwith20UT4RNALigase1in1xRNAligasebuffer andreversetranscribedasdescribedbelow.TheobtainedcDNAswereamplifiedby PCRandseparatedon8%(w/v)acrylamidegelsin1xTBE.Thecorrectproducts wereelutedfromgelslicesbyincubationin300mMNaClovernightat4Cwith constantrotation.ThereleasedDNAwaspurifiedbyphenol/chloroformextraction, precipitatedandfurtheramplifiedinasecondPCRreaction.Itwasagainpurifiedby
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phenol/chloroformextractionandprecipitatedwithisopropanol.Theproductswere clonedandsequencedusingvectorspecificprimers. 2.2.7.2 LinkerLigation ToverifythepositionoftheadditionalnucleotidesofchloroplasticrRNAspecies,200 pmol3ddCmodifiedlinkerwerephosphorylatedwith1UPNKandligatedwith 20UT4RNAligase1tothe3endsoftotalRNA.Thereactionwascarriedoutin1x T4RNAligasebuffercontaining10%(v/v)DMSOand1URNasin.Theproductwas thenreversetranscribedwithalinkerspecificprimer.TheobtainedcDNAwasam plifiedbyPCRandgelextractedwithNucleoSpin.ThepurifiedPCRproductswere clonedandsequencedusingvectorspecificprimers. 2.2.7.3 ReverseTranscription(RT) 15gtotalRNAwereincubatedwith1Mfirststrandprimerat70Cfor10min. ThecDNAwassynthesizedbysupplementingthereactionwith1xRTbuffer,1mM ofeachdNTP,1URNasinand2Ureversetranscriptaseandincubatedat42Cfor 4560min.Thereactionwasterminatedbyafinalincubationat85Cfor5min. 2.2.7.4 PolymeraseChainReaction(PCR) ForspecificamplificationofaDNAfragmentareactioncontaining1xTaqbuffer, 1.252.5mMMgCl2,0.2Mforwardandreverseprimer,200MofeachdNTP,1U TaqpolymeraseandtheappropriateamountoftemplateDNAdependingonthe applicationwereincubatedinathermocycler.Atypicalprogramconsistedofanini tialdenaturationstepat95Cfor3min,followedby35cyclesofdenaturationfor15 30secat95C,annealingfor1530secat4560C(dependingonthenatureofthe primers)andextensionfor30secperkbat72C. ForamorespecificamplificationofthelinkerligatedRNAsatouchdownprotocol wasused.Itstartedwithahighannealingtemperatureofthecorrespondingprimer meltingtemperatureplusfivedegreesCelsius(Tm+5C)whichwasreducedgradu

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allyuntilanannealingtemperatureofthecorrespondingprimerTm5Cwas reached.Theprogramcycledatthistemperature25timesasdescribedabove.

2.2.8 RapidAmplificationofcDNAends(RACE)
3RACEwasperformedwith1gtotalRNAusingtheTaKaRa3FullRACECore Setaccordingtothemanufacturersprotocol.Thereactionwasincubatedfor10min at30C,1530minat50C,5minat95Candfinally5minat5C.10Lwereused forPCR,clonedandsentforsequencing.

2.2.9 QuantitativerealtimePCR(qPCR)
TotalRNAwastreatedwith10UDNaseIin1xDNasebuffertoremovetracesof genomicDNA.TheRTreactionwasperformedasabovewith2.5MOligo(dT)re verseprimer.ThecDNAwastreatedwith1URNaseHtoremoveRNA/DNAhy brids.Thereactionwasthendilutedonetotenand5LwereusedforaqPCRwith theFermentasMaximaSYBRGreenqPCRKitaccordingtothemanufacturers documentation. Atypicalprogramconsistedofaninitialdenaturationstepat94Cfor10min,fol lowedby40cyclesofdenaturationfor5secat94C,annealingfor5secat55Cand extensionfor12secat72C.Theresultingamountoffluorescencewasrecordedat72 C,81Cand84C.Themeltingcurvewasrecordedbyincreasingthetemperature to94Cin1.0Cstepsandholdingeachtemperaturefor2sec. TheresultingcurveswereanalyzedwiththeRotorGeneVersion6programbycom parisonwithastandardcurveofknowntemplateamounts.

2.2.10 Protoplasts
YoungNicotianabenthamianaleavesweresubmergedinprotoplastextractionbuffer andcutintothin,lessthan1mmwideslices.Thesampleswereincubatedforap proximately5hoursatroomtemperaturewithslightshakingat50rpm.Thesamples werefilledintolongtubeswithasmalldiameterandleftforsedimentationat4C forsomehoursorpreferablyovernight.Intactprotoplastsfloatedontopandcould beuseddirectlyformicroscopicanalysis.
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2.2.11 Preparationformicroscopicanalysis
Nicotianabenthamianaleavesweresubmergedinfixingbufferandinfiltratedbyap plyingvacuum.Thefixedtissuewaswashed,stainedwith1%(w/v)OsO4andsec tionspreparedforelectronmicroscopy.Afteranalysistransverseleafsectionswere stainedwith1%(w/v)toluidineblueandusedforopticalmicroscopy.

2.2.12 Fluorescencemeasurement
LeafdiscsofNicotianabenthamianaplantswerecutandleftonwetWhatmanpapers inapetridish;thishumidchamberwasthencoveredwithaluminumfoil.Aftera darkadaptationof15mintheleafdiscswereexcitedwithlightandtheresulting fluorescencemeasuredwithaHandyPEAfluorometer.Theresultswereanalyzed withtheBiolyzerHP3software.

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3. Results

3.1 Localization
Asdescribedinchapter1.2.10plantERL1sequencespossessanNterminalsequence motifpossiblyleadingtotheimportoftheproteinsequenceintochloroplasts.This leaderpeptideispresentinvariousplantvarietiesincludingmonoanddicotyledo nousspecies(Figure3.1a),twoexceptionsareMedicagotruncatulaandSorghumbi color,butthelattersequencecanbeconsideredasincompletesinceitstartswithSer ineinsteadofMethionine. ToverifythepredictedchloroplasticlocalizationofArabidopsisthalianaERL1,itsfull lengthcDNAwasfusedtotheNterminusofGFP(ERL1:GFP)andtransientlyover expressedbyagroinfiltrationintoNicotianabenthamianaleaves.AssoonastheGFP expressioncouldbeobservedasgreenfluorescenceunderahandheldUVlamp,pro toplastswerepreparedandanalyzedwithaconfocalmicroscope.PlainGFPinfil tratedintoadifferentspotonthesameleafwasusedasacontrolforthespecificityof chloroplastimport.ProtoplastpreparationsofNicotianabenthamianaline16Cwith stablyintegratedGFPservedasapositivetransgenicGFPcontrol.Nicotianabentha mianawildtypetissuewasusedasanegativecontrol. ExpressionoftheERL1:GFPconstructresultedinastrongGFPsignaldetectablein definedspotsinsidethecells.Theseorganellescouldbeidentifiedaschloroplastsby detectionoftheredautofluorescenceofchlorophyllinthesecondchannelsinceboth showedaperfectoverlap.ThecytoplasmandvacuolewerefreeofGFPfluorescence, allexpressedERL1:GFPwastargetedtothechloroplast(Figure3.1b). Incontrast,whenplainGFPwasinfiltrated,thegreenfluorescencewasexcluded fromthechloroplastsandcouldonlybedetectedinthecytoplasm(Figure3.1d).In protoplastspreparedfromleavesofNicotianabenthamianaline16Cstronggreenfluo rescencecouldbedetectedthroughouttheEndoplasmaticReticulum(Figure3.1e). ThiscorrespondstothelocalizationoftheintegratedGFPinthisline,ashasbeen
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describedbefore(Voinnetetal.,1999).Protoplastpreparationsofwildtypeshowed nodetectablegreenfluorescenceandconfirmedthespecificityofthedetectedsig nals. Todeterminewhetherthepredictedleaderpeptidewasabletodrivetheimportof proteinsintochloroplasts,itscDNAwasfusedtoGFP(leader:GFP)andinfiltratedas above.TheexpressionpatternwasidenticaltotheresultobtainedwiththeERL1:GFP construct(Figure3.1c).Inaddition,fusionofGFPtothe5endofERL1resultedin strongGFPfluorescenceinthecytoplasm(Eckhardt,2007)thereforeanaccessibleN terminalleadersequenceisindispensableforchloroplastimport.
(A)
A.thaliana M.truncatula N.benthamiana O.sativa P.trichocarpa S.bicolor V.vinifera Z.mays (1) (1) (1) (1) (1) (1) (1) (1)

1 100 MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCN--------------SSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSEN-----ARWRP ---------------------------------------------------------------------------------------------------------SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRK-ISISASRSTTEESTSSLIQPTPSR------TRWKP ---------MAIARVSPPAFS--SPFLIHSLLRPFSSPSSVL-------RPRVTRVPHHRGFAIAAALSQASPLPSADGDGAVMEAPPRPSSR--RPWKP ----MSFPRIPLSRVPSYLHNSNN--CFHLLHPPFIPVSKTP------------SLPTYQTARTYTDFNSQTQTQPPLSLPSLIPSPPVNNPNATHRWKP ---------------------------------------------------------------------------SAASSATVRASG-SVG-----------------MAFYRVSPFRYGSLS-S---LIPYVS-------------SP-----SSLSPPVRT-FTLSASISTPHPSPPSLLTASPKAS-----DRWRP ---------MALARVSPSSLANLIPPLLQSFFRPFSS-------------DFPIRNSRRRSSPVAAAFSLTSQSAHAAREGLVMEAP-RPSSR--YPWKP

Caption:identical,similar (B) GFP Chlorophyll Merge

10m

10m

10m

(C)

10m

10m

10m

(D)

10m

10m

10m

(E)

5m

5m

5 m

Figure3.1:LocalizationofplantERL1 (A)AnalignmentoftheNterminalERL1 proteinregionsrevealsdifferencesbe tweenplantspecies.WhiletheS.bicolor ERL1sequenceisincomplete,M.trunca tulaERL1doesnotcontainachloroplast signalpeptide.(B)(C)Confocalmicros copyofprotoplastpreparationsfrom leavesagroinfiltratedwithGFP containingconstructs:(B)FusionofERL1 toGFPcDNAprovesperfectco localizationofERL1withthechlorophyll ofchloroplasts.(C)FusionoftheERL1 leadersequencetoGFPshowsthesame effectasaboveandidentifiestheleaderto becapableofchloroplastimport.(D)Infil trationofplainGFPresultsingreenfluo rescencewhichisexcludedfromchloro plastsandonlydetectableinthecyto plasm.(E)ProtoplastpreparationsofN. benthamianaline16CwheretheGFPpro teinislocalizedintheEndoplasmaticRe ticulum.

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3.2 RACE
UntilnownocompletesequenceinformationisavailableforERL1ofNicotianasp. ThereexisttwopublishedESTsfromNicotianatabacumintheDFCITobaccoGene Project(http://compbio.dfci.harvard.edu/tgi/cgibin/tgi/Blast/index.cgi)withtheac cessionsEB681897with732ntandBP529372consistingof632nt.Theyhaveaniden tityof75%withtheir5endsbeingalmostidentical,whereastheir3endsaremore dissimilar.SinceSouthernanalysisrevealedtwocopiesoftheERL1geneinNicotiana benthamiana(comparechapter3.3.1)bothESTscouldbetheresultoftranscriptionof twoindividualgenes. WhenalignedwithArabidopsisthalianaERL1cDNA,bothESTshaveanidentityof morethan70%onastretchof30%ofthesequence(Figure3.2a). ToidentifytheERL1sequenceinNicotianabenthamianaRACEexperimentswereper formed.3RACEidentifiedanadditionalstretchof362nt.Theobtainedconsensus sequencecorrespondedtoESTEB681897,noneoftheclonedsequencescorresponded toESTBP529372atthe3end.Theresultingproductshowed55.3%identitywith ArabidopsisthalianaERL1onthenucleotidelevel,aswellas53.2%identityand64.2% similarityontheproteinlevel.5RACEcouldnotbeperformedsuccessfully,butthe sequencecomparisonwithArabidopsisthalianaERL1cDNAsuggestsanearlycom plete5endoftheESTs.TheobtainedNicotianabenthamianasequencesarepresented inthesupplementaryresultsection(chapter6.5.1),analignmentofthegainedse quencesisprovidedinFigure3.2bandc:
Figure3.2:AlignmentsofNicotianasp.ERL1sequence (A)AlignmentofthepublishedN.tabacumESTsEB681897.1andBP529372.1togetherwithA.thaliana ERL1cDNA.Fromthelatteronlythefirst800nucleotidesaredepicted,correspondingtothelengthof theESTs.ThetwoESTsshare75.2%,EB681897.1andAt_ERL137.2%andBP529372.1shares32.2% sequenceidentitywithAt_ERL1.(B)Theconsensusofobtained3RACEsequencesanditsalignment withthelaterclonedN.benthamianasequencesidentifiesESTEB681897.1asthecorrespondingEST;it has98%identitywiththenewlyidentifiedN.benthamianasequenceonthelengthofthealreadypub lishedstretch.(C)AlignmentoftheA.thalianaERL1proteinsequenceandthetranslatedN.bentha mianacDNA.Thelatterisincompleteatthe5endwhichcanbeconcludedfromamissingmethion ine.Thesequencesshare53.2%identityand64.2%similarity,intheexonucleasedomain(highlighted byanarrow)thesequencesareevenmorerelatedsharing68.3%identityand76.9%similarity. Caption:At_ERL1=ERL1ofA.thaliana,Nb_ERL1=ERL1ofN.benthamiana

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(A)
At_ERL1_cDNA BP529372.1 EB681897.1 At_ERL1_cDNA BP529372.1 EB681897.1 1 100 (1) --------AGTTCCCAGTCCCTGTACTCGAAAGGA-AGATCTTCATCTTCAATCTTCATGCTAATCGACGAAAATGGCGTCCGCATTCTCTGCATTTAGG (1) ----------------------------------------CTTCTTCAGGAAAACTCATAC----CTCAGAGA--GCCGTTCAATTCCAATGGCTAT-GG (1) GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAAC-TCACTC----CTCAAAGGA-GCCGTTTAATTCCAATGGCTAC-GG 101 200 (92) GTTTCGTTGTCCAGAATCAGTCCTTTCCGTGATACCCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAAT-CATGTGCAACTCTTCG (54) GATTT----TCTAGGGTCC--CCTTGCTGCGG----CGTTTCCTTGGTATCTCCTC--CGGTACTACCTTCTTCGTACTCACTTCAGGCCCAACCGTAAA (94) GATTT----TGTAGGGTCC--CCTTGCTGCGG----CGGTTCCTTG-TATCTCCGC--CGGTACTACCTTTTTCGTACTCACTTCA-GCCCAGCCGTAAA

201 300 At_ERL1_cDNA (191) CATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTTCTTCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGTGAAA BP529372.1 (142) ATGAATATCTCAGCCTCTCTTTCTACCACCGAAGAATCTACTTCTTC--C-----C---TAATTCAGCCCACACCTTCCCGT--------ACCCGT---EB681897.1 (180) ATCAGTATCTCCGCCTCTCTTTCTACCACCGAAGAATCTACTCCTTC--C-----C---TAATTCAGCCCACAACTTCCCGT--------ACCCGT---301 400 At_ERL1_cDNA (291) ATGCAAGGTGGAGACCCATGTGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGATGATCCTGCCCATTTGGAGATTTTTAACCACGATTGTTCAAA BP529372.1 (220) --------TGGAAGCCAACGTGTCTCTACTTTACTCAAGGTAAGTGCACCAAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCGCT EB681897.1 (258) --------TGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATGATCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCT 401 500 At_ERL1_cDNA (391) GGAACTTCGAGTGGCTGCTGCTGATCTTGAGAGAAAGAAGTCACAAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGAAAAGTTGAGATTCTTGAG BP529372.1 (312) GGAGTTTATGCAAAATGCTGCGGGACTTGAGAATTTGCGGAAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAG EB681897.1 (350) TGAGCTTATGCAAAATGTTGCGGGACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAG 501 600 At_ERL1_cDNA (491) TTTCCTATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGACTTATTCCACAGGTTTGTAAGACCCACCAAAATGAGCGAGCAAGCAATTAACAAAT BP529372.1 (412) TTTCCAGTTCTCCTCTTTGATGCTAAAACCATGGATGTGGTTGACTTGTTCCATAGGTTTGTGAGGCCAACAAAAATGCACGAAGAAAGAATAAACGAAT EB681897.1 (450) TTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTTTTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAAT 601 700 At_ERL1_cDNA (591) ACATCGAAGGCAAGTATGGGGAACTCGGGGTTGATCGTGTGTGGCATGACA--CAGCTATTCCATTTAAGCAAGTTGTTGAGGAGTTTGAAGTTTGGTTA BP529372.1 (512) ATATAGAAGGGAAATATGGAAAACTAGGAGTTGATCG-GTATGGTATCTAATTCAGTAGTTCGAAATA--CAA----TTCAGCAGTTGGAACTT-----A EB681897.1 (550) ATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGATA--CAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTG 701 800 At_ERL1_cDNA (689) GCTGAGCATGACTTGTGGGATAAAGATACAGATTGGGGTCTGAACGATGCAGCTTTTGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTCCTGAGC BP529372.1 (600) GC-------GCCTTTTGGT-TCTATATGAAGAATTGCATG-----------------------------------------------------------EB681897.1 (648) GGTGAACGTCAATTGTGGAGAAATGAACTGGGCGGCTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGA---------------

(B)
3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 3'RACE EB681897.1 Nb_ERL1_1 Nb_ERL1_2 (1) (1) (1) (1) (1) (100) (101) (101) (1) (200) (201) (201) (48) (300) (301) (301) (148) (400) (401) (401) (248) (500) (501) (501) (348) (600) (601) (601) (448) (700) (701) (701) (548) (733) (801) (801) (648) (733) (901) (901) (748) (733) (1001) (1001)

1 100 ----------------------------------------------------------------------------------------------------GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAAGGAGCCGTTTAATTCCAATGGCTACGGGATTTT GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTT GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCTCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTT 101 200 ---------------------------------------------------------------------------------------------------GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCT GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCG GTAGGGTCCCCTTGCTGCGGCGGTTCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTATCTCCGCCTCTCG 201 300 -----------------------------------------------------CCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATCTACTCCTTCCCTAATTCAGCCCACAACTTCCCGTACCCGTTGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATTTACTTCTTCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT TTCTACCACCGAAGAATCTACTTCTTCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACT 301 400 AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCTTGAGCTTATGCAAAATGTTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT AAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCGGGACTTAAGAATTTGCGGCAGCAGGAGT 401 500 TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT TGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAAGTCGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACTTTTT 501 600 CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT CCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAGGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGAT 601 700 ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGTGAACGTCAATTGTGGAGAAATGAACTGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG ACAGCTATCCCATTTGGAGAAGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCGGCTGTCTAAATAAAGCTG 701 800 CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAA CCTTTGTTACTTGTGGGAACTGGGATCTGAAGA------------------------------------------------------------------CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAA CCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCTCAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAACGGATTAA 801 900 TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT ---------------------------------------------------------------------------------------------------TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT TTTGAAGGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAACTCCAGATGCCTTTGTTAGGGAGTCATCACCTT 901 1000 GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCATTCTCCTG ---------------------------------------------------------------------------------------------------GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAACCCTCATTCTCCTGA GGAATAGATGATGCAAAAAACATAGCAAGAGTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCATTCTCCTG 1001 1097 AAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTTCTTCTGAACCATTTTGTTATCACCTAAACATTTTTAGAAAAAAAAAAAAAAA ------------------------------------------------------------------------------------------------AAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTT----------------------------------------------------AAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTT----------------------------------------------------

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(C)
At_ERL1 Nb_ERL1 At_ERL1 Nb_ERL1 1 100 (1) ---------MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSSSSPSTFSLMETSENARWRPMCLYYTHGKC (1) SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQ---PSRKISISASRSTTEESTSSLIQPTPSRTRWKPTCLYFTQGKC

101 200 (92) TKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGVDRVWH (98) TKMDDPMHIDKFNHNCSLELMQNAAGLKNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGKYGKLGVDRVWH EXOIII 201 300 At_ERL1 (192) DTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWINLKDVYLNFYGREARGMVSMMRQCGIKLMGSHH Nb_ERL1 (198) DTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQCKVAGTKLPPYFMEWINLKDVFLNFYKRRAKGMLSMMRELQMPLLGSHH 301 346 At_ERL1 (292) LGIDDTKNITRVVQRMLSEGAVLKLTARRSKSNMRNVEFLFKNRIK Nb_ERL1 (298) LGIDDAKNIARVLQHMLSDGALVQITARRNPHSPEKVEYLFEDRIV

Caption: identical, conserved, similar,

Exonuclease domain

3.3 TransgenicNicotianabenthamianaplants
Agrobacteriumtumefaciensmediatedleafdisctransformationwasusedtoproduce transgenicNicotianabenthamianaplantswhichweremisexpressingERL1.Thestable integrationwasusedtostudytheeffectofERL1duringthewholelifecycleofthe plants.

3.3.1 KnockdownofERL1
SixindividuallineswerecreatedbytransformingNicotianabenthamianaleaveswitha hairpindesignedfromNicotianatabacumESTEB681897(Schumacher,2009).Thiscon structwassupposedtodownregulatetheendogenousNicotianasp.ERL1mRNAby RNAsilencing. Theplantsshowedresistancetokanamycinandslightbleachinginayoungstage (Figure3.3a).Thisphenomenonrevertedtoawildtypelikephenotypeaftersome weeks(Figure3.3b),notonlymacroscopicallybutalsowhenobservedwiththelight andelectronmicroscope(datanotshown).Fiveoutofthesesixlineshadasegrega tionpatternmostlikelycorrespondingtoasingleinsertion(compareTable3.1).In addition,fouroutofthesixlinesdidnotproduceanyseedsorif,thenataverylate

(A)

(B)

(C)

wt 103

Figure3.3:Analysisofpresum ableERL1suppressorplants (A) Slight bleaching in young plants(B)wildtypelikephenotype inadultleaves(C)Southernanaly sis revealed the same ERL1 copy number in wildtype (wt) and the transformed plants () suggesting nopresenceoftheERL1hairpin.

3.Results
stageandonlyinsmallamounts.CrossingoftheselineswithNicotianabenthamiana wildtypeinbothdirectionsshowed,thatthemalesexualorganswerenotfunctional, whereastheplantsproducedseedswhenpollinatedwithwildtypepollen.Thepres enceoftheERL1hairpin,however,couldnotbeproven,Northernanalysisfailedto detectthehairpinorsmallRNAsresultingfromit.qPCRanalysisshowedthesame ERL1expressionlevelasinwildtypeleaves(datanotshown).FinallySouthernhy bridizationwithanERL1probeshowedthesametwocopiesoftheERL1geneasin wildtype(Figure3.3c).Theplantswerethereforeconsideredastransgenic,butthere wasprobablynodownregulationofERL1present.Theobservedphenotypeisnot consideredasaresultofdownregulationofERL1.Furthersequenceanalysisofthe originalvectorproved,thattheconstructdesignedbyapriorlabmemberwasnot correct.
Table3.1:SegregationofNicotianabenthamianaT2plantlinestransformedwitha hairpinconstructdesignedfordownregulationofERL1

line ERL1KD1 ERL1KD2 ERL1KD3 ERL1KD4 ERL1KD5 ERL1KD6

segregation 1:4.5 1:5.5 1:18.7 1:3.8 1:4.0 1:4.5

3.3.2 OverexpressionofERL1
anArabidopsisthalianaERL1cDNAconstructdrivenbytheconstitutiveCaMV35S promoterwasusedtotransformleavesofNicotianabenthamiana. Intheresultingtwelvelinestheplantsshowedalreadyinthefirstleavesmulti facetedphenotypeswhichweresometimesnoteasytodistinguishfromsusceptibil itytokanamycin.ThereforeallplantsweretransferredtosoilandtheirERL1expres sionlevelsdeterminedbyNorthernanalysis.Fromthisdatathesegregationratio couldbecalculated(compareTable3.2).Intotal,RNAof344plantswasanalyzed andrevealedadirectrelationoftheERL1expressionlevelandtheseverityofthe
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phenotype(Figure3.4f).Mosttransgeniclinesshowedmorethanoneofthedifferent phenotypes.Thereforetheycannotbeattributedsolelytothenumberandlocationof theinsertionsintotheNicotianabenthamianagenomes,buttheyareconsideredtobea directconsequenceofERL1overexpression.Ingeneral,themacroscopicphenotypes couldbesubdividedintofourdistinctgroups: ThetermBleachwasusedforaphenotypevaryingfromplantswithleaves whichwereslightlybrighterthanwildtypetoplantswithanalmostcomplete lossofchlorophyllandwhiteleaves.Thelattershowedastuntedgrowthwith matchingsmallsizedleaves.Comparedtowildtypetheyreachedthematurity statestronglydelayedandproducedonlyfewseeds(Figure3.4b).Inaddi tion,bleachplantsappearedtohaveaprolongedlifecyclecomparedtowild type. SomebleachplantswithastrongphenotypeenteredapathwayofERL1 transgenesilencingandrevertedtoawildtypelikephenotype.Itsprogression wascomparabletosystemicsilencingspread.Theseplantscontainedcom pletelywhiteandgreentissuewhichcouldbecollectedindependently.The reversionofthebleachphenotypecouldalsobeobservedwhichmayhave beentriggeredbyenvironmentalfactors.Onlythestrongestbleachplantsof distinctlinesshowedthisbehaviorbutthephenomenonwasinheritableand appearedinconsecutivegenerations.ThelinewasfurthernamedSelf silencer(Figure3.4dande).Itispossible,butnottested,thattheendoge nousERL1expressionisalsosilencedinthisline. Mosaicplantshadaphenotypewithspeckledleavesofgreenandwhite patches;itcannotbedistinguishedwhetherthegreenpatchesarearesultof endogenousERL1transgenesilencingorpossessedwildtypecharacteristics. Thesizeoftheplantsandtheirlifecycleshowednodifferencewhencom
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paredtowildtypealthoughtheyshowedstrongoverexpressionofERL1 (Figure3.4aandf). TheNophenotype(NP)linewastheexceptiontotheabovementionedrela tionofERL1transgeneexpressionandthebleachingphenotype.Theseplants showedanERL1expressioncomparabletomosaicandbleachplants,butnei therthemorphologynorthelifecycleoftheplantdifferedtowildtype.There wasonlyonelinethatshowedthisbehavior(incontrasttotheotherpheno types),thereforethephenomenonwasattributedtopositionaleffects(Figure 3.4c).Inconsecutivegenerationsindividualsofthislineexhibitedmild bleachingofsomeleaves.

(A)

(B)

(C)

(D)

(E)

(F)

mosaic (G)

bleach

nophenotype

selfsilencer

49,1kDa

34,8kDa 28,9kDa

M 1

Figure3.4:AnalysisofNicotianabenthamiana plantsoverexpressingERL1 (A)Mosaicphenotype(B)Bleachphenotype(C)No phenotype(NP)(D,E)Selfsilencingphenotype(F) Northernanalysisoftransgenicplantlinesrevealed strongoverexpressionofERL1inmostindividuals. Thedegradedsignalinselfsilencinglinescouldbe duetosilencingoftheERL1mRNA(G)Western analysisofplantsoverexpressingERL1leadtoa strongbandofapproximately42kDa.Inmosaic andwhitetissuethestrongsignalmightresultfrom twobandsasinArabidopsisthalianatransgeniclines; inselfsilencedtissuethesignalwasveryweak. Coomassiestainingisdepictedasaloadingcontrol.

Caption:M=BioRadPrestainedSDSPAGEstandard,1=wildtype,24=agroinfiltratedERL1+FLAG ofanunsuccessfulpulldownexperiment,5=nophenotype(NP),6=mosaic,7=selfsilencergreen tissue,8=selfsilencerwhitetissue 106

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Table3.2:SummaryofsegregationandphenotypicpatternofNicotianabenthamianaT1plantlines overexpressingERL1

line ERL1over1 ERL1over2 ERL1over3 ERL1over4 ERL1over5 ERL1over6 ERL1over7 ERL1over8 ERL1over10 ERL1over11 ERL1over12 ERL1over13

phenotype bleach bleach bleach,selfsilencing nooverexpression bleach,selfsilencing nooverexpression bleach mosaic,bleach nophenotype bleach,selfsilencing mosaic,bleach mosaic,bleach

segregation 1:8.8 1:3.4 1:0.3 nodata 1:1.6 nodata 1:11.3 1:3.1 1:1.8 1:0.4 1:2.7 1:5.7

3.3.2.1 Microscopy Tobetterdescribethemacroscopicallyobservedphenotypesleafsectionswerepre paredandtheorganellestructurewasanalyzedbytransmissionelectronmicroscopy (TEM).Someofthesesectionswerethenstainedwithtoluidineblueandtheleaf morphologyfurtheranalyzedbylightmicroscopy. Themorphologyofawildtypeleaf(asdepictedinFigure3.6a)canbedescribedas following: theepidermis,onelayerofchloroplastfreecellscoveringtheupperandthe lowersideoftheleaves. themesophyllwhichisassembledoftwodistinctchloroplastrichcelltypes: theupperpalisadelayerwithelongatedcellsarrangedinparallelandthe lowerspongylayerwithroundercellsandlargerintercellularspaces.The spongylayeralsocontainsthevasculaturecells. Wildtypechloroplastsareovalshapedorganelleswithintercellularmembranes(thy lakoids)whicharearrangedinstacks(grana)andresponsibleforphotosynthesis.In
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(A) cyt (B) cw st pg gra cw
0.7 m

pla chl

cyt

(C)

(D)

pg cyt chl st

cw chl gra pg cyt

0.8 m 0.5 m

cw pla

gra

cyt

Figure3.5:TEManalysisofphenotypesofNicotianabenthamianaplantsoverexpressingERL1 (A)Inwildtypeplantschloroplastsareovalshapedandcontainstoragemoleculespackedintostarch granulesandplastoglobuli.Uptotenthylakoidmembranesarepackedpergranum.(B)Undifferenti atedproplastidsinbleachplantsshowonlyunorganizedintercellularmembranestructuresandno storagemolecules.(C)Chloroplastsingreentissueofselfsilencingplantspossessroundishchloro plastsfilledwithgranastacks,whichareassembledbymorethantenthylakoidmembranes.Usually therearenostarchgranulesdetectable,butthenumberofplastoglobuliappearstobenormal.(D) Mosaicplantsshowthecharacteristicsofwhitetissue(leftcell,lower)andgreentissue(rightcell,up per)inneighboringcells. Caption:cw=cellwall,cyt=cytoplasm,chl=chloroplasts,gr=granum,st=starchgranules,pl= plasmamembrane

addition,theycontainlipiddropletsnamedplastoglobuliandstarchgranuleswhich serveasstoragecompoundforexcessenergy(Figure3.5a). InERL1overexpressingtransgenicplantsseveralstructuralalterationscouldbeob servedwhichdidnotonlyinfluencetheleafarchitecturebutdistortedthewholeor ganizationofthechloroplasts.WhitetissuecausedbystrongERL1overexpression showedaslightlydisorderedtransverseleafstructurewhenobservedinthelight microscope.Thepalisadecellswerelesselongatedandlosttheirparallelorganiza tionpattern.Leavesfrombleachplantswerealsothinnercomparedtowildtype (Figure3.6b).Anevenmoredramaticeffectcouldbeobservedintheelectronmicro

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scope.Thecellspossessedonlyfewplastidswhichresembledundifferentiatedpro plastidsofmeristematictissues.Theycontainedonlyundevelopedthylakoidmem braneswhichweredispersedinsidethestromawithoutorganization.Consistent withtheinabilityforproperphotosynthesis,nostarchgranulescouldbedetectedas therewasnoexcessenergypresent(Figure3.5b). GreentissueinareaswithsilencedERL1expressionresembledwildtypesections whenobservedinthelightmicroscope.Thepalisadecellshadtheusualelongated shapewithsometimesslightlyenlargedspacesbetweenthem.Theleafthicknesswas similartowildtype(Figure3.6c),whichcouldalsobeobservedmacroscopically whenagreenpatchdevelopedwithinawhiteleaf.Whenexaminedingreaterdetail
(A) pal chl spo mes epi (B) epi

mes

epi 50m (C) pal epi chl mes spo vasc epi 50m 50m vasc (D) pal chl 50m

epi

epi

spo

mes

epi

Figure3.6:PhenotypicanalysisbylightmicroscopyofNicotianabenthamianaplantsoverexpress ingERL1 (A)Inwildtypetwolayersofepidermalcellswithoutchloroplastscoverthemesophyllcellswith manychloroplastsalignedalongthecellwalls.Itconsistsoftheupperpalisadelayerwithtightly packedelongatedcellsarrangedinparallelandthespongylayerwithroundercellswithoutorganiza tion.(B)Inbleachtissuetheorganizationofthemesophyllislost,thereisnopalisadelayerdetectable andthecellscontainnochloroplasts.Inadditiontheleafdiameterissignificantlydecreasedcompared towildtype.(C)Ingreentissuethemesophyllorganizationispartlyrestoredwithmoreelongated palisadecells,anormalnumberofchloroplastsandaleafdiametercomparabletowildtype.(D)Mo saicplantsshowamixtureofwhite(margins)andgreen(middle)characteristics. Caption:epi=epidermis(lowerandupper),mes=mesophyll,pal=palisademesophyll,spo=spongy mesophyll,vasc=vasculature,chl=chloroplast 109

