Citrus tristeza virus : molecular characterization of isolates for use in mild strain cross protection, localization of the 5'-terminus and heterologous encapsidation

101 Pages·2001·4.1 MB·English

by  Ochoa-Corona, Francisco Manuel, 1958-

CITRUSTRISTEZAVIRUS:MOLECULARCHARACTERIZATIONOFISOLATES
FORUSEINMILDSTRAINCROSSPROTECTION,LOCALIZATIONOFTHE5’-
TERMINUSANDHETEROLOGOUSENCAPSIDATION.
By
FRANCISCOMANUELOCHOA-CORONA
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2001
TomybestfriendandbelovedwifeOlimpiaEmilia,andourjoyfullness:Luisana
andFrancisco.
Tomyparents,FranciscoandSilfa,fortheirvaluesandlove,andforteachingme
thatlifegoalsarenotdreamsandcanbemadetrue.
Withlovetomysisterandbrother.
ACKNOWLEDGMENTS
Ismydesireto express agratefullyacknowledge and sincere gratitudetothe
followingpersonsandinstitutionsthatsupportedmygraduatestudiesattheUniversityof
Florida.
ThefinancialsupportfromtheFloridaCitrusProductionResearchCouncil,and
CitrusResearchandEducationCenterofUniversityofFlorida.
Majorprofessor, Dr. R. F. Lee, forhis encouragement, guidance and support
throughoutthecourseofmygraduateprogramandresearch,fortrustinginmypotential,
andforhispersonalinterestinmyacademicpreparation.
Co-majorprofessorDr.C.L.Niblett,forco-adviceandsupportofmyresearchin
Gainesville.
Drs.K.Derrick,J.GrosserandC.Powell,membersofmysupervisorycommittee
forvaluableconversation,suggestions,ideasandhelpwithrevisionofmanuscripts.
Drs.S.M.Gamsey,USDA,Orlando; M.Cambra,IVIA,Valencia,Spain,andV.
J.Febres,UniversityofFlorida,forsupplyingtheCREC109,3E10,4G12,3DFI,3CA5,
andUF34antibodies.
Drs.W.DawsonandS.Gowda,forsupplyingtheprimersC121,C335,C193,
Cl73,the5’-terminusprobe,supportfortheNorthernblotanalyses,andforvaluable
interaction,guidance,andrevisionofmanuscripts.
Drs.R.Albiach-Marti,M.Ayllon,M.Kayim,M.Mawassi,andT.Satyanarayana,
forvaluabletechnicalconversation,interactionandexchangeofideasandarticles.
Dr. R. Brlansky and D. Howd, University ofFlorida, CREC, for technical
assistanceandreceptivityduringCTVpurificationandextraction.
Drs.V.Febres,K.ManjunathandR.ChandrikafortechnicalassistanceatDr.
NibletfsLab.
G.Barthe,M.Dekkers,F.Funk,N.Berger,andT.Ashchifortheircontinual
assistanceandwarmfriendship.
M. Petersen, D. Achor, andC. Davis fortechnical assistance, friendship, and
valuablesupportduringE.M.sessions.
Dr. Rachel Shireman for her responsible commitment with students and for
personalandprofessionalsacrificesmadeinthenameoftheethicsandacademicvalues
attheGraduateSchool,CollegeofAgriculture.UniversityofFlorida.
The warm friendship I found in all my fellow students, laboratorypersonnel,
administrativestaffandfacultymembersinthePlantPathologyDepartment,Gainesville,
andattheCitrusResearchandEducationCenter,LakeAlfred. Thesefriendshipswillnot
beforgotten.
