The Open Reading Frame VI Product of Cauliflower mosaic viru(15)

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

Full-lengthP6andtheP6fragmentsA,A1,andA2expressedfrompETKaKSandpGEX-2TKvectors,respectively,werelabeledinthepresenceof[g-32P]ATP(3000Ci/mmole)andbovineheartmuscleproteinkinase(10units)for2hatroomtemperature,accordingtotheinstructionsofthemanufacturer(Sigma-Aldrich,St.Louis,MO).ExcessradioactiveATPwaseliminatedby ltrationthroughaSephadexG50orG25column(Amersham-PharmaciaBiotech)dependingonthemolecularmassofthelabeledfusionproteins.ProteinGelBlotAnalysis

ProteinsfromrecombinantbacteriawereseparatedbySDS-PAGEandelectrophoreticallytransferredontoanitrocellulosemembrane(SchleicherandSchuell,Dassel,Germany).Themembraneswereblockedovernightin5%nonfatdriedmilkinPBSbuffer(140mMNaCl,2.7mMKCl,and8.1mMNa2HPO4,pH7.3)containing0.1%Tween20andthenincubatedfor4hatroomtemperaturewithspeci crabbitorsheeppolyclonalantibodiesraisedagainstP6(1:10,000dilution)orGST(1:5,000dilution),respectively.ThemembraneswerewashedwithPBSbufferandtreatedwithgoatanti-rabbitIgGantibodies,respectively,conjugatedeithertoalkalinephosphataseorperoxidase,atthedilutionrecommendedbythemanufacturer.FarProteinGelBlotAssays

Aproteinblottingoverlaytechniquewasusedtodetectinteractionsbetweenproteins.ProteinswereresolvedbySDS-PAGEandtransferredontoanitrocellulosemembrane.Membraneswerewashedseveraltimesat48CinHMbuffer(10mMTris-HCl,pH7.5,100mMNaCl,and25mMMgCl2)containing5%nonfatdriedmilkandincubatedfor12hat48Cwithgentleshakinginthesamebuffercontainingthe[32P]-labeledproteinintheoverlay.AfterthreewashesinHMbuffer,themembranesweredriedandradioactivecomplexesweredetectedbyautoradiography.TransientExpressioninTobaccoBY-2Cells

TheCaMVP6proteinanditsdeletedversionsfusedtoEGFPweretransientlyexpressedinBY-2tobaccosuspensioncells(NicotianatabacumcvBrightYellow2)maintainedasdescribedbyBanjokoandTrelease(1995).Cellsweresubculturedeach7dandharvested3daftermediumrenewalforbiolistictransfection.Cellswere lteredontoWhatmandisksandplacedfor2to4hon0.8%agarMSmediaplatessupplementedwith0.1Mmannitoland0.1Msorbitol.Particleprepara-tionandbombardmentassayswereperformedasdescribedbyHunoldetal.(1995)withmodi cations:2mgof1.1mmtungstenparticles(Bio-Rad,Hercules,CA)wereimmersedin1mLofabsolutealcoholfor20min.Driedparticleswerethensuccessivelymixedwith10mgofrecombinantplasmidDNA(pCK-EGFPvector)supplementedwith18%glycerol,0.75MCaCl2,and90mMspermidineina nalvolumeof90mL.The r-ingdistancewas11cmandtheheliumpressurewas7bars.Afterbom-bardment,cellsweretransferredto0.8%agarMSmediaplatesandincubatedinthedarkat288C.BY-2transfectedcellswerecollectedunderHBObinoculars(excitation/emissionwavelength488/505to545nm)20hafterbombardmentandculturedinMSliquidmediumbeforefurthertreatmentand/orCLSMobservations.VirusandHostPlant

Turnips(BrassicarapacvJustRightF1hybrid,providedbyTakiiandCo.,Kyoto,Japan)weremechanicallyinoculatedatthefourleafstage(Jacquotetal.,1998)withCaMVCabb-JIandgrowninagreenhouseat228Cfor5weeksbeforepreparationofprotoplastsfrominfectedleaves.

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.