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intheelectronmicroscopeonecouldobservesomedifferencesbetweenwildtypeand greentissue.Theycontainedacomparableamountofchloroplastswithdrastically enlargedgranainthelatter,consistingofmorethandoubletheamountofthylakoid membranesthaninwildtype.Sincegreenandwhitetissueswerecollectedfromthe sameleaf,theincreasedamountofphotosyntheticcompartmentsingreenareas probablycompensatesforthewhitesectors.Thefactthatgreentissueisabletore generatefromwhitetissuesupportsthehypothesisofplastidsinwhiteareasbeing meristematicproplastidswhichcanfurtherdifferentiateintochloroplastswhennor malERL1levelsarerestored(Figure3.5c). Mosaictissuecombinedbothoftheabovedescribedphenomena.Ingreenpatches theleaveswereorganizednormallywhereasinwhitepatchestheyshowedthedis tortedpalisademesophyll.Chloroplastsfromgreentissuehadenlargedgranastacks nexttocellscontainingchloroplastswithrandomlydispersedthylakoids(Figure3.6 dandFigure3.5d). 3.3.2.2 EffectofERL1overexpressiononchloroplastmRNAs ThestrongeffectofERL1overexpressiononchloroplastswasfurtherinvestigatedby Northernanalysisofchloroplastrelatedgenetranscripts.Forthispurposegreenand whitetissuewasagaincollectedindependentlyandusedforRNAextraction.The mRNAlevelswerecomparedtoNicotianabenthamianawildtypetotalRNA,inaddi tion,someofthegeneswerealsoinvestigatedinwildtypeplantsafterinfiltration andconsecutivetransientERL1overexpression.Asnegativecontrolsfortransient overexpressionservedwildtypematerialinfiltratedwithGFPortheerroneousERL1 hairpinconstruct(seechapter3.3.1),asnegativecontrolfortransgenicplantsand constitutivegenemisexpressionservedalinewhichhadalsobeentransformedwith thewrongERL1hairpinconstruct(comparechapter3.3.1). ERL1overexpressionwasinvestigatedinwildtype,greenandwhitetissue;from theseonlythelattershoweddetectableexpressionofERL1.Severalothergenes whicharealsoencodedinthenucleusandtranscribedbyPolIIhadbeeninvesti gatedfortheirtranscriptlevels(Figure3.7a).TheNicotianagenePFTFcodesforthe
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homologueofthemetalloproteaseFTSH2fromArabidopsisthalianawhichisrespon siblefortheturnoverofphotosystemIIcomponentsbyproteolyticdegradation (Summer&Cline,1999).Theexpressionofthegenewasslightlyincreasedinwhite tissuebutevenfurtherupregulatedingreentissue.Thisincreasecouldcorrespond withthehigheramountofphotosyntheticallyactivemembranesingreentissue (compareFigure3.5c).Incontrast,itsantagonistCLPCshowedcomparableexpres sioningreenandwildtypetissue,butslightupregulationintissueoverexpressing ERL1.MisexpressionofunrelatedgenesleadtothesameupregulationofCLPC, thereforethisresultwasnotconsideredasspecific.Finallythelevelofthenucleus
Figure3.7:Northernanalysisof selectedchloroplastrelatedgenes ERL1 (A)Chloroplastrelatedproteins (A) transcribedinthenucleusbyRNA PTFT Nucleusencoded PolymeraseII(POLII).Allthree POLIItranscripts genesofthisgrouptestedhere CLPC (PTFT,CLPCandNEP)showeda slightlystrongerexpressionin NEP whitetissuecomparedtowildtype. Therewasnocleartrendingreen (B) CLPP tissue.(B)mRNAlevelsofchloro Plastidencoded plasticgenestranscribedbythe NEPtranscripts PEP chloroplastimportednuclear encodedRNApolymerase(NEP) (C) RBCL werestronglyupregulatedinwhite Plastidencoded tissue,allothersamplesshowedan PEPtranscripts PSB expressioncomparabletowildtype. (C)ThemRNAlevelsoftwogenes (RBCLandPSB)transcribedbythe rRNA plastidencodedRNApolymerase (PEP)werestronglyimpairedin whitetissue.Sincechloroplastic rRNAswerealmostmissing(com paretherRNAloadingcontrol), translationoftheupregulatedPEP transcripthadbeenlowandPEP dependentgeneswereprobablynot 1 2 3 4 5 6 7 transcribednormally.Duetothe problemswiththe transgenicplantssupposedlysuppressedforERL1(seetext)thetissuefromthese plantsisregardedascontrol. Caption:1=infiltratedGFPoverexpressioninNicotianabenthamianawildtypeplants,2=infiltrated ERL1hairpininNicotianabenthamianawildtypeplants,3=presumabletransgenicERL1suppressor plants,4=Nicotianabenthamianawildtypeplants,5=selfsilencergreentissue,6=selfsilencerwhite tissue,7=infiltratedERL1overexpressioninNicotianabenthamianaplants 111

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encodedpolymerase(NEP)whichistranscribedandtranslatedoutsideofthe chloroplastandthenimportedthereinwasassessedaswell.Itwasmarginally upregulatedinwhitetissuebutthesameamountofupregulationcouldalsobede tectedinplantsbeingtransgenicforanunrelatedgene;thereforethisresultwasnot consideredasspecificforconstitutiveERL1overexpression. NEPdependenttranscriptswerealsoinvestigated(Figure3.7b),oneofthemisthe plastidencodedpolymerase(PEP)whichisthesecondactiveRNApolymeraseinthe chloroplast.Itsexpressionwasstronglyupregulatedinwhitetissuewhencompared towildtype.Anothergene,CLPP,whichiscodingforaproteasesubunitconsidered asindispensibleforchloroplastdevelopment(Shikanaietal.,2001),showedaninter estingtranscriptionpatterninwhitetissue:insteadofasinglebandasecondlarger onecouldbedetectedwithbothsignalsbeingstrongerthaninallotheranalyzedtis sueswhichshowedapproximatelyequalexpression. FinallyPEPdependenttranscriptswerealsoinvestigated(Figure3.7c).Thesetran scriptsaremainlycompromisedofphotosynthesisrelatedgenesandshowedase verelyreducedexpressioninwhitetissue.Allothersampleshadacomparableex pressionwiththeonlyexceptionofgreentissueshowingupregulatedlevelsofthe Ribulose1,5bisphosphatecarboxylaseoxygenase(RuBisCO)beingresponsiblefor thefirststepofcarbonfixationinthechloroplasts. ThefactthatchloroplasticribosomalRNAsarenearlyabsentinwhitetissueresulted inreducedtotalproteinlevelsintheselines(compareFigure3.4gwhereinlane8the otherwiseprominentRuBisCOproteinbandof52.7kDaisalmostmissing). 3.3.2.3 EffectofERL1overexpressiononthephotosyntheticapparatus SinceoverexpressionofERL1inNicotianabenthamianaplantsmacroscopicallyleads toalossofchlorophyllandmicroscopicallyresultsinchloroplastalterations,itsef fectonthephotosyntheticapparatuswasinvestigatedinmoredetail.Leafdiscsof NicotianabenthamianaERL1overexpressorsandwildtypeplantswerecutanddark adaptedinahumidchamber.Afteratleast15minutesthefluorescenceofchloro

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(A) (B)

(C)

(D)

Figure3.8:DeterminationofphotosyntheticparametersinNicotianabenthamianaplantsoverex pressingERL1 Thevaluesarenormalizedtowildtypetissue,whichisdepictedasthecircleinblack.(A)Themeas uredfluorescencevaluesofgreentissuealmostcorrespondtowildtypetissueexceptforTF(max),which issignificantlylower.(B)Incontrast,nophenotypetissueshowsmoredifferences,themostpromi nentbeingSm,NandPI(csm).(C)Whitetissueshowsnosimilaritywithwildtypetissue,withmajor peaksforTF(max),F0/Fm,PHI(D0)andDI0/CS0.(D)Mosaictissuelookslikeamixtureofwhiteandgreen orwildtypetissuewhichisnoteasytointerpret,butthemostprominentparameterisPI(csm)whichis almostnotexistent.

phyllawasmeasuredwithaHandyPEAfluorophotometer.Theresultsareshown inFigure3.8;onlythealteredparametersarediscussedexplicitly(forfurtherinfor mationpleaserefertoBusottietal.,2007). Parametersresultingfromwildtypetissuewereusedasareferenceanddefinedas value1.0.Asexpected,whitetissuefromaselfsilencingplantdidnotshareany

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similaritywithphotosyntheticbehaviorofwildtype.Allparametersdifferedsignifi cantlyanditwasthereforeassumedthatnopartofthephotosyntheticapparatuswas functionalinwhitetissue(Figure3.8c). Interestingly,greentissueofthesameleafshowedanalmostwildtypelikephotosyn theticbehavior,withonesignificantexception:themaximalfluorescencewas reachedlaterthaninwildtype,describedbytheparameterTF(max)(Figure3.8a).Since themaximalfluorescenceFmandthenumberofchlorophyllamolecules(which couldbeconcludedfromparameterF0,thefluorescenceinthedarkadaptedstate) werecomparabletowildtype,thisextendedperiodmusthavebeentheresultofan otherintrinsicfactor.Togetherwiththeresultsfromabove(seeFigure3.7)itcanbe statedthatthechlorophyllamoleculesofgreensectorsshowanenhancedphotosyn theticactivitycomparedtowildtype. Mosaictissuehadbeenanalyzedaswellbutitsresultsweredifficulttointerpretits resultsinceitcontainsamixtureofwhitecellswithchaoticfluorescenceparameters andgreentissuewithprobablynormalphotosyntheticbehavior.Mostprominent wastheparameterPI(csm)whichhadbeencompletelylost;thisparameterdescribes thephotosyntheticperformanceperleafarea(Figure3.8d). Nophenotype(NP)tissue,althoughmacroscopicallynotdistinguishablefromwild typetissue,showedmoredifferencesthantherevertedgreentissue(Figure3.8b). NotonlythebasicfluorescenceF0butalsothemaximalfluorescenceFmwereslightly decreased.Sm,theenergywhichisneededforacompletereductionofallreaction centersandN,thenumberofreactionsuntilthemaximalfluorescenceisreached, werehigherthaninwildtype.Thephotosyntheticperformancewaslowerthanin wildtype. SummarizeditcanbestatedthatthephotosynthesiswaslessefficientinNPplants overexpressingERL1thaningreentissuewithsilencedERL1expression.Thelatter evenshowedanenhancedphotosyntheticactivity.Bleachandmosaicdidnotshow anynormalphotosyntheticbehavior.

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3.4 TransgenicArabidopsisthalianaplants
ArabidopsisthalianaplantlinesmisexpressingERL1wereusedtoconfirmtheresults whichhadbeenobtainedbeforeinNicotianabenthamianaplants(comparechapter 3.3).Presumableknockoutlineshadbeenorderedfrompublicseedcollectionsand theoverexpressinglineswerecreatedbyfloraldiptransformation.

3.4.1 KnockdownofERL1
ThreeindividualTDNAinsertionlines(namedCS834430,SALK_579265and SALK_544378)generatedbyvacuuminfiltrationofA.thalianaecotypeColumbia (Col0)werereceivedfromNASC,theEuropeanArabidopsisStockCentre.Line CS834430(McElveretal.,2001)containstheBARgenecodingforphosphinothricin acetyltransferase,aselectivemarkerprovidingresistancetotheherbicideBASTA. ThetwoSALKlines(Alonsoetal.,2003)containtheNPTIIgenecodingforneomycin phosphotransferase,aselectivemarkerprovidingresistancetotheantibiotickana mycin. Noneoftheselinesshowedanymacroscopicphenotypecomparedtotheirback groundlineCol0,butlinesSALK_579265andSALK_544378revealedincreasingsi lencingofthekanamycinresistancegene,observableasbleachingofthefirstleaves andconsecutivedeathoftheseedlings.Sincethiseffectpreventedthesegregationon aselectivemediumseedsofthesetransgeniclinesweregrownonplateswithoutan tibioticsandselectedbyPCRanalysisoftheinsertionsite.LineCS834430wasgermi natedonsoilandtransgenicindividualswereselectedbysprayingtheseedlings with100g/mlofBASTAontwoconsecutivedays;homozygositywasfurtherveri fiedbyPCRanalysisinthesurvivingplants. TheTDNAinsertionsresideindifferentpositionsoftheERL1sequenceandshowed adistinctsegregationbehavior:inlineSALK_579265theinsertionliesinthepro moteroftheERL1geneandshowedasegregationof1:2.7.LineCS834430contains theTDNAinanintronoftheERL1geneandsegregatedwitharatioof1:5.5.Line SALK_544378hastheinsertioninanexonoftheERL1DNAsequence,whichalso correspondstothepromoterofthenextgene;thislinedidnotsegregateatall.
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(A) (B)

265378wt430wt

ERL1 expression

TUB9expression

Figure3.9:CharacterizationofselectedpubliclyavailableArabidopsisthalianaERL1knockout plants (A)SouthernanalysisoflinesSALK_579265,SALK_544378andCS834430withaprobespecificforthe respectiveresistancegene.TheleftpictureshowslinesSALK_579265,SALK_544378andwildtype DNAprobedfortheNPTIIgene.Therewasasinglelineofbackgroundhybridizationdetectablein wildtypewhichisconsideredasplasmidcontamination.InlineSALK_579265oneadditionalband canbedetected,thereisapossiblesecondbandbutduetothesegregationpatternthisisunlikely. LineSALK_544378showsthreestrongbandscorrespondingtothreecopiesofthekanamycinresis tancegene.Intheright,lineCS834430andwildtypeDNAareprobedfortheBARgene.Inthemutant planteightbandscouldbedetectedwhilethewildtypeDNAisfreeofbackgroundhere.Thehigh copynumberdoesnotcorrespondtothedetectedsegregationrateof1:5.5(B)Quantitativerealtime analysisofERL1expressioninmutantandwildtypeplantscomparedtotheexpressionofTUB9,melt ingcurve,quantitationcurveandstandardcurveareshown.Duetotheverylowexpressionrateof ERL1thethresholdhadtobesetveryhigh.InthecaseofERL1thestandardcurvepossessesanR2 valueof0.99894,inthecaseofTUB9thestandardcurvehasanR2valueof0.99924.Exactquantitation isprovidedinthetextandTable3.3. Caption:265=SALK_579265,378=SALK_544378,430=CS834430,wt=wildtype

SouthernanalysiswasusedtodeterminethecopynumberoftheinsertedTDNA sequences,theprobeswerespecificforthecorrespondingresistanceproviding genes.Inthecaseofkanamycinresistanceacontaminatingbandcouldbedetectedin wildtype,forthefurtherdeterminationofthecopynumberthisbandwassubtracted inthesetwomutantlines.Incontrast,therewasnocontaminationofBARDNAob servableinwildtype.BasedonthisanalysislineSALK_579265containsasinglein sertion,lineSALK_544378threeinsertionsandlineCS834430eightinsertions(com

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pareFigure3.9a).Crosshybridizationcannotbeexcludedinthelattercasesincethe linewassubjecttosegregation,thereforeitwasnotexcludedfromfurtheranalysis. LinesSALK_579265andCS834430wereanalyzedfortheexactamountofdownregu lationofERL1byqPCR,theformerhavingaremainingERL1expressionof13%and thelatter6%comparedtowildtype.Thereforetheobtainedlinesonlyprovideda reducedERL1activity,butnocompleteknockout(Figure3.9b).AnERL1hairpin expressinglinehasbeencreatedbyfloraldiptransformationattheendofthiswork, butisnotanalyzedyet.
Table3.3:SummaryofcharacteristicsofArabidopsisthalianaERL1knockdownplantlines

Line SALK_579265 SALK_544378 CS834430

Segregation 1:2.7 1:5.5

Insertions 1 3 8

Downregulation 13%ofwt nodata 6%ofwt

3.4.2 OverexpressionofERL1
Agrobacteriumtumefaciensmediatedfloraldiptransformationwasusedtoproduce16 individualtransgenicArabidopsisthalianalinesconstitutivelyoverexpressingERL1 withthestrongCaMV35Spromoter.Theplantsshowedresistancetokanamycin whichwasdeterminedbygerminationandconsecutivesegregationonselective platesandPCRanalysisoftheNPTIIgene.Fewindividualsoutofthreelinesexhib itedsomebleachingandaslightlystuntedgrowthwhencomparedtowildtype (Figure3.10c). TheoverexpressionlevelsofERL1weredeterminedbyNorthernandWesternanaly sis.PlantsoftheT1generationofthe16individualArabidopsisthalianalinessuppos edlyoverexpressingERL1werepooledforRNAextraction.15goftotalleafRNA wereseparatedonagelandinvestigatedbyNorthernanalysis,radioactivelylabeled ERL1DNAservedasaprobe.Outofthese16linesfourdidnotshowanydetectable ERL1expression,sevenlinesshowedweakoverexpressionandfivelineswerecon sideredasstrongoverexpressors(Figure3.10a).Fromthesethelinesnamed1,2and 4werefurtherpropagated.Slightlyunequalloadingcouldbeobservedintheformer
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twowhencomparedtotheother14lines(compareFigure3.10a,lower),butboth hadalsoshowntheabovedescribedphenotypeandwerethereforechosenaswellfor furtheranalysis. Theprogenyofthesethreelineswasagainselectedforoverexpressionandthechar acteristicsofERL1overexpressionfurtheridentifiedbyWesternanalysis(Figure3.10 b).Line1wasfoundtobethestrongestoverexpressor,followedbyline2and4, whichshowedapproximatelythesameexpressionlevels.Thelatterlinewasstillseg regatingsincetherewasnoexpressionofERL1detectableinplant41.Thetrans genicoverexpressionofERL1resultedintwodistinctproteinbands,amajorbandof approximately42kDaandaminorbandofapproximately35kDawithlessthanhalf oftheexpressionoftheotherband.Thesetwobandsmostlikelycorrespondtoplant ERL1beforeandaftermaturation,consequentlycontainingormissingtheapproxi mately8kDaleaderpeptide.
(A) (B)

49,1 kDa 34,8kDa 28,9 kDa

01020304050607080910111213141516 (C) wt 111221224142M Figure3.10:AnalysisofArabidopsisthalianaplantsoverexpressingERL1 (A)Northernanalysisof16ArabidopsisthalianalinesoverexpressingERL1revealeddetectableERL1 levelsintwelvelines,asaloadingcontrolservedEthidiumbromidestainingof28SrRNA.(B)Lines1, 2and4werepropagatedtotheT2generationandtheirERL1expressionlevelwasinvestigatedwith Westernblotting;asaloadingcontrolservedCoomassiestainingofthegelaftertransfer.Transgenic overexpressionofERL1resultedintwobandsdetectableinalllines,amajorbandofapproximately42 kDaandaminorbandofapproximately35kDa.Thestrongestoverexpressioncouldbedetectedin line1.(C)AsampleplantofArabidopsisthalianaline1overexpressingERL1showsslightbleachingin theleaftips(leftplant,arrows)andadelayedgrowthcomparedtowildtype(rightplant). Caption:0116=T1lines,11&12T2linesof1,21&22T2linesof2,41&42T2linesof4,M=Bio RadPrestainedSDSPAGEstandard

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3.5 EffectofERL1onsilencing
TheCaenorhabditiseleganshomologueofERL1wasfirstdescribedtohaveaneffecton RNAsilencing.NeuronalcellswhichareusuallyRNAirecalcitrantcouldbesub jectedtoRNAsilencingineri1mutants,henceitsnameERI1(enhancedRNAi1) (comparechapter1.2.9oftheintroduction). Inaddition,theeffectofexonucleasesonthedegradationofmiRNAs,anothermole culeofthesilencingpathways,waspublishedrecently(Ramachandran&Chen, 2008),thereforeERL1wasinvestigatedforbothphenomena.

3.5.1 Crosses
TotesttheinvolvementofERL1inRNAsilencingprocesses,ERL1overexpressing NicotianabenthamianaplantswerecrossedwiththeGFPexpressingline6.4thathad beencreatedanddescribedearlierbyourlab(Kalantidisetal.,2006).Line6.4shows frequentspontaneoussilencingofitsGFPtransgenewhichusuallyalsoleadstosys temicGFPsilencingspread.Thereforeitcanbeusedasareporterlinefortestingthe effectofotherfactorsonRNAsilencing.
(A) (B)

wt

Figure3.11:EffectofplantERL1ondifferentmoleculesoftheRNAsilencingapparatus. (A)TwoindividualcrossesofERL1overexpressingplantswiththemetastableGFPexpressingline6.4 revealedthattherewasnoeffectofERL1overexpressiononsiRNAmediatedRNAsilencinginplants. Spontaneoussilencingonset(redspotintheleftpicture)wasnotinfluenced;inaddition,systemic silencingspreadcouldbeobservedleadingtoacompletelysilencedplantinthebackgroundofthe rightpicturewithasinglebranchintheforegroundstillexpressingGFP.(B)Northernanalysisof20 gtotalRNAandhybridizationwithmiRNA159LNAshowedasimilarsignalinallanalyzedplant lines.Theslightlyweakersignalingreentissuewasnotconsideredasspecificsincetherewashigh resistanceinthispartofthegel,probablyleadingtothesmearysignalwhichcanbeobservedabove thedetectedband.EtBrstainingof5.8ScytoplasmicrRNAisdepictedasaloadingcontrol. Caption:wt=wildtype,G=greentissueofSelfsilencerplants,W=whitetissueofBleachplants, A=tissuecollectedaftertransientERL1overexpressionbyagroinfiltration. 119

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Twoindividualcrossesinbothdirections(lines6.4xERL1+andERL1+x6.4)were performedandtheirF1generationwasselectedforexpressionofbothtransgenes.In F2doublehomozygousplantswereselectedandmonitoredfortheirGFPsilencing behavior. Figure3.11ashowstwoexamplesofGFPsilencingidenticaltoline6.4beforecross ingwithERL1overexpression.AllplantsofthesixdoublehomozygousF2plant crossesshowedonsetofGFPsilencing,threeofthemalreadyinearlystages.Outof fiveN.benthamianaplantsofline6.4(withoutinbredERL1)whichweregrownin parallel,allshowedonsetofGFPsilencing,threeofthemalreadyinearlystages.The introductionofERL1intothereporterlinedidnotalteritssilencingcharacteristics, thereforethegenewasconsideredtohavenoeffectonendogenousgenesilencingin plants.Thesameresulthadbeenobservedaftertransientoverexpressioninco infiltrationassays,whereERL1,incontrasttotheidentifiedviralsilencingsuppres sorP19,didnotresultinsuppressionoftransgenesilencing.Interestingly,inanother experimentoverexpressionofERL1wasabletoquenchtheamountsofviroidrelated siRNAs(seesupplementaryresults;Schumacher,2009)

3.5.2 LNA159
TotestforthepossibilityofERL1havinganeffectonmiRNAs,totalRNAofplants overexpressingERL1wasinvestigatedfortheexpressionlevelofmiR159,anabun dantmiRNAfamilyrepressingMYBdomaincontainingtranscriptionfactors(Mette etal.,2002). TheresultsaredepictedinFigure3.11b,theexpressionwascomparableinwhite tissueconstitutivelyoverexpressingERL1andwildtypetissuetransientlyoverex pressingERL1byagroinfiltration.InbothoverexpressingtissuesmiR159expression seemedtobeevenabitstrongerthaninwildtype.InthegreentissuemiR159lev elsappeartobemarginallyreducedcomparedtowildtype,butthelowqualityofthe hybridizationsignalforthissampledoesnotallowdrawingsafeconclusions.

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SummarizeditcanbestatedthatERL1overexpressiondidnothaveasignificant negativeeffectontheexpressionofmiR159,otherplantmiRNAshadnotbeentested sincetherewasnoeffectexpectedeither.

3.6 EffectofERL1onribosomalRNA
InbleachplantsstronglyoverexpressingERL1animpairedribosomalRNApattern couldbedetected.SinceitwasreportedthathomologuesofERI1takepartin5.8S rRNAripeninginCaenorhabditiselegansandMusmusculusthiseffectwasalsoinves tigatedinplants.Twodifferentapproacheswereused:theimmediateeffectoftran sientERL1overexpressionwasdetectedbyNorthernanalysis.Inadditionribosomal RNAoftransgenicplantsmisexpressingERL1wereanalyzedbycloninganalysis.

3.6.1 rRNAblots
InafirstapproachtheexpressionlevelsofsmallplantrRNAswereassessedbycom paringNorthernhybridizationsofNicotianabenthamianawildtypetissuewithtissue overexpressingERL1(greentissueofselfsilencingplantlines,whitetissueofbleach plantsandwildtypetissueaftertransientoverexpressionofERL1).Oneshouldnote thatinthecaseofwhitetissuetheamountofloadedRNAwasreducedbyanempiri callydeterminedarbitraryfactorof0.7,preventinganexcessiveloadingoftheresid ualRNAsduetothealmosttotallackofchloroplasticRNA. Cytoplasmic5.8SrRNAdidnotshowanyeffectaftertransientERL1overexpression (Figure3.12a,right&d).Chloroplastic4.5SrRNAwassignificantlyreducedinwhite tissuebutdidnotshowanyalterationsinexpressionlevelsfortheothertissues whencomparedtowildtype(Figure3.12a,left&b).Chloroplastic5SrRNA,how ever,wasnotonlyalmostlostinwhitetissuebutalsoshoweddecreasedexpression levelsintissuewhereERL1wastransientlyoverexpressedbyagroinfiltration(Figure 3.12a,middle&c).16SrRNAappearedtohavebeenlostafterGFPexpression,while itwasunaffectedafterERL1overexpression(Figure3.12e).Sinceoverexpressionof GFPshouldnothaveaneffectonchloroplastic16SrRNAthisresultwasnotconsid eredasspecific.AstheexpressionafterERL1overexpressionwascomparableonthe

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firstandfifthdayafterinfiltration,aneffectofERL1on16SrRNAisunlikely.23S rRNAseemedslightlyupregulatedafterERL1overexpressionwhichcanprobablybe attributedtounequalloadingofthesesamples(Figure3.12f).

(A)

wt (B)

W 4.5S

wt (C)

G 5S

wt (D)

W 5.8S

D1 D5 D1 D5 ERL1 GFP 4.5S (E)

D1 D5 ERL1 5S

D1

D5 GFP

D1 D5 ERL1 5.8S

D1 D5 GFP

(F)

D1 D5 D1 D5 ERL1 GFP 16S

D1 D5 D1 D5 ERL1 GFP 23S

Figure3.12:NorthernanalysisofchloroplasticribosomalRNAsandcytosolic5.8SrRNAfollowing overexpressionofERL1 IntheupperpanelthehybridizationwiththerespectiverRNAprobeisdepicted,inthelowerpanel 28SmitochondrialRNAisusedasaloadingcontrol. (A)TotalRNAoftissueoverexpressingERL1wasseparatedonaNortherngelandprobedforthe correspondingsmallrRNA.Whitetissueshowednoexpressionofchloroplastic4.5Sand5SrRNA,in additionwildtypetissueaftertransientoverexpressionofERL1haddecreasedlevelsof5SrRNA.(B) (F)AgrobacteriaoverexpressingERL1orGFP(actingasacontrolconstruct)wereinfiltratedintoNico tianatabacumleavesandtissuewascollectedafteroneandfivedays.IntheleftERL1overexpression, intherightGFPoverexpressionisdepicted:(B)Chloroplastic4.5SrRNAwasnotaffectedbythetran sientoverexpressionofanyconstruct.(C)5SrRNAhadadecreasedexpressionlevelonthefifthday aftertransientERL1overexpression.(D)Cytosolic5.8SrRNAshowednoeffectaftertransientoverex pression.(E)Chloroplastic16SrRNAwasnotaffectedaftertransientERL1overexpressionbutcould notbedetectedafterGFPoverexpressionwhichwasprobablyduetoaprobleminNorthernhybridi zation.(F)Chloroplastic23SrRNAshowedupregulationafteroverexpressionofERL1.However, unequalloadingisvisibleintheloadingcontrol,thereforeitisconsideredtobeunaffected. Caption:D1=firstdayafteragroinfiltration,D5=fifthdayafteragroinfiltration,wt=RNAofwild typeNicotianabenthamianatissue,G=RNAofgreentissueofselfsilencingplants,W=whitetissueof selfsilencerandbleachplants,A=overexpressionofERL1byagroinfiltration 122

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3.6.2 Linkerligations
TodeterminealterationsinthesequenceofribosomalRNAs,ithadtobecloned withoutimposingbiasontheclonedends(aswouldbethecasethroughdirectPCR). Forthisreasontwodifferentapproacheswereused:totestforageneraleffectthe totalRNAwasselfligatedandtheresultingproductwasclonedandsequencedto seeanextensionattheends.Tospecificallydeterminethe3locationoftheexten sionslinkerfragmentsweredesignedandsynthesizedusingnonplantsequences(in thiscasethebilbotransposonsequencefromDrosophilamelanogaster).Itwasmodi fiedatits3endwithdideoxyCytosintoprotectthisendfromligation.Thislinker wasthenspecificallyligatedtothe3endsoftotalrRNAextractedfromNicotiana benthamianaandArabidopsisthalianaleaves. 3.6.2.1 Effecton5.8SrRNA AlthoughERL1wasfoundtobetargetedtothechloroplasts,theroleofitsortholo gousgenesinanimalsincytoplasmic5.8SrRNAmetabolism(Gabel&Ruvkun,2008; Anseletal.,2008)promptedustoaddressthisissuealsoinplants. TotestifplantERL1has5.8SrRNAasasubstrateitwasanalyzedbyselfligation andsubsequentcloningwithspecificprimersforthisribosomalRNA.Therewasno effectdetectableinsevensequencesderivedfromplantseithertransientlyorconsti tutivelyoverexpressingERL1(compareFigure3.13).
1 Nb_wt_5.8s-01 Nb_wt_5.8s-02 Nb_wt_5.8s-03 Nb_wt_5.8s-04 Nb_wt_5.8s-05 Nb_wt_5.8s-06 Nb_ERL1-OE_5.8s-01c Nb_ERL1-OE_5.8s-02c Nb_ERL1-OE_5.8s-03c Nb_ERL1-OE_5.8s-04c Nb_ERL1-OE_5.8s-01t Nb_ERL1-OE_5.8s-02t Nb_ERL1-OE_5.8s-03t Consensus (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) (1) 98 CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT CGATGGTTCACGGGATTCTGCAATTCACACCAAGTATCGCATTTCGCTACGTTCTTCATCGATGCGAGAGCCGAGATATCCGTTGCCGAGAGTCGTTT

Figure3.13:Alignmentofcytosolic5.8SrRNAfromNicotianabenthamianaplantsoverexpressing ERL1 5.8SrRNAwasanalyzedbyselfligationoftotalRNAofN.benthamianawildtypeandoverexpressing ERL1plantsandsubsequentcloning.Neitherthesequenceofthemutantwithaseverephenotypenor thesequenceclonedaftertransientoverexpressionshowedanydifferencescomparedtowildtype. Caption:Nb_wt=N.benthamianawildtype,Nb_ERL1OE=overexpressionofERL1inN.benthamiana, c=constitutiveoverexpression,t=transientoverexpression

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Since5.8SrRNAislocatedinthecytoplasmandtheenzymeissupposedtobetar getedtothechloroplast(seechapter3.1)itisconcludedthattheplanthomologueis unlikelytopossessthesamesubstrateasintheanimalkingdom. 3.6.2.2 EffectonchloroplasticrRNAs SinceplantERL1istargetedtothechloroplastitseffectonallfourchloroplastic rRNAswastested.Transientoverexpressionwasusedaswellinordertopreventthe detectionofartifactsduetothedetrimentaleffectsofstrongconsecutiveoverexpres sionofERL1inNicotianabenthamiana.Severalsequencealterationscouldbedetected, predominantlyinchloroplastic5SrRNA.Theobtainedresultsaresummarizedin Table3.4,theeffectofERL1overexpressionwasonlyassessedinNicotianabentha mianaplants,whereastheeffectofERL1suppressioncouldonlybeassessedinArabi dopsisthalianaplants.ThiswasduetothefactthatArabidopsisthalianaplantsoverex pressingERL1andNicotianabenthamianaplantssuppressingERL1werenotavailable atthetimeoftheanalysis. OverexpressionofERL1showedstrongereffectsthantheknockdownofERL1 whichonlyhadconsequenceson16SrRNAprocessing.Twooutofsix16SrRNA sequencesshowedanextraThymidineattheir3endcomparedtotheclonedwild typesequences,thiscorrespondedto33%ofthesequences(Figure3.14c).Interest ingly,overexpressionofERL1didnothaveanyeffecton16SchloroplasticrRNA (compareFigure3.12e).Itshouldbenotedthatthepublished16SrRNAsequence containstwoextraThymidinesatits3end,butcloningof16SrRNAfromNicotiana benthamianaandArabidopsisthalianatotalRNAofwildtypeplantsprovedthatthe correct16SrRNAsequenceinourexperimentalsetupdidnotcontainthesetwo Thymidinesatits3end. Asalreadydescribedinchapter3.6.1thestrongesteffectofERL1overexpression couldbedetectedin5SchloroplasticrRNA(Figure3.14b).5SrRNAsequencescould beroughlydividedintotwogroups:onewithadditionalnucleotidesderivedfrom the5Sprecursorsequencewhichwerepresumablenottrimmedproperlyandone withadditionalnovelnucleotides.TherewasnoeffectinERL1KDArabidopsis
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thalianaplantsdetectable.InNicotianabenthamianaplantsconstitutivelyoverexpress ingERL1fiveoutofeightsequencesshowedanadditionofnovelnucleotidesatthe 3endoftherRNA.
Table3.4:SummaryofsequencealterationsinchloroplasticrRNAafterERL1misexpressionin Nicotianabenthamiana(Nb)andArabidopsisthaliana(At)plants

rRNA TypeofERL1 Species species misexpression constitutive overexpression transientover expression constitutive knockdown constitutive overexpression transientover expression constitutive knockdown constitutive overexpression transientover expression constitutive knockdown constitutive overexpression transientover expression constitutive knockdown Total: Nb Nb At Nb Nb At Nb Nb At Nb Nb At

Numberof Typeof Numberof Ratio analyzed alteration alterations clones 5 4 4 8 12 9 10 nodata 6 4 nodata 6 68 none none none extra A/CC(C) extraCC extraGA none none nodata extraT deletion ofCT nodata none 0 0 0 5 1 3 0 0 nodata 2 1 nodata 0 12 0% 0% 0% 63% 8% 25% 0% 0% no data 33% 25% no data 0% 18%

4.5S

5S

16S

23S

InthreecasesthiswasaclearextraAC,inonescaseitcouldnotbeconcludedfrom thesequencingdatawhetherthefirstoftheadditionalnucleotideswasanAdenosine oraCytosine.Inonecasetherewerethreeextranucleotidesdetected,comprisedof anextraACCmotif.Thesummarizeddescriptionofthissequencealterationisan

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3.Results
extraA/CC(C).Fromtwelvesequencesderivedaftertransientoverexpressionof ERL1fourexhibitedsequencealterations.Oneofthesequencesshowedthesame behaviorastheconstitutivelyoverexpressingplantsbycontaininganextraCCmo tif.TheotherthreealteredsequencespossessedanextraGAwhichcorrespondsto theprecursorsequenceof5SchloroplasticrRNA. In23SchloroplasticrRNAonlyonesequencefromconstitutivelyoverexpressing ERL1planttissueshowedasequencealterationbymissingtwonucleotidesatthe3 endcomparedtothewildtypesequence(Figure3.14d). TherewasnoeffectofplantERL1detectableontheprocessingofchloroplastic4.5S rRNA(Figure3.14a).Intotal68clonedsequenceshadbeenanalyzedandshowed sequencealterationsin18%ofallcases.
Figure3.14:AlignmentofchloroplasticrRNAsofArabidopsisthalianaandNicotianabenthamiana plantsmisexpressingplantERL1 ChloroplasticrRNAsofwildtypeplantsaswellasplantsmisexpressingERL1wereligatedtoamodi fiedlinkersequenceattheir3end.Theyarecomparedtothepublishedsequencesoftherespective rRNAplusthelinkersequence(namedaccordingtothecorrespondingrRNA+linker).Forbettervisu alizationonlythelast100nucleotidesofthesequencesaredepicted. (A)FoursequenceswithdownregulatedERL1andninesequencesoverexpressingERL1wereana lyzedforchloroplastic4.5SrRNAanddidnotshowanysequencealterationscomparedtowildtype. (B)Fivewildtypesequences,ninesequenceswithdownregulatedERL1andtwentysequencesover expressingERL1wereanalyzedforchloroplastic5SrRNA.Twosequencesshowedasingledeletion. Sinceoneofthemisawildtypesequence,thisdeletionisnotconsideredasspecificforERL1misex pression.OftheanalyzedplantswithdownregulatedERL1noneshowedasequencealterationcom paredtowildtype.FromeightplantsconstitutivelyoverexpressingERL1fivesequencesshowedan extraA/CC(C)sequencemotif.FromtwelveplantstransientlyoverexpressingERL1foursequences possessedanextratwonucleotidemotifwhichwaseitherCCorGA.(C)Sixsequenceswithdown regulatedERL1andtensequencesoverexpressingERL1wereanalyzedforchloroplastic16SrRNA.In addition,threewildtypesequenceswereanalyzedandshowedatwonucleotidedeletioncomparedto thepublishedsequence.ComparedtothissequencetwosequenceswithdownregulatedERL1pos sessedanextraThymidineattheir3end.(D)SixsequenceswithdownregulatedERL1andfourse quencesoverexpressingERL1wereanalyzedforchloroplastic23SrRNAandshowedasinglese quencealterationcomparedtowildtype.OneplantconstitutivelyoverexpressingERL1hadatwo nucleotidedeletionatthe3end. Caption:At_wt=Arabidopsisthalianawildtype,Nb_wt=Nicotianabenthamianawildtype,AT_ERL1KD =knockdownofERL1inArabidopsisthaliana,Nb_ERL1OE=overexpressionofERL1inNicotiana benthamiana,c=constitutiveoverexpression,t=transientoverexpression (A)4.5s
14 113 4.5s+linker (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAACATCGTCACAACAAATGGCATC (14) (14) (14) (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC At_ERL1-KD_4.5s-01 At_ERL1-KD_4.5s-02 At_ERL1-KD_4.5s-03 At_ERL1-KD_4.5s-04 Nb_wt_4.5s-01