IV
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
iii
LISTOFTABLES vii
LISTOFFIGURES viii
CITRUSTRISTEZADISEASE:THECAUSALAGENTANDVECTOR 1
Citrustristezadisease 1
TheCTVgenome 5
Thevector 10
CTVintheCaribbeanandcrossprotection 10
MOLECULARCHARACTERIZATIONOFFLORIDACITRUSTRISTEZAVIRUS
ISOLATESTOAIDINTHESELECTIONOFISOLATESWITHPOTENTIALUSE
INMILDSTRAINCROSSPROTECTION 14
Introduction 14
Materialsandmethods 16
Results 19
Discussion 23
LOCALIZATIONOFTHECPANDMINORCPOFCTVINRELATIONTOTHE
GENOMICRNA 28
Introduction 28
MaterialsandMethods 30
Results 37
Discussion 41
STUDIESOFHETEROLOGOUSENCAPSIDATIONINTRANSFORMEDPLANTS
EXPRESSINGTHECAPSIDPROTEINOFCITRUSTRISTEZAVIRUS 46
Introduction 46
MaterialandMethods 49
Results 53
Discussion 60
v
APPENDIXA IMMUNO-CAPTURERT-PCROFCTVFROMCITRUS 62
APPENDIXB INFECTIOUSCTVPURIFICATIONPROCEDURE 65
APPENDIXC CTVRNAFROMSUCROSEGRADIENTFRACTIONS 68
APPENDIXD DESIGNINGPRIMERSFORPCRBASEDEXPERIMENTS 69
LISTOFREFERENCES 72
BIOGRAPHICALSKETCH 87
vi
LISTOFTABLES
Table Page
Table2-1. OligonuclotideprimersusedforCPandp27RT-PCRamplification 20
Table.2-2.SerologicalandmoleculardifferencesamongsomeFloridaisolates 22
Table.3-1.Oligonucleotideprimersusedfortheamplificationof5’-and3’-terminiof
citrustristezavirusbyIC-RT-PCRusingUF34andCREC31antisera 33
Table4-1.Serologicalreactionsoftransformedplantsandnon-transformedcontrolplants
afterinoculationwithT36,T30aorT1lausingUF34ascoatingantiserumin
DAS-indirect ELISA. Top) ELISA reactivity using G604 as secondary
antibody. Bottom)SpecificdetectionbyMCA 13monoclonalofsevereCP
components 58
vii
LISTOFFIGURES
Figure Page
Fig.1-1. Citrustristezavirussymptoms.A)DeclineofValenciasweetorangegraftedon
sourorangerootstock. B)Bulgeabovethebudunionofsweetorangegrafted
onsourorangerootstock. C)Phloemsideofasourorangebarkpiececutnear
thebudunionshowing“honeycombing”.Therearecorrespondingbristlesor
pegsprotrudingfromthexylemtissuebeneath thebarkpiece(magnification
2X). D) StempittingonVolkamerlemon(Citrusvolkameriana Ten. and
Pasq.) rootstock ofa four year old Valencia tree. E) Stem pitting on a
ValenciasweetorangebranchduetoCTVisolateB-249. F)Veinclearingon
MexicanlimeleafduetoinfectionbyCTV,isolateB248.G)Veincorkingon
ValenciasweetorangeleaveduetoCTVisolateB-249. Allpicturesareby
theauthorandfromVenezuela 3
Fig. 1-2. Schematicrepresentationofthegeneexpressionandgenomeorganizationof
citrustristezavirus. The ORFs 1 to 11 arerepresentedbyboxes andthe
putativedomainsonORF la, lbareindicated:papain-likeproteases1and2
(PRO),methyltransferase(MT),Helicase(HEL),andRNA-dependentRNA
polymerase(RdRp). Thetwo3’-modulesaredifferentiallyhighlightedas
wellthetwoCPs.ThegenomicandsubgenomicRNAsareshownbysolid
linesatthebottom-right. Thesize,inkilobases,isindicatedbythescaleatthe
top. Thedefective-RNArecombinationstrategyisshowbydashedlinesat,
thebottomleft 6
Fig.2-1. Hybridization blots of Florida isolates with CP-CTV biotin-labeled
probes.Probe0,fromaconservedCPregion. ProbeI,T36andT66(QD).
ProbeII,B1 andB53 (SPfromAustraliaandJapan). ProbeV,B128and
B249(SPfromColombiaandVenezuela).ProbeVI,T26andT30(Florida
mild). ProbeVII,B188andB215 (Orientmild) 24
Fig.2-2. SSCPpatternsofsomeFlorida CTVstrains.SelectedcontrolsareT36(Florida
QD),T30(FloridaMild),B249(VenezuelaSP) 25
Fig.3-1. CTVpurifiedbandsafterCS2SO4isopycnicgradientcentrifugation. A)Bands
2Tand2Bfromsucrosestepgradientfractionnumber2. B)Bands3Tand3B
fromsucrosegradientfractionnumber3 34
viii
Fig.3-2. StrategyusedfortheIC-RT-PCRamplificationof5’and3’terminibyspecific
primers. Laneonleft,100bpladder;lanecenter-left,the510nt5’-terminus
product;lanecenter-right,the899nt3’-terminusproduct;laneright,the672
ntofp25CPproductascontrol.ForprimersequencesrefertoTable2-1and
Table3-1 36
Fig.3-3. IC-RT-PCR,usingUF34antiseraspecificforthep27minorcapsidprotein
(CP) from bands generated afterCS2SO4isopycnicgradient centrifugation.
