Development of infectious papaya mosaic virus using a cDNA clone fused to the 35S CaMV promoter

113 Pages·2000·3.9 MB·English

by  Ferwerda-Licha, Margarita, 1956-

DEVELOPMENTOFINFECTIOUSPAPAYAMOSAICVIRUSUSINGAcDNA
CLONEFUSEDTOTHE35SCaMVPROMOTER
By
MARGARITAFERWERDA-LICHA
ADISSERTATIONPRESENTEDTOTHEGRADUATESCHOOL
OFTHEUNIVERSITYOFFLORIDAINPARTIALFULFILLMENT
OFTHEREQUIREMENTSFORTHEDEGREEOF
DOCTOROFPHILOSOPHY
UNIVERSITYOFFLORIDA
2000
Inmemoryofmygrandmother,TeofilaBetancourt
Tomyparents,forshowingmetheway.
Tomysister,forbeingthebestexampletofollow.
Tomyhusbandandchildren,forremindingmewhatisreallyimportantinlife.
ACKNOWLEDGMENTS
IwouldliketoexpressmysinceregratitudeandappreciationtoDr.Ernest
Hiebert,chairmanofthesupervisorycommittee;Dr.MichaelJDavis,cochairof
thesupervisorycommittee; Dr. DanE. Purcifull, and Dr. ChristineChase, for
theirconstantsupportandguidanceduringthecourseofthisprogram.Iwould
also liketo extend my gratitude to Dr. ChandrikaRamadugu, Dr. Manjunath
Keremane,andFeikoFerwerdafortheirsupportandsuggestions. Specialthanks
aregiventoKrisBeckhamforherinvaluabletechnicalassistanceandfriendship.
I wish to thank Dr. Michael J. Davis and the Tropical Research and
Education Center in Homestead that granted part ofthe assistantship ofthis
graduateprogramforthesupportprovided. IalsowishtothanktheAgricultural
ExperimentStation,UniversityofPuertoRico,thatgrantedthefundingforthis
studyleaveinsupportofthepursuitofthisgraduatedegree.
IwouldliketothankDr.Chin-AnChang,Dr.JulianneNagel,Dr.Gustavo
Frias,Dr.CarlosLopes,Dr.DaveMitchell,Dr.R.Charudattan,GeneCrawford,
andLuciousMitchell,andallmyfriendsintheplantpathologydepartmentfor
their unconditional friendship, support, and encouragement through all these
years.
in
Finally,Iwouldliketoexpressmydeepestappreciationandgratitudeto
myhusbandFeiko,mydaughterAnaMargarita,andmysonHedzerFeikofor
theirencouragement,love,andsupport,andtoourparentsandfamilyinPuerto
Rico,TheNetherlands,andCuracaofortheirfaithinmyendeavors,andtheir
unconditionalsupportandloveduringtheseyears.
IV
TABLEOFCONTENTS
page
ACKNOWLEDGMENTS
iii
LISTOFTABLES vii
LISTOFFIGURES viii
KEYTOABBREVIATIONS x
ABSTRACT xi
CHAPTERS
1 INTRODUCTION 1
PapayaGeneralCharacteristics 2
PapayaRingspotVirus 4
PapayaMosaicVirus 14
2 DEVELOPMENTOFAPAPAYAMOSAICVIRUScDNACLONE 18
Introduction 18
MaterialsandMethods 19
Results 26
Discussion 33
3 ADDITIONOFTHECAULIFLOWERMOSAICVIRUS35S
PROMOTERTOPAPAYAMOSAICVIRUS 34
Introduction 34
MaterialsandMethods 35
Results 42
Discussion 44
v
4 TESTINGOFTHECONSTRUCTOFPAPAYAMOSAIC
VIRUSWITHTHE35S CaMVPROMOTERFORINFECTIVITY 49
Introduction 49
MaterialsandMethods 50
Results 54
Discussion 79
5 SUMMARYANDCONCLUSIONS 82
LISTOFREFERENCES 86
BIOGRAPHICALSKETCH 100
vi
2-
LISTOFTABLES
3-
Table page
4-
1 SequenceofspecificoligonucleotidesusedforPCRamplification
of6.7kbcDNAofPMV 23
1 SequenceofspecificoligonucleotidesusedforoverlappingPCR
foradditionofCaMV35Spromoter 38
1 BallisticinoculationofG.globosa,N.benthamianaandC.papaya
withthePMVinfectiousclone 57
4-2 DetectionofPMVbyI-ELISA1inextractsofplantsballistically
