CaMVP6IsaNucleocytoplasmicProtein941
IsolationofTurnipNuclei
ProtoplastspreparedfromCaMV-infectedandnoninfectedturnipleaves(Kobayashietal.,1998)wereusedtoisolatenuclei.Approximately83106protoplastswerewashedtwiceinnucleibuffer(250mMsucrose,25mMMes,0.5mMEDTA,1mMMgCl2,1mMEGTA,pH5.5,andacompletecocktailofproteaseinhibitors[Roche,Indianapolis,IN]).Aftercentrifugationfor5minat100g,theprotoplastswereresuspendedin50mLofcoldnucleibuffercontaining1mMDTT,0.025%NonidetP-40,and1mMphenylmethylsulfonyl uorideandshakenslowlyfor20minat48C.Nucleiwereisolatedby lteringthesuspensionthrough50-mmmeshnylonandcollectedat48Cbycentrifugationfor5minat550g.Theywereresuspendedinthenucleibufferandcentrifugedat48Cthroughadiscontinuousgradientcomposedof18%Ficolland85%Percollfor15minat8000g.Thebandcontainingthenuclei,locatedbetweentheFicollandPercolllayers,wasdilutedthreefoldwith10mMPipes-KOH,pH7.0,andcentrifugedat600gduring10min.
FluorescenceAnalysis
FluorescentBY-2tobaccocells,transfectedwithEGFPoraproteinfusedtoEGFP,wereobservedbetweenaslideandcoverslipwithaZeissLSM510confocalmicroscope(Jena,Germany).EGFPwasviewedbyexcitationat488nmwithanargonlaserusinganappropriateemission ltertocollectthegreensignalfromtheopticalsection.Fluorescentcellswerealsoobservedunderthesameconditionsafterincubationfor8hat248CwithgentleshakingintheBY-2cellculturemediumcontaining100nMleptomycinB.
Forimmuno uorescencestudies,protoplastsornucleipreparedasdescribedabovewereharvestedand xedfor15minwithgentleshakinginprotoplastornuclei-speci cmedium,respectively,containing4%glutaraldehyde.Thereafter,theywerewashedthreetimeswiththeappropriatemedium,oncewiththemediumdilutedvolumetovolumewithPBS,thenagainwithPBSand nallyresuspendedinPBSbuffer.Asampleofprotoplastsornucleiwasmountedonapoly-L-Lys–coatedcoverslip,allowedtosettlefor1hatroomtemperature,andthentreatedovernightat48Cina0.1%sodiumborohydridesolution.Protoplastsandnucleiwereincubatedfor1hinablockingsolution(5%acetylatedBSA[Aurion,Wageningen,TheNetherlands],5%normalgoatserum,and0.1%coldwater shskingelatinpreparedinPBS)andthenovernightwiththepolyclonalanti-P6antibodies.Aftersixwasheswith0.1%BSAcinPBS,protoplastsornucleiweretreatedwithgoatanti-rabbitantibodiescoupledtoAlexa488(MolecularProbes,Eugene,OR),respectively,for12h.Afterremovalofexcesssecondaryantibodiesbysixwashesin0.1%BSAcinPBS,theprotoplastsandnucleiweresubsequentlyexaminedwithaZeissLSM510confocalmicroscope.
ACKNOWLEDGMENTS
WethankMarcBergdollforthethree-dimensionalmodelingofP6andJohnStanleyforprovidinguswiththeCaMVCabb-JIgenomese-quence.WearemostgratefultoChristianeGaraudandJe
´ro meMuttererforadviceonCLSMandtoKenRichardsforcriticalreadingofthemanuscript.TheInter-InstituteConfocalMicroscopyPlatformwas
co nancedbytheRe
´gionAlsace,CentreNationaldelaRechercheScienti que,theUniversite
´LouisPasteur,andtheAssociationdelaRecherchepourleCancer.ThisworkwassupportedbytheCentre
NationaldelaRechercheScienti queandbytheUniversite
´LouisPasteurofStrasbourg.
ReceivedNovember1,2004;acceptedDecember9,2004.
The Cauliflower mosaic virus (CaMV) open reading frame VI product (P6) is essential for the viral infection cycle. It controls translation reinitiation of the viral polycistronic RNAs and forms cytoplasmic inclusion bodies (viroplasms) where virus replicat
942ThePlantCell
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