(1) ------------------------------------------------TAGGCATCCTAACAGACCGGTAGACTTGAAC

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Nb_ERL1-OE_4.5s_01c (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_02c (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_03c (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_04c (1) ---------------------------------------------GCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_05c (14) TTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGATGTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_01t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_02t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_03t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC Nb_ERL1-OE_4.5s_04t (1) -------------------------------------------------AGGCATCCTAACAGACCGGTAGACTTGAAC

(B)5s

19 119 5s+linker (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT---ATCGTCACAACAAATGGCATC (6) (15) (9) (9) (17) (17) (17) (16) (1) ACTTGGTGGGTTAAACTCTATCTGCGGTGACGATGCTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGCGTCGACGCTAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGAAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTKGGTTAAACTCTA-CTGCGGTGACGATACTGTCGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTC-TGCGGAAAAATAGC-TCGACGCCAGGAT--ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT-------------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT---

At_ERL1-KD_5s-01 At_ERL1-KD_5s-02 At_ERL1-KD_5s-03 At_ERL1-KD_5s-04 At_ERL1-KD_5s-05 At_ERL1-KD_5s-06 At_ERL1-KD_5s-07 At_ERL1-KD_5s-08 At_ERL1-KD_5s-09

Nb_wt_5s-01 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGA---Nb_wt_5s-02 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCG-AAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-03 (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-04 (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_wt_5s-05 (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-01c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-02c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATMCNb_ERL1-OE_5s-03c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-04c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACNb_ERL1-OE_5s-05c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGA---Nb_ERL1-OE_5s-06c (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATACC Nb_ERL1-OE_5s-07c (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGAGAGGTCCTGCGGAAAAATAGC-TCTACGCCAGGAT--Nb_ERL1-OE_5s-08c (16) ACTTGGTGG-TTAAACTCTA-CT-CGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-01t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-02t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-03t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-04t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-05t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-06t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCTGGATCCNb_ERL1-OE_5s-07t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TTGACGCCAGGAT--Nb_ERL1-OE_5s-08t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-09t (1) -----------TAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGATGANb_ERL1-OE_5s-10t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-11t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGC-TCGACGCCAGGAT--Nb_ERL1-OE_5s-12t (16) ACTTGGTGG-TTAAACTCTA-CTGCGGTGACGAGACTGTAGGGGAGGTCCCGCGGAAAAATAGC-TCGACGCCAGGAT---

(C)16s

182 281 16s+linker (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTTATCGTCACAACAAATGGCATC (174) (182) (161) (182) (182) (162) (182) (182) (182) (182) (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTGCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCTTGCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGNGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGTCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGGAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGCAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT

At_ERL1-KD_16s-01 At_ERL1-KD_16s-02 At_ERL1-KD_16s-03 At_ERL1-KD_16s-04 At_ERL1-KD_16s-05 At_ERL1-KD_16s-06 At_ERL1-OE_16s-01 At_ERL1-OE_16s-02 At_ERL1-OE_16s-03 At_ERL1-OE_16s-04 At_ERL1-OE_16s-05

At_wt_16s-01 (166) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-Nb_wt_16s-01 (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-Nb_wt_16s-02 (95) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT Nb_ERL1-OE_16s_01c Nb_ERL1-OE_16s_02c Nb_ERL1-OE_16s_03c Nb_ERL1-OE_16s_04c Nb_ERL1-OE_16s_05c (182) (182) (161) (125) (182) GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGAAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGGAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT-GCCGAAGGCAGGGCTAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCCT--

(D)23s

198 297 23s+linker (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCTATCGTCACAACAAATGGCATC (198) (198) (198) (198) (198) (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGGATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT

At_ERL1-KD_23s-01 At_ERL1-KD_23s-02 At_ERL1-KD_23s-03 At_ERL1-KD_23s-04 At_ERL1-KD_23s-05 At_ERL1-KD_23s-06

Nb_wt_23s-01 (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT Nb_ERL1-OE_23s_01c Nb_ERL1-OE_23s_02c Nb_ERL1-OE_23s_03c Nb_ERL1-OE_23s_04c (198) (198) (198) (198) CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGACGAGTGCTCTC-CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT CGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCACCCCAAGATGAGTGCTCTCCT

Caption: identical, conserved, linker

127

128

4. Discussion
4.1 ImplicationsofplantERL1inRNAsilencingprocesses
InthepastyearsRNAsilencingmechanismshaveproventobeinvolvedintheregu lationofnumerouscellularpathways.Thisubiquitousinvolvementcertainlyraises thequestionabouttheendogenouscontrolofRNAsilencing.Sofaronlyviralsup pressorsofsilencinghavebeenidentifiedtospecificallycounteractthesilencing mechanismoftheirhostsinordertocircumventitsfunctionasantiviralimmunesys tem.Theyfollowvariousstrategies,onebeingthespecificsequesteringofsiRNAs. Althoughtherehavebeenseveralreportsofendogenoussuppressorsofsilencing (comparechapter1.2.8oftheintroduction),theobservedeffectsupontheirlossof functionaremainlysecondaryandnottheresultofaspecificlossofthesuppression ofRNAsilencingsuppression. AnexceptionpresentstheproteinERI1fromC.elegansanditshomologueswhich havebeenshowntospecificallydegradesiRNAsinvitro(Kennedyetal.,2004;Iidaet al.,2006;Kupscoetal.,2006;Yangetal.,2006)andthereforesuppressingtheextentof silencingindiversetissuesinvivo.Theproteinsarecharacterizedbyanexonuclease domainwithafunctionalDEDDhmotif,whichhasbeentermedERI1_3hExo_like EXOIIIdomain. SofarnoplanthomologueofER11hasbeendescribedintheliterature.Asinglelo cuscouldbeidentifiedinArabidopsiswhichsharessignificanthomologytotheal readydescribedERI1likeproteinsandcontainstheERI1_3hExo_likeEXOIIIdo main.ThereforeasimilarfunctioninplantRNAsilencingprocesseshasbeenimpli cated.Thishypothesiswasinitiallysupportedbyseveralhints(Schumacher,2009; supplementaryresults6.2.1,6.2.3&6.2.4).Oneofthefirstobservationshadbeenthe significantreductionofviroidderivedsiRNAsinPSTVdinfectedplantsafterover expressionofERL1.ThisimplicatedaspecificsequesteringofsiRNAsbyERL1. However,onecannotexcludethepossibilitythatthestrongoverexpressionlevel whichprevailsafteragroinfiltrationwouldhavethesameeffectwithanyotherex
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onuclease.AnotherfindinghadbeenthefactthatN.benthamianaplantsoverexpress ingERL1showedahypersensitivitytoinfectionbyPlumpoxviruswhichusuallyhas onlymildsymptomsinplanta.Takingintoaccounttheweakconstitutionofthese vereERL1overexpressingplantsthishypersensitivitymightnotspecificallybeare sultofERL1overexpressionbutratherasecondaryeffect.Thestrongestresultimpli catinganeffectofplantERL1onRNAsilencingprocessesisthefactthatthesystemic silencingoftheERL1overexpressionbyintroducinganERL1hairpinbyagroinfiltra tionisonlysuccessfulin50%oftheplants.Therestfailstomaintainsystemicsilenc ingandrevertsbacktothebleachphenotypetypicalofERL1overexpression (Schumacher,2009). Despitethesefindingsseveralotherresultscontraindicatedanimplicationofplant ERL1inRNAsilencing:agroinfiltrationassayshaveearlierbeendescribedasa methodforthedescriptionofRNAsilencingsuppressoractivity(Brignetietal.,1998). Twodistinctmechanismsareexploited,theadditionaloverexpressionofaGFP transgeneintoaGFPexpressingplantlineleadstocosuppressionbyusingthein trinsicRNAsilencingpathways.TheuseofaGFPsuppressinghairpincircumvents thenecessityofRNAsilencingamplificationbyRDRdependentmechanisms.Inthe caseofERL1noeffectcouldbedetectedafteritstransientoverexpressionbyco infiltrationwiththerespectiveGFPconstruct(Schumacher,2009;supplementaryre sults6.2.2).Onehastonote,however,thattheseassaysareratherinsensitiveand havealowtempospatialresolution.Thereforeanotherapproachhasbeenusedby crossingERL1overexpressingN.benthamianaplantswithlineGFP6.4whichisde scribedbyinitiationofspontaneousGFPsilencingandsubsequentsystemicGFPsi lencingspread(Kalantidisetal.,2006).TheonsetofthisGFPsilencingisprobably characterizedbyovercomingathresholdofaberranttransgeneRNA,thereforepro vidingamoresensitivesystemtoinvestigateanymildsilencingsuppressoreffects. Theresultingcrosses,however,didnotshowanydifferencesinGFPsilencing,nei thertheinitialonset,noritsspreadincludingthesystemicspreadofsilencingare negativelyinfluencedbytheoverexpressionofplantERL1(comparechapter3.5.1).
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4.Discussion
TheonlyresultpointingtowardsaninvolvementofERL1inRNAsilencinginplants, isthedistortedsystemicsilencinginERL1overexpressingplants.Itcanpossiblybe accountedtothefactthatthelevelsofDCL1andDCL4transcriptsare,incontrast toothernuclearmRNAs,severelydownregulatedinplantswhichoverexpressERL1 (Schumacher,2009).Uptonowthereisnoclearexplanationwhetherthisisaspecific resultofERL1overexpressionorasecondaryeffectofthepoorconstitutionofthe plants. Inconclusion,itcanbestatedthattheproteinlikelydoesnothaveaneffectonsup pressionofRNAsilencinginplantsbutratheradistinctfunctioncomparedtoERI1 inotherspecies.Interestingly,fromthealreadypublishedERI1homologuesalso SnipperinDrosophiladidnotshowanenhancedRNAiphenotypeuponitslossof function,thereforeitprobablyalsopossessesafunctionapartfromRNAsilencing (Kupscoetal.,2006).BasedonphylogeneticanalysistwodistinctsubclassesofERI1 homologueshavebeenidentifiedinastudyforRNAirelatedproteinsinTribolium castaneum(Tomoyasuetal.,2008).BothgroupscontaintheERI1_3hExo_likeEXOIII domainwithahighdegreeofconservation.TheSAPdomain,whichconfersbinding tonucleicacids,isonlypresentinasubsetofERI1homologues.InthisgroupIall memberswithdescribedeffectsofERI1onRNAsilencingareincluded,e.g.homo logueswhicharefoundinC.elegans,humansandalsoayettobeanalyzedproteinof seaurchin(Strongylocentrotuspurpuratus).Thesecondgroup(groupII)ischaracter izedbytheDrosophilaSnipperprotein;otheryettobeanalyzedhomologuesorigi natefromTribolium,seaurchinandhumans(Tomoyasuetal.,2008).Snipperhasnot proventobeinvolvedinRNAsilencingprocessesinvivo.Interestingly,theArabidop sishomologuewouldalsoassociateswiththegroupIIproteins,maybeexplaining thefactthatitisnotinvolvedinplantRNAsilencingprocesses.

4.2 InvolvementsofERL1inchloroplastmetabolism
DuetothelowexpressionofplantERL1aminorregulatoryimpactinvivoisimagin able.Ingeneral,acompletelossofageneishardtoaccomplisheitherwithinsertion mutantsorwithhairpinconstructs,sincesometranscriptsmayalwaysescapethe
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downregulation.Inthecaseoflowgenetranscriptionadetectableeffectofthis knockdownishardlyobvious(comparechapter3.4.1oftheresultsection).Therefore ourapproachusedtheoverexpressionofthegeneinadditiontotheincomplete knockout,inordertoidentifypossibletargetsoftheprotein.Strongeffectsfollowing overexpressionofERI1homologueshavebeenreportedearlier(Bhleretal.,2005; Almeidaetal.,2006).Secondaryeffectsofthisoverexpressioncannotbeexcludedof course,butfirstconclusionstotheinfluencedpathwayscanbedrawn. Afterconstitutiveoverexpressiontheresultingtransgenicplantsshowedadistortion inthechlorophylllevels.Subsequentinsilicoanalysisoftheproteinrevealedapossi blechloroplasttargetingsignalatitsaminoterminus.Thishasbeenprovenbypre paringacarboxyterminalfusiontoGFPfollowedbyconfocalmicroscopy.Interest inglythechloroplastisconsideredasanintracellularcompartmentwhichisfreeof RNAsilencingprocesses(Hegemanetal.,2005).Thereforethischloroplasticlocaliza tionisconsideredasanadditionalproofforERL1havingafunctionapartfromRNA silencinginplants. Eversincetheendosymbioticincorporationofphotosyntheticallyactiveprokaryotes, duringevolutionchloroplastshavetransferredmostoftheirgenestothenucleus.In addition,chloroplastscontainmanyeukaryoticproteinsindependentofthechloro plastprokaryoticorigin.Thecorrespondingproteinsareimportedintothechloro plastsbyseveralimportmechanismsandtakepartintheirregulatorypathways. Nevertheless,chloroplastsstillcontainafullyfunctionalgeneexpressionmachinery whichismostlyencodedinthechloroplastitself.Inaddition,theycodeforphoto synthesisrelatedproteins.Thegeneexpressionretainedmanyprokaryoticcharacter isticsleadingtopolycistronictranscriptswhicharemainlyregulatedpost transcriptionally.Thematurationofchloroplastictranscriptsincludesseveralendo andexonucleolyticcleavagesteps(comparechapter1.3.2oftheintroduction).Dueto itschloroplasticlocalizationanditsexonucleaseactivityitislikelythatERL1isa playerinthisposttranscriptionaleditingofchloroplasticRNAs.

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FrompublishedESTcollectionsprobablyallplantERL1sequencesexceptS.bicolor andP.trichocarpacontainachloroplasttargetingsignal.Althoughthismayrepresent afunctionaldiversificationofplantERL1proteins,incompleteannotationcannotbe excluded.Inthecaseofsorghumthisisalreadyobviousfromthefactthatanamino terminalmethionineismissingfromthesequence.InthecaseofP.trichocarpathisis notthecasebutsincethecompletegenomicsequenceofpoplarisavailable,theup streamregionoftheannotatedgenewasanalyzedandapossiblechloroplaststarget ingsignalimmediatelyupstreamoftheannotatedERL1sequencescouldbeidenti fied.Duetothefactthatthechloroplasttargetingsignalsshownoconservationbe tweenthedifferentplantspecies(compareFigure1.13oftheintroduction)theywere probablyaddedindependentlyaftersplittingintothedifferentplantlineages.There foreitcannotbeexcludedthatERL1proteinsfromsorghumandpoplarservedis tinctfunctions.However,itisinterestingthatbothmajorplantlineagesofmonocoty ledonsanddicotyledonscontainmemberswhichpossessapredictedchloroplastic localizationofERL1,thereforemakingpurecoincidenceunlikely.

4.3 SeverephenotypicalterationsafteroverexpressionofERL1suggest aninvolvementinchloroplastdevelopment

TheoverexpressionofERL1resultedinvariousphenotypeswithaslowandstunted growth.Inaddition,allphenotypeswerecharacterizedbythepartialorfullbleach ingoftheplants.Thisvariegationhasbeendescribedbeforeasaresultofmutations inphotosynthesisrelatedandotherplastidgenes.Theresultingleavescontainnor malgreensectorsandwhitesectorswithnoncanonicalchloroplastsresemblingun differentiatedproplastids.Sincechloroplastsarenotsynthesizedfromscratchafter plantcelldivisionbutratherdivide,cellsusuallycontaineithercanonicalordis tortedplastidsinthevariegatedphenotypes.Thevariegationpatternhadbeen showntobeinfluencedbyenvironmentalfactorssuchaslightandtemperature(re viewedinSakamoto,2003),thisbehaviorcouldalsobeseeninourexperimentsal thoughduetothegreenhouseconditionsitcannotbestatedifintenselightorhigher temperatureorbothcausedthemoreintensebleachinginthesummermonths.
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TheanalysisofERL1transcriptlevelsinthemutantplantlinesrevealedthatthese verityofthebleachingclearlycorrespondstotheamountofERL1presentinthe plants.Thecommonlyobservedphenomenonofphenotypereversionbacktowild typephenotypewasaccompaniedbythedegradationoftheERL1mRNA(compare Figure3.4&Figure3.7oftheresults),comparabletosystemicsilencingofatrans gene(comparechapter1.2.6oftheintroduction).However,itisnotclearwhetherthe endogenousERL1ofN.benthamianaissilencedaswellinthisphenotype,sincethe Arabidopsissequencehadbeenusedforthepreparationoftheoverexpressingplants. Thetwosequencesdonotshareanidenticalstretchof21ntwhichwouldresultin siRNAsabletosilencetheendogenoussequencetoo.Interestingly,greentissueap pearstohaveanenhancedphotosyntheticactivity(comparechapter3.3.2.2and 3.3.2.3oftheresultssection),eithertocompensatefortheresidualplantwhichisstill characterizedbywhiteleavesor,inthecaseifendogenousERL1issilencedaswell, thehyperactivephotosyntheticapparatusmightbearesultoflossofERL1.Thepos siblereversionofthephenotypeisalsointeresting,sinceitsuggeststhatchloroplast developmentisonlyblockedreversiblyinwhitetissueandnotlostinthesecells. Inadditiontothemorphologicalphenotypethetranscriptionofchloroplasticgenes isdistortedinthemutantplantlines.Twoindividualpolymerasesareresponsiblefor chloroplasticgenetranscription:thenucleusencodedplastidRNApolymerase (NEP)isimportedintothechloroplastsand,amongstothers,responsiblefortran scriptionofthesecondpolymerase,theplastidencodedRNApolymerase(PEP).The transcriptionbytheformerisindependentofchloroplasticmoleculeswhereasthe translationofthegeneproductsdependsontheribosomesofthechloroplasts.There forethereisanaccumulationofPEPtranscriptdetectable,accompaniedbyalackof ribosomalRNAswhicharethemselvesaproductofPEPtranscription.Inaddition, immaturerRNAsareconsideredtobenonfunctionalforribosomeassembly.Inter estingly,distortedchloroplasticrRNAshavepreviouslybeenreportedtocausevarie gatedphenotypes(Barkan,1993;Bisanzetal.,2003;Bellaoui&Gruissem,2004;Bol lenbachetal.,2005;Rodioetal.,2007).IthastobenotedthatPEPdependenttran
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scriptionisnotcompletelylostintheoverexpressingtissue.Thisfactmaybeaccounts fortheobservationthatwhitetissueisabletorecoverwhentheERL1expressionis silencedbytheplant. Literaturesearchrevealedseveralexamplesofmutantswiththeobservedmorpho logicalphenotypes[(comparefigureFigure3.5&Figure3.6oftheresultsection) (Reiteretal.,1994;Chatterjeeetal.,1996;Babiychuketal.,1997;Wangetal.,2000)],as wellasthetranscriptionalalterationswhichcouldbedetectedafterconstitutive overexpressionofERL1inN.benthamiana[(compareFigure3.7oftheresultsection) (Hessetal.,1993;Allisonetal.,1996;DeSantisMacIosseketal.,1999;Zubko&Day, 2002)].Anotherexampleoftheobservedphenotypeistheinfectionofpeachwitha variantofthepeachlatentmosaicviroid(PLMVd).ItbelongstothegroupofAvsun viroidaewhichreplicateinthechloroplast.ThePCC40strainleadstopeachcalico,a severechlorosisoftheleaflamina.Thesesymptomsareassociatedwithastablesec ondarystructurewhichiscomprisedofa3stemloop.Intriguingly,adisturbancein chloroplasticrRNAgenerationhasbeenattributedtobethecauseofthephenotype (Rodioetal.,2007). Onelinepresentedanexceptiontothedirectconnectionbetweentheseverityofthe phenotypeandtheERL1overexpressionlevels.Inthebeginningitdidnotshowany phenotypealbeithavingahighexpressionlevelofERL1,thiswasexplainedbyposi tionaleffects.Anotherscenariocouldbeamutationinthechloroplastleadersignal preventinganimportintotheplastids.However,insubsequentgenerationstheline showedanincreasingamountofbleachingreminiscentoftheotherlineswithhigh transgeneexpression.

4.4 ERL1isinvolvedinchloroplasticribosomalRNAprocessing
InadditiontotheirinvolvementinRNAsilencingprocessesERI1homologueshave latershowntobeinvolvedinthe3processingofcytosolic5.8SrRNA.ERI1knock outmutantsofC.elegans,S.pombeandM.musculusfrequentlyshoweda2ntex tendedversionoftheribosomalRNA(Anseletal.,2008;Gabel&Ruvkun,2008).Itis believedthattheSAPassistsintheinteraction,butwhenamutantversionofERI1
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withoutSAPdomainhasbeenoverexpressedinmicetheenzymewasstillableto interactwithribosomalproteins(Anseletal.,2008).SincethisfunctionofERI1seems tobeconserved,aninvolvementofERL1insimilarplantpathwayshasbeenconsid eredaslikely. Despiteapparentdifferencesthetwoidentifiedinvivosubstratesshareamajorsimi larity:5.8SrRNAinvivobindsto28SrRNAleadingtoadoublestrandedstemfol lowedbya2ntoverhang.ThisstructureresemblesthestructureofsiRNAs.Another identifiedsubstrate,albeitnotbeingverifiedinvivo,istheshort3terminalstem loopofhistonemRNAwhichisprocessedbyhuman3hExo(Dominskietal.,2003, Dominskietal.,2005).DrosophilaSnphasalsobeenshowntoprocesssubstrateswith 3overhangsofminimumtwonucleotides(Kupscoetal.,2006). TakentogetherwiththeresultthatplantERL1localizestothechloroplasts,chloro plasticribosomalRNAshavebeenconsideredasalikelysubstratefortheprocessing activityofERL1.Insilicopredictionoftheirsecondarystructuresrevealsthatonly5S rRNAhasaprecursorstructurecomparabletoothersubstratesofERI1homologues. TheotherchloroplasticrRNAseitherpossessextendedoverhangsordonotfoldinto a3terminalstematall(compareFigure4.1). 5.8SrRNA,incontrasttootherspecies,probablydoesnotprovideasuitablesub strateforplantERL1;itislocalizedinthecytoplasmandinadditionitshowsa slightlydifferentfoldingcomparedtoitsanimalcounterpartwithanextended3 overhanginplants. FrominitialexperimentswithprobingfortherRNAlevelsaftertransientoverex pressionofERL1byagroinfiltration,only5SrRNAshowedadetectabledownregula tionalreadyaftersomedaysofERL1overexpression.Unfortunatelytherearenore sultsavailableforthetransientsuppressionofERL1.Amoredetailedpicturecould beobtainedaftermappingtheexactendsoftherRNAsbyacloningapproach(com pareTable3.4oftheresultsection).Indeed,alterationsatthe3endscouldbede tectedin18%ofallanalyzedsamples.Asexpectedfromtheinitialtransientassays thestrongesteffectcouldbeseenin5SrRNA;interestinglyonlyafterERL1overex
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(A) 3 (B) 3

(C)

(D)

Figure4.1:SecondarystructuresofchloroplasticrRNA(predictedbyRNAfold;Gruberetal.,2008) (A)4.5SrRNAdoesnotpossessa3terminalstem.(B)5SrRNAendswitha3terminalstemleaving anyadditionalprecursornucleotidesavailableforERL1processing.(C)16SrRNAhasanextended singlestranded3end.Forvisualizationonlythelast161ntaredepicted,afulllength16Sstructure predictiongivesasimilarresult.(D)23SrRNApossessesonlyatwonucleotidedoublestrandbefore the3endprobablybeingtooshortforapresumablesubstrateofERL1.Againonlythelast150ntare depicted.

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pressionbutnotuponitssuppressionintransgenicA.thalianaplants.In45%ofall casesanadditionoftwonucleotideshadbeenpresent.Surprisingly,theseextensions belongedtotwodistinctgroups:inonethirdofthealteredsequencestheextension correspondedtotheprecursorof5SrRNA;thistypeofextensioncouldonlybede tectedaftertransientoverexpression.However,intwothirdsofallextended5S rRNAversionstheextensioncorrespondedtoanuntemplatedextraA/CC(C)mo tif,withthefirstbasebeingcomprisedofeitheradenosineorcytosine,thesecond basebeingalwayscytosine;inrarecasesathirdcytosinecouldoccur.Intriguingly, untemplatedadditionofasimilarmotifhasbeenreportedfortobaccochloroplast transcripts(ZanduetaCriado&Bock,2004)andmitochondrialtranscriptsinmaize (Williamsetal.,2000). Sofar,thereisnoexplanationwhyoverexpressionofERL1wouldresultinaddi tionalnucleotidesof5SrRNA.ItcanonlybespeculatedhowadditionalERL1might interferewith5SrRNAprocessing.Thescenariowithtransientoverexpressionof ERL1representstheearlystepsofthepathway,allalteredsequencescorresponded totemplatedextensionscomingfromtheprecursorsequence.Thismaymeanthat misexpressionofERL1firstinfluencesthecanonicalprocessingof5SrRNA.After constitutiveoverexpressiononlythenontemplatedextensioncouldbedetected.It canbehypothesizedthattheadditionalnucleotidesareaddedafterthenormalproc essingofthe5SrRNAprecursorhasbeendisturbed. Inaddition,eveninthemutantbackgroundmorethanhalfofallsequencescorre spondtothecorrectwildtypeversionsoftheribosomalRNA.Thissuggeststhat ERL1probablyonlyactsredundantlytootherproteinsin5SrRNAmaturation.A similareffecthadbeenobservedearlierinlossoffunctionmutantsofthe RIBONUCLEOTIDEREDUCTASE1(RNR1)inArabidopsis.Thernr1mutantshowsa strongphenotypewithextensionsin4.5Sand5SchloroplasticrRNAparalleltowild typeversionsofthesemolecules.Theauthorsbythenproposedanunknown35 exonucleasetoactredundantlyinthe3endmaturationoftheserRNAs(Bollenbach etal.,2005).
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Recently,Sharwoodetal.haveproposedadsRNAspecificribonucleasetodestabilize 5SrRNAinthernr1mutantbackground(Sharwoodetal.,2011);itisimaginablethat ERL1mightexertthisfunction. However,5SrRNAwasnottheonlychloroplasticrRNAmoleculeshowingsequence alterationsattheir3ends.16SrRNAwastheonlymoleculewithsequencealtera tionsintheArabidopsiserl1suppressionbackground.Intwooutofsixofthetested sequencestheypossessedanextrathymidinecomparedtothewildtypesequences.It hastobenotedatthispointthatthesequencedwildtypeclonesinourexperimental setupshowedatwonucleotidedeletionattheir3endscomparedtothepublished sequence.Sincethiscouldbedetectedinallwildtypeandmostmutantsequences thisshortenedversionhadbeenconsideredasthewildtypesetup.TheextraTcor respondstothe16SrRNAprecursorsequence.Thefoldofthe16SrRNAprecursor doesnotsuggestittobeasuitablesubstrateforERL1andasideeffectoftheinser tionmutantcannotbeexcluded.IntheArabidopsisgenometheERL1geneliesimme diatelyupstreamofasequencecodingforaPPRproteinwhosepromoterispartof theERL1sequence.Itcanthereforenotbeexcludedthatamutationcouldinfluence bothtranscripts.PPRproteinsareimplicatedineditingofchloroplastictranscripts (comparechapter1.3.2oftheintroduction).IfthelevelofthePPRtranscriptisinflu encedinthemutantline,itisimpossibletoarguethattheelongated16SrRNAis specificforthesuppressionofERL1.Nevertheless,thegenomicrelationoftheERL1 andPPRgeneareaninterestingaspect,sincetherelativedistanceofgenesmightalso beanindicatorforafunctioninsimilarprocesses.ERL1missesamotifproviding bindingtoRNA,thisfunctioncouldberescuedbythePPRprotein. Inthecaseof23SrRNAa3deletioncouldbedetectedinonecase.Althoughthis behaviorwouldbeinaccordancewiththeexpectedfunctionofERL1byshowinga deletioninthecaseofoverexpressionoftheexonuclease,itishardtointerpretatthis timepointsince,sofar,ithadnotbeenobservedrepeatedly.Thesecondarystructure predictsoneofthedeletednucleotidestobeboundinadoublestrandedstem,there foremakingitanunlikelysubstrateofERL1.However,itcannotbeexcludedthatin
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4.Discussion
thematureribosomeadditionaldoublestrandedRNAstemscouldformbetween differentRNAspecies. Thefactthat4.5SrRNAhasnotbeenchangedinanyofthetestedsequencessuggests thattheobservedsequencealterationsintheotherchloroplasticrRNAs,especially5S rRNAarenotpurelyrandombutaspecificresultofERL1misexpression.

4.5 Conclusions
ManyhintspointtowardsacontributionofplantERL1intheprocessingofchloro plastic5SrRNA.Theproteinislikelytolocalizetothechloroplastinvivo.Therehave beennoresultsobtained,whichspecificallysupportaninvolvementofplantERL1in RNAsilencingprocesses.ReportsfromotherspeciessuggestaninvolvementofERI 1homologuesinrRNAprocessingand5SrRNAisclearlyinfluencedbythemisex pressionofERL1inplants.However,amajordisadvantageistheunavailabilityof dataofcompleteknockoutsofthegene.TheanalyzedinsertionmutantsofArabidop sisthalianastillpossessadetectableresidualERL1transcriptlevel.Inaddition,they donotshowaclearphenotype,thespecificelongationofchloroplastic16SrRNAstill needstobeprovenbyanalysisofthePPRtranscriptlevels.Itwillbeimportantto createandanalyzeplantlineswithminimalresidualERL1expression. Theproposedsubstrateshouldalsobeprovenbyinvitroassays.Sofarallattempts withplantERL1havebeenunsuccessful.Severalpossiblereasonsforthiscanbead dressed:onemaybethatthefulllengthproteinhasbeenusedforproteinproduction andpurification.Thissequencepossessesachloroplasticleadersignalwhichwillbe cleavedinvivoaftertheimport.Theleadersignalmightinterferewithsubstratebind ingandprocessingintheinvitroexperiments,amatureproteinwithouttheleader peptideshouldthereforebeexpressed.Preliminaryresultswiththematureprotein inshiftingassaysshowbindingtodsRNAwith2ntoverhangs(Vlatakis,unpub lishedresults).Inaddition,ERL1mightrequireotherfactorsforitsfunction.Itis verylikelythatitworksincooperationwithaproteinprovidingbindingtonucleic acids,sinceitlacksaSAPmotif.Theseproteinsmightbeindispensableforitsfunc tionalsoinvitroandneedtobeidentifiedbyimmunocoprecipitation.Homologues
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4.Discussion
ofERI1havebeenshowntointeractwithDicerinC.elegans(Duchaineetal.,2005) andwithribosomalproteinsinmice(Anseletal.,2008). Alastquestiontoaddressis,whetherplantERL1wouldhavetheabilitytodegrade siRNAsifitdidnotlocalizetothechloroplast?Thiscouldbetestedintheabove mentionedinvitroassayswiththerespectivecofactors.Anotherapproachhadbeen thegenerationoftransgenicplantsoverexpressingthematureproteinwithoutthe leaderpeptide.However,itwasnotpossibletoregeneratetheselines(Vamvaka, 2010).Itwillbeinterestingtoaddressifthisisrepeatableandthereforemaybeatoxic effectoftheoverexpressionofERL1inthecytoplasm.

141

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5. References
AdenotX,ElmayanT,LauresserguesD,BoutetS,BouchN,GasciolliV, VaucheretH.DRB4dependentTAS3transactingsiRNAscontrolleafmorphology throughAGO7.CurrBiol.2006May9;16(9):92732. AlbertsB,JohnsonA,LewisJ,RaffM,RobertsK,Walter,P.Molecularbiologyof thecell.Fifthedition,2008,GarlandScience,Taylor&FrancisGroup.ISBN:08153 41059. AllenE,XieZ,GustafsonAM,CarringtonJC.microRNAdirectedphasingduring transactingsiRNAbiogenesisinplants.Cell.2005Apr22;121(2):20721. AllenE,HowellMD.miRNAsinthebiogenesisoftransactingsiRNAsinhigher plants.SeminCellDevBiol.2010Oct;21(8):798804.Epub2010Mar30. AllisonLA,SimonLD,MaligaP.DeletionofrpoBrevealsaseconddistincttran scriptionsysteminplastidsofhigherplants.EMBOJ.1996Jun3;15(11):28029. AlmeidaR,BuscainoA,AllshireRC.Molecularbiology:silencingunlimited.Curr Biol.2006Aug22;16(16):R6358. AlonsoJM,StepanovaAN,LeisseTJ,KimCJ,ChenH,ShinnP,StevensonDK, ZimmermanJ,BarajasP,CheukR,GadrinabC,HellerC,JeskeA,KoesemaE, MeyersCC,ParkerH,PrednisL,AnsariY,ChoyN,DeenH,GeraltM,HazariN, HomE,KarnesM,MulhollandC,NdubakuR,SchmidtI,GuzmanP,Aguilar HenoninL,SchmidM,WeigelD,CarterDE,MarchandT,RisseeuwE,BrogdenD, ZekoA,CrosbyWL,BerryCC,EckerJR.Genomewideinsertionalmutagenesisof Arabidopsisthaliana.Science.2003Aug1;301(5633):6537. AltschulSF,MaddenTL,SchfferAA,ZhangJ,ZhangZ,MillerW,LipmanDJ. GappedBLASTandPSIBLAST:anewgenerationofproteindatabasesearchpro grams.NucleicAcidsRes.1997Sep1;25(17):3389402. AmbrosV,BartelB,BartelDP,BurgeCB,CarringtonJC,ChenX,DreyfussG, EddySR,GriffithsJonesS,MarshallM,MatzkeM,RuvkunG,TuschlT.Auni formsystemformicroRNAannotation.RNA.2003Mar;9(3):2779. AnandalakshmiR,MaratheR,GeX,HerrJMJr,MauC,MalloryA,PrussG, BowmanL,VanceVB.Acalmodulinrelatedproteinthatsuppressesposttranscrip tionalgenesilencinginplants.Science.2000Oct6;290(5489):1424. AnselKM,PastorWA,RathN,LapanAD,GlasmacherE,WolfC,SmithLC,Papa dopoulouN,LampertiED,TahilianiM,EllwartJW,ShiY,KremmerE,RaoA,He issmeyerV.MouseEri1interactswiththeribosomeandcatalyzes5.8SrRNAproc essing.NatStructMolBiol.2008May;15(5):52330.Epub2008Apr27.