inoculatedwiththe
infectious.clone 61
4-3 MechanicalinoculationofG.globosa,N.benthamiana
andC.papayawiththePMVinfectiousclone 63
4-4 DetectionofPMVbyI-ELISA1inextractsofplantsmechanically
inoculatedwiththeinfectiousclone 64
4-5 TheeffectofconcentrationontheinfectivityofthePMV
infectiousclonebyrestrictiondigestwithKpnl 65
4-6 DetectionofPMVbyindirectELISA 74
Vll
LISTOFFIGURES
Figure page
2-1 Schematicrepresentationoffirststrandsynthesis 22
2-2 AgarosegelelectrophoresisofPMVRNAisolatedfrompurified
virions 27
2-3 PCRproductsofPMVamplifiedwithdifferentprimer
2- combinationsfromthefirststrandseparatedbyagarosegel
electrophoresis 29
23--4 Cutanduncutplasmidextractedfromcolonycontaining
full-lengthPMVinsertseparatedbyagarosegelelectrophoresis 30
2-5 Similarityofthenucleotidesequenceofthe5’terminalregion
3- ofthePMVclone#10withapreviouslydescribedsequenceofthe
region 31
4-
6 Similarityofthenucleotidesequenceofthe3’terminalregion
ofthePMVclone#10withapreviouslydescribedsequence
oftheregion 32
1 pSecrhfeomartmiecdrtoepartetsaecnhtatthieo3n5oSfpthreomsoetqeurenocfeCoafMPVCRtortehaect5i’ons
endofPMV 37
3-2 PCRproductsofPMVcDNAandPMVcDNAwiththefused
35SCaMVpromoter 45
3 RestrictiondigestsofPMVclone#124 46
1 GomphrenaglobosainoculatedwithPMVinfectiousclone 56
4-2 Localizedregionwithsymptomsinpapayainoculated
withinfectiousclone 58
4-3 TissueblotsofC.papayaandG.globosaplants
ballisticallyinoculatedwithinfectiousclone 59
viii
4-4 Viralparticlesstainedwithuranylacetateobservedinleafdipsof
plantsinoculatedwiththePMVinfectiousclone 60
4-5 LocallesionsonG.globosamechanicallyinoculated
withtheinfectiousclone 65
4-6 ComparisonofsymptomscausedbyPMVinfectiousclone
andPMVinpapayaplants 66
4-7 TissueblotsofplantsofC.papaya,N.benthamianaand
G.globosamechanicallyinoculatedwithPMVinfectious
clone 68
4-8 Tissueblotsofplantsback-inoculatedwithPMV
infectiousclone 71
4-9 Papayaleafinoculatedwith3pg/plofuncutinfectious
cloneofPMVshowingveinclearing 75
4-10 Papayaplantinoculatedwith3pg/plofPMVclonecut
withKpnIshowingmildmosaicsystemicsymptoms 76
4-11 Systemicsymptomsonolderpapayaplantsinoculated
with2pg/plofPMVinfectiousclonecutwithKpn1 77
4-12 Papayainoculatedwithinfectiouscloneshowing
deformationsymptoms 78
IX
KEYTOABBREVIATIONS
ATCC AmericanTypeCultureCollection
bp basepair
CaMV35S cauliflowermosaicvirus35Spromoter
CP coatprotein
C-terminus carboxy-terminus
cDNA complementaryDNA
CMV cucumbermosaicvirus
ELISA enzyme-linkedimmunosorbentassay
HC/Pro helpercomponent/protease
IPTG isopropyl-P-D-thiogalactopyranoside
kb kilobase
kDa kilodalton
LB Luriabroth
P-ME p-mercaptoethanol
MW
molecularweight
3'-NCR 3'non-codingregion
N-terminus amino-terminus
nm nanometer
nt nucleotide
oligodT oligonucleotidedeoxythymidine
PCR polymerasechainreaction
PDR pathogenderivedresistance
PMV papayamosaicvirus
pPsRiSV-W ppaopuanydasrpienrgsspqoutarveiriunschtypeW
PRSV-P papayaringspotvirustypeP
PVY potatovirusY
RT-PCR reversetranscription-polymerasechainreaction
TMV tobaccomosaicvirus
X-Gal 5-bromo-4-chloro-3-infolyl-|3-D-galactopyranoside
KPa kilopascals
x