Notethatthe5’-,and3’-termini,andp25majorCPwerenotamplifiedfrom
bands2Tand 38
Fig. 3-4. CTVdistributionpatternthroughalinear 10-40%ratezonalcentrifugation
(RZC) sucrose gradient as determined by ELISA. CTV reactivity was
estimated by ELISA using UF 34 antiserum, specific for p27 minor coat
protein (CP) as trapping antiserum (1:500 from 1:1 glycerol stock) and
3DF1+3CA5 (0.05 pg/ml each) as secondary antibody. CREC 31, made
againstpurifiedCTVpreparationsandwhichrecognizesbothCPsp25 and
p27(1:1000from 1:1 glycerolstock),wasusedascontroltoestimatetotal
amount of virus. Top: without sonication of band 2B. Bottom: after
sonicationofband2B. Fraction1isatthebottomoftheRZC 39
Fig.3-5. Specificamplificationofthe5’-terminusofCTVRNAbyIC-RT-PCRof10-40
%RZC-sucrosegradientfractionswithspecificantiseraUF34specifictop27
andCREC31,whichrecognizesbothp25 andp27. *F/referstofraction
numberfromRZCsucrosegradients,refertoFig.3-4 40
Fig.3-6. CTVdefectiveRNAs(D-RNAs)detectedbyNorthernblothybridizationusing
a5’-specificriboprobe Left:analysisofthedsRNAextractsfromdifferent
MexicanlimesplantsinfectedwithCTVisolateT36. Right:analysisoftotal
RNA extracted from fractions collected from a rate zonal centrifugation
sucrosegradientfollowingsonication. *F/indicatesfractionnumberfroma
RZCsucrosegradient;refertoFig.3-4 44
Fig.4-1. ExpressionofCTVT36isolatecoatprotein(CP)intransformedsourorange
plants. A. DTBIAimpressionsdevelopedusingMCA13(1:20,000),notea
purplestainonT36andthetransformedcitrus.T36=sourorangeinoculated
with T36; T30a= sour orange inoculated with T30a; Transformed=
transformedsourorange,T1la=sourorangeinoculatedwithT1la;Healthy=
sourorangewithoutinoculation. B. RT-PCRandPCRamplificationofthe
T36CPgenefrominfectedandtransformedsourorangeplantsusingprimers
CN119andCN120. LaneonleftcontainsastandardofXDNAdigestedby
HindIII; lane2 containstheT36 CP geneproductamplifiedfromasour
orangeplantinfectedwithT36; lane3 containsnoproductfromahealthy
control plant; lane 4 contains the T36 CP gene product amplified from a
transformedsourorangeplantcarryingtheT36plantCPgene 55
IX
Fig.4-2. EffectoftheconcentrationofUF34specificforp27capsidprotein(CP)on
detectionofCTVisolatesT36andT30a. O.D.readingsfromDAS-indirect
ELISAat405nmusingextractsfromplantsinfectedwithCTVisolatesT36
andT30a,usingUF34asthecoatingantiseraatthedilutionsindicated. G604
wasusedasthesecondaryantibody 56
Fig.4-3. ReactivityofUF34and1051forp27andp25coatprotein(CP)respectively.
T1o:p3)000O.fDor.405conamtirnegadainndgsexftrroamctsELfIrSomApalsasnatyssiunsfiencgteUdFw3i4thatdiaffedrielnuttioCnToVf
isolatesasindicated,plustransformedplantsexpressingtheT36CPgene,and
healthyplantsascontrol. Bottom)O.D.readingsfromELISAusingextracts
fromplantsinfectedwithCTVisolatesT36andT30ausing1051 ascoating
antiseraatthedilutionof1:1000. G604wasusedasthesecondaryantiserum
forallELISAtests 57
Fig.4-4.HybridizationwithstrainspecificprobesofIC-RT-PCRproductsobtainedfrom
T1lasamplespositivetoMCA13fromtransformedplantsexpressingp25CP
followinginoculationwithTlla. A)Probe0(universal). B)ProbeI(quick
decline). C)ProbeVI(Floridamild) 59
x