143

5.References
AravinAA,NaumovaNM,TulinAV,VaginVV,RozovskyYM,GvozdevVA. DoublestrandedRNAmediatedsilencingofgenomictandemrepeatsandtranspos ableelementsintheD.melanogastergermline.CurrBiol.2001Jul10;11(13):101727. AravinAA,KlenovMS,VaginVV,BantigniesF,CavalliG,GvozdevVA.Dissec tionofanaturalRNAsilencingprocessintheDrosophilamelanogastergermline.Mol CellBiol.2004Aug;24(15):674250. AravinAA,SachidanandamR,GirardA,FejesTothK,HannonGJ.Developmen tallyregulatedpiRNAclustersimplicateMILIintransposoncontrol.Science.2007 May4;316(5825):7447.Epub2007Apr19. AusinI,MocklerTC,ChoryJ,JacobsenSE.IDN1andIDN2arerequiredforde novoDNAmethylationinArabidopsisthaliana.NatStructMolBiol.2009 Dec;16(12):13257.Epub2009Nov15. AxtellMJ,BowmanJL.EvolutionofplantmicroRNAsandtheirtargets.Trends PlantSci.2008Jul;13(7):3439.Epub2008May22. BabiychukE,FuangthongM,VanMontaguM,InzD,KushnirS.Efficientgene tagginginArabidopsisthalianausingagenetrapapproach.ProcNatlAcadSciUSA. 1997Nov11;94(23):127227. BannaiH,TamadaY,MaruyamaO,NakaiK,MiyanoS.Extensivefeaturedetection ofNterminalproteinsortingsignals.Bioinformatics.2002Feb;18(2):298305. BarkanA.NuclearMutantsofMaizewithDefectsinChloroplastPolysomeAssem blyHaveAlteredChloroplastRNAMetabolism.PlantCell.1993Apr;5(4):389402. BatistaPJ,RubyJG,ClaycombJM,ChiangR,FahlgrenN,KasschauKD,Chaves DA,GuW,VasaleJJ,DuanS,ConteDJr,LuoS,SchrothGP,CarringtonJC,Bartel DP,MelloCC.PRG1and21URNAsinteracttoformthepiRNAcomplexrequired forfertilityinC.elegans.MolCell.2008Jul11;31(1):6778.Epub2008Jun19. BaulcombeD.RNAsilencinginplants.Nature.2004Sep16;431(7006):35663. BaumbergerN,BaulcombeDC.ArabidopsisARGONAUTE1isanRNASlicerthat selectivelyrecruitsmicroRNAsandshortinterferingRNAs.ProcNatlAcadSciUS A.2005Aug16;102(33):1192833.Epub2005Aug4. BaumbergerN,TsaiCH,LieM,HaveckerE,BaulcombeDC.ThePolerovirussi lencingsuppressorP0targetsARGONAUTEproteinsfordegradation.CurrBiol. 2007Sep18;17(18):160914. BayneEH,RakitinaDV,MorozovSY,BaulcombeDC.Celltocellmovementof potatopotexvirusXisdependentonsuppressionofRNAsilencing.PlantJ.2005 Nov;44(3):47182. BehmAnsmantI,RehwinkelJ,DoerksT,StarkA,BorkP,IzaurraldeE.mRNA degradationbymiRNAsandGW182requiresbothCCR4:NOTdeadenylaseand DCP1:DCP2decappingcomplexes.GenesDev.2006Jul15;20(14):188598.Epub2006 Jun30.
144

5.References
BellaouiM,KeddieJS,GruissemW.DCLisaplantspecificproteinrequiredfor plastidribosomalRNAprocessingandembryodevelopment.PlantMolBiol.2003 Nov;53(4):53143. BellaouiM,GruissemW.AlteredexpressionoftheArabidopsisorthologueofDCL affectsnormalplantdevelopment.Planta.2004Sep;219(5):81926.Epub2004Jun10. BernsteinE,CaudyAA,HammondSM,HannonGJ.Roleforabidentateribonucle aseintheinitiationstepofRNAinterference.Nature.2001Jan18;409(6818):3636. BiesEtheveN,PontierD,LahmyS,PicartC,VegaD,CookeR,LagrangeT.RNA directedDNAmethylationrequiresanAGO4interactingmemberoftheSPT5elon gationfactorfamily.EMBORep.2009Jun;10(6):64954.Epub2009Apr3. BisanzC,BgotL,CarolP,PerezP,BlignyM,PeseyH,GalloisJL,LerbsMacheS, MacheR.TheArabidopsisnuclearDALgeneencodesachloroplastproteinwhichis requiredforthematurationoftheplastidribosomalRNAsandisessentialfor chloroplastdifferentiation.PlantMolBiol.2003Mar;51(5):65163. BlevinsT,RajeswaranR,ShivaprasadPV,BeknazariantsD,SiAmmourA,Park HS,VazquezF,RobertsonD,MeinsFJr,HohnT,PoogginMM.FourplantDicers mediateviralsmallRNAbiogenesisandDNAvirusinducedsilencing.NucleicAcids Res.2006;34(21):623346.Epub2006Nov7. BleysA,VanHoudtH,DepickerA.Downregulationofendogenesmediatedbya transitivesilencingsignal.RNA.2006Sep;12(9):16339. BohmertK,CamusI,BelliniC,BouchezD,CabocheM,BenningC.AGO1defines anovellocusofArabidopsiscontrollingleafdevelopment.EMBOJ.1998Jan 2;17(1):17080. BollenbachTJ,LangeH,GutierrezR,ErhardtM,SternDB,GagliardiD.RNR1,a 35exoribonucleasebelongingtotheRNRsuperfamily,catalyzes3maturationof chloroplastribosomalRNAsinArabidopsisthaliana.NucleicAcidsRes.2005May 12;33(8):275163. BorsaniO,ZhuJ,VersluesPE,SunkarR,ZhuJK.EndogenoussiRNAsderived fromapairofnaturalcisantisensetranscriptsregulatesalttoleranceinArabidopsis. Cell.2005Dec29;123(7):127991. BortolamiolD,PazhouhandehM,ZieglerGraffV.ViralsuppressionofRNAsi lencingbydestabilisationofARGONAUTE1.PlantSignalBehav.2008Sep;3(9):657 9. BouchN,LauresserguesD,GasciolliV,VaucheretH.Anantagonisticfunctionfor ArabidopsisDCL2indevelopmentandanewfunctionforDCL4ingeneratingviral siRNAs.EMBOJ.2006Jul26;25(14):334756.Epub2006Jun29. BrutigamA,WeberAP.Proteomicanalysisoftheproplastidenvelopemembrane providesnovelinsightsintosmallmoleculeandproteintransportacrossproplastid membranes.MolPlant.2009Nov;2(6):124761.Epub2009Aug25.
145

5.References
BrazAS,FinneganJ,WaterhouseP,MargisR.AplantorthologueofRNaseLin hibitor(RLI)isinducedinplantsshowingRNAinterference.JMolEvol.2004 Jul;59(1):2030. BrenneckeJ,AravinAA,StarkA,DusM,KellisM,SachidanandamR,HannonGJ. DiscretesmallRNAgeneratinglociasmasterregulatorsoftransposonactivityin Drosophila.Cell.2007Mar23;128(6):1089103.Epub2007Mar8. BrignetiG,VoinnetO,LiWX,JiLH,DingSW,BaulcombeDC.Viralpathogenicity determinantsaresuppressorsoftransgenesilencinginNicotianabenthamiana.EMBO J.1998Nov16;17(22):673946. BrodersenP,SakvarelidzeAchardL,BruunRasmussenM,DunoyerP,Yamamoto YY,SieburthL,VoinnetO.WidespreadtranslationalinhibitionbyplantmiRNAs andsiRNAs.Science.2008May30;320(5880):118590.Epub2008May15. BhlerM,MohnF,StalderL,MhlemannO.Transcriptionalsilencingofnonsense codoncontainingimmunoglobulinminigenes.MolCell.2005Apr29;18(3):30717. BhlerM,VerdelA,MoazedD.TetheringRITStoanascenttranscriptinitiates RNAiandheterochromatindependentgenesilencing.Cell.2006Jun2;125(5):87386. BussottiF,StrasserRJ,SchaubM.Photosyntheticbehaviorofwoodyspeciesunder highozoneexposureprobedwiththeJIPtest:areview.EnvironPollut.2007 Jun;147(3):4307.Epub2006Oct10. CampbellNA.Biology.1996.TheBenjamin/CummingsPublishingCompanyInc. CaoX,ZhouP,ZhangX,ZhuS,ZhongX,XiaoQ,DingB,LiY.Identificationofan RNAsilencingsuppressorfromaplantdoublestrandedRNAvirus.JVirol.2005 Oct;79(20):1301827. CarmellMA,HannonGJ.RNaseIIIenzymesandtheinitiationofgenesilencing. NatStructMolBiol.2004Mar;11(3):2148. CarthewRW,SontheimerEJ.OriginsandMechanismsofmiRNAsandsiRNAs. Cell.2009Feb20;136(4):64255. CeruttiL,MianN,BatemanA.Domainsingenesilencingandcelldifferentiation proteins:thenovelPAZdomainandredefinitionofthePiwidomain.TrendsBio chemSci.2000Oct;25(10):4812. ChalfieM,HorvitzHR,SulstonJE.Mutationsthatleadtoreiterationsinthecell lineagesofC.elegans.Cell.1981Apr;24(1):5969. ChapinA,CorreaP,MaguireM,KohnR.SynapticneurotransmissionproteinUNC 13affectsRNAinterferenceinneurons.BiochemBiophysResCommun.2007Mar 23;354(4):10404.Epub2007Jan29. ChatterjeeM,SparvoliS,EdmundsC,GarosiP,FindlayK,MartinC.DAG,agene requiredforchloroplastdifferentiationandpalisadedevelopmentinAntirrhinum majus.EMBOJ.1996Aug15;15(16):4194207.
146

5.References
ChatterjeeS,GrosshansH.ActiveturnovermodulatesmaturemicroRNAactivityin Caenorhabditiselegans.Nature.2009Sep24;461(7263):5469.Epub2009Sep6. ChenJ,LiWX,XieD,PengJR,DingSW.ViralvirulenceproteinsuppressesRNA silencingmediateddefensebutupregulatestheroleofmicrornainhostgeneexpres sion.PlantCell.2004May;16(5):130213.Epub2004Apr20. ChenPY,ManningaH,SlanchevK,ChienM,RussoJJ,JuJ,SheridanR,JohnB, MarksDS,GaidatzisD,SanderC,ZavolanM,TuschlT.Thedevelopmental miRNAprofilesofzebrafishasdeterminedbysmallRNAcloning.GenesDev.2005 Jun1;19(11):128893. ChendrimadaTP,GregoryRI,KumaraswamyE,NormanJ,CoochN,NishikuraK, ShiekhattarR.TRBPrecruitstheDicercomplextoAgo2formicroRNAprocessing andgenesilencing.Nature.2005Aug4;436(7051):7404.Epub2005Jun22. ChengY,PatelDJ.Crystallographicstructureofthenucleasedomainof3hExo,a DEDDhfamilymember,boundtorAMP.JMolBiol.2004Oct15;343(2):30512. CheloufiS,DosSantosCO,ChongMM,HannonGJ.AdicerindependentmiRNA biogenesispathwaythatrequiresAgocatalysis.Nature.2010Jun3;465(7298):5849. ChitwoodDH,NogueiraFT,HowellMD,MontgomeryTA,CarringtonJC, TimmermansMC.PatternformationviasmallRNAmobility.GenesDev.2009Mar 1;23(5):54954. ChomczynskiP,SacchiN.SinglestepmethodofRNAisolationbyacidguanidin iumthiocyanatephenolchloroformextraction.AnalBiochem.1987Apr;162(1):1569. CifuentesD,XueH,TaylorDW,PatnodeH,MishimaY,CheloufiS,MaE,Mane S,HannonGJ,LawsonND,WolfeSA,GiraldezAJ.AnovelmiRNAprocessing pathwayindependentofDicerrequiresArgonaute2catalyticactivity.Science.2010 Jun25;328(5986):16948.Epub2010May6. CloughSJ,BentAF.Floraldip:asimplifiedmethodforAgrobacteriummediated transformationofArabidopsisthaliana.PlantJ.1998Dec;16(6):73543. CokusSJ,FengS,ZhangX,ChenZ,MerrimanB,HaudenschildCD,PradhanS, NelsonSF,PellegriniM,JacobsenSE.ShotgunbisulphitesequencingoftheArabi dopsisgenomerevealsDNAmethylationpatterning.Nature.2008Mar 13;452(7184):2159.Epub2008Feb17. CsorbaT,BoviA,DalmayT,BurgynJ.Thep122subunitofTobaccoMosaicVirus replicaseisapotentsilencingsuppressorandcompromisesbothsmallinterfering RNAandmicroRNAmediatedpathways.JVirol.2007Nov;81(21):1176880.Epub 2007Aug22. DalmayT,HorsefieldR,BraunsteinTH,BaulcombeDC.SDE3encodesanRNA helicaserequiredforposttranscriptionalgenesilencinginArabidopsis.EMBOJ.2001 Apr17;20(8):206978.

147

5.References
DasPP,BagijnMP,GoldsteinLD,WoolfordJR,LehrbachNJ,SapetschnigA,Bu hechaHR,GilchristMJ,HoweKL,StarkR,MatthewsN,BerezikovE,KettingRF, TavarS,MiskaEA.PiwiandpiRNAsactupstreamofanendogenoussiRNApath waytosuppressTc3transposonmobilityintheCaenorhabditiselegansgermline.Mol Cell.2008Jul11;31(1):7990.Epub2008Jun19. DaxingerL,KannoT,BucherE,vanderWindenJ,NaumannU,MatzkeAJ, MatzkeM.Astepwisepathwayforbiogenesisof24ntsecondarysiRNAsand spreadingofDNAmethylation.EMBOJ.2009Jan7;28(1):4857.Epub2008Dec11. DelerisA,GallegoBartolomeJ,BaoJ,KasschauKD,CarringtonJC,VoinnetO. HierarchicalactionandinhibitionofplantDicerlikeproteinsinantiviraldefense. Science.2006Jul7;313(5783):6871.Epub2006Jun1. DenliAM,TopsBB,PlasterkRH,KettingRF,HannonGJ.Processingofprimary microRNAsbytheMicroprocessorcomplex.Nature.2004Nov11;432(7014):2315. Epub2004Nov7. DeSantisMacIossekG,KoferW,BockA,SchochS,MaierRM,WannerG,Rdi gerW,KoopHU,HerrmannRG.TargeteddisruptionoftheplastidRNApoly merasegenesrpoA,BandC1:molecularbiology,biochemistryandultrastructure. PlantJ.1999Jun;18(5):47789. DiazPendonJA,LiF,LiWX,DingSW.Suppressionofantiviralsilencingbycucum bermosaicvirus2bproteininArabidopsisisassociatedwithdrasticallyreducedaccu mulationofthreeclassesofviralsmallinterferingRNAs.PlantCell.2007 Jun;19(6):205363.Epub2007Jun22. DazPendnJA,DingSW.DirectandindirectrolesofviralsuppressorsofRNA silencinginpathogenesis.AnnuRevPhytopathol.2008;46:30326. DingL,SpencerA,MoritaK,HanM.ThedevelopmentaltimingregulatorAIN1 interactswithmiRISCsandmaytargettheargonauteproteinALG1tocytoplasmicP bodiesinC.elegans.MolCell.2005Aug19;19(4):43747. DingL,HanM.GW182familyproteinsarecrucialformicroRNAmediatedgene silencing.TrendsCellBiol.2007Aug;17(8):4116.Epub2007Sep4. DiederichsS,HaberDA.DualroleforargonautesinmicroRNAprocessingand posttranscriptionalregulationofmicroRNAexpression.Cell.2007Dec 14;131(6):1097108. DlakiM.DUF283domainofDicerproteinshasadoublestrandedRNAbinding fold.Bioinformatics.2006Nov15;22(22):27114.Epub2006Sep5. DominskiZ,YangXC,KaygunH,DadlezM,MarzluffWF.A3exonucleasethat specificallyinteractswiththe3endofhistonemRNA.MolCell.2003Aug;12(2):295 305.

148

5.References
DominskiZ,YangXC,PurdyM,MarzluffWF.Differencesandsimilaritiesbetween Drosophilaandmammalian3endprocessingofhistonepremRNAs.RNA.2005 Dec;11(12):183547.Epub2005Oct26. DonaireL,BarajasD,MartnezGarcaB,MartnezPriegoL,PagnI,LlaveC. Structuralandgeneticrequirementsforthebiogenesisoftobaccorattlevirusderived smallinterferingRNAs.JVirol.2008Jun;82(11):516777.Epub2008Mar19. DuchaineTF,WohlschlegelJA,KennedyS,BeiY,ConteDJr,PangK,Brownell DR,HardingS,MitaniS,RuvkunG,YatesJR3rd,MelloCC.Functionalpro teomicsrevealsthebiochemicalnicheofC.elegansDCR1inmultiplesmallRNA mediatedpathways.Cell.2006Jan27;124(2):34354. DunoyerP,HimberC,VoinnetO.DICERLIKE4isrequiredforRNAinterference andproducesthe21nucleotidesmallinterferingRNAcomponentoftheplantcell tocellsilencingsignal.NatGenet.2005Dec;37(12):135660.Epub2005Nov6. DunoyerP,HimberC,RuizFerrerV,AliouaA,VoinnetO.Intraandintercellular RNAinterferenceinArabidopsisthalianarequirescomponentsofthemicroRNAand heterochromaticsilencingpathways.NatGenet.2007Jul;39(7):84856.Epub2007Jun 10. EbhardtHA,ThiEP,WangMB,UnrauPJ.Extensive3modificationofplantsmall RNAsismodulatedbyhelpercomponentproteinaseexpression.ProcNatlAcadSci USA.2005Sep20;102(38):13398403.Epub2005Sep12. EckhardtS.FunctionalAnalysisofERI1.KasselUniversity.2007. ElbashirSM,LendeckelW,TuschlT.RNAinterferenceismediatedby21and22 nucleotideRNAs.GenesDev.2001aJan15;15(2):188200. ElbashirSM,MartinezJ,PatkaniowskaA,LendeckelW,TuschlT.Functional anatomyofsiRNAsformediatingefficientRNAiinDrosophilamelanogasterembryo lysate.EMBOJ.2001bDec3;20(23):687788. ElShamiM,PontierD,LahmyS,BraunL,PicartC,VegaD,HakimiMA,Jacobsen SE,CookeR,LagrangeT.ReiteratedWG/GWmotifsformfunctionallyandevolu tionarilyconservedARGONAUTEbindingplatformsinRNAirelatedcomponents. GenesDev.2007Oct15;21(20):253944. EulalioA,BehmAnsmantI,IzaurraldeE.Pbodies:atthecrossroadsofpost transcriptionalpathways.NatRevMolCellBiol.2007Jan;8(1):922. EulalioA,HelmsS,FritzschC,FauserM,IzaurraldeE.ACterminalsilencingdo maininGW182isessentialformiRNAfunction.RNA.2009Jun;15(6):106777.Epub 2009aApr21. EulalioA,TritschlerF,IzaurraldeE.TheGW182proteinfamilyinanimalcells:new insightsintodomainsrequiredformiRNAmediatedgenesilencing.RNA.2009b Aug;15(8):143342.Epub2009Jun17.

149

5.References
EystathioyT,ChanEK,TenenbaumSA,KeeneJD,GriffithK,FritzlerMJ.Aphos phorylatedcytoplasmicautoantigen,GW182,associateswithauniquepopulationof humanmRNAswithinnovelcytoplasmicspeckles.MolBiolCell.2002 Apr;13(4):133851. FabianMR,MathonnetG,SundermeierT,MathysH,ZipprichJT,SvitkinYV, RivasF,JinekM,WohlschlegelJ,DoudnaJA,ChenCY,ShyuAB,YatesJR3rd, HannonGJ,FilipowiczW,DuchaineTF,SonenbergN.MammalianmiRNARISC recruitsCAF1andPABPtoaffectPABPdependentdeadenylation.MolCell.2009 Sep24;35(6):86880.Epub2009Aug27. FagardM,BoutetS,MorelJB,BelliniC,VaucheretH.AGO1,QDE2,andRDE1 arerelatedproteinsrequiredforposttranscriptionalgenesilencinginplants,quell inginfungi,andRNAinterferenceinanimals.ProcNatlAcadSciUSA.2000Oct 10;97(21):116504. FagardM,VaucheretH.Systemicsilencingsignal(s).PlantMolBiol.2000Jun;43(2 3):28593. FahlgrenN,MontgomeryTA,HowellMD,AllenE,DvorakSK,AlexanderAL, CarringtonJC.RegulationofAUXINRESPONSEFACTOR3byTAS3tasiRNAaf fectsdevelopmentaltimingandpatterninginArabidopsis.CurrBiol.2006May 9;16(9):93944. FangY,SpectorDL.Identificationofnucleardicingbodiescontainingproteinsfor microRNAbiogenesisinlivingArabidopsisplants.CurrBiol.2007May1;17(9):81823. Epub2007Apr19. FelippesFF,WeigelD.TriggeringtheformationoftasiRNAsinArabidopsisthaliana: theroleofmicroRNAmiR173.EMBORep.2009Mar;10(3):26470.Epub2009Jan30. FilipowiczW,BhattacharyyaSN,SonenbergN.Mechanismsofposttranscriptional regulationbymicroRNAs:aretheanswersinsight?NatRevGenet.2008 Feb;9(2):10214. FireA,XuS,MontgomeryMK,KostasSA,DriverSE,MelloCC.Potentandspe cificgeneticinterferencebydoublestrandedRNAinCaenorhabditiselegans.Nature. 1998Feb19;391(6669):80611. FrstemannK,TomariY,DuT,VaginVV,DenliAM,BratuDP,KlattenhoffC, TheurkaufWE,ZamorePD.NormalmicroRNAmaturationandgermlinestemcell maintenancerequiresLoquacious,adoublestrandedRNAbindingdomainprotein. PLoSBiol.2005Jul;3(7):e236.Epub2005May24. FriedmanAM,FischmannTO,SteitzTA.Crystalstructureoflacrepressorcore tetrameranditsimplicationsforDNAlooping.Science.1995Jun23;268(5218):17217. GabelHW,RuvkunG.TheexonucleaseERI1hasaconserveddualrolein5.8S rRNAprocessingandRNAi.NatStructMolBiol.2008May;15(5):5313.Epub2008 Apr27.
150

5.References
GarciaD,CollierSA,ByrneME,MartienssenRA.Specificationofleafpolarityin ArabidopsisviathetransactingsiRNApathway.CurrBiol.2006May9;16(9):9338. GarciaD.AmiRacleinplantdevelopment:roleofmicroRNAsincelldifferentiation andpatterning.SeminCellDevBiol.2008Dec;19(6):58695.Epub2008Jul30. GasciolliV,MalloryAC,BartelDP,VaucheretH.Partiallyredundantfunctionsof ArabidopsisDICERlikeenzymesandaroleforDCL4inproducingtransacting siRNAs.CurrBiol.2005Aug23;15(16):1494500. GazzaniS,LawrensonT,WoodwardC,HeadonD,SablowskiR.Alinkbetween mRNAturnoverandRNAinterferenceinArabidopsis.Science.2004Nov 5;306(5698):10468. GleaveAP.AversatilebinaryvectorsystemwithaTDNAorganisationalstructure conducivetoefficientintegrationofclonedDNAintotheplantgenome.PlantMol Biol.1992Dec;20(6):12037. GregoryRI,YanKP,AmuthanG,ChendrimadaT,DoratotajB,CoochN,Shiek hattarR.TheMicroprocessorcomplexmediatesthegenesisofmicroRNAs.Nature. 2004Nov11;432(7014):23540.Epub2004Nov7. GriffithsJonesS,SainiHK,vanDongenS,EnrightAJ.miRBase:toolsformi croRNAgenomics.NucleicAcidsRes.2008Jan;36(Databaseissue):D1548.Epub2007 Nov8. GrishokA,PasquinelliAE,ConteD,LiN,ParrishS,HaI,BaillieDL,FireA,Ru vkunG,MelloCC.GenesandmechanismsrelatedtoRNAinterferenceregulateex pressionofthesmalltemporalRNAsthatcontrolC.elegansdevelopmentaltiming. Cell.2001Jul13;106(1):2334. GruberAR,LorenzR,BernhartSH,NeubckR,HofackerIL.TheViennaRNA websuite.NucleicAcidsRes.2008Jul1;36(WebServerissue):W704.Epub2008Apr 19. GunawardaneLS,SaitoK,NishidaKM,MiyoshiK,KawamuraY,NagamiT,Sio miH,SiomiMC.AslicermediatedmechanismforrepeatassociatedsiRNA5end formationinDrosophila.Science.2007Mar16;315(5818):158790.Epub2007Feb22. GuoHS,DingSW.Aviralproteininhibitsthelongrangesignalingactivityofthe genesilencingsignal.EMBOJ.2002Feb1;21(3):398407. GyI,GasciolliV,LauresserguesD,MorelJB,GombertJ,ProuxF,ProuxC, VaucheretH,MalloryAC.ArabidopsisFIERY1,XRN2,andXRN3areendogenous RNAsilencingsuppressors.PlantCell.2007Nov;19(11):345161.Epub2007Nov9. HaaseAD,JaskiewiczL,ZhangH,LainS,SackR,GatignolA,FilipowiczW. TRBP,aregulatorofcellularPKRandHIV1virusexpression,interactswithDicer andfunctionsinRNAsilencing.EMBORep.2005Oct;6(10):9617. HamiltonAJ,BaulcombeDC.AspeciesofsmallantisenseRNAinposttranscrip tionalgenesilencinginplants.Science.1999Oct29;286(5441):9502.
151

5.References
HammondSM,BernsteinE,BeachD,HannonGJ.AnRNAdirectednucleaseme diatesposttranscriptionalgenesilencinginDrosophilacells.Nature.2000Mar 16;404(6775):2936. HanJ,LeeY,YeomKH,NamJW,HeoI,RheeJK,SohnSY,ChoY,ZhangBT,Kim VN.MolecularbasisfortherecognitionofprimarymicroRNAsbytheDrosha DGCR8complex.Cell.2006Jun2;125(5):887901. HarrisEH,BoyntonJE,GillhamNW.Chloroplastribosomesandproteinsynthesis. MicrobiolRev.1994Dec;58(4):70054. HaseloffJ,SiemeringKR,PrasherDC,HodgeS.Removalofacrypticintronand subcellularlocalizationofgreenfluorescentproteinarerequiredtomarktransgenic Arabidopsisplantsbrightly.ProcNatlAcadSciUSA.1997Mar18;94(6):21227. HaveldaZ,HornyikC,CrescenziA,BurgynJ.InsitucharacterizationofCymbi diumRingspotTombusvirusinfectioninducedposttranscriptionalgenesilencingin Nicotianabenthamiana.JVirol.2003May;77(10):60826. HegemanCE,HalterCP,OwensTG,HansonMR.Expressionofcomplementary RNAfromchloroplasttransgenesaffectseditingefficiencyoftransgeneandendoge nouschloroplasttranscripts.NucleicAcidsRes.2005Mar8;33(5):145464. HenkeJI,GoergenD,ZhengJ,SongY,SchttlerCG,FehrC,JnemannC,Niep mannM.microRNA122stimulatestranslationofhepatitisCvirusRNA.EMBOJ. 2008Dec17;27(24):330010.Epub2008Nov20. HenzSR,CumbieJS,KasschauKD,LohmannJU,CarringtonJC,WeigelD, SchmidM.DistinctexpressionpatternsofnaturalantisensetranscriptsinArabidop sis.PlantPhysiol.2007Jul;144(3):124755.Epub2007May11. HerrAJ,MolnrA,JonesA,BaulcombeDC.DefectiveRNAprocessingenhances RNAsilencingandinfluencesfloweringofArabidopsis.ProcNatlAcadSciUSA. 2006Oct10;103(41):149945001.Epub2006Sep28. HessWR,PrombonaA,FiederB,SubramanianAR,BrnerT.Chloroplastrps15 andtherpoB/C1/C2geneclusterarestronglytranscribedinribosomedeficientplas tids:evidenceforafunctioningnonchloroplastencodedRNApolymerase.EMBOJ. 1993Feb;12(2):56371. HimberC,DunoyerP,MoissiardG,RitzenthalerC,VoinnetO.Transitivity dependentandindependentcelltocellmovementofRNAsilencing.EMBOJ.2003 Sep1;22(17):452333. HiraguriA,ItohR,KondoN,NomuraY,AizawaD,MuraiY,KoiwaH,SekiM, ShinozakiK,FukuharaT.SpecificinteractionsbetweenDicerlikeproteinsand HYL1/DRBfamilydsRNAbindingproteinsinArabidopsisthaliana.PlantMolBiol. 2005Jan;57(2):17388. HfgenR,WillmitzerL.StorageofcompetentcellsforAgrobacteriumtransformation. NucleicAcidsRes.1988Oct25;16(20):9877.
152

5.References
HongJ,QianZ,ShenS,MinT,TanC,XuJ,ZhaoY,HuangW.Highdosesof siRNAsinduceeri1andadar1geneexpressionandreducetheefficiencyofRNA interferenceinthemouse.BiochemJ.2005Sep15;390(Pt3):6759. HorschRB,FryJE,HoffmannNL,EichholtzD,RogersSG,FraleyRT.Asimpleand generalmethodfortransferringgenesintoplants.Science.1985Mar 8;227(4691):122931. HortonP,ParkKJ,ObayashiT,FujitaN,HaradaH,AdamsCollierCJ,NakaiK. WoLFPSORT:proteinlocalizationpredictor.NucleicAcidsRes.2007Jul;35(Web Serverissue):W5857.Epub2007May21. HouwingS,KammingaLM,BerezikovE,CronemboldD,GirardA,vandenElst H,FilippovDV,BlaserH,RazE,MoensCB,PlasterkRH,HannonGJ,DraperBW, KettingRF.AroleforPiwiandpiRNAsingermcellmaintenanceandtransposon silencinginzebrafish.Cell.2007Apr6;129(1):6982. HowellMD,FahlgrenN,ChapmanEJ,CumbieJS,SullivanCM,GivanSA,Kass chauKD,CarringtonJC.GenomewideanalysisoftheRNADEPENDENTRNA POLYMERASE6/DICERLIKE4pathwayinArabidopsisrevealsdependencyon miRNAandtasiRNAdirectedtargeting.PlantCell.2007Mar;19(3):92642.Epub 2007Mar30. HuntzingerE,BraunJE,HeimstdtS,ZekriL,IzaurraldeE.TwoPABPC1binding sitesinGW182proteinspromotemiRNAmediatedgenesilencing.EMBOJ.2010Dec 15;29(24):414660.Epub2010Nov9. HutvgnerG,McLachlanJ,PasquinelliAE,BlintE,TuschlT,ZamorePD.Acel lularfunctionfortheRNAinterferenceenzymeDicerinthematurationofthelet7 smalltemporalRNA.Science.2001Aug3;293(5531):8348.Epub2001Jul12. HutvgnerG,SimardMJ.Argonauteproteins:keyplayersinRNAsilencing.Nat RevMolCellBiol.2008Jan;9(1):2232. IidaT,KawaguchiR,NakayamaJ.Conservedribonuclease,Eri1,negativelyregu latesheterochromatinassemblyinfissionyeast.CurrBiol.2006Jul25;16(14):145964. Epub2006Jun22. InabaT.Bilateralcommunicationbetweenplastidandthenucleus:plastidprotein importandplastidtonucleusretrogradesignaling.BiosciBiotechnolBiochem. 2010;74(3):4716.Epub2010Mar7. InoueH,NojimaH,OkayamaH.HighefficiencytransformationofEscherichiacoli withplasmids.Gene.1990Nov30;96(1):238. JinH,VacicV,GirkeT,LonardiS,ZhuJK.SmallRNAsandtheregulationofcis naturalantisensetranscriptsinArabidopsis.BMCMolBiol.2008Jan14;9:6. JorgensenRA,AtkinsonRG,ForsterRL,LucasWJ.AnRNAbasedinformationsu perhighwayinplants.Science.1998Mar6;279(5356):14867.

