TheDNAsequenceencodingtheNterminusoftheCaMVP6protein(nucleotides1to336ofORFVI)wasampli edfromthepETKaKS.6recombinantplasmidencompassingthecompleteORFVIusingprimerscarryinganNcoIrestrictionsiteattheir59end.ThePCRfragmentwasdigestedwithNcoIandclonedintopETP42(Lauberetal.,1998),whichhadbeencleavedwiththesameenzymetoproducepET-A:P42.PlasmidspGST:A(ORFVInucleotides1to336),pGST:A1(ORFVInucleotides1to249),andpGST:A2(ORFVInucleotides250to336),codingfordifferentregionsoftheP6NterminusfusedtoGST,wereobtainedbyPCRampli cationofdifferentORFVIsequenceswithprimerscontaining59terminalBamHIandEcoRIsites(Table1).Theampli edDNAfragmentsweredigestedwiththeappropriaterestrictionendonucleasesandclonedintolinearizedpGEX-2TK.
ThepCK-EGFPvectorwasusedtoconstructtherecombinantplas-midscodingforfusionproteinsbetweenEGFPandwild-typeCaMVP6orP6mutants.ThecorrespondingORFVIsequenceswereampli edbyPCRfrompMD324usingtwoprimerscarryingattheir59endsBsrGIandXbaIsites,respectively(Table1).
DeletionsandpointmutationswereintroducedinEGFP:P6andEGFP:Abysite-directedmutagenesis(Stratagene,LaJolla,CA).Therecombinantplasmidswereampli edbyPCRusingpfuTurbopoly-meraseaccordingtothemanufacturer’sinstructionsanddesignedinternaloligonucleotidesasprimers(Table1).ThemixturewasthenincubatedwithtwounitsofDpnIfor2hat378Ctodestroythetemplate.The59endsofPCRproductswerephosphorylatedbyT4polynucleotidekinaseinthepresenceof1mMATPforsubsequentligation.Error-freerecombinantplasmidswereidenti edbyDNAsequencing.
ProductionandPhosphorylationofRecombinantProteins
E.coliBL21/DE3(pLysS)strainwastransformedbyelectroporationwithpETKaKSandpGEX-2TKrecombinantplasmidscodingforfull-lengthP6orP6mutants.Expressionoftheheterologousproteinswasinducedwith1mMisopropylb-thiogalactosidefor2honcethebacterialculturehadreachedtheexponentialphase.Bacteriawerecollectedbycentrifu-gationat5000gfor5min,resuspendedinHMKbuffer(20mMTris-HCl,pH7.5,100mMNaCl,and12mMMgCl2),andlysedbysonication(threepulsesfor20sat50W).Aftercentrifugationat12,000gfor10min,thesupernatantwasdiscardedandtheinclusionbodiesresuspendedin500mLofHMKbuffer.
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.