153

5.References
KalantidisK,TsagrisM,TablerM.SpontaneousshortrangesilencingofaGFP transgeneinNicotianabenthamianaispossiblymediatedbysmallquantitiesofsiRNA thatdonottriggersystemicsilencing.PlantJ.2006Mar;45(6):100616. KalantidisK,DentiMA,TzortzakakiS,MarinouE,TablerM,TsagrisM.Virp1isa hostproteinwithamajorroleinPotatospindletuberviroidinfectioninNicotiana plants.JVirol.2007Dec;81(23):1287280.Epub2007Sep26. KalantidisK,SchumacherHT,AlexiadisT,HelmJM.RNAsilencingmovementin plants.BiolCell.2008Jan;100(1):1326. KarimiM,InzeD,DepickerA.GATEWAYvectorsforAgrobacteriummediated planttransformation.TrendsPlantSci.2002May;7(5):1935. KasschauKD,CarringtonJC.Longdistancemovementandreplicationmaintenance functionscorrelatewithsilencingsuppressionactivityofpotyviralHCPro.Virology. 2001Jun20;285(1):7181. KatiyarAgarwalS,MorganR,DahlbeckD,BorsaniO,VillegasAJr,ZhuJK, StaskawiczBJ,JinH.ApathogeninducibleendogenoussiRNAinplantimmunity. ProcNatlAcadSciUSA.2006Nov21;103(47):180027.Epub2006Oct27. KennedyS,WangD,RuvkunG.AconservedsiRNAdegradingRNasenegatively regulatesRNAinterferenceinC.elegans.Nature.2004Feb12;427(6975):6459. KettingRF,FischerSE,BernsteinE,SijenT,HannonGJ,PlasterkRH.Dicerfunc tionsinRNAinterferenceandinsynthesisofsmallRNAinvolvedindevelopmental timinginC.elegans.GenesDev.2001Oct15;15(20):26549. KhvorovaA,ReynoldsA,JayasenaSD.FunctionalsiRNAsandmiRNAsexhibit strandbias.Cell.2003Oct17;115(2):20916. KippM,GhringF,OstendorpT,vanDrunenCM,vanDrielR,PrzybylskiM, FackelmayerFO.SAFBox,aconservedproteindomainthatspecificallyrecognizes scaffoldattachmentregionDNA.MolCellBiol.2000Oct;20(20):74809. KirinoY,MourelatosZ.ThemousehomologofHEN1isapotentialmethylasefor PiwiinteractingRNAs.RNA.2007Sep;13(9):1397401.Epub2007Jul24. KlattenhoffC,TheurkaufW.BiogenesisandgermlinefunctionsofpiRNAs.Devel opment.2008Jan;135(1):39.Epub2007Nov21. KobayashiK,ZambryskiP.RNAsilencinganditscelltocellspreadduringArabi dopsisembryogenesis.PlantJ.2007May;50(4):597604.Epub2007Apr5. KomodaK,MawatariN,HagiwaraKomodaY,NaitoS,IshikawaM.Identification ofaribonucleoproteinintermediateoftomatomosaicvirusRNAreplicationcomplex formation.JVirol.2007Mar;81(6):258491.Epub2006Nov15. KsselH,NattE,StrittmatterG,FritzscheE,GozdzickaJozefiakA,PrzybylD. StructureandexpressionofrRNAoperonsfromplastidsofhigherplants.Molecular

154

5.References
FormandFunctionofthePlantGenome(vanVlotenDoting,L.,Groot,G.S.P.and Hall,T.C.,eds).NewYork:PlenumPress,1985pp.183198 KotakisC,VrettosN,KotsisD,TsagrisM,KotzabasisK,KalantidisK.Lightinten sityaffectsRNAsilencingofatransgeneinNicotianabenthamianaplants.BMCPlant Biol.2010Oct12;10:220. KrolJ,LoedigeI,FilipowiczW.ThewidespreadregulationofmicroRNAbiogene sis,functionanddecay.NatRevGenet.2010Sep;11(9):597610.Epub2010Jul27. KumakuraN,TakedaA,FujiokaY,MotoseH,TakanoR,WatanabeY.SGS3and RDR6interactandcolocalizeincytoplasmicSGS3/RDR6bodies.FEBSLett.2009Apr 17;583(8):12616.Epub2009Mar28. KupscoJM,WuMJ,MarzluffWF,ThaparR,DuronioRJ.Geneticandbiochemical characterizationofDrosophilaSnipper:Apromiscuousmemberofthemetazoan 3hExo/ERI1familyof3to5exonucleases.RNA.2006Dec;12(12):210317.Epub 2006Oct24. KuriharaY,TakashiY,WatanabeY.TheinteractionbetweenDCL1andHYL1is importantforefficientandpreciseprocessingofprimiRNAinplantmicroRNAbio genesis.RNA.2006Feb;12(2):20612. LagiotisGD.FunctionalanalysisofplantERI1:ERL1(ERI1LIKE1).Universityof Crete.2009. LakatosL,SzittyaG,SilhavyD,BurgynJ.MolecularmechanismofRNAsilencing suppressionmediatedbyp19proteinoftombusviruses.EMBOJ.2004Feb 25;23(4):87684.Epub2004Feb19. LakatosL,CsorbaT,PantaleoV,ChapmanEJ,CarringtonJC,LiuYP,DoljaVV, CalvinoLF,LpezMoyaJJ,BurgynJ.SmallRNAbindingisacommonstrategyto suppressRNAsilencingbyseveralviralsuppressors.EMBOJ.2006Jun 21;25(12):276880.Epub2006May25. LawJA,JacobsenSE.Establishing,maintainingandmodifyingDNAmethylation patternsinplantsandanimals.NatRevGenet.2010Feb9;11(3):204220. LeeRC,FeinbaumRL,AmbrosV.TheC.elegansheterochronicgenelin4encodes smallRNAswithantisensecomplementaritytolin14.Cell.1993Dec3;75(5):84354. LeeY,JeonK,LeeJT,KimS,KimVN.MicroRNAmaturation:stepwiseprocessing andsubcellularlocalization.EMBOJ.2002Sep2;21(17):466370. LeeY,AhnC,HanJ,ChoiH,KimJ,YimJ,LeeJ,ProvostP,RdmarkO,KimS, KimVN.ThenuclearRNaseIIIDroshainitiatesmicroRNAprocessing.Nature.2003 Sep25;425(6956):4159. LeeY,KimM,HanJ,YeomKH,LeeS,BaekSH,KimVN.MicroRNAgenesare transcribedbyRNApolymeraseII.EMBOJ.2004bOct13;23(20):405160.Epub2004 Sep16.
155

5.References
LeeYS,NakaharaK,PhamJW,KimK,HeZ,SontheimerEJ,CarthewRW.Distinct rolesforDrosophilaDicer1andDicer2inthesiRNA/miRNAsilencingpathways. Cell.2004aApr2;117(1):6981. LeuschnerPJ,AmeresSL,KuengS,MartinezJ.CleavageofthesiRNApassenger strandduringRISCassemblyinhumancells.EMBORep.2006Mar;7(3):31420.Epub 2006Jan20. LiJ,YangZ,YuB,LiuJ,ChenX.MethylationprotectsmiRNAsandsiRNAsfroma 3enduridylationactivityinArabidopsis.CurrBiol.2005Aug23;15(16):15017. LimAK,KaiT.Uniquegermlineorganelle,nuage,functionstorepressselfishge neticelementsinDrosophilamelanogaster.ProcNatlAcadSciUSA.2007Apr 17;104(16):67149.Epub2007Apr11. LinH,SpradlingAC.Anovelgroupofpumiliomutationsaffectstheasymmetric divisionofgermlinestemcellsintheDrosophilaovary.Development.1997 Jun;124(12):246376. LingelA,SimonB,IzaurraldeE,SattlerM.Structureandnucleicacidbindingofthe DrosophilaArgonaute2PAZdomain.Nature.2003Nov27;426(6965):4659.Epub 2003Nov16. ListerR,OMalleyRC,TontiFilippiniJ,GregoryBD,BerryCC,MillarAH,Ecker JR.HighlyintegratedsinglebaseresolutionmapsoftheepigenomeinArabidopsis. Cell.2008May2;133(3):52336. LiuQ,RandTA,KalidasS,DuF,KimHE,SmithDP,WangX.R2D2,abridgebe tweentheinitiationandeffectorstepsoftheDrosophilaRNAipathway.Science. 2003Sep26;301(5641):19215. LiuJ,CarmellMA,RivasFV,MarsdenCG,ThomsonJM,SongJJ,HammondSM, JoshuaTorL,HannonGJ.Argonaute2isthecatalyticengineofmammalianRNAi. Science.2004Sep3;305(5689):143741.Epub2004Jul29. LoveAJ,LairdJ,HoltJ,HamiltonAJ,SadanandomA,MilnerJJ.Cauliflowermosaic virusproteinP6isasuppressorofRNAsilencing.JGenVirol.2007Dec;88(Pt 12):343944. LuR,FolimonovA,ShintakuM,LiWX,FalkBW,DawsonWO,DingSW.Three distinctsuppressorsofRNAsilencingencodedbya20kbviralRNAgenome.Proc NatlAcadSciUSA.2004Nov2;101(44):157427.Epub2004Oct25. LuoZ,ChenZ.Improperlyterminated,unpolyadenylatedmRNAofsensetrans genesistargetedbyRDR6mediatedRNAsilencinginArabidopsis.PlantCell.2007 Mar;19(3):94358.Epub2007Mar23. LurinC,AndrsC,AubourgS,BellaouiM,BittonF,BruyreC,CabocheM,De bastC,GualbertoJ,HoffmannB,LecharnyA,LeRetM,MartinMagnietteML, MireauH,PeetersN,RenouJP,SzurekB,TaconnatL,SmallI.Genomewide

156

5.References
analysisofArabidopsispentatricopeptiderepeatproteinsrevealstheiressentialrolein organellebiogenesis.PlantCell.2004Aug;16(8):2089103.Epub2004Jul21. LynnK,FernandezA,AidaM,SedbrookJ,TasakaM,MassonP,BartonMK.The PINHEAD/ZWILLEgeneactspleiotropicallyinArabidopsisdevelopmentandhas overlappingfunctionswiththeARGONAUTE1gene.Development.1999 Feb;126(3):46981. MacraeIJ,ZhouK,LiF,RepicA,BrooksAN,CandeWZ,AdamsPD,DoudnaJA. StructuralbasisfordoublestrandedRNAprocessingbyDicer.Science.2006Jan 13;311(5758):1958. MaitiM,LeeHC,LiuY.QIP,aputativeexonuclease,interactswiththeNeurospora ArgonauteproteinandfacilitatesconversionofduplexsiRNAintosinglestrands. GenesDev.2007Mar1;21(5):590600.Epub2007Feb20. MalloryAC,MlotshwaS,BowmanLH,VanceVB.Thecapacityoftransgenicto baccotosendasystemicRNAsilencingsignaldependsonthenatureoftheinducing transgenelocus.PlantJ.2003Jul;35(1):8292. MalloryAC,HinzeA,TuckerMR,BouchN,GasciolliV,ElmayanT,Laur esserguesD,JauvionV,VaucheretH,LauxT.Redundantandspecificrolesofthe ARGONAUTEproteinsAGO1andZLLindevelopmentandsmallRNAdirected genesilencing.PLoSGenet.2009Sep;5(9):e1000646.Epub2009Sep18. MaloneCD,BrenneckeJ,DusM,StarkA,McCombieWR,SachidanandamR, HannonGJ.SpecializedpiRNApathwaysactingermlineandsomatictissuesofthe Drosophilaovary.Cell.2009bMay1;137(3):52235.Epub2009Apr23. MaloneCD,HannonGJ.SmallRNAsasguardiansofthegenome.Cell.2009aFeb 20;136(4):65668. MarrisonJL,RutherfordSM,RobertsonEJ,ListerC,DeanC,LeechRM.Thedis tinctiverolesoffivedifferentARCgenesinthechloroplastdivisionprocessinArabi dopsis.PlantJ.1999Jun;18(6):65162. MatrangaC,TomariY,ShinC,BartelDP,ZamorePD.Passengerstrandcleavage facilitatesassemblyofsiRNAintoAgo2containingRNAienzymecomplexes.Cell. 2005Nov18;123(4):60720.Epub2005Nov3. MatzkeM,KannoT,DaxingerL,HuettelB,MatzkeAJ.RNAmediatedchromatin basedsilencinginplants.CurrOpinCellBiol.2009Jun;21(3):36776.Epub2009Feb 23. McElverJ,TzafrirI,AuxG,RogersR,AshbyC,SmithK,ThomasC,SchetterA, ZhouQ,CushmanMA,TossbergJ,NickleT,LevinJZ,LawM,MeinkeD,Patton D.InsertionalmutagenesisofgenesrequiredforseeddevelopmentinArabidopsis thaliana.Genetics.2001Dec;159(4):175163.

157

5.References
MegrawM,BaevV,RusinovV,JensenST,KalantidisK,HatzigeorgiouAG.Mi croRNApromoterelementdiscoveryinArabidopsis.RNA.2006Sep;12(9):16129. Epub2006Aug3. MeisterG,LandthalerM,PatkaniowskaA,DorsettY,TengG,TuschlT.Human Argonaute2mediatesRNAcleavagetargetedbymiRNAsandsiRNAs.MolCell. 2004Jul23;15(2):18597. MraiZ,KernyiZ,MolnrA,BartaE,VlcziA,BisztrayG,HaveldaZ,Burgyn J,SilhavyD.AureusvirusP14isanefficientRNAsilencingsuppressorthatbinds doublestrandedRNAswithoutsizespecificity.JVirol.2005Jun;79(11):721726. MraiZ,KernyiZ,KertszS,MagnaM,LakatosL,SilhavyD.Doublestranded RNAbindingmaybeageneralplantRNAviralstrategytosuppressRNAsilencing. JVirol.2006Jun;80(12):574756. MetteMF,vanderWindenJ,MatzkeM,MatzkeAJ.ShortRNAscanidentifynew candidatetransposableelementfamiliesinArabidopsis.PlantPhysiol.2002 Sep;130(1):69. MiS,CaiT,HuY,ChenY,HodgesE,NiF,WuL,LiS,ZhouH,LongC,ChenS, HannonGJ,QiY.SortingofsmallRNAsintoArabidopsisargonautecomplexesis directedbythe5terminalnucleotide.Cell.2008Apr4;133(1):11627.Epub2008Mar 13. MiyoshiK,TsukumoH,NagamiT,SiomiH,SiomiMC.SlicerfunctionofDroso philaArgonautesanditsinvolvementinRISCformation.GenesDev.2005Dec 1;19(23):283748.Epub2005Nov14. MiyoshiK,OkadaTN,SiomiH,SiomiMC.CharacterizationofthemiRNARISC loadingcomplexandmiRNARISCformedintheDrosophilamiRNApathway.RNA. 2009Jul;15(7):128291.Epub2009May18. MlotshwaS,VoinnetO,MetteMF,MatzkeM,VaucheretH,DingSW,PrussG, VanceVB.RNAsilencingandthemobilesilencingsignal.PlantCell.2002;14 Suppl:S289301. MoazedD.SmallRNAsintranscriptionalgenesilencingandgenomedefense.Na ture.2009Jan22;457(7228):41320. MoissiardG,ParizottoEA,HimberC,VoinnetO.TransitivityinArabidopsiscanbe primed,requirestheredundantactionoftheantiviralDicerlike4andDicerlike2, andiscompromisedbyviralencodedsuppressorproteins.RNA.2007 Aug;13(8):126878.Epub2007Jun25. MontgomeryTA,HowellMD,CuperusJT,LiD,HansenJE,AlexanderAL,Chap manEJ,FahlgrenN,AllenE,CarringtonJC.SpecificityofARGONAUTE7miR390 interactionanddualfunctionalityinTAS3transactingsiRNAformation.Cell.2008a Apr4;133(1):12841.Epub2008Mar13.

158

5.References
MontgomeryTA,YooSJ,FahlgrenN,GilbertSD,HowellMD,SullivanCM,Alex anderA,NguyenG,AllenE,AhnJH,CarringtonJC.AGO1miR173complexiniti atesphasedsiRNAformationinplants.ProcNatlAcadSciUSA.2008bDec 23;105(51):2005562.Epub2008Dec9. MosherRA,SchwachF,StudholmeD,BaulcombeDC.PolIVbinfluencesRNA directedDNAmethylationindependentlyofitsroleinsiRNAbiogenesis.ProcNatl AcadSciUSA.2008Feb26;105(8):314550.Epub2008Feb19. MourrainP,BclinC,ElmayanT,FeuerbachF,GodonC,MorelJB,JouetteD,La combeAM,NikicS,PicaultN,RmouK,SanialM,VoTA,VaucheretH.Arabi dopsisSGS2andSGS3genesarerequiredforposttranscriptionalgenesilencingand naturalvirusresistance.Cell.2000May26;101(5):53342. NapoliC,LemieuxC,JorgensenR.IntroductionofaChimericChalconeSynthase GeneintoPetuniaResultsinReversibleCoSuppressionofHomologousGenesin trans.PlantCell.1990Apr;2(4):279289. NakazawaY,HiraguriA,MoriyamaH,FukuharaT.ThedsRNAbindingprotein DRB4interactswiththeDicerlikeproteinDCL4invivoandfunctionsinthetrans actingsiRNApathway.PlantMolBiol.2007Apr;63(6):77785.Epub2007Jan14. NicholsonAW.Function,mechanismandregulationofbacterialribonucleases. FEMSMicrobiolRev.1999Jun;23(3):37190. NishimuraK,AshidaH,OgawaT,YokotaA.ADEADboxproteinisrequiredfor formationofahiddenbreakinArabidopsischloroplast23SrRNA.PlantJ.2010 Sep;63(5):76677. OharaT,SakaguchiY,SuzukiT,UedaH,MiyauchiK,SuzukiT.The3terminiof mousePiwiinteractingRNAsare2Omethylated.NatStructMolBiol.2007 Apr;14(4):34950.Epub2007Mar25. OkamuraK,HagenJW,DuanH,TylerDM,LaiEC.Themirtronpathwaygenerates microRNAclassregulatoryRNAsinDrosophila.Cell.2007Jul13;130(1):89100.Epub 2007Jun28. OlivieriD,SykoraMM,SachidanandamR,MechtlerK,BrenneckeJ.Aninvivo RNAiassayidentifiesmajorgeneticandcellularrequirementsforprimarypiRNA biogenesisinDrosophila.EMBOJ.2010Oct6;29(19):330117.Epub2010Sep3. OmarovRT,CiomperlikJJ,ScholthofHB.RNAiassociatedssRNAspecificribonu cleasesinTombusvirusP19mutantinfectedplantsandevidenceforadiscretesiRNA containingeffectorcomplex.ProcNatlAcadSciUSA.2007Jan30;104(5):17149. Epub2007Jan23. romUA,NielsenFC,LundAH.MicroRNA10abindsthe5UTRofribosomalpro teinmRNAsandenhancestheirtranslation.MolCell.2008May23;30(4):46071.

159

5.References
PaneA,WehrK,SchpbachT.zucchiniandsquashencodetwoputativenucleases requiredforrasiRNAproductionintheDrosophilagermline.DevCell.2007 Jun;12(6):85162. PantaleoV,SzittyaG,BurgynJ.MolecularbasesofviralRNAtargetingbyviral smallinterferingRNAprogrammedRISC.JVirol.2007Apr;81(8):3797806.Epub 2007Jan31. PappI,MetteMF,AufsatzW,DaxingerL,SchauerSE,RayA,vanderWindenJ, MatzkeM,MatzkeAJ.EvidencefornuclearprocessingofplantmicroRNAand shortinterferingRNAprecursors.PlantPhysiol.2003Jul;132(3):138290. ParkW,LiJ,SongR,MessingJ,ChenX.CARPELFACTORY,aDicerhomolog,and HEN1,anovelprotein,actinmicroRNAmetabolisminArabidopsisthaliana.Curr Biol.2002Sep3;12(17):148495. ParkMY,WuG,GonzalezSulserA,VaucheretH,PoethigRS.Nuclearprocessing andexportofmicroRNAsinArabidopsis.ProcNatlAcadSciUSA.2005Mar 8;102(10):36916.Epub2005Feb28. PartridgeJF,DeBeauchampJL,KosinskiAM,UlrichDL,HadlerMJ,Noffsinger VJ.Functionalseparationoftherequirementsforestablishmentandmaintenanceof centromericheterochromatin.MolCell.2007May25;26(4):593602. PazhouhandehM,DieterleM,MarroccoK,LechnerE,BerryB,BraultV,Hemmer O,KretschT,RichardsKE,GenschikP,ZieglerGraffV.Fboxlikedomaininthe polerovirusproteinP0isrequiredforsilencingsuppressorfunction.ProcNatlAcad SciUSA.2006Feb7;103(6):19949.Epub2006Jan30. PeragineA,YoshikawaM,WuG,AlbrechtHL,PoethigRS.SGS3and SGS2/SDE1/RDR6arerequiredforjuveniledevelopmentandtheproductionoftrans actingsiRNAsinArabidopsis.GenesDev.2004Oct1;18(19):236879. PfefferS,DunoyerP,HeimF,RichardsKE,JonardG,ZieglerGraffV.P0ofbeet Westernyellowsvirusisasuppressorofposttranscriptionalgenesilencing.JVirol. 2002Jul;76(13):681524. PierleoniA,MartelliPL,FariselliP,CasadioR.BaCelLo:abalancedsubcellularlo calizationpredictor.Bioinformatics.2006Jul15;22(14):e40816. PikaardCS,HaagJR,ReamT,WierzbickiAT.RolesofRNApolymeraseIVingene silencing.TrendsPlantSci.2008Jul;13(7):3907. PouchPlissierMN,PlissierT,ElmayanT,VaucheretH,BokoD,JantschMF, DeragonJM.SINERNAinducesseveredevelopmentaldefectsinArabidopsis thalianaandinteractswithHYL1(DRB1),akeymemberoftheDCL1complex.PLoS Genet.2008Jun13;4(6):e1000096. QuF,MorrisTJ.EfficientinfectionofNicotianabenthamianabyTomatobushystunt virusisfacilitatedbythecoatproteinandmaintainedbyp19throughsuppressionof genesilencing.MolPlantMicrobeInteract.2002Mar;15(3):193202.
160

5.References
QuF,YeX,HouG,SatoS,ClementeTE,MorrisTJ.RDR6hasabroadspectrum buttemperaturedependentantiviraldefenseroleinNicotianabenthamiana.JVirol. 2005Dec;79(24):1520917. RajagopalanR,VaucheretH,TrejoJ,BartelDP.Adiverseandevolutionarilyfluid setofmicroRNAsinArabidopsisthaliana.GenesDev.2006Dec15;20(24):340725. RamachandranV,ChenX.DegradationofmicroRNAsbyafamilyofexoribonucle asesinArabidopsis.Science.2008Sep12;321(5895):14902. ReamTS,HaagJR,WierzbickiAT,NicoraCD,NorbeckAD,ZhuJK,HagenG, GuilfoyleTJ,PasaToliL,PikaardCS.SubunitcompositionsoftheRNAsilencing enzymesPolIVandPolVrevealtheiroriginsasspecializedformsofRNApoly meraseII.MolCell.2009Jan30;33(2):192203.Epub2008Dec24. RehwinkelJ,BehmAnsmantI,GatfieldD,IzaurraldeE.AcrucialroleforGW182 andtheDCP1:DCP2decappingcomplexinmiRNAmediatedgenesilencing.RNA. 2005Nov;11(11):16407.Epub2005Sep21. ReinhartBJ,SlackFJ,BassonM,PasquinelliAE,BettingerJC,RougvieAE,Hor vitzHR,RuvkunG.The21nucleotidelet7RNAregulatesdevelopmentaltimingin Caenorhabditiselegans.Nature.2000Feb24;403(6772):9016. ReinhartBJ,WeinsteinEG,RhoadesMW,BartelB,BartelDP.MicroRNAsin plants.GenesDev.2002Jul1;16(13):161626. ReiterRS,CoomberSA,BourettTM,BartleyGE,ScolnikPA.Controlofleafand chloroplastdevelopmentbytheArabidopsisgenepalecress.PlantCell.1994 Sep;6(9):125364. RhoadesMW,ReinhartBJ,LimLP,BurgeCB,BartelB,BartelDP.Predictionof plantmicroRNAtargets.Cell.2002Aug23;110(4):51320. RobertsonHD,WebsterRE,ZinderND.Purificationandpropertiesofribonuclease IIIfromEscherichiacoli.JBiolChem.1968Jan10;243(1):8291. RodioME,DelgadoS,DeStradisA,GmezMD,FloresR,DiSerioF.Aviroid RNAwithaspecificstructuralmotifinhibitschloroplastdevelopment.PlantCell. 2007Nov;19(11):361026.Epub2007Nov30. RogersSO,BendichAJ.ExtractionofDNAfrommilligramamountsoffresh,her bariumandmummifiedplantissues.PlantMolBiol.1985.5:6976 RothsteinSJ,DimaioJ,StrandM,RiceD.Stableandheritableinhibitionoftheex pressionofnopalinesynthaseintobaccoexpressingantisenseRNA.ProcNatlAcad SciUSA.1987Dec;84(23):843943. RubyJG,JanC,PlayerC,AxtellMJ,LeeW,NusbaumC,GeH,BartelDP.Large scalesequencingreveals21URNAsandadditionalmicroRNAsandendogenous siRNAsinC.elegans.Cell.2006Dec15;127(6):1193207.

161

5.References
RubyJG,JanCH,BartelDP.IntronicmicroRNAprecursorsthatbypassDrosha processing.Nature.2007Jul5;448(7149):836.Epub2007Jun24. RuizMT,VoinnetO,BaulcombeDC.Initiationandmaintenanceofvirusinduced genesilencing.PlantCell.1998Jun;10(6):93746. SaitoK,IshizukaA,SiomiH,SiomiMC.ProcessingofpremicroRNAsbythe Dicer1LoquaciouscomplexinDrosophilacells.PLoSBiol.2005Jul;3(7):e235.Epub 2005May24. SaitoK,NishidaKM,MoriT,KawamuraY,MiyoshiK,NagamiT,SiomiH,Siomi MC.SpecificassociationofPiwiwithrasiRNAsderivedfromretrotransposonand heterochromaticregionsintheDrosophilagenome.GenesDev.2006Aug 15;20(16):221422.Epub2006Aug1. SahaD,PrasadAM,SrinivasanR.Pentatricopeptiderepeatproteinsandtheir emergingrolesinplants.PlantPhysiolBiochem.2007Aug;45(8):52134.Epub2007 Mar24. SakamotoW.LeafvariegatedmutationsandtheirresponsiblegenesinArabidopsis thaliana.GenesGenetSyst.2003Feb;78(1):19. SambrookJ,RusselDW.MolecularCloningALaboratoryManual.NewYork: ColdSpringHarboLaboratoryPress.2001. SarmientoC,NigulL,KazantsevaJ,BuschmannM,TruveE.AtRLI2isanendoge noussuppressorofRNAsilencing.PlantMolBiol.2006May;61(12):15363. SatyanarayanaT,GowdaS,AyllnMA,AlbiachMartMR,RabindranS,Dawson WO.Thep23proteinofcitrustristezaviruscontrolsasymmetricalRNAaccumula tion.JVirol.2002Jan;76(2):47383. ScheinAI,KissingerJC,UngarLH.Chloroplasttransitpeptideprediction:apeek insidetheblackbox.NucleicAcidsRes.2001Aug15;29(16):E82. SchmitzLinneweberC,SmallI.Pentatricopeptiderepeatproteins:asocketsetfor organellegeneexpression.TrendsPlantSci.2008Dec;13(12):66370.Epub2008Nov 12. SchbH,KunzC,MeinsFJr.Silencingoftransgenesintroducedintoleavesby agroinfiltration:asimple,rapidmethodforinvestigatingsequencerequirementsfor genesilencing.MolGenGenet.1997Nov;256(5):5815. SchumacherHT.InvolvementsofthePlant35ExonucleaseERL1inChloroplast RibosomalRNABiogenesisandRNASilencingPathways.KasselUniversity.2009. SchpbachT,WieschausE.Femalesterilemutationsonthesecondchromosomeof Drosophilamelanogaster.II.Mutationsblockingoogenesisoralteringeggmorphology. Genetics.1991Dec;129(4):111936.

162

5.References
SchwabR,MaizelA,RuizFerrerV,GarciaD,BayerM,CrespiM,VoinnetO,Mar tienssenRA.EndogenousTasiRNAsmediatenoncellautonomouseffectsongene regulationinArabidopsisthaliana.PLoSOne.2009Jun19;4(6):e5980. SchwachF,VaistijFE,JonesL,BaulcombeDC.AnRNAdependentRNApoly merasepreventsmeristeminvasionbypotatovirusXandisrequiredfortheactivity butnottheproductionofasystemicsilencingsignal.PlantPhysiol.2005 Aug;138(4):184252.Epub2005Jul22. SchwarzDS,HutvgnerG,DuT,XuZ,AroninN,ZamorePD.Asymmetryinthe assemblyoftheRNAienzymecomplex.Cell.2003Oct17;115(2):199208. SharwoodRE,HottoAM,BollenbachTJ,SternDB.Overaccumulationofthe chloroplastantisenseRNAAS5iscorrelatedwithdecreasedabundanceof5SrRNA invivoandinefficient5SrRNAmaturationinvitro.RNA.2011Feb;17(2):23043. Epub2010Dec9. ShibolethYM,HaronskyE,LeibmanD,AraziT,WasseneggerM,WhithamSA, GabaV,GalOnA.TheconservedFRNKboxinHCPro,aplantviralsuppressorof genesilencing,isrequiredforsmallRNAbindingandmediatessymptomdevelop ment.JVirol.2007Dec;81(23):1313548.Epub2007Sep26. ShikanaiT,ShimizuK,UedaK,NishimuraY,KuroiwaT,HashimotoT.The chloroplastclpPgene,encodingaproteolyticsubunitofATPdependentprotease,is indispensableforchloroplastdevelopmentintobacco.PlantCellPhysiol.2001 Mar;42(3):26473. SijenT,FleenorJ,SimmerF,ThijssenKL,ParrishS,TimmonsL,PlasterkRH,Fire A.OntheroleofRNAamplificationindsRNAtriggeredgenesilencing.Cell.2001 Nov16;107(4):46576. SilhavyD,MolnrA,LucioliA,SzittyaG,HornyikC,TavazzaM,BurgynJ.A viralproteinsuppressesRNAsilencingandbindssilencinggenerated,21to25 nucleotidedoublestrandedRNAs.EMBOJ.2002Jun17;21(12):307080. SimmerF,TijstermanM,ParrishS,KoushikaSP,NonetML,FireA,AhringerJ, PlasterkRH.LossoftheputativeRNAdirectedRNApolymeraseRRF3makesC. eleganshypersensitivetoRNAi.CurrBiol.2002Aug6;12(15):13179. SmallID,PeetersN.ThePPRmotifaTPRrelatedmotifprevalentinplantorganel larproteins.TrendsBiochemSci.2000Feb;25(2):467. SmallI,PeetersN,LegeaiF,LurinC.Predotar:Atoolforrapidlyscreeningpro teomesforNterminaltargetingsequences.Proteomics.2004Jun;4(6):158190. SmithLM,PontesO,SearleI,YelinaN,YousafzaiFK,HerrAJ,PikaardCS,Baul combeDC.AnSNF2proteinassociatedwithnuclearRNAsilencingandthespread ofasilencingsignalbetweencellsinArabidopsis.PlantCell.2007May;19(5):150721. Epub2007May25.

163

5.References
SongJJ,SmithSK,HannonGJ,JoshuaTorL.CrystalstructureofArgonauteandits implicationsforRISCsliceractivity.Science.2004Sep3;305(5689):14347.Epub2004 Jul29. SouretFF,KastenmayerJP,GreenPJ.AtXRN4degradesmRNAinArabidopsisand itssubstratesincludeselectedmiRNAtargets.MolCell.2004Jul23;15(2):17383. SternDB,GoldschmidtClermontM,HansonMR.ChloroplastRNAmetabolism. AnnuRevPlantBiol.2010Jun2;61:12555. SummerEJ,ClineK.Redbellpepperchromoplastsexhibitinvitroimportcompe tencyandmembranetargetingofpassengerproteinsfromthethylakoidalsecand DeltapHpathwaysbutnotthechloroplastsignalrecognitionparticlepathway.Plant Physiol.1999Feb;119(2):57584. SzittyaG,SilhavyD,MolnrA,HaveldaZ,LovasA,LakatosL,BnfalviZ,Bur gynJ.LowtemperatureinhibitsRNAsilencingmediateddefensebythecontrolof siRNAgeneration.EMBOJ.2003Feb3;22(3):63340. TablerM,SngerHL.ClonedsingleanddoublestrandedDNAcopiesofpotato spindletuberviroid(PSTV)RNAandcoinoculatedsubgenomicDNAfragmentsare infectious.EMBOJ.1984Dec20;3(13):305562. TakedaA,IwasakiS,WatanabeT,UtsumiM,WatanabeY.Themechanismselect ingtheguidestrandfromsmallRNAduplexesisdifferentamongargonautepro teins.PlantCellPhysiol.2008Apr;49(4):493500.Epub2008Mar14. ThierryMiegD,ThierryMiegJ.AceView:acomprehensivecDNAsupportedgene andtranscriptsannotation.GenomeBiol.2006;7Suppl1:S12.114.Epub2006Aug7. TillS,LejeuneE,ThermannR,BortfeldM,HothornM,EnderleD,HeinrichC, HentzeMW,LadurnerAG.AconservedmotifinArgonauteinteractingproteins mediatesfunctionalinteractionsthroughtheArgonautePIWIdomain.NatStruct MolBiol.2007Oct;14(10):897903.Epub2007Sep23. TimmonsL.EndogenousinhibitorsofRNAinterferenceinCaenorhabditiselegans. Bioessays.2004Jul;26(7):7158. TrinksD,RajeswaranR,ShivaprasadPV,AkbergenovR,OakeleyEJ,Veluthambi K,HohnT,PoogginMM.SuppressionofRNAsilencingbyageminivirusnuclear protein,AC2,correlateswithtransactivationofhostgenes.JVirol.2005 Feb;79(4):251727. TomoyasuY,MillerSC,TomitaS,SchoppmeierM,GrossmannD,BucherG.Ex ploringsystemicRNAinterferenceininsects:agenomewidesurveyforRNAigenes inTribolium.GenomeBiol.2008Jan17;9(1):R10. ToliaNH,JoshuaTorL.Slicerandtheargonautes.NatChemBiol.2007Jan;3(1):36 43.

164

5.References
TournierB,TablerM,KalantidisK.Phloemflowstronglyinfluencesthesystemic spreadofsilencinginGFPNicotianabenthamianaplants.PlantJ.2006Aug;47(3):383 94.Epub2006Jun12. TritschlerF,HuntzingerE,IzaurraldeE.RoleofGW182proteinsandPABPC1inthe miRNApathway:asenseofdjvu.NatRevMolCellBiol.2010May;11(5):37984. Epub2010Apr9. VaginVV,SigovaA,LiC,SeitzH,GvozdevV,ZamorePD.AdistinctsmallRNA pathwaysilencesselfishgeneticelementsinthegermline.Science.2006Jul 21;313(5785):3204.Epub2006Jun29. Vamvaka,E.Invivofunctionalanalysisoftheplant3exonucleaseERL1.University ofCrete.2010. vanderKrolAR,MurLA,BeldM,MolJN,StuitjeAR.Flavonoidgenesinpetunia: additionofalimitednumberofgenecopiesmayleadtoasuppressionofgeneex pression.PlantCell.1990Apr;2(4):2919. VargasonJM,SzittyaG,BurgynJ,HallTM.SizeselectiverecognitionofsiRNAby anRNAsilencingsuppressor.Cell.2003Dec26;115(7):799811. VasudevanS,TongY,SteitzJA.Switchingfromrepressiontoactivation:microR NAscanupregulatetranslation.Science.2007Dec21;318(5858):19314.Epub2007 Nov29. VaucheretH,VazquezF,CrtP,BartelDP.TheactionofARGONAUTE1inthe miRNApathwayanditsregulationbythemiRNApathwayarecrucialforplantde velopment.GenesDev.2004May15;18(10):118797.Epub2004May6. VaucheretH,MalloryAC,BartelDP.AGO1homeostasisentailscoexpressionof MIR168andAGO1andpreferentialstabilizationofmiR168byAGO1.MolCell.2006 Apr7;22(1):12936. VaucheretH.PlantARGONAUTES.TrendsPlantSci.2008Jul;13(7):3508.Epub2008 May26. VazquezF,VaucheretH,RajagopalanR,LepersC,GasciolliV,MalloryAC,Hil bertJL,BartelDP,CrtP.EndogenoustransactingsiRNAsregulatetheaccumula tionofArabidopsismRNAs.MolCell.2004Oct8;16(1):6979. VazquezF,BlevinsT,AilhasJ,BollerT,MeinsFJr.EvolutionofArabidopsisMIR genesgeneratesnovelmicroRNAclasses.NucleicAcidsRes.2008Nov;36(20):6429 38.Epub2008Oct8. ViswanathanM,LovettST.ExonucleaseXofEscherichiacoli.Anovel35DNase andDnaqsuperfamilymemberinvolvedinDNArepair.JBiolChem.1999Oct 15;274(42):30094100. VlatakisIX.InvitrostudyofAtERILIKE1:purificationandtryingtodiscoverits naturalsubstrate.UniversityofCrete.2010.
165

5.References
VoglerH,AkbergenovR,ShivaprasadPV,DangV,FaslerM,KwonMO,Zhany bekovaS,HohnT,HeinleinM.ModificationofsmallRNAsassociatedwithsup pressionofRNAsilencingbytobamovirusreplicaseprotein.JVirol.2007 Oct;81(19):1037988.Epub2007Jul18. VoinnetO,BaulcombeDC.Systemicsignallingingenesilencing.Nature.1997Oct 9;389(6651):553. VoinnetO,VainP,AngellS,BaulcombeDC.Systemicspreadofsequencespecific transgeneRNAdegradationinplantsisinitiatedbylocalizedintroductionofectopic promoterlessDNA.Cell.1998Oct16;95(2):17787. VoinnetO,PintoYM,BaulcombeDC.Suppressionofgenesilencing:ageneral strategyusedbydiverseDNAandRNAvirusesofplants.ProcNatlAcadSciUSA. 1999Nov23;96(24):1414752. VoinnetO,LedererC,BaulcombeDC.Aviralmovementproteinpreventsspreadof thegenesilencingsignalinNicotianabenthamiana.Cell.2000Sep29;103(1):15767. VoinnetO.Origin,biogenesis,andactivityofplantmicroRNAs.Cell.2009Feb 20;136(4):66987. VothknechtUC,WesthoffP.Biogenesisandoriginofthylakoidmembranes.Bio chimBiophysActa.2001Dec12;1541(12):91101. WangY,DubyG,PurnelleB,BoutryM.TobaccoVDLgeneencodesaplastidDEAD boxRNAhelicaseandisinvolvedinchloroplastdifferentiationandplantmorpho genesis.PlantCell.2000Nov;12(11):212942. WangG,ReinkeVA.C.elegansPiwi,PRG1,regulates21URNAsduringspermato genesis.CurrBiol.2008Jun24;18(12):8617.Epub2008May22. WasseneggerM,KrczalG.NomenclatureandfunctionsofRNAdirectedRNApo lymerases.TrendsPlantSci.2006Mar;11(3):14251.Epub2006Feb13. WierzbickiAT,HaagJR,PikaardCS.NoncodingtranscriptionbyRNApolymerase PolIVb/PolVmediatestranscriptionalsilencingofoverlappingandadjacentgenes. Cell.2008Nov14;135(4):63548. WilliamsMA,JohzukaY,MulliganRM.Additionofnongenomicallyencodednu cleotidestothe3terminusofmaizemitochondrialmRNAs:truncatedrps12mRNAs frequentlyterminatewithCCA.NucleicAcidsRes.2000Nov15;28(22):444451. WilliamsL,CarlesCC,OsmontKS,FletcherJC.Adatabaseanalysismethodidenti fiesanendogenoustransactingshortinterferingRNAthattargetstheArabidopsis ARF2,ARF3,andARF4genes.ProcNatlAcadSciUSA.2005Jul5;102(27):97038. Epub2005Jun24. Wingard,S.A.Hostsandsymptomsofringspot,avirusdiseaseofplants.J.Agric. Res.37,127153(1928).

166

5.References
WinterD,VinegarB,NahalH,AmmarR,WilsonGV,ProvartNJ.AnElectronic FluorescentPictographbrowserforexploringandanalyzinglargescalebiological datasets.PLoSOne.2007Aug8;2(1):e718. XieZ,KasschauKD,CarringtonJC.NegativefeedbackregulationofDicerLike1in ArabidopsisbymicroRNAguidedmRNAdegradation.CurrBiol.2003Apr 29;13(9):7849. XieZ,JohansenLK,GustafsonAM,KasschauKD,LellisAD,ZilbermanD, JacobsenSE,CarringtonJC.GeneticandfunctionaldiversificationofsmallRNA pathwaysinplants.PLoSBiol.2004May;2(5):E104.Epub2004Feb24. XieZ,AllenE,FahlgrenN,CalamarA,GivanSA,CarringtonJC.Expressionof ArabidopsisMIRNAgenes.PlantPhysiol.2005aAug;138(4):214554.Epub2005Jul22. XieZ,AllenE,WilkenA,CarringtonJC.DICERLIKE4functionsintransacting smallinterferingRNAbiogenesisandvegetativephasechangeinArabidopsisthaliana. ProcNatlAcadSciUSA.2005bSep6;102(36):129849.Epub2005Aug29. YaegashiH,TakahashiT,IsogaiM,KoboriT,OhkiS,YoshikawaN.Applechlorotic leafspotvirus50kDamovementproteinactsasasuppressorofsystemicsilencing withoutinterferingwithlocalsilencinginNicotianabenthamiana.JGenVirol.2007 Jan;88(Pt1):31624. YangZ,EbrightYW,YuB,ChenX.HEN1recognizes2124ntsmallRNAduplexes anddepositsamethylgroupontothe2OHofthe3terminalnucleotide.Nucleic AcidsRes.2006Jan30;34(2):66775.Print2006. YangXC,PurdyM,MarzluffWF,DominskiZ.Characterizationof3hExo,a3ex onucleasespecificallyinteractingwiththe3endofhistonemRNA.JBiolChem.2006 Oct13;281(41):3044754.Epub2006Aug15. YangX,BalijiS,BuchmannRC,WangH,LindboJA,SunterG,BisaroDM.Func tionalmodulationofthegeminivirusAL2transcriptionfactorandsilencingsuppres sorbyselfinteraction.JVirol.2007Nov;81(21):1197281.Epub2007Aug22. YeK,MalininaL,PatelDJ.RecognitionofsmallinterferingRNAbyaviralsuppres sorofRNAsilencing.Nature.2003Dec18;426(6968):8748.Epub2003Dec3. YiR,QinY,MacaraIG,CullenBR.Exportin5mediatesthenuclearexportofpre microRNAsandshorthairpinRNAs.GenesDev.2003Dec15;17(24):30116.Epub 2003Dec17. YigitE,BatistaPJ,BeiY,PangKM,ChenCC,ToliaNH,JoshuaTorL,MitaniS, SimardMJ,MelloCC.AnalysisoftheC.elegansArgonautefamilyrevealsthatdis tinctArgonautesactsequentiallyduringRNAi.Cell.2006Nov17;127(4):74757. YooBC,KraglerF,VarkonyiGasicE,HaywoodV,ArcherEvansS,LeeYM,Lough TJ,LucasWJ.AsystemicsmallRNAsignalingsysteminplants.PlantCell.2004 Aug;16(8):19792000.Epub2004Jul16.

167

5.References
YoshikawaM,PeragineA,ParkMY,PoethigRS.Apathwayforthebiogenesisof transactingsiRNAsinArabidopsis.GenesDev.2005Sep15;19(18):216475.Epub2005 Aug30. YuB,ChapmanEJ,YangZ,CarringtonJC,ChenX.Transgenicallyexpressedviral RNAsilencingsuppressorsinterferewithmicroRNAmethylationinArabidopsis. FEBSLett.2006May29;580(13):311720.Epub2006May2. YuB,BiL,ZhengB,JiL,ChevalierD,AgarwalM,RamachandranV,LiW,La grangeT,WalkerJC,ChenX.TheFHAdomainproteinsDAWDLEinArabidopsis andSNIP1inhumansactinsmallRNAbiogenesis.ProcNatlAcadSciUSA.2008 Jul22;105(29):100738.Epub2008Jul15. ZamorePD,TuschlT,SharpPA,BartelDP.RNAi:doublestrandedRNAdirectsthe ATPdependentcleavageofmRNAat21to23nucleotideintervals.Cell.2000Mar 31;101(1):2533. ZamorePD.SomaticpiRNAbiogenesis.EMBOJ.2010Oct6;29(19):321921. ZanduetaCriadoA,BockR.SurprisingfeaturesofplastidndhDtranscripts:addition ofnonencodednucleotidesandpolysomeassociationofmRNAswithanunedited startcodon.NucleicAcidsRes.2004Jan26;32(2):54250.Print2004. ZekriL,HuntzingerE,HeimstdtS,IzaurraldeE.ThesilencingdomainofGW182 interactswithPABPC1topromotetranslationalrepressionanddegradationofmi croRNAtargetsandisrequiredfortargetrelease.MolCellBiol.2009Dec;29(23):6220 31.Epub2009Sep21. ZhangB,PanX,CannonCH,CobbGP,AndersonTA.Conservationanddiver genceofplantmicroRNAgenes.PlantJ.2006Apr;46(2):24359. ZhangL,DingL,CheungTH,DongMQ,ChenJ,SewellAK,LiuX,YatesJR3rd, HanM.SystematicidentificationofC.elegansmiRISCproteins,miRNAs,and mRNAtargetsbytheirinteractionswithGW182proteinsAIN1andAIN2.MolCell. 2007Nov30;28(4):598613. ZhangD,TrudeauVL.TheXSdomainofaplantspecificSGS3proteinadoptsa uniqueRNArecognitionmotif(RRM)fold.CellCycle.2008Jul15;7(14):226870. Epub2008May14. ZhengX,ZhuJ,KapoorA,ZhuJK.RoleofArabidopsisAGO6insiRNAaccumula tion,DNAmethylationandtranscriptionalgenesilencing.EMBOJ.2007Mar 21;26(6):1691701.Epub2007Mar1. ZhengX,PontesO,ZhuJ,MikiD,ZhangF,LiWX,IidaK,KapoorA,PikaardCS, ZhuJK.ROS3isanRNAbindingproteinrequiredforDNAdemethylationinArabi dopsis.Nature.2008Oct30;455(7217):125962.Epub2008Sep24. ZhengB,WangZ,LiS,YuB,LiuJY,ChenX.IntergenictranscriptionbyRNApo lymeraseIIcoordinatesPolIVandPolVinsiRNAdirectedtranscriptionalgenesi lencinginArabidopsis.GenesDev.2009Dec15;23(24):285060.
168

5.References
ZilbermanD,CaoX,JacobsenSE.ARGONAUTE4controloflocusspecificsiRNA accumulationandDNAandhistonemethylation.Science.2003Jan31;299(5607):716 9.Epub2003Jan9. ZipprichJT,BhattacharyyaS,MathysH,FilipowiczW.ImportanceoftheC terminaldomainofthehumanGW182proteinTNRC6Cfortranslationalrepression. RNA.2009May;15(5):78193.Epub2009Mar20. ZubkoMK,DayA.Differentialregulationofgenestranscribedbynucleusencoded plastidRNApolymerase,andDNAamplification,withinribosomedeficientplastids instablephenocopiesofcerealalbinomutants.MolGenetGenomics.2002 Mar;267(1):2737.Epub2002Feb8.

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6.1 Supplementarymethods
6.1.1 Virus/viroidinfectionsinNicotianasp.plants
YoungNicotianasp.plantswereinfectedasdescribedbefore(Tabler&Snger,1984; Kalantidisetal.,2007). 100mgtomatoleafmaterialinfectedwithPotatospindletuberviroid(PSTVd)strain PH106weregroundin1mLof1%K2HPO4.Alternatively100mgtobaccoleafmate rialinfectedwithPlumpoxvirus(PPV)wasgroundin1mL50mMphosphatebuffer, pH8.0.LeavesofNicotianasp.werepretreatedwithcarborundumandmechanically inoculatedwith100Lofthebeforecreatedinfectioussapperleaf.Alternatively50 ngofinvitrotranscribedviroidcouldbeused.Theleaveswererinsedwithwaterand grownunderstandardgreenhouseconditions.Fullsystemicinfectioncouldbeob servedafter37weekspostinfection.

6.1.2 Invitrotranscription
FortheproductionofsmallrRNAspeciesT7promotercontainingoligonucleotides wereusedtocreatedsDNAwhichservedastemplateforinvitrotranscription.1g dsRNA,8mMNTPmix,10mMDTT,10URNaseinhibitorin1xT7RNApoly merasereactionbufferwereincubatedat37Cfor1h.Subsequently250UT7RNA polymerasewereaddedandthemixturewasincubatedat37Cfor2h.Afterafinal DNaseItreatmentfor15mintheinvitrotranscribedRNAwaspurifiedbyphe nol/chloroformextractionandprecipitatedwithisopropanol.

6.1.3 PurificationofrecombinantERL1protein
ERL1cDNAcontaininganNterminalHistagwasclonedintopET15bandex pressedinE.colistrainJM109.Thecellsweregrownat37CinselectiveLBtoan OD600ofapproximately0.7.Proteinexpressionwasinducedbyadditionof0.5mM IPTGandincubationat37Cfor3h.Thecellswereharvestedbycentrifugationat
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2500xgat4Cfor20minandresuspendedinproteinpurificationbuffer.Thecells werelyzedwith1mg/mllysozymeonicefor1handpelletedat15000xgat4Cfor 10min.ThesupernatantwasloadedonapreequilibratedNiNTAcolumn.After twowasheswithwashingbuffer(proteinpurificationbuffercontaining20and30 mMimidazole)theproteinwaselutedwithelutionbuffer(proteinpurificationbuffer containing400mMimidazole).

6.1.4 InvitroassaysforrecombinantERL1protein
SyntheticsiRNAsorinvitrotranscribedrRNAswereradiolabeledandpurified.50 fmolwereincubatedwithrecombinantERL1proteinincleavagebuffer[10mMTris pH8.0,27mMKCl,0.5mMMgCl2,0.5%glycerol(Iidaetal.,2006)]atroomtempera tureforempiricallydeterminedincubationtimes.ThereactionswererunonPAA gelsandexposedtoXrayfilms.

6.2 Supplementaryresults
TheprojectofcharacterizingERL1inN.benthamianaandA.thalianawasconducted byseverallabmembersaspartoftheirMasterandPhDtheses.Thereforerelevant additionalresultsforthisthesisarepresentedbelowandpartlycitedliterally.For additionalinformationpleaserefertothefollowingtheses: Eckhardt,StephanieFunctionalAnalysisofERI1(KasselUniversity,2007) Schumacher,HeikoInvolvementsofthePlant35ExonucleaseERL1inChloro plastRibosomalRNABiogenesisandRNASilencingPathways(KasselUniversity, 2009) Vamvaka,EvgeniaInvivofunctionalanalysisoftheplant3exonuclease ERL1[originaltitle:,Invivo 3ERI1(UniversityofCrete,2010)] Vlatakis,IoannisInvitrostudyofAtERILIKE1:purificationandtryingtodiscover itsnaturalsubstrate[originaltitle:,InvitroAtERI LIKE1:, (UniversityofCrete,2010)]
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Lagiotis,GeorgiosFunctionalanalysisofplantERI1:ERL1(ERI1LIKE1)[original title:,ERI1:ERL1(ERI1 LIKE1)(UniversityofCrete,2009)]

6.2.1 PSTVdderivedsiRNAsaresuppresseduponERL1overexpression
UponinfectionwithPotatospindletuberviroid(PSTVd),Nicotianaplantsproducelarge amountsof2124ntsiRNAlikeRNAsderivedfromtheviroidsequence,butareoth erwisesymptomfree.ThesePSTVdsiRNAsareeasilydetectableinnorthernhy bridisationsandhencepresentasuitablereportersystemtostudypossibleeffectsof ERL1misexpressiononsiRNAsteadystatelevels. Comparativeagroinfiltrationtimecourseexperimentswereconductedbyoverex pressingArabidopsisERL1insystemicallyPSTVdinfectedN.tabacum.Infiltrations withanemptybinaryvectorservedascontrols.TostudyPSTVdsiRNAsteadystate levelsovertime,sampleswerecollectedevery4thday,andsmallRNAfractionswere subsequentlyelectrophoresedon20%PAAgels,northernblotted,andanalysedby hybridisationswithPSTVdspecificprobes.Noninfiltratedsamplesofthesameplant wereusedastimepointszero.Theresultsofthisexperimentaresummarisedin Figure6.1.8dayspostinfiltrationPSTVdsiRNAlevelsarereducedapproximately4 foldinsamplesthatweretreatedwithERL1overexpression(Figure6.1,leftpanel). ThereductionseemstoaffectthedifferentsiRNAsizeclasses(21,22and24nt) equally(Figure6.1,leftpanel).InnontreatedsamplesofthesameplantPSTVd siRNAlevelsremainconstantovertime(Figure6.1,rightpanel),rulingoutanun specificeffectduetoagingoftheplantordifferentialprogressionofthePSTVdinfec tionoverthecourseoftheexperiment.Agroinfiltrationwithacontrolplasmidsimi larlyhadnomeasurableeffectonPSTVdsiRNAlevels(Figure6.1,middlepanel). ThisexperimentshowsthatthesteadystatelevelsofsiRNAsproducedfromPSTVd uponinfectionofN.tabacumarebeingnegativelyaffectedbyectopicagro infiltrationmediatedArabidopsisERL1overexpression.Itcannotbeundoubtedlyde duced,however,ifthisreductioninsiRNAlevelsiscausedbyansiRNAdegrading

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activityofERL1,orifthisistheresultofasecondaryeffect(literallycitedfrom Schumacher,2009).

Figure6.1:ComparativeagroinfiltrationtimecourseinsystemicallyPSTVdinfectedtobacco. IntheleftpanelERL1overexpressionreducedthenumberofPSTVdsiRNAssignificantly8dpi whereasafterinfiltrationofacontrolplasmid(middlepanel)andinuntreatedplants(rightpanel) remainconstant(resultandpicturebySchumacher,2009)

6.2.2 ERL1failstoaffectRNAsilencinginAgrobacteriumcoinfiltration assays

Inplantviruses,pathogenicitydeterminentshavebeenshowntoactasRNAsilenc ingsuppressors(Brignetietal.,1998).Traditionally,viralgenesaretestedforsilenc ingsuppressoractivitybyassayingtheireffectsonsenseinducedGFPsilencinginN. benthamianaplants.Todetermine,ifERL1showsRNAsilencingsuppressoractivity, Agrobacteriumcoinfiltrationassayswereperformed,essentiallyasdescribedbefore (Luetal.,2004).Inthisassay,GFPsilencingisinducedbyoverexpressionofGFPin analreadyGFPexpressingplant.EctopicGFPexpressiontriggersthecosuppression pathwayandleadstoRNAimediatedsilencingofthetransgenicallyproducedGFP. BonafidesuppressorsofsilencingareabletoinhibitordelaytheinitiationofRNA silencingwhenexpressedalongwiththesilencinginducer.Theassayswereper formedinN.benthamianaline16cthatischaracterisedbystrongandstableGFPex pression(Ruizetal.,1998).TocreateERL1overexpression(35SAtERL1gen)andsup pression(35SNtERL1hp)constructs,thegenomicsequenceoftheArabidopsisERL1 geneandahairpinconstructderivedfromatobaccoERL1EST(ESTIDEB681897), respectively,wereclonedinthebinaryvectorpART27(Gleave,1992)andtrans formedintoA.tumefaciensstrainC58C1.EqualvolumesofanA.tumefaciensstrain
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carryinga35SGFPconstruct[pBIN35SmGFP4(Haseloffetal.,1997)]servingasthe silencinginducerwerethenmixedwithAgrobacteriacarryingtheplasmidsforERL1 overexpressionorERL1suppression.Thefinalconcentrationsofallstrainsweread justedto0.25atOD600.1:1mixtureswithanemptybinaryplasmidorwithacon structexpressingthesilencingsuppressorP19ofCymbidiumringspotvirus[35SP19 CymRSV(Haveldaetal.,2003;Lakatosetal.,2004)]servedasnegativeandpositive controls,respectively.TheAgrobacteriummixtureswereusedtoagroinfiltratedis tinctpatcheson16cleaves,andGFPexpressionandsilencinginitiationweremoni toredovertimeusingahandheldBlakRaylongwaveUVlamp.Underthecondi tionstested,ERL1wasnotabletosuppresstheonsetofGFPsilencing(Figure6.2). UponcoexpressionofERL1andGFP(Figure3.4a),theredringoflocalGFPsilenc ingspread(Himberetal.,2003;Kalantidisetal.,2008)appearedatthesametimeasin theemptyplasmidnegativecontrol(Figure6.2a).ERL1suppressionwithahairpin constructsimilarlyhadnoeffectontheRNAsilencingtimecourse(Figure6.2a).The redringisnotonlyindicativeofRNAsilencinginitiationbutalsoshowsthatSLSS wasnotaffectedbyERL1overexpressionorsuppression.CoexpressionofP19onthe otherhandwasabletosuppressRNAsilencinginitiation,exemplifiedbystrongGFP signalintheinfiltratedpatchandtheabsenceoftheredringoflocalGFPsilencing spreadoverthecourseoftheexperiment(Figure6.2a).

Figure6.2:AgrobacteriumcoinfiltrationassaysinN.benthamianaline16CtotestERL1forRNA silencingsuppressoractivity (A)IncontrasttotheverifiedsilencingsuppressorproteinP19(upperleftspot)ERL1overexpression (upperrightspot)doesnotresultinsuppressionofGFPsilencingsimilartothecontrolconstructs (lowerpanel),whichisvisiblebytheformationofaredringaroundthegreenspotofGFPinfiltration. (B)InductionofGFPsilencingbytheinfiltrationofaGFPhairpinconstructisnotaffectedbyoverex pressionofERL1identicaltothecontrolconstructs(resultsandpicturebySchumacher,2009). 175

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Inanequivalentexperiment,ERL1overexpressionandsuppressionalsofailedtoaf fectGFPsilencingwhenaGFPhairpinconstructwasusedtoinducesilencing(Figure 6.2b). Takentogether,theseassaysshownoabilityofERL1tonegativelyaffectRNAsilenc ing.Givenitslowexpressionlevelsandpredominantlychloroplasticlocalisationa possibleroleofERL1innegativeRNAsilencingregulationmay,however,betoo weaktobedetectedinAgrobacteriumcoinfiltrationassaysthatrequireratherrobust silencingsuppressorslikeP19tocounteragroinfiltrationinducedRNAsilencing (literallycitedfromSchumacher,2009).

6.2.3 ExogenouslyinducedsilencingspreadmaybesuppressedafterERL1 overexpression

ToconfirmthatwhitesectorformationisadirectresultofERL1overexpression, youngERL1overexpressingplantswithamildbleachphenotype(i.e.palegreen leaves)wereagroinfiltratedwithanArabidopsisERL1hairpinconstruct(35S AtERL1hp)toinduceRNAsilencingofthetransgene(Figure3.7f,pointofinfiltra tionindicatedbyblackarrowhead).Severaldayspostinfiltrationwildtypelikegreen tissuestartedspreadingfromtheveinsofsystemicleaves,stronglyresemblingthe phenotypeofsystemicGFPsilencingspread(Figure3.7f). Itwasobservedthatinabout50%ofthecases,systemicspreadofinducedERL1si lencingdidnotpropagatemorethanafewleaves(Figure3.7f,redarrowheadindi catinganewlyemergingleafvoidofRNAsilencingtypespreadingofgreentissue), whichsuggeststhatsystemicRNAsilencingmaytosomeextentbesuppressedin ERL1overexpressorplants.Theremaining50%ofplantscontinuedtosystemically silencetheERL1dependentbleachingphenotypeandregainedwildtypeappearance untilsenescence(literallycitedfromSchumacher,2009).

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Figure6.3:SilencingoftheERL1phenotypeinducedby agroinfiltration SystemicERL1silencinginableachtypeplant,inducedby agroinfiltrationwithanERL1hairpinconstruct(theblackarrowhead inthelowerpartofthepictureindicatesthepointofagro infiltration).Theredarrowhead(inthemiddleofthepicure)showsa newlyemergingleafvoidofsystemicsilencingtypespreadof wildtypeliketissue(resultandpicturebySchumacher,2009).

6.2.4 ERL1overexpressingplantsarehypersensitivetowardsviralinfection
SinceantiviraldefenseconstitutesoneofthemajorfunctionsofRNAsilencingin plants,itwasinvestigatedhowplantswithdifferentERL1backgroundsbehaveupon viralinfection.TothisendwildtypeN.benthamianaandERL1overexpressorplants wereinfectedwithPlumpoxvirus(PPV). Undertheconditionstested,wildtypeN.benthamianaplantsshowedatypicalpro gressionofPPVinfection,withmildmosaicsymptomsdevelopingafter12weeks thatpersisteduntilsenescence(Figure6.4a).ERL1overexpressorplantsincontrast developedmuchstrongersymptomswithseverelycrippledleaves(Figure6.4b). Onlyapproximately33%ofthePPVinfectedERL1overexpressingplantssurvived theinfection,whiletheremaining67%haddieduntilsixweekspostinfection.Given thatPPVinfectionsaretypicallynonlethalinNicotianaplants,suchahighlethality rateimpliesahypersensitivityofERL1overexpressorplantstowardsPPVinfection. ThesurvivingERL1overexpressorplantsremaineddwarveswithcrippledleaves untilsenescenceandproducedonlyfewunderdevelopedflowersthatfailedtopro duceanyseeds.Theslowgrowthandreducedfertilitymaytosomeextentbeex plainedbyERL1overexpressionitself.Agrowth/fertilitydefectaspronouncedasin thePPVinfectedindividuals,however,hasneverbeenobservedinthespecific bleachtypeERL1overexpressorlineusedintheinfectionexperiments. 12weekspostinfectiontotalRNAwasextractedfromwildtypeN.benthamianaanda survivingERL1overexpressorplantandtestedfortherespectiveviralloadsinnorth ernhybridisations(Figure6.4c).IncomparisontowildtypeN.benthamiana,ERL1 overexpressorplantsaccumulateapproximately35xhighervirustitres.Theincrease
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inviralloadmayexplaintheobservedaggravatedsymptoms.Whetherthishyper sensitivityisaccountabletoansiRNAdegradingactivityofERL1could,however, notbedeterminedinthisexperiment.NorthernanalysesofvariegatedERL1overex pressortissuesshowedasignificantdecreaseinthesteadystatemRNAlevelsoftwo ofthefourDICERLIKEproteins,namelyDCL1andDCL4(Figure6.4d).Sincethe DICERLIKEproteinsarecrucialcorecomponentsofRNAimediatedantiviralde fenseinplants,thedescribedhypersensitivitytowardsPPVinfectionmaybecaused byareductioninDCL14productionandhencenotconstituteaprimaryeffectof ERL1overexpression.ThefactthatatleastDCL1andDCL4aredownregulatedin ERL1overexpressingtissue,however,givesleewaytoapossibleconnectionbetween ERL1functionandRNAsilencingpathways(literallycitedfromSchumacher,2009).

Figure6.4:ERL1overexpressorplantsarehypersensitivetowardsinfectionbyPPV (A)SymptomsofPPVinfectiononbleachplantswhichshowcrippledleavesandastuntedgrowth. (B)ThesymptomofPPVinfectiononN.benthamianawildtypeplantsisamildmosaicphenotype.(C) ERL1overexpressorplantsaccumulateahigherviralload12weekspostinfection.(D)DCL1and DCL4transcriptlevelsaredownregulatedinwhitetissuewhereastheyremainnormalingreentissue overexpressingERL1(resultandpicturebySchumacher,2009).

6.2.5 InvitroexperimentswithERL1
SeveralattemptshavebeenconductedtocharacterizeplantERL1invitro.Increasing amountsofrecombinantERL1wereincubatedwiththefollowingradiolabeledsub strates:synthetic21ntsiRNA,invitrotranscribed5.8S,5S,4.5SrRNAandseveralof theirnaturalprocessingintermediates,aswellasextractednaturalrRNAs.Thesam
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pleswerethenanalyzedonPAAgels.Neitheranyshortenedprocessingproducts norashiftofthesubstrates,whichwouldbearesultofbindingtoERL1,couldbe detectedintheseassays.ThissuggestsnoERL1processingactivityatthechosenre actionconditions(Eckhardt,2007;Lagiotis,2009;Vlatakis,2010).

6.2.6 PreparationofaNbERL1suppressionconstructandanalysisofitsef fectsaftertransientandtransgenicexpression

FortheanalysisofERL1suppressioninNicotianabenthamianaahairpinconstructwas preparedfromtheNbERL1sequencewithapproximatelengthof3000bp.Afterco infiltrationwithanNbERL1overexpressionconstructofapproximately1000bpan immediatesuppressioncouldbeobserved.Incontrast,whentheNbERL1overex pressionconstructwasinfiltrated,itsdegradationbytheplantstartedafterthesec ondday(Figure6.5a). TheeffectofNbERL1suppressiononchloroplastrelatedtranscriptshasbeeninves tigatedbytransientexpressionoftheconstructandconsecutiveanalysis.Theresults werecomparedwithplantsconstitutivelyoverexpressingERL1andshowingamo saicphenotype.Inadditionwildtypeplantsservedasacontrol.Theexpressionof CLPC,NEPandPSBAwerenotinfluencedbythemisexpressionofERL1.Inthecase ofPFTFaslightreductioncouldbedetectedinthesuppressingtissueincontrastto overexpressortissuewheretheexpressionwascomparabletowildtype.PEPand CLPPweredownregulatedinsuppressingtissuewhileanupregulationcouldbeob servedinoverexpressingtissue.FinallyRBCLwasstronglydownregulatedinERL1 overexpressingtissueandabitlessdownregulatedinERL1suppressingtissuecom paredtowildtype(Figure6.5b). TheanalysisoftransgenicplantssuppressingERL1startedwiththreechloroplast relatedgenesandwascomparedwiththetransientsuppression.Incontrasttothe resultsdescribedbefore,thedownregulationofPFTFandPEPcouldnotberepeated inthetransgenicplantsandtheirexpressionlevelswerecomparabletotransgenic plantsoverexpressingERL1[(Figure6.5c)(Vamvaka,2010)].
179

6.Supplements
(A) (B)

3000bp 1000bp

(C)

Figure6.5:AnalysisofthesuppressionofERL1inNicotianabenthamiana. (A)ConstructsforoverexpressionandsuppressionofERL1wereinfiltratedintoN.benthamianaand theirexpressionfollowedoverfourdays.Theoverexpressingconstructiseasilydetectableonthefirst dayandvanishesoverthetimecourse.Thecoinfiltrationoftheoverexpressionandhairpincon structspreventsthedetectionoftheoverexpressionfromthebeginning.(B)Analysisofchloroplast relatedtranscriptsinwildtypeN.benthamiana,mosaicplantsoverexpressingERL1andaftertransient suppressionofERL1byagroinfiltration.(C)Controloftheearlieridentifiedinfluencedtranscriptsin transgenicERL1suppressorplantsfailstodetectspecificexpressionalterationsafterknockdownof ERL1(resultsandpicturesbyVamvaka,2010).

180

6.Supplements
6.3 Oligonucelotides
Thefollowingprimerswereusedduringthecourseoftheworkfortherespective cloningpurposes,mutantanalysisandforprobepreparation:
PrimersusedforanalysisofArabidopsisthalianaKDplants GABI_KAT LB3_SAIL Lbb1_SALK N544378F N544378R N579265_For N579265_Rev N834430_For N834430_Rev clpPProbe NEPFor NEPRev NtCLPBamF NtCLPNotR psbAFor psbARev rbcLFor rbcLRev rpoBFor rpoBRev TobPftfForward TobPftfReverse AtEriBamF AtEriNotR AthEriqPCRF AthEriqPCRR EriFLAGRev ErinoStop+TCRev Leader+TCRev mature+Bam+start NbeeriFOR NbeeriREV ATATTGACCATCATACTCATTGC TAGCATCTGAATTTCATAACCAATCTCGATACAC GCGTGGACCGCTTGCTGCAACT ACACCTTGTAAGCCATCAACG TTTCAAACATCAATCTTCCGC CCAATCTCGTTCAGACATTCC TTTCTTGGACCGGGTTTTAAG TTTACGTTCATTTCCTGAGGC TTCCATGGTTTTGGCATCTAC CCTTGTGAGGGTTTCACGCAGTTTCAGCAGTTCTTCCGCTTCCAG TTTGATGCGCACTCATGGATCTA GCAAATTCAAGGATTCCTCGACA GGATCCGAAAGGAGGCCGTCGTATAGGTT GCGGCCGCATCTGAGGGAGTTCCGCTAGTGC TAACCATGAGCGGCTACGATGTT GTAGCTTGTTACATGGGTCGTG TGCGAATCCCTCCTGCTTATGTT CCAAGCTAGTATTTGCGGTGAAT CTTCCAGCTACTTTATCGCCTAC CTTATATGCCGTGGGAGGGTTAC GGATCCCCCGAGAGGTTTACCGCAGTG GCGGCCGCGGTGTCCTCATGGCTATAACTT GGATCCATGGCGTCCGCATTCTCTGC GCGGCCGCTTACTTGATCCTGTTCTTGAAG CAAAATGAGCGAGCAAGCAATTA ATCCCAGTTTCCACAGGTTACAA GCGGCCGCCGATGAATAGTAGTAGTAGGAACATTAGGTATTGAT CCTGTTCTTGAAG GCGGCCGCCTCTTGATCCTGTTCTTGAAGAGA GCGGCCGCCTACTTGTTTCCATTAACGAAAAAG GGATCCATGGAAAATGCAAGGTGGAGACCCATG AGCAGTCACAAGACTCGC GGTGATAACAAAATGGTTCAG 181

Primersusedfordetectingchloroplastrelatedtranscripts

PrimersusedforthecloninganddetectionofERL1

6.Supplements
NbeerinewqRTF NbeerinewqRTR Nteri_RACEA1 Nteri_RACEA2 Nteri_RACES1 Nteri_RACES2 Nteri_RTprimer 16SFor 16SRev 16S+linkFor 18SFor 18SRev 23s_For_new 23s_Rev_new 23S+linkFor 4.5SProbe 4.5S+linkFor 4.5ScircularF 4.5ScircularR 5.8sprobeFOR 5.8sprobeRev 5.8srRNA 5.8srRNAstartFor 5.8ScircularF 5.8ScircularR 5SProbe 5S+linkFor 5ScircularF 5ScircularR linkermod linkerREV Miscellaneous 35SpART7&27F 35SpART7&27R AthUBQ_For AthUBQ_Rev IntronFor IntronRev NicoUBQ_For NicoUBQ_Rev RacerdT AAGTTTGGCTGGGTGAACGTC AAGGCCCTCCTCTTGTAGAAG TGCTCGCTGGAGTTTATGCAA ACTTGAGAATTTGCGGAAGC GTGTGGGCTGAATTAGGGA TTGGGCCTGAAGTGAGTACGA GGCCTCACAAACCTATG TCATGGAGAGTTCGATCCTGGCT ATCCTCACCTTCCTCCGGCTTATCA CAAAAACCCGTCCTCAGTTC TCAACCATAAACGATGCCGACCA GCGTGCGGCCCAGAACATCTAA ACAGGTCTCCGCAAAGTCGTA GCCCTTCCAGAGTGGTATCTCA CCACCTGGGGCTGTAGTATG ATCCTGGCGTCGAGCTATTTTTCCGCAGGACCTCCCCTAC CGAGACGAGCCGTTTATCAT AGGCATCCTAACAGACCGGTAG TCCACTTGACACCTATCGTAATG GCAACGGATATCTCGGCTCT TAATGGCTTCGGGCGCAACT CGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATAC CGGATATCTCGGCTCT CGCCCGAAGCCATTAGG CGATGGTTCACGGGATTC GGATGCCTCAGCTGCATACATCACTGCACTTCCACTTGAC ACCAATCCATCCCGAACTT TAAACTCTACTGCGGTGAC CAAGTTCGGGATGGATTGG ATCGTCACAACAAATGGCATddC GATGCCATTTGTTGTGACGAT TATAGAGGAAGGGTCTTGCGAAGG CAACAAAGGATAATTTCGGGAAAC TAACCCTTGAGGTTGAATCATCC AACTCCTTCTTTCTGGTAAACGT CTTGGAGCAGAACATGAGATTCG ATTGAGCCAGGGCATTTACCTC GAGGTTGAATCTTCCGACACAAT AACATAGGTGAGCCCACACTTAC GCTGTCAACGATACGCTACGTAACGGCATGACAGTGTTTTTTTTTT TTTTTTTT 182

PrimersusedforthedetectionandcloningofribosomalRNAs

6.Supplements
6.4 Vectormaps
6.4.1 AtERL1GFP(11243bp;Spec);basedonpB7FWG2(Karimietal.,2002)
GFP 0 attB2

ERL1cDNA

attB1

35S

6.4.2 AtleaderGFP(10451bp;Spec);basedonpB7FWG2(Karimietal., 2002) GFP

0 attB2

ERL1leader
attB1

35S

183

6.Supplements
6.4.3 AtERL1over(15987bp;Kan/Spec);Schumacher,2009

AtERL 1genomic

35S Restrictionsites:963(NotI),1747(BamHI),3895(HindIII),5283(NotI),5774(BamHI)

6.4.4 NtERL1hp(15776bp;Kan/Spec);Schumacher,2009
0

NtERL1sense
spacer

NtERL1antisense

35S

Restrictionsites:960(PstI),963(NotI),1026(PstI),1761(EcoRI),2410,2823&2956 (BamHI),3021(PstI),3023&3672(EcoRI),3684(HindIII),3713(EcoRI),5072(NotI), 5563(BamHI),5728&8266(PstI)

184

6.Supplements
6.5 Sequences
6.5.1 Newlyidentifiedsequences
>N.benthamiana_ERL1_cDNA(incomplete) GCCCTTAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCC TCAAGGGAGCCGTTTAATTCCAATGGCTACGGGATTTTGTAGGGTCCCCTTGCTGCGGCGGT TCCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGT ATCTCCGCCTCTCGTTCTACCACCGAAGAATCTACTTCTTCCCTAATTCAGCCCACACCTTC CCGTACCCGTTGGAAGCCAACGTGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATG ATCCTATGCATATTGACAAGTTTAATCATAATTGCTCCCTTGAGCTTATGCAAAATGCTGCG GGACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAA AGTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCAACT TTTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAA GGGAAATATGGAAAGCTAGGAGTTGATCGCGTCTGGCATGATACAGCTATCCCATTTGGAGA AGTTATCGAGCAGTTTGAAGTTTGGCTGGGGGAACGTCAATTGTGGAGAAATGAACCGGGCG GCTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGACTAAAGTTCCT CAGCAATGCAAAGTAGCAGGGACGAAATTGCCACCGTATTTCATGGAATGGATTAATTTGAA GGATGTGTTTTTGAACTTCTACAAGAGGAGGGCCAAAGGAATGCTTTCAATGATGAGGGAAC TCCAGATGCCTTTGTTAGGGAGTCATCACCTTGGAATAGATGATGCAAAAAACATAGCAAGA GTACTGCAACACATGCTTAGTGATGGTGCCCTTGTGCAAATCACAGCTAGAAGAAACCCTCA TTCTCCTGAAAAAGTTGAATATCTTTTTGAGGATCGCATTGTATAACTAGTTTCTTCTGAAC CATTTTGTTATCACCTAAACATTTTTAGAAAAAAAAAAAAAAA >N.benthamiana_ERL1_protein(incomplete) SSHKTRHRILHFFFRKLTPQGSRLIPMATGFCRVPLLRRFLVSPPVLPFSYSLQPSRKISIS ASRSTTEESTSSLIQPTPSRTRWKPTCLYFTQGKCTKMDDPMHIDKFNHNCSLELMQNAAGL KNLRQQELEYFLVLDLEGKVEILEFPVLLFDAKTMDVVNFFHRFVRPTKMHEDRINEYIEGK YGKLGVDRVWHDTAIPFGEVIEQFEVWLGERQLWRNEPGGCLNKAAFVTCGNWDLKTKVPQQ CKVAGTKLPPYFMEWINLKDVFLNFYKRRAKGMLSMMRELQMPLLGSHHLGIDDAKNIARVL QHMLSDGALVQITARRNPHSPEKVEYLFEDRIV >A.thaliana_ERL1_leader_cDNA ATGGCGTCCGCATTCTCTGCATTTAGGGTTTCGTTGTCCAGAATCAGTCCTTTCCGTGATAC CCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAATCATGTGCAACTCTT CGCATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTTCT TCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGT >A.thaliana_ERL1_leader_protein MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSS SSPSTFSLMETS >A.thaliana_ERL1_mature_cDNA GAAAATGCAAGGTGGAGACCCATGTGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGA TGATCCTGCCCATTTGGAGATTTTTAACCACGATTGTTCAAAGGAACTTCGAGTGGCTGCTG CTGATCTTGAGAGAAAGAAGTCACAAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGA AAAGTTGAGATTCTTGAGTTTCCTATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGA CTTATTCCACAGGTTTGTAAGACCCACCAAAATGAGCGAGCAAGCAATTAACAAATACATCG AAGGCAAGTATGGGGAACTCGGGGTTGATCGTGTGTGGCATGACACAGCTATTCCATTTAAG CAAGTTGTTGAGGAGTTTGAAGTTTGGTTAGCTGAGCATGACTTGTGGGATAAAGATACAGA TTGGGGTCTGAACGATGCAGCTTTTGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTC CTGAGCAATGCGTAGTTTCAAACATCAATCTTCCGCCATATTTTATGGAGTGGATCAATCTC AAAGACGTCTACTTGAATTTCTATGGCCGTGAGGCAAGAGGAATGGTGTCAATGATGAGGCA
185

6.Supplements
GTGTGGAATAAAACTCATGGGAAGCCACCATCTGGGCATTGATGACACAAAGAACATCACGA GGGTGGTGCAACGGATGCTCTCAGAAGGTGCAGTTCTCAAGCTCACAGCTCGAAGGAGCAAA TCCAATATGAGAAACGTCGAGTTTCTCTTCAAGAACAGGATCAAGTAA >A.thaliana_ERL1_mature_protein SENARWRPMCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQEFNFFLVIDLE GKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGVDRVWHDTAIPF KQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNINLPPYFMEWIN LKDVYLNFYGREARGMVSMMRQCGIKLMGSHHLGIDDTKNITRVVQRMLSEGAVLKLTARRS KSNMRNVEFLFKNRIK

6.5.2 Publishedsequencesusedforinsilicoanalysisandprimerdesign
>C.elegans_ERI1_protein(NCBIRef.Seq.NP_741293.1) MSADEPSPEDEKYLESLRDLLKISQEFDASNAKQNDEPEKTAVEVESAETRTDESEKSIDIP REQQLLPSERVEPLKSMVEPEYVKKVIRQMDTMTAEQLKQALMKIKVSTGGNKKTLRKRVAQ YYRKENALLNRKMEPNADKTARFFDYLIAIDFECTCVEIIYDYPHEIIELPAVLIDVREMKI ISEFRTYVRPVRNPKLSEFCMQFTKIAQETVDAAPYFREALQRLYTWMRKFNLGQKNSRFAF VTDGPHDMWKFMQFQCLLSNIRMPHMFRSFINIKKTFKEKFNGLIKGNGKSGIENMLERLDL SFVGNKHSGLDDATNIAAIAIQMMKLKIELRINQKCSYKENQRSAARKDEERELEDAANVDL TSVDISRRDFQLWMRRLPLKLSSVTRREFINEEYLDCDSCDDLTDDKVKHLHSCDIYEIFDE KTSASFTDSKCLIC >H.sapiens_3hExo_protein(NCBIRef.Seq.NP_699163.2) MEDPQSKEPAGEAVALALLESPRPEGGEEPPRPSPEETQQCKFDGQETKGSKFITSSASDFS DPVYKEIAITNGCINRMSKEELRAKLSEFKLETRGVKDVLKKRLKNYYKKQKLMLKESNFAD SYYDYICIIDFEATCEEGNPPEFVHEIIEFPVVLLNTHTLEIEDTFQQYVRPEINTQLSDFC ISLTGITQDQVDRADTFPQVLKKVIDWMKLKELGTKYKYSLLTDGSWDMSKFLNIQCQLSRL KYPPFAK >M.musculus_Eri1_protein(NCBIRef.Seq.Q7TMF2.2) MEDERGRERGGDAAQQKTPRPECEESRPLSVEKKQRCRLDGKETDGSKFISSNGSDFSDPVY KEIAMTNGCINRMSKEELRAKLSEFKLETRGVKDVLKKRLKNYYKKQKLMLKESSAGDSYYD YICIIDFEATCEEGNPAEFLHEIIEFPVVLLNTHTLEIEDTFQQYVRPEVNAQLSEFCIGLT GITQDQVDRADAFPQVLKKVIEWMKSKELGTKYKYCILTDGSWDMSKFLSIQCRLSRLKHPA FAKKWINIRKSYGNFYKVPRSQTKLTIMLEKLGMDYDGRPHSGLDDSKNIARIAVRMLQDGC ELRINEKILGGQLMSVSSSLPVEGAPAPQMPHSRK >D.rerio_Eri1_protein(NCBIRef.Seq.NP_001018450.1) METKEKSRKPPNKTPQSEGDQEDQPCPDTSCEKNEDQEPSSPKQGEFSDPVYKEIALANGAI NRMNREELRAKCTELKLDTRGVNDVLRKRLKSYYKKQKLMHSPAAEGNSDMYFDYICVVDFE ATCEENNPPDYLHEIIEFPMVLIDTHTLEIVDSFQEYVKPVLHPQLSEFCVKLTGITQEMVD EAKTFHQVLKRAISWLQEKELGTKYKYMFLTDGSWDMGKFLHTQCKLSRIRYPQFARKWINI RKSYGNFYKVPRTQTKLICMLENLGMEYDGRPHCGLDDSRNIARIAIHMLKDGCQLRVNECL HSGEPRSVPISAPIEGAPAPQPPKKRD >X.laevis_Eri1_protein(NCBIRef.Seq.NP_001089554.1) MEEQKENRPLDTEDSVVEEDLCKKLSRNLDLVGVKQRCRFDGQEDNGTSTVSSNTSDFSDPV YKEIAIANGCVNRMTKDELKAKLVEHKLDTRGVKDVLRKRLKNYYKKQKLTHALHKDSNTDC YYDYICVIDFEATCEAGNSLDYPHEIIEFPIVLLNTHTLEIEDVFQCYVRPEINPQLSEFCV NLTGITQDTVDKSDTFPNVLRSVVEWMREKELGSKYKYAILTDGSWDMSKFLNMQCRISRLK YPRFAKKWINIRKSYGNFYKVPRTQTKLTTMLEKLGMTYNGRLHSGLDDSKNIARIAAHMLQ DGCELRVNERMHAGQLMTVSSSLPFEGAPVPQNPHLKN
186

6.Supplements
>D.melanogaster_Snp_protein(NCBIRef.Seq.NP_611632.3) MALIKLARQLGLIDTIYVDGARPDPNNDPEESFNEDEVTEANSVPAKSKKSRKSKRLAMQPY SYVIAVDFEATCWEKQAPPEWREAEIIEFPAVLVNLKTGKIEAEFHQYILPFESPRLSAYCT ELTGIQQKTVDSGMPLRTAIVMFNEWLRNEMRARNLTLPKMNKSNILGNCAFVTWTDWDFGI CLAKECSRKGIRKPAYFNQWIDVRAIYRSWYKYRPCNFTDALSHVGLAFEGKAHSGIDDAKN LGALMCKMVRDGALFSITKDLTPYQQLNPRFVL >S.pombe_Eri1_protein(NCBIRef.Seq.XP_001713129.1) MESPVQILVWPFPCDEMNQKTPSTVEEIRIALQELGLSTNGNKRYLLIVDVEATCEEGCGFS FENEIIELPCLLFDLIEKSIIDEFHSYVRPSMNPTLSDYCKSLTGIQQCTVDKAPIFSDVLE ELFIFLRKHSNILVPSVDEIEIIEPLKSVPRTQPKNWAWACDGPWDMASFLAKQFKYDKMPI PDWIKGPFVDIRSFYKDVYRVPRTNINGMLEHWGLQFEGSEHRGIDDARNLSRIVKKMCSEN VEFECNRWWMEYEKNGWIPNRSYPPYFAS >N.crassa_Qip_protein(GenBank:ABQ45366.1) MEDEQFMQQLRNLTCQTVDWSGFDPSKWRDEDINDWDISDDAQGDEDDNYASDASILSARHL DPFNVKPATRPHHTGPTSLRIEDVTDEQEEYRDASDLENISWPEVSVEQGEIDPITELFTPW RMVLEYPNLFVGKRNGARARPLFTLESLHENRIWDLFYLYRPSNEGNNNPLIFVPTYQMQHL LDVINRKLDVEFTFPRGHQDMFAMPFGQSNTAKPRFLGRSRSAEEWKQLTNNVPARKPGDTS ENAPFLAKQELTRRLNSIFSIQDKSKKTKNNQYKRSNLHRAWGKNIKRVQRYLGLRRRVLSD PEVSSYTPLDLTQPTGIQPEKSVVFVAIDLEAYELDQSIITEVGLAILDTAEITNVAPGEGS KNWFDFIKARHIRVKEFSWAQNSRHVQGRAEYFDFGESEFIEVAKIASVLKETIEGESSIGG EGAKRPVVLVFHDQSQDLKYIRMLGYDVASADNILEVVDTREMYQYLSRSNNASKLSNVCGY LDIPWKNMHNAGNDAVYTLQAMMGLAIDMRQKSLERAAAKASKANTSNDGYVTYSEFTATKE DVDEGWISTGELSDGGEPSLVMAASTVPNSVVETTVCENWEL >V.vinifera_ERL1_protein(NCBIRef.Seq.XP_002282697.1) MAFYRVSPFRYGSLSSLIPYVSSPSSLSPPVRTFTLSASISTPHPSPPSLLTASPKASDRWR PMCLYYTQGKCTKMDDPTHLETFNHNCSRELQVNAANFQHLQSQHLDFFLVLDLEGKIEILE FPVLMINAKTMDVVDLFHRFVRPSEMSEQRINEYIEGKYGKLGVDRVWHDTSIPFKEVIQQF EAWLTQHHLWTKEMGGRLDQAAFVTCGNWDLKTKVPQQCKVSKMKLPPYFMEWINLKDVYLN FYKRRATGMMTMMKELQIPLLGSHHLGIDDTKNIARVLQRMLADGALLQITARRNADSPENV EFLFKNRIR >O.sativa_ERL1_protein(NCBIRef.Seq.NM_001187847.1) MALARVSPPAFSSPFLIHSLLRPFSSPSSVLRPRVTRVPHHRGFAIAAALSQASPLPSADGD GAVMEAPPRPSSRRPWKPTCLYYTQGKCTMLNDTLHLEKFNHNLPTDLPVNYSAADKVKSQK LDYFLVLDLEGKVEILEFPVVMIDAQSMEFVDSFHRFVHPTAMSEQRIREYIEGKYGKFGVD RVWHDTAIPFMEVLQEFEDWIEHHKFWKKEQGGALNSAAFITWFRILVEQELWKILEVRVD >S.bicolor_ERL1_protein(NCBIRef.Seq.XP_002436970.1) SAASSATVRASGSVGCHMNDAMHLEKFGHNLKMDLPVNASATDKFKPQKLEYFLILDLEGRV EILEFPVVMIDAQSTEFIDSFHRFVRPTAMSEQRTTEYIEGKYGKFGVDRVWHDTAVPFKEV LQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKATGMMTMMRELQLPIIGNHHLGID DSKNIARVVQRMIADGAVIEITAKRQSTTGNVKFLFKDRIR >Z.mays_ERL1_protein(NCBIRef.Seq.NP_001131746.1) MALARVSPSSLANLIPPLLQSFFRPFSSDFPIRNSRRRSSPVAAAFSLTSQSAHAAREGLVM EAPRPSSRYPWKPTCLYYTQGKCTMMNDAMHLEKFSHNLKMDLPVNASATDKSKPQKLEYLL ILDLEGRVEILEFPVMMIDAQNREFVDSFHRFVRPTAMSEQRTTEYIEGKYGKFGVDRVWHD TATPFKQVLQEFEDWLGNHNLWKKEQGGSLNRGAFVTCGNWDLKTKVPEQCKVSKINLPTYF MEWINLKDIYLNFYNRRATGMMTMMRELQLPIVGNHHLGIDDSKNIARVVQRMLADGAVIQI TAKRQSATGDVRFLFKDRIR
187

6.Supplements
>P.trichocarpa_ERL1_protein(NCBIRef.Seq.AC217034.1) MCLYHTHGKCTKIDDPVHVERFNHDCSRDFQVSAADFERKRPQDFDFFLVFDLEGKVEILEF PVLIIDAKTMGVVDLFHRFVRPTAMSEERVNEYIYNKYGKFGVDRVWHDTALPFNEVLQQFE SWLTQHNLWEKTRGGRLNRAAFVTCGNWDVKTQVPHQCSVSKLKLPPYFMEWINLKDVYQNF YNPRNEARGMRTMMSQLKIPMVGSHHLGLDDTKNIARVLLRMLADGAVLPITARRKPESPGS VNFLYKNRI >P.trichocarpa_putativeERL1_chloroplast_leader,translated(NCBIRef.Seq. AC217034.1:3208432329) MSFPRIPLSRVPSYLHNSNNCFHLLHPPFIPVSKTPSLPTYQTARTYTDFNSQTQTQPPLSL PSLIPSPPVNNPNATHRWKP >A.thaliana_ERL1_cDNA(NCBIRef.Seq.NM_112377.1) AGTTCCCAGTCCCTGTACTCGAAAGGAAGATCTTCATCTTCAATCTTCATGCTAATCGACGA AAATGGCGTCCGCATTCTCTGCATTTAGGGTTTCGTTGTCCAGAATCAGTCCTTTCCGTGAT ACCCGGTTCTCTTATCCCGCCACGTTGGCTTTAGCTCATACCAAACGAATCATGTGCAACTC TTCGCATTCTGTATCTCCATCTCCTTCTCCCTCTGACTTTTCTTCTTCTTCTTCTTCTTCTT CTTCTTCTCCTTCTACTTTTTCGTTAATGGAAACAAGTGAAAATGCAAGGTGGAGACCCATG TGCTTGTATTACACCCACGGAAAGTGTACAAAGATGGATGATCCTGCCCATTTGGAGATTTT TAACCACGATTGTTCAAAGGAACTTCGAGTGGCTGCTGCTGATCTTGAGAGAAAGAAGTCAC AAGAATTCAATTTTTTCTTGGTTATTGACTTGGAAGGAAAAGTTGAGATTCTTGAGTTTCCT ATTTTGATCGTAGATGCCAAAACCATGGAAGTCGTAGACTTATTCCACAGGTTTGTAAGACC CACCAAAATGAGCGAGCAAGCAATTAACAAATACATCGAAGGCAAGTATGGGGAACTCGGGG TTGATCGTGTGTGGCATGACACAGCTATTCCATTTAAGCAAGTTGTTGAGGAGTTTGAAGTT TGGTTAGCTGAGCATGACTTGTGGGATAAAGATACAGATTGGGGTCTGAACGATGCAGCTTT TGTAACCTGTGGAAACTGGGATATAAAGACAAAGATTCCTGAGCAATGCGTAGTTTCAAACA TCAATCTTCCGCCATATTTTATGGAGTGGATCAATCTCAAAGACGTCTACTTGAATTTCTAT GGCCGTGAGGCAAGAGGAATGGTGTCAATGATGAGGCAGTGTGGAATAAAACTCATGGGAAG CCACCATCTGGGCATTGATGACACAAAGAACATCACGAGGGTGGTGCAACGGATGCTCTCAG AAGGTGCAGTTCTCAAGCTCACAGCTCGAAGGAGCAAATCCAATATGAGAAACGTCGAGTTT CTCTTCAAGAACAGGATCAAGTAAAGCTCTCAAGGAAAAATGACAAACCCAACAAGCTCCAT GATCCATAAATTAGTTACTTAGTCAAGTCTTTTGAGTATTAAGATGATAACTCTTAGAAGAC AATGTGGTTACGCCTAAATGTGATGTGGTGAGAAGGTTTTGTAGATTCACATGTTTCAGAGC TATGATATTTAGATTACG >A.thaliana_ERL1_protein(NCBIRef.Seq.NP_566502.1) MASAFSAFRVSLSRISPFRDTRFSYPATLALAHTKRIMCNSSHSVSPSPSPSDFSSSSSSSS SSPSTFSLMETSENARWRPMCLYYTHGKCTKMDDPAHLEIFNHDCSKELRVAAADLERKKSQ EFNFFLVIDLEGKVEILEFPILIVDAKTMEVVDLFHRFVRPTKMSEQAINKYIEGKYGELGV DRVWHDTAIPFKQVVEEFEVWLAEHDLWDKDTDWGLNDAAFVTCGNWDIKTKIPEQCVVSNI NLPPYFMEWINLKDVYLNFYGREARGMVSMMRQCGIKLMGSHHLGIDDTKNITRVVQRMLSE GAVLKLTARRSKSNMRNVEFLFKNRIK >N.tabacum_4.5S_rRNA(NCBIRef.Seq.NC_001879:109242109344) GAAGGTCACGGCGAGACGAGCCGTTTATCATTACGATAGGTGTCAAGTGGAAGTGCAGTGAT GTATGCAGCTGAGGCATCCTAACAGACCGGTAGACTTGAAC >N.tabacum_5S_rRNA(NCBIRef.Seq.NC_001879:109601109721) TATTCTGGTGTCCTAGGCGTAGAGGAACCACACCAATCCATCCCGAACTTGGTGGTTAAACT CTACTGCGGTGACGATACTGTAGGGGAGGTCCTGCGGAAAAATAGCTCGACGCCAGGAT >N.tabacum_5.8S_rRNA(GenBank:AJ492448.1)

188

6.Supplements
GCAACGGATATCTCGGCTCTCGCATCGATGAAGAACGTAGCGAAATGCGATACTTGGTGTGA ATTGCAGAATCCCGTGAACCATCGAGTCTTTGAACGCAAGTTGCGCCCGAAGCCATTAGGCC GAGGGCACGTCTGCCTGGGCGTCACGCATCGCGTCGCCCCCCGCAC >N.tabacum_16S_rRNA(NCBIRef.Seq.NC_001879:102762104252) TCTCATGGAGAGTTCGATCCTGGCTCAGGATGAACGCTGGCGGCATGCTTAACACATGCAAG TCGGACGGGAAGTGGTGTTTCCAGTGGCGGACGGGTGAGTAACGCGTAAGAACCTGCCCTTG GGAGGGGAACAACAGCTGGAAACGGCTGCTAATACCCCGTAGGCTGAGGAGCAAAAGGAGGA ATCCGCCCGAGGAGGGGCTCGCGTCTGATTAGCTAGTTGGTGAGGCAATAGCTTACCAAGGC GATGATCAGTAGCTGGTCCGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGAC TCCTACGGGAGGCAGCAGTGGGGAATTTTCCGCAATGGGCGAAAGCCTGACGGAGCAATGCC GCGTGGAGGTAGAAGGCCCACGGGTCGTGAACTTCTTTTCCCGGAGAAGAAGCAATGACGGT ATCTGGGGAATAAGCATCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGATGCAAG CGTTATCCGGAATGATTGGGCGTAAAGCGTCTGTAGGTGGCTTTTTAAGTCCGCCGTCAAAT CCCAGGGCTCAACCCTGGACAGGCGGTGGAAACTACCAAGCTGGAGTACGGTAGGGGCAGAG GGAATTTCCGGTGGAGCGGTGAAATGCGTAGAGATCGGAAAGAACACCAACGGCGAAAGCAC TCTGCTGGGCCGACACTGACACTGAGAGACGAAAGCTAGGGGAGCGAATGGGATTAGATACC CCAGTAGTCCTAGCCGTAAACGATGGATACTAGGCGCTGTGCGTATCGACCCGTGCAGTGCT GTAGCTAACGCGTTAAGTATCCCGCCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAA TTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAAAGCGAAGAACCT TACCAGGGCTTGACATGCCGCGAATCCTCTTGAAAGAGAGGGGTGCCTTCGGGAACGCGGAC ACAGGTGGTGCATGGCTGTCGTCAGCTCGTGCCGTAAGGTGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCGTGTTTAGTTGCCATCGTTGAGTTTGGAACCCTGAACAGACTGCCGGTGAT AAGCCGGAGGAAGGTGAGGATGACGTCAAGTCATCATGCCCCTTATGCCCTGGGCGACACAC GTGCTACAATGGCCGGGACAAAGGGTCGCGATCCCGCGAGGGTGAGCTAACCCCAAAAACCC GTCCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGCCGGAATCGCTAGTAATCGC CGGTCAGCCATACGGCGGTGAATTCGTTCCCGGGCCTTGTACACACCGCCCGTCACACTATG GGAGCTGGCCATGCCCGAAGTCGTTACCTTAACCGCAAGGAGGGGGATGCCGAAGGCAGGGC TAGTGACTGGAGTGAAGTCGTAACAAGGTAGCCGTACTGGAAGGTGCGGCTGGATCACCTCC TTT >N.tabacum_23S_rRNA(NCBIRef.Seq.NC_001879:106331109140) TTCAAACGAGGAAAGGCTTACGGTGGATACCTAGGCACCCAGAGACGAGGAAGGGCGTAGTA ATCGACGAAATGCTTCGGGGAGTTGAAAATAAGCATAGATCCGGAGATTCCCGAATAGGGCA ACCTTTCGAACTGCTGCTGAATCCATGGGCAGGCAAGAGACAACCTGGCGAACTGAAACATC TTAGTAGCCAGAGGAAAAGAAAGCAAAAGCGATTCCCGTAGTAGCGGCGAGCGAAATGGGAG CAGCCTAAACCGTGAAAACGGGGTTGTGGGAGAGCAATACAAGCGTCGTGCTGCTAGGCGAA GCAGCCCGAATGCTGCACCCTAGATGGCGAAAGTCCAGTAGCCGAAAGCATCACTAGCTTAT GCTCTGACCCGAGTAGCATGGGGCACGTGGAATCCCGTGTGAATCAGCAAGGACCACCTTGC AAGGCTAAATACTCCTGGGTGACCGATAGCGAAGTAGTACCGTGAGGGAAGGGTGAAAAGAA CCCCCATCGGGGAGTGAAATAGAACATGAAACCGTAAGCTCCCAAGCAGTGGGAGGAGCCAG GGCTCTGACCGCGTGCCTGTTGAAGAATGAGCCGGCGACTCATAGGCAGTGGCTTGGTTAAG GGAACCCACCGGAGCCGTAGCGAAAGCGAGTCTTCATAGGGCAATTGTCACTGCTTATGGAC CCGAACCTGGGTGATCTATCCATGACCAGGATGAAGCTTGGGTGAAACTAAGTGGAGGTCCG AACCGACTGATGTTGAAGAATCAGCGGATGAGTTGTGGTTAGGGGTGAAATGCCACTCGAAC CCAGAGCTAGCTGGTTCTCCCCGAAATGCGTTGAGGCGCAGCAGTTGACTGGACATCTAGGG GTAAAGCACTGTTTCGGTGCGGGCCGCGAGAGCGGTACCAAATCGAGGCAAACTCTGAATAC TAGATATGACCTCAAAATAACAGGGGTCAAGGTCGGCTAGTGAGACGATGGGGGATAAGCTT CATCGTCGAGAGGGAAACAGCCCGGATCACCAGCTAAGGCCCCTAAATGATCGCTCAGTGAT AAAGGAGGTAGGGGTGCAGAGACAGCCAGGAGGTTTGCCTAGAAGCAGCCACCCTTGAAAGA GTGCGTAATAGCTCACTGATCGAGCGCTCTTGCGCCGAAGATGAACGGGGCTAAGCGATCTG CCGAAGCTGTGGGATGTAAAAATACATCGGTAGGGGAGCGTTCCGCCTTAGAGAGAAGCCTC CGCGCGAGCGGTGGTGGACGAAGCGGAAGCGAGAATGTCGGCTTGAGTAACGCAAACATTGG
189

6.Supplements
TGAGAATCCAATGCCCCGAAAACCTAAGGGTTCCTCCGCAAGGTTCGTCCACGGAGGGTGAG TCAGGGCCTAAGATCAGGCCGAAAGGCGTAGTCGATGGACAACAGGTGAATATTCCTGTACT GCCCCTTGTTGGTCCCGAGGGACGGAGGAGGCTAGGTTAGCCGAAAGATGGTTATCGGTTCA AGAACGTAAGGTGTCCCTGCTTTGTCAGGGTAAGAAGGGGTAGAGAAAATGCCTCGAGCCAA TGTTCGAATACCAGGCGCTACGGCGCTGAAGTAACCCATGCCATACTCCCAGGAAAAGCTCG AACGACTTTGAGCAAGAGGGTACCTGTACCCGAAACCGACACAGGTGGGTAGGTAGAGAATA CCTAGGGGCGCGAGACAACTCTCTCTAAGGAACTCGGCAAAATAGCCCCGTAACTTCGGGAG AAGGGGTGCCTCCTCACAAAGGGGGTCGCAGTGACCAGGCCCGGGCGACTGTTTACCAAAAA CACAGGTCTCCGCAAAGTCGTAAGACCATGTATGGGGGCTGACGCCTGCCCAGTGCCGGAAG GTCAAGGAAGTTGGTGACCTGATGACAGGGGAGCCGGCGACCGAAGCCCCGGTGAACGGCGG CCGTAACTATAACGGTCCTAAGGTAGCGAAATTCCTTGTCGGGTAAGTTCCGACCCGCACGA AAGGCGTAACGATCTGGGCACTGTCTCGGAGAGAGGCTCGGTGAAATAGACATGTCTGTGAA GATGCGGACTACCTGCACCTGGACAGAAAGACCCTATGAAGCTTCACTGTTCCCTGGGATTG GCTTTGGGCCTTTCCTGCGCAGCTTAGGTGGAAGGCGAAGAAGGCCTCCTTCCGGGGGGGCC CGAGCCATCAGTGAGATACCACTCTGGAAGGGCTAGAATTCTAACCTTGTGTCAGGACCTAC GGGCCAAGGGACAGTCTCAGGTAGACAGTTTCTATGGGGCGTAGGCCTCCCAAAAGGTAACG GAGGCGTGCAAAGGTTTCCTCGGGCCGGACGGAGATTGGCCCTCGAGTGCAAAGGCAGAAGG GAGCTTGACTGCAAGACCCACCCGTCGAGCAGGGACGAAAGTCGGCCTTAGTGATCCGACGG TGCCGAGTGGAAGGGCCGTCGCTCAACGGATAAAAGTTACTCTAGGGATAACAGGCTGATCT TCCCCAAGAGCTCACATCGACGGGAAGGTTTGGCACCTCGATGTCGGCTCTTCGCCACCTGG GGCTGTAGTATGTTCCAAGGGTTGGGCTGTTCGCCCATTAAAGCGGTACGTGAGCTGGGTTC AGAACGTCGTGAGACAGTTCGGTCCATATCCGGTGTGGGCGTTAGAGCATTGAGAGGACCTT TCCCTAGTACGAGAGGACCGGGAAGGACGCACCTCTGGTGTACCAGTTATCGTGCCCACGGT AAACGCTGGGTAGCCAAGTGCGGAGCGGATAACTGCTGAAAGCATCTAAGTAGTAAGCCCAC CCCAAGATGAGTGCTCTCCT >N.tabacum_similartoUniRef100_A7P1T2Cluster:Chromosomechr19scaffold_4, tobacco(EB681897;KP1B.105O08F.060116T7) GACTCAGCAGTCACAAGACTCGCCACCGTATCCTTCATTTCTTCTTCAGGAAACTCACTCCT CAAAGGAGCCGTTTAATTCCAATGGCTACGGGATTTTGTAGGGTCCCCTTGCTGCGGCGGTT CCTTGTATCTCCGCCGGTACTACCTTTTTCGTACTCACTTCAGCCCAGCCGTAAAATCAGTA TCTCCGCCTCTCTTTCTACCACCGAAGAATCTACTCCTTCCCTAATTCAGCCCACAACTTCC CGTACCCGTTGGAAGCCAACATGTCTCTATTTTACTCAAGGTAAGTGCACTAAGATGGATGA TCCTACGCATATTGACAAGTTTAATCATAGTTGCTCCCTTGAGCTTATGCAAAATGTTGCGG GACTTAAGAATTTGCGGCAGCAGGAGTTGGAATACTTTTTGGTGCTTGATTTGGAGGGTAAA GTTGAGATTCTTGAGTTTCCAGTTCTCCTATTTGATGCCAAAACCATGGACGTCGTCGAGTT TTTCCATAGGTTTGTGAGGCCGACAAAAATGCATGAAGACAGAATAAATGAATATATAGAAG GGAAATATGGAAAGCTAGGAGTTGATCGCGTCTAACATGATACAGCTATCCCATTTGGAGAA GTTATCGAGCAGTTTGAAGTTTGGCTGGGTGAACGTCAATTGTGGAGAAATGAACTGGGCGG CTGTCTAAATAAAGCTGCCTTTGTTACTTGTGGGAACTGGGATCTGAAGA >N.tabacum_similartoUniRef100_A7P1T2Cluster:Chromosomechr19scaffold_4 (BP529372;BY14266) CTTCTTCAGGAAAACTCATACCTCAGAGAGCCGTTCAATTCCAATGGCTATGGGATTTTCTA GGGTCCCCTTGCTGCGGCGTTTCCTTGGTATCTCCTCCGGTACTACCTTCTTCGTACTCACT TCAGGCCCAACCGTAAAATGAATATCTCAGCCTCTCTTTCTACCACCGAAGAATCTACTTCT TCCCTAATTCAGCCCACACCTTCCCGTACCCGTTGGAAGCCAACGTGTCTCTACTTTACTCA AGGTAAGTGCACCAAGATGGATGATCCTATGCATATTGACAAGTTTAATCATAATTGCTCGC TGGAGTTTATGCAAAATGCTGCGGGACTTGAGAATTTGCGGAAGCAGGAGTTGGAATACTTT TTGGTGCTTGATTTGGAGGGTAAAGTTGAGATTCTTGAGTTTCCAGTTCTCCTCTTTGATGC TAAAACCATGGATGTGGTTGACTTGTTCCATAGGTTTGTGAGGCCAACAAAAATGCACGAAG AAAGAATAAACGAATATATAGAAGGGAAATATGGAAAACTAGGAGTTGATCGGTATGGTATC
190

6.Supplements
TAATTCAGTAGTTCGAAATACAATTCAGCAGTTGGAACTTAGCGCCTTTTGGTTCTATATGA AGAATTGCATG >N.tabacum_FtsHlikeproteinPftfprecursor(Pftf)mRNA,nucleargeneencoding chloroplastprotein,completecds(GenBank:AF117339.1) ATGGCTACTTCATCAGTATGCATAGCAGGAAATAGTTTGTCTACTCATAGAAGGCAGAAAGT TTTCAGGAAGGACATTTATGGCAGGAAAATTTTATTTTCCTCAAATCTTCCATCGTCTAGTA AAACATCGAGAATAGCTGTAAAAGCATCCCTTCAGCAAAGGCCAGATGAAGGAAGAAGAGGT TTTCTCAAATTATTGCTTGGAAATGTTGGGCTTGGAGTACCGGCTTTGTTAGGTGATGGAAA AGCCTACGCTGATGAGCAAGGTGTGTCTAACTCAAGGATGTCGTATTCTAGATTTTTGGAGT ATTTGGATAAGGATAGGGTGCAAAAAGTAGATTTGTTTGAAAACGGAACCATAGCTATTGTT GAGGCTATATCTCCAGAATTAGGAAACCGGGTTCAGAGGGTTCGGGTACAACTACCTGGGCT CAGCCAGGAACTCCTTCAGAAGTTGCGAGAAAAGAACATTGACTTTGCTGCTCACAATGCCC AAGAGGACTCGGGTTCTTTCCTATTCAACTTGATTGGGAATCTGGCATTCCCGCTTATTTTG ATTGGTGGTCTTTTCCTGCTATCAAGGCGGTCTCCCGGAGGAATGGGAGGTCCTGGTGGGCC TGGTAACCCATTAGCATTTGGTCAATCAAAGGCTAAATTCCAAATGGAGCCAAACACTGGTG TAACATTTGATGATGTTGCTGGTGTAGATGAAGCAAAACAAGATTTTATGGAGGTCGTAGAA TTTTTGAAGAAGCCCGAGAGGTTTACCGCAGTGGGGGCTCGTATTCCAAAAGGTGTTCTTCT TGTTGGTCCTCCTGGTACTGGGAAGACCCTGCTAGCAAAGGCAATTGCTGGTGAAGCGGGTG TTCCATTTTTCTCAATTTCAGGTTCAGAATTTGTTGAGATGTTTGTTGGTGTAGGAGCCTCT CGAGTCCGTGATCTTTTCAAGAAGGCCAAGGAAAATGCTCCCTGCATTGTATTTGTTGATGA AATTGATGCTGTTGGGCGGCAAAGAGGGACTGGAATTGGAGGAGGGAATGATGAAAGGGAAC AGACCCTGAACCAACTATTGACAGAAATGGACGGTTTCGAAGGAAATACTGGTATAATAGTT GTTGCGGCAACCAATCGTGCAGATATTCTTGACTCTGCTTTGCTGAGACCAGGGCGATTTGA TAGACAAGTAAGTGTGGATGTTCCAGATATCAAGGGAAGAACAGAGATCTTAAAGGTTCACG CGGGCAACAAGAAGTTCGATTCTGATGTTTCTCTTGAAGTTATAGCCATGAGGACACCCGGT TTTAGTGGTGCAGATCTTGCTAACCTCTTAAATGAAGCAGCCATTCTTGCTGGTCGGCGTGG TAAGACAGCAATCGCATCCAAAGAGATTGATGATTCAATTGATAGGATAGTGGCTGGAATGG AAGGAACAGTCATGACTGATGGCAAGAGCAAGAGTCTGGTGGCATACCACGAAGTTGGACAT GCCATCTGTGGAACTCTCACTCCAGGGCATGATGCTGTTCAAAAGGTCACATTAATCCCACG TGGTCAGGCAAAAGGTTTGACCTGGTTCATTCCTGCAGATGATCCAACCTTAATATCCAAGC AGCAACTCTTTGCTAGAATTGTCGGAGGACTTGGGGGAAGAGCTGCAGAGGAAGTTATCTTT GGTGAACCTGAGGTGACCACTGGTGCTGCAGGCGATTTGCAGCAGATCACCGGTTTGGCAAA ACAGATGGTTGTCACTTTTGGGATGTCTGAACTTGGCCCATGGTCACTCATGGATTCTTCTG CCCAAAGTGGTGATGTAATCATGAGAATGATGGCTAGGAATTCTATGTCAGAAAAGCTAGCT GAAGACATTGATGGTGCTGTGAAGAGGCTTTCAGACAGCGCATATGAGATTGCATTGACCCA TATCCGCAACAACCGTGAAGCAATTGATAAGATTGTGGAAGTCCTCCTTGAAAAGGAGACGA TGACCGGAGATGAATTCCGCGCTATTCTCTCAGAATTTGTTGAAATTCCTGCTGAAAACCGA GTTGCTCCTGTTGTACCTACCCCAGCAACTGTATAA >ATPdependentClpproteaseNicotianatabacumhomologue(NtGI:TC85451) CATTGAGAAAGATCCTGCGTTGGAGAGGAGGTTCCAACCAGTTAAAGTCCCTGAACCTACTG TGGATGAAACCATACAGATCTTGAAAGGGCTTCGGGAGAGATATGAGATTCATCACAAGCTC CGTTACACCGATGAGGCATTAGAAGCTGCTGCCCAGCTTTCTTATCAGTACATCAGTGACCG TTTTCTGCCTGATAAAGCAATTGATTTGATTGATGAAGCTGGTTCCCGTGTTAGACTACGCC ATGCACAGCTCCCTGAGGAAGCAAGAGAGCTCGAGAAAGAACTTCGTCAGATTACAAAGGAG AAAAATGAAGCTGTGCGAGGTCAAGATTTTGAAAAGGCGGGGGAACTGCGTGATAGAGAAAT GGATCTTAAGGCACAGATCTCAGCCCTGATAGACAAAAACAAAGAGATGAGCAAGGCTGAAT CCGAGGCTGGAGATACAGGTCCACTCGTTACAGAGGCAGATATTCAGCACATTGTGTCTTCA TGGACTGGCATCCCTGTCGAGAAGGTTTCAACAGATGAATCTGATCGCCTCTTAAAAATGGA AGAAACACTTCACACCAGAATCATTGGCCAGGATGAAGCTGTGAAAGCCATTAGTCGCGCTA TCCGACGTGCTCGTGTTGGGCTCAAGAATCCCAACCGACCTATTGCCAGTTTCATCTTTTCT GGTCCAACTGGTGTTGGGAAATCAGAACTGGCCAAGGCTTTAGCAGCGTACTACTTTGGTTC
191

6.Supplements
TGAAGAAGCAATGATCCGGCTTGATATGAGTGAGTTTATGGAGAGACACACTGTCTCTAAAC TCATTGGTTCACCCCCTGGTTATGTTGGTTACACTGAAGGTGGTCAACTGACTGAAGCTGTG AGGCGTCGACCTTACACTGTTGTGCTCTTTGATGAGATTGAGAAGGCTCATCCTGATGTCTT CAACATGATGCTTCAAATTCTTGAAGATGGAAGATTGACAGACAGCAAGGGCAGAACTGTCG ACTTCAAGAATACACTTCTCATCATGACATCGAATGTCGGAAGCAGTGTGATAGAGAAAGGA GGCCGTCGTATAGGTTTTGATCTAGATTATGACGAGAAGGATAGCAGTTACAACCGTATCAA GAGCTTGGTGACTGAGGAGTTGAAACAGTACTTTAGGCCAGAGTTCTTGAACAGATTGGATG AGATGATTGTATTCCGTCAGCTCACTAAGTTAGAGGTGAAGGAGATAGCTGATATCATGCTT AAGGAGGTCTTTGAGAGGTTGAAAAATAAGGAGATAGAACTTCAAGTGACGGAGAGGTTTAG AGACAGGGTGGTTGACGAAGGGTACAACCCAAGCTACGGAGCAAGACCGTTGAGGAGAGCTA TTATGAGACTGCTGGAAGACAGCATGGCCGAGAAGATGCTTGCAGGTGAGATCAAAGAAGGT GATTCAGTAATTGTGGACGTGGACTCTGATGGTAATGTGACCGTCCTCAATGGCACTAGCGG AACTCCCTCAGATCCAGCTCCTGAGCCTATCCCTGTGTAGATCCGCTCTGCTGCTTGCTTTA GCTCTGCAAATTTGTTGTTTGTAATGTTGCTTTCATTTGTCTTGGCCACTAAGCTCTCCTGG GGTTATGAAGCAACTTGTGAGTAATTTATGGGGTCATTGGGTGATGAAATTCTGCCAAGTTG AGAAGGGTAGCACCTCCATTATATCAGACCTTTCAGGATTACACACTATATACTGAATTTCT TTAAGATTGTAGTGACCTCGCAAGATAGTGTAAAATTGCGACGAAGACTATGTAGAACC >N.tabacum_mRNAforchloroplastRNApolymerase,rpoT3tomgene(GenBank: AJ416570.2) CTGGCTTCCACAGCTTCTTACTCTCCAAGTCCAACATCTCAATGGAGAACTCAAAAACTCCC AAAAAGATTCAATTTTTATGTCATTCACAATCAAGAATTTGGAAAATTATCCCAAAGTTCAT CACTTTCCACTTCTTCATTTCCCAAAACTCTTAAATTACCTGTAATTCATATGCCAATTAAT AATATTCAGTCCCAAACAACAGTGTGTGTATCCACTGATGAGAATCTTGAAGAATTGGTGAA TTTGCAGAAAATCCCAAATGGGTTTTTGAATAAAGAATCAAATAAGAGAGTTTTTATTCAAG ACCCACCTTGGGTTTCTTCACTTTTTATGAATAGTTTGTTTGTTAGAGCTAAACAGGTTCAA GGGGTGAGAAGGGAATTTAGGGAAATTGAGAGAAGGAGAAGGTATGCTATGTTGAGGAGGAG ACAAATAAAGGCGGAAACTGAGGCTTGGGAGCAAATGGTGGAGGAATATAGGGAGTTGGAGA GGGAAATGTGTGAGAAGAAACTAGCACCCAATTTGCCTTATGTTAAAAAGCTGTTATTGGGT TGGTTTGAGCCATTGAGACAAGCTATAGAGAAGGAGCAGAACGCTGAGACGACCGTGAAACA TAGGGCGGCGTTTGCGCCACATATTGATTCTTTACCTGCTGATAAAATGGCTGTGATTGTGA TGCATAAGTTGATGGGATTGTTGATGATGGGTGGTAAAGAAGAGAGATGTGTTCAGGTCGTC CAAGCTGCGGTGCAGATTGGCATGGCAGTCGAGAATGAAGTTAGGATTCATAATTTCTTGGA GAAAACAAAGAAACTCCAGAAACATATGACTGGAGCTCAAAGTCAAGAAGATATGAGTAAGG AGACAATGATTCTAAGGAAACGGGTCAAAAGCTTGATTAAAAGGAATCGAGTAGTTGAGGTG AGAAAGCTGATGAAAAGTGAAGAACCCGAGTCTTGGGGTCGGGATACACAGGCTAAGTTAGG ATGCCGGCTTTTAGAATTATTAACAGAAACAGCTTATGTGCAACGTCCAGTGGATCAGTCTG CTGATACTCCTCCTGATATTAGGCCTGCGTTCAGGCACGTATTCAAAATTGCTACAAGAGAT CCGGGGAAGAACATTGTCAAGAAGTATGGTGTCATTGAATGTGATCCATTGGTTGTTGCGGG AGTTGACAGAACAGTGAAACAGATGATGATTCCTTATGTGCCTATGTTGGTGCCACCCAAAA AATGGAGAGGGTATGACAAAGGCGGATACTTATTCTTGCCCTCCTATTTGATGCGCACTCAT GGATCTAGGAGGCAACAAGATGCTGTAAGAGGTGTTCCCACGAAACAAATGCAGCAAGTTTA TGAGGCCTTGGATACCTTAGGAAGCACTAAATGGAGAGTGAATAAAAGGATACTAAATGTGG TTGAGAGTATTTGGGCTGGAGGAGGAAATATTGCTGGCCTAGTGGATCGCAAAGATGTTCCC ATACCGGAGTTGAGCAACTCCGATGATATAATGGAAGTGAAAAAGTGGAAATGGAAAGTGAG AAAATCCAAGAAAATCAACCAAGAGTTGCATTCCCAAAGATGTGACACAGAGCTCAAGCTTT CAGTTGCTCGGAAATTGAAAGATGAGGAAGGATTTTATTATCCTCACAATCTTGATTTTCGA GGACGTGCATACCCTATGCATCCTCATTTGAATCACTTGAGCTCTGATCTCTGTCGAGGAAT CCTTGAATTTGCGGAAGGACGACCACTAGGAAAGTCAGGATTGCGTTGGCTGAAAATACATT TAGCAAGTCTTTATGCAGGGGGGGTAGAGAAGCTCTGCTACGATGCACGCCTTGCATTTGTA GAAAACCACATTCATGACATATTAGATTCAGCAAACAATCCTCTAAATGGAAATCGATGGTG GTTAAATGCTGAGGATCCTTTTCAGTGCTTAGCAGCTTGCATCAACCTATCAGAAGCTTTAA
192

6.Supplements
AAAGCTCGTCACCACATACTGTCATCTCCCATCTGCCTATTCATCAGGATGGTTCATGCAAT GGCCTACAGCACTATGCTGCTCTGGGGAGAGATAGCATGGAGGCCGCAGCCGTCAACTTAGT TGCTGGAGAGAAACCAGCTGATGTTTATACTGAAATTGCTCTGAGGGTTGATCATATTATCA GAGGAGATAGTATCAAGGACCCTGCAATTGATCCTAATGCTTTACTAGCCAAACTCCTAATT GACCAGGTTGACAGGAAATTGGTGAAGCAGACAGTAATGACCTCAGTGTATGGTGTTACCTA TGTCGGAGCACGTGAGCAAATCAAAAGAAGATTGGAGGAGAAGGGTCTTATCGATGATGATA GGCTGCTATTTACTGCATCTTGCTATGCTGCTAAAGTGACATTAGCTGCTTTGGGGGAGTTA TTTCAAGCGGCACGTGGCACAATGACTTGGCTTGGTGACTGTGCTAAGGTGATTGCTTTAGA AAATCAGCCAGTGCGATGGACGACACCACTGGGGCTCCCTGTTGTGCAGCCTTACTTTAAAA CTCAGCGGCATGTTATAAGAACTTCTCTTCAAATTTTGGCTTTGCAGCGCGAGGGTGATGCA GTTGAGGTCAGGAAACAGAGAACTGCTTTTCCTCCAAATTTCGTGCACTCACTTGATGGTTC GCATATGATGATGACTGCTGTTGCTTGTAGGGATGCTGGACTACAATTTGCAGGGGTACATG ATTCCTTCTGGACTCATGCATGTGATGTCGACCAGATGAACAGGATACTCCGCGAAAAGTTT GTGGAGCTGTACAGTTTGCCTATTCTTGAAGATTTGCTCGAAAACTTCCAGAAGTCATATCC AGCATTAACATTTCCTCCTCTACCAAAAAGAGGTGATTTCAATTTAAGGGAAGTTCTCGAGT CGCCCTACTTCTTTAACTGA >N.tabacum_ClpPprotease(ClpP)mRNA,chloroplastgeneencodingchloroplast protein,completecds(GenBank:U32397.1) ATGCCTATTGGTGTTCCAAAAGTCCCTTTCCGAAGTCCTGGAGAGGAAGATGCATCTTGGGT TGACGTATACAACCGACTTTATCGAGAAAGATTACTTTTTTTAGGCCAAGAGGTTGATAGCG AGATTTCGAATCAACTTATTGGTCTTATGGTATATCTCAGTATCGAGGATGAGACCAAAGAT CTGTATTTGTTTATAAACTCTCCTGGGGGCTGGGTAATACCTGGGGTGGCTATTTATGATAC TATGCAATTTGTGCGACCAGATGTCCATACAATATGCATGGGATTAGCCGCTTCAATGGGAT CTTTTATCCTGGTCGGAGGAGAAATTACCAAACGTCTAGCATTCCCTCACGCTAGGGTAATG ATCCATCAACCTGCTAGTTCTTTTTATGAGGCACAAACAGGCGAATTTGTCCTGGAAGCGGA AGAACTGCTGAAACTGCGTGAAACCCTCACAAGGGTTTATGTACAAAGAACGGGGAAACCCT TATGGGTTGTATCCGAAGATATGGAAAGAGATGTTTTTATGTCAGCAACAGAAGCCCAAGCT TATGGAATTGTTGATCTTGTAGCGGTTGAATGA >N.tabacum_chloroplastDNAforputativeRNApolymerasebetasubunit(ORF 1070)(GenBank:X12745.1) ATGCTCGGGGATGGAAATGAGGGAATATCTACAATACCTGGATTTAATCAGATACAATTTGA AGGATTTTGTAGGTTCATTGATCAAGGTTTGACGGAAGAACTTTATAAGTTTCCAAAAATTG AAGATACAGATCAAGAAATTGAATTTCAATTATTTGTGGAAACATATCAATTGGTCGAACCC TTGATAAAGGAAAGAGATGCTGTGTATGAATCACTCACATATTCTTCTGAATTATATGTATC CGCGGGATTAATTTGGAAAAACAGTAGGGATATGCAAGAACAAACAATTTTTATCGGAAACA TTCCTCTAATGAATTCCCTGGGAACTTCTATAGTCAATGGAATATATAGAATTGTGATCAAT CAAATATTGCAAAGTCCCGGTATTTATTACCGATCAGAATTGGACCATAACGGAATTTCGGT CTATACCGGCACCATAATATCAGATTGGGGAGGAAGATCAGAATTAGAAATTGATAGAAAAG CAAGGATATGGGCTCGTGTAAGTAGGAAACAAAAAATATCTATTCTAGTTCTATCATCAGCT ATGGGTTTGAATCTAAGAGAAATTCTAGAGAATGTTTGCTATCCTGAAATTTTTTTGTCTTT TCTGAGTGATAAGGAGAGAAAAAAAATTGGGTCAAAAGAAAATGCCATTTTGGAGTTTTATC AACAATTTGCTTGTGTAGGTGGCGATCCGGTATTTTCTGAATCCTTATGTAAGGAATTACAA AAGAAATTCTTTCAACAAAGATGTGAATTAGGAAGGATTGGTCGACGAAATATGAACCGAAG ACTGAACCTTGATATACCCCAGAACAATACATTTTTGTTACCACGAGATATATTGGCAGCCG CCGATCATTTGATTGGGCTGAAATTTGGAATGGGTGCACTTGACGATATGAATCATTTGAAA AATAAACGTATTCGTTCTGTAGCAGATCTTTTACAAGATCAATTCGGATTGGCTCTGGTTCG TTTAGAAAATGTGGTTCGGGGGACTATATGTGGAGCAATTCGGCATAAATTGATACCGACAC CTCAGAATTTGGTAACCTCAACTCCATTAACAACTACTTATGAATCCTTTTTCGGTTTACAC CCATTATCTCAAGTTTTGGATCGAACTAATCCATTGACACAAATAGTTCATGGGAGAAAATT AAGTTATTTGGGCCCTGGAGGACTGACAGGGCGCACTGCTAGTTTTCGGATACGAGATATCC
193

6.Supplements
ATCCTAGTCACTATGGACGTATTTGCCCAATTGACACATCTGAAGGAATCAATGTTGGACTT ATTGGATCCTTAGCAATTCATGCGAGGATTGGTCATTGGGGATCTCTAGAAAGCCCTTTTTA TGAAATTTCTGAGAGGTCAACCGGGGTACGGATGCTTTATTTATCACCAGGTAGAGATGAAT ACTATATGGTAGCGGCAGGAAATTCTTTAGCCTTAAATCAGGATATTCAGGAAGAACAGGTT GTTCCAGCTCGATACCGTCAAGAATTCTTGACTATTGCATGGGAACAGGTTCATCTTCGAAG TATTTTTCCTTTTCAATATTTTTCTATTGGAGCTTCCCTCATTCCTTTTATCGAACATAATG ATGCGAATCGAGCTTTAATGAGTTCTAATATGCAACGTCAAGCAGTTCCTCTTTCTCGCTCC GAGAAATGCATTGTTGGAACTGGGTTGGAACGACAAGCAGCTCTAGATTCGGGGGCTCTTGC TATAGCCGAACGCGAGGGAAGGGTCGTTTATACCAATACTGACAAGATTCTTTTAGCAGGTA ATGGAGATATTCTAAGCATTCCATTAGTTATATATCAACGTTCCAATAAAAATACTTGTATG CATCAAAAACTCCAGGTTCCTCGGGGTAAATGCATTAAAAAGGGACAAATTTTAGCGGATGG TGCTGCTACGGTTGGTGGCGAACTTGCTTTGGGGAAAAACGTATTAGTAGCTTATATGCCGT GGGAGGGTTACAATTCTGAAGATGCAGTACTTATTAGCGAGCGTTTGGTATATGAAGATATT TATACTTCTTTTCACATACGGAAATATGAAATTCAGACTCATGTGACAAGCCAAGGCCCTGA AAAAGTAACTAATGAAATACCGCATTTAGAAGCCCATTTACTCCGCAATTTAGATAAAAATG GAATTGTGATGCTGGGATCTTGGGTAGAGACAGGTGATATTTTAGTAGGTAAATTAACACCC CAGGTCGTGAAAGAATCGTCGTATGCCCCGGAAGATAGATTGTTACGAGCTATACTTGGTAT TCAGGTATCTACTTCAAAAGAAACTTGTCTAAAACTACCTATAGGTGGCAGGGGTCGGGTTA TTGATGTGAGGTGGATCCAGAAGAGGGGTGGTTCTAGTTATAATCCCGAAACGATTCGTGTA TATATTTTACAGAAACGTGAAATCAAAGTAGGCGATAAAGTAGCTGGAAGACACGGAAATAA AGGTATCATTTCCAAAATTTTGCCTAGACAAGATATGCCTTATTTACAAGATGGAAGATCCG TTGATATGGTCTTTAACCCATTAGGAGTACCTTCACGAATGAATGTAGGACAGATATTTGAA TGTTCACTAGGGTTAGCAGGGAGTCTGCTAGACAGACATTATCGAATAGCACCTTTTGATGA GAGATATGAACAAGAAGCTTCGAGAAAACTTGTGTTTTCTGAATTATATGAAGCCAGTAAGC AAACAGCGAATCCATGGGTATTTGAACCCGAATATCCAGGAAAAAGCAGAATATTTGATGGA AGGACGGGGAATCCTTTTGAACAACCCGTTATAATAGGAAAGCCTTATATCTTGAAATTAAT TCATCAAGTTGATGATAAAATCCATGGGCGCTCCAGTGGACATTATGCGCTTGTTACACAAC AACCCCTTAGAGGAAGAGCCAAACAGGGGGGACAGCGGGTAGGAGAAATGGAGGTTTGGGCT CTAGAAGGGTTTGGGGTTGCTCATATTTTACAAGAGATGCTTACTTATAAATCGGATCATAT TAGAGCTCGCCAGGAAGTACTTGGTACTACGATCATTGGGGGAACAATACCTAATCCCGAAG ATGCTCCAGAATCTTTTCGATTGCTCGTTCGAGAACTACGATCTTTAGCTCTGGAACTGAAT CATTTCCTTGTATCTGAGAAGAACTTCCAGATTAATAGGAAGGAAGCT >N.tabacum_couplingfactorbetasubunitgene,partialcds;andribulose1,5 biphosphatecarboxylase/oxygenaselargesubunitgene,completecds;chloroplast genesforchloroplastproducts(GenBank:J01450.1) ATGTCACCACAAACAGAGACTAAAGCAAGTGTTGGATTCAAAGCTGGTGTTAAAGAGTACAA ATTGACTTATTATACTCCTGAGTACCAAACCAAGGATACTGATATATTGGCAGCATTCCGAG TAACTCCTCAACCTGGAGTTCCACCTGAAGAAGCAGGGGCCGCGGTAGCTGCCGAATCTTCT ACTGGTACATGGACAACTGTATGGACCGATGGACTTACCAGCCTTGATCGTTACAAAGGGCG ATGCTACCGCATCGAGCGTGTTGTTGGAGAAAAAGATCAATATATTGCTTATGTAGCTTACC CTTTAGACCTTTTTGAAGAAGGTTCTGTTACCAACATGTTTACTTCCATTGTAGGTAACGTA TTTGGGTTCAAAGCCCTGCGCGCTCTACGTCTGGAAGATCTGCGAATCCCTCCTGCTTATGT TAAAACTTTCCAAGGTCCGCCTCATGGGATCCAAGTTGAAAGAGATAAATTGAACAAGTATG GTCGTCCCCTGTTGGGATGTACTATTAAACCTAAATTGGGGTTATCTGCTAAAAACTACGGT AGAGCCGTTTATGAATGTCTTCGCGGTGGACTTGATTTTACTAAAGATGATGAGAACGTGAA CTCACAACCATTTATGCGTTGGAGAGATCGTTTCTTATTTTGTGCCGAAGCACTTTATAAAG CACAGGCTGAAACAGGTGAAATCAAAGGGCATTACTTGAATGCTACTGCAGGTACATGCGAA GAAATGATCAAAAGAGCTGTATTTGCTAGAGAATTGGGCGTTCCGATCGTAATGCATGACTA CTTAACGGGGGGATTCACCGCAAATACTAGCTTGGCTCATTATTGCCGAGATAATGGTCTAC TTCTTCACATCCACCGTGCAATGCATGCGGTTATTGATAGACAGAAGAATCATGGTATCCAC TTCCGGGTATTAGCAAAAGCGTTACGTATGTCTGGTGGAGATCATATTCACTCTGGTACCGT
194

6.Supplements
AGTAGGTAAACTTGAAGGTGAAAGAGACATAACTTTGGGCTTTGTTGATTTACTGCGTGATG ATTTTGTTGAACAAGATCGAAGTCGCGGTATTTATTTCACTCAAGATTGGGTCTCTTTACCA GGTGTTCTACCCGAGGCTTCAGGAGGTATTCACGTTTGGCATATGCCTGCTCTGACCGAGAT CTTTGGGGATGATTCCGTACTACAGTTCGGTGGAGGAACTTTAGGACATCCTTGGGGTAATG CGCCAGGTGCCGTAGCTAATCGAGTAGCTCTAGAAGCATGTGTAAAAGCTCGTAATGAAGGA CGTGATCTTGCTCAGGAAGGTAATGAAATTATTCGCGAGGCTTGCAAATGGAGCCCGGAACT AGCTGCTGCTTGTGAAGTATGGAAAGAGATCGTATTTAATTTTGCAGCAGTGGACGTTTTGG ATAAGTAA >N.tabacum_chloroplastgeneP32forthylakoidmembraneprotein(GenBank: X00616.1) ATGACTGCAATTTTAGAGAGACGCGAAAGCGAAAGCCTATGGGGTCGCTTCTGTAACTGGAT AACTAGCACTGAAAACCGTCTTTACATTGGATGGTTTGGTGTTTTGATGATCCCTACCTTAT TGACGGCAACTTCTGTATTTATTATTGCCTTCATTGCTGCTCCTCCAGTAGACATTGATGGT ATTCGTGAACCTGTTTCAGGGTCTCTACTTTACGGAAACAATATTATTTCCGGTGCCATTAT TCCTACTTCTGCAGCTATAGGTTTACATTTTTACCCAATCTGGGAAGCGGCATCCGTTGATG AATGGTTATACAACGGTGGTCCTTATGAACTAATTGTTCTAGACTTCTTACTTGGCGTAGCT TGTTACATGGGTCGTGAGTGGGAGCTTAGTTTCCGTCTGGGTATGCGACCTTGGATTGCTGT TGCATATTCAGCTCCTGTTGCAGCTGCTACCGCAGTTTTCTTGATCTACCCAATTGGTCAAG GAAGTTTTTCTGATGGTATGCCTCTAGGAATCTCTGGTACTTTCAATTTCATGATTGTATTC CAGGCTGAGCACAACATCCTTATGCACCCATTTCACATGTTAGGCGTAGCTGGTGTATTCGG CGGCTCCCTATTCAGTGCTATGCATGGTTCCTTGGTAACTTCTAGTTTGATCAGGGAAACCA CAGAAAATGAATCTGCTAATGAAGGTTACAGATTCGGTCAAGAGGAAGAAACTTATAACATC GTAGCCGCTCATGGTTATTTTGGCCGATTGATCTTCCAATATGCTAGTTTCAACAACTCTCG TTCGTTACACTTCTTCCTAGCTGCTTGGCCTGTAGTAGGTATCTGGTTTACCGCTTTAGGTA TCAGCACTATGGCTTTCAACCTAAATGGTTTCAATTTCAACCAATCTGTAGTTGACAGTCAA GGCCGTGTAATTAATACTTGGGCTGATATCATTAACCGTGCTAACCTTGGTATGGAAGTTAT GCATGAACGTAATGCTCACAACTTCCCTCTAGACCTAGCTGCTATCGAAGCTCCATCTACAA ATGGATAA >TDNAvectorpDsLox,completesequence,bastaresistance:3195to3746 (AY836546.1) ATGAGCCCAGAACGACGCCCGGCCGACATCCGCCGTGCCACCGAGGCGGACATGCCGGCGGT CTGCACCATCGTCAACCACTACATCGAGACAAGCACGGTCAACTTCCGTACCGAGCCGCAGG AACCGCAGGAGTGGACGGACGACCTCGTCCGTCTGCGGGAGCGCTATCCCTGGCTCGTCGCC GAGGTGGACGGCGAGGTCGCCGGCATCGCCTACGCGGGCCCCTGGAAGGCACGCAACGCCTA CGACTGGACGGCCGAGTCAACCGTGTACGTCTCCCCCCGCCACCAGCGGACGGGACTGGGCT CCACGCTCTACACCCACCTGCTGAAGTCCCTGGAGGCACAGGGCTTCAAGAGCGTGGTCGCT GTCATCGGGCTGCCCAACGACCCGAGCGTGCGCATGCACGAGGCGCTCGGATATGCCCCCCG CGGCATGCTGCGGGCGGCCGGCTTCAAGCACGGGAACTGGCATGACGTGGGTTTCTGGCAGC TGGACTTCAGCCTGCCGGTACCGCCCCGTCCGGTCCTGCCCGTCACCGAGATTTGA

195

6.6 Curriculumvitae(March,2011)
PersonalInformation DIJuttaMariaHelm Bornthe11thofJuly,1981inGmunden,Austria Brcklweg20,4663Laakirchen,Austria Mobile:+4369910402842 EMail:Jutta.Helm@gmx.at Austriancitizenship,unmarried,nochildren Education DoctorateStudiesinAgriculturalSciencesattheUniversity ofNaturalResourcesandAppliedLifeSciencesinVienna, Austria;supervisedbyAo.Univ.Prof.DIDr.MarieTheres Hauser expectedend:2011 PhDthesis:TheputativeRNAsilencingproteinERL1is involvedinchloroplastribosomalRNAprocessingin plants,fundedbyaMarieCurieFellowshipattheInstitute ofMolecularBiologyandBiotechnology,FoundationofRe searchandTechnologyHellas(FoRTHIMBB)inHeraklion, Greece;supervisedbyAss.Prof.Dr.KritonKalantidis GraduationtoDiplomingenieurininFoodScienceand Biotechnology(comparabletoMSc)withexcellentsuccess Masterthesis:Characterizationofthemyeloperoxidase mutantMet243ThrproducedinCHOcells;supervisedby Ao.Univ.Prof.Mag.Dr.ChristianObinger Grade:excellent ExchangesemesterattheAristoteleioPanepistimioThessa lonikisinThessaloniki,Greece StudyofFoodScienceandBiotechnologywithemphasison biochemistryandcellbiologyattheUniversityofNatural ResourcesandAppliedLifeSciencesinVienna,Austria GCSEAlevelwithgoodsuccessattheBG/BRGGmunden, Austria

09/2006current

14thofOctober,2005

10/200302/2004 10/199910/2005

22thofJune,1999

196

6.Supplements
Workexperience EbewePharmaGesmbHNfgKG(SandozBusinessUnit OncologyInjectables)inUnterach,Austria RegulatoryAffairsManager:productexpert,regionalex pertCIS/SEE,regulatorymaintenance UniversityofNaturalResourcesandAppliedLifeSciences inVienna,Austria Teachingassignment:ExercisesinMolecularBiology(Eng lish) UniversityofNaturalResourcesandAppliedLifeSciences inVienna,Austria AssistantwithintheGENAUProject:Heritablestressef fectsinplants SCAGraphicSundsvallAB(OrtvikenPaperMillDevel opmentDepartment)inSundsvall,Sweden Evaluationandvalidationofaprotocolforthedetermina tionofCODreleaseduringthethermomechanicalpulping processanditsapplicationatdifferentrefinerloads. 03/200505/2005 UniversityofNaturalResourcesandAppliedLifeSciences inVienna,Austria Laboratorysupportinanalyticchemistryforundergradu atestudents. MerckLifeScience&Analytics(Research&Development Department)inDarmstadt,Germany Reflectometric,photometricandpotentiometricdetermina tionofwinequalityparametersandoptimizationofthe probepreparationfortheReflectoquantdeterminationof yeastusablenitrogen. SCALaakirchen(QualityAssurance)inLaakirchen,Austria Sewageandphysicalpaperanalysis. AustriaFrostNahrungsmittelGmbH(QualityAssurance)in GroEnzersdorf,Austria Chemicalanalysisoffoodrawmaterial,validationofcali brationfunctionsandassistanceinthemicrobiologicallabo ratory.

04/2010current

01/2010

04/200608/2006

06/200507/2005

07/200409/2004

08/2003

07/200208/2002

197

6.Supplements
Additionalskills Languages Methods German:mothertongue;English:excellent;Greek:very good;French,Spanish:good PCR,Cloning,DNA/RNA/ProteinExtraction,Generation oftransgenicplants,Agroinfiltration,Isola tion/Transformationofprotoplasts,realtimePCR,3 RACE,SDSPAGE,Southern/Northern/WesternBlotting, workingwithradioactivity,animalcellculture(CHOcells), liquidchromatography,FPLC,StoppedFlowSpectroscopy, FlowCytometry,ELISA,Confocal/Electron/Lightmicro scopy MSOffice2003&2007,AdobePhotoshopCS3,SAP,COI, eCTDManager,competentwithinternetapplications,ba sicsinSPSS A,B(owncaravailable) Studyinglanguagesandtravelling,toexperienceforeign culturesandhabits,mydogMavrodafni,sportsespecially bicycling,swimming,hikingandvolleyball,reading,play ingguitar

Software

Drivinglicenses Interests

Publications

HelmJM,SchumacherHT,VamvakaE,KalantidisK.AroleforplantERL1inrRNA biogenesisinpreparation HelmJM,DadamiE,KalantidisK.(2011)LocalRNAsilencingmediatedbyagroin filtration.RNAi&PlantGeneFunctionAnalysis.MethodsinMolecularBiology744 inpress KalantidisK,SchumacherHT,AlexiadisT,HelmJM.(2008).RNAsilencingmove mentinplants.BiolCell.100(1):1326 FurtmllerPG,ZederbauerM,JantschkoW,HelmJ,BognerM,JakopitschC,Obin gerC.(2006).Activesitestructureandcatalyticmechanismsofhumanperoxidases. ArchBiochemBiophys.445(2):199213

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