UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Transformation of Brassica napus cv. Westar with the beet western yellows virus coat protein gene Lee, Lawrence 1993

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-ubc_1993_fall_lee_lawrence.pdf [ 4.93MB ]
Metadata
JSON: 831-1.0086246.json
JSON-LD: 831-1.0086246-ld.json
RDF/XML (Pretty): 831-1.0086246-rdf.xml
RDF/JSON: 831-1.0086246-rdf.json
Turtle: 831-1.0086246-turtle.txt
N-Triples: 831-1.0086246-rdf-ntriples.txt
Original Record: 831-1.0086246-source.json
Full Text
831-1.0086246-fulltext.txt
Citation
831-1.0086246.ris

Full Text

TRANSFORMATION OF BRASSICA NAPUS cv. WESTAR WITH THE BEETWESTERN YELLOWS VIRUS COAT PROTEIN GENEByLawrence LeeB.Sc., The University of British Columbia, 1989A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFMASTER OF SCIENCEinTHE FACULTY OF GRADUATE STUDIESDepartment ofPLANT SCIENCEWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIAJuly 1993© Lawrence Lee, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.Department of FLA-1,JY 6C1F-44c.E.The University of British ColumbiaVancouver, CanadaDate <--3 IA-544^Ill 3DE-6 (2/88)AbstractDouble stranded (ds) complementary DNA (cDNA) copies of the beet western yellowsvirus (BWYV) coat protein (CP) gene were synthesized from genomic BVVYV RNAusing reverse transcriptase (RT) followed by the polymerase chain reaction (PCR).The ds cDNA copy of the BWYV CP was then cloned into plasmid pRT103, a plantexpression vector which contains the cauliflower mosaic virus (CaMV) 35S promoterand polyadenylation signal. The CP cDNA was inserted in the plus-sense orientation(clone BW102D) and in the anti-sense orientation (clone BW137D) relative to theCaMV 35S promoter. The CP coding regions of both pRT103 constructs weresequenced and found to correspond closely to those of previously published BWYVCP sequences. In vitro translation analysis in wheat germ extracts of synthetictranscripts of the cloned BWYV CP DNA produced only one major protein of ca. 22.5kDa, the expected size for the BWYV coat protein. The CaMV 35S promoter-BWYVCP-polyadenylation cassette from clones BW102D and BW137D were then clonedseparately into the Hind III restriction enzyme site between the left and right bordersof the T-DNA region and next to the npt II cassette of the two binary plasmids,pCGN1548 and pCGN1557 (Calgene). After mobilization of the four resulting binaryplasmid constructs (pCGN154802, pCGN154837, pCGN155702 and pCGN155737)into Agrobacterium tumefaciens EHA101, the A. tumefaciens were used in the planttransformation procedure described by Moloney et al. (1989) utilizing cotyledonary11explants of Brassica napus cv. Westar. Thirty eight regenerated plants were recoveredunder the selection of the antibiotic, kanamycin. Southern blot analysis of Hind IIIdigested plant DNA indicated that the BWYV CP gene was present in the genome ofthree transformed plants (154802-1, 154802-3 and 1557-7). PCR analysis of the sameplant genomic DNA followed by Southern blot analysis of the PCR products indicatedthat several plants contained the BWYV CP gene integrated into the plant genome(plants 155702-1 to -8, -10 to -14, 1557-7 and 154802-1 to -5). RNA transcripts ofeither the BWYV CP gene or the co-transformed npt II gene could not be detectedusing Northern blot analysis of the RNA extracted from regenerated plants. However,germination of the R1 seeds from the regenerated plants on kanamycin indicated thatthe npt II gene product which confers kanamycin resistance was functional in many ofthe progeny of the regenerated plants. BWYV CP subunits could not be detected byenzyme-linked immunosorbent assay (ELISA) using a polyclonal antiserum whichreacts with BWYV CP subunits. Preliminary evaluation of the progeny of twopromising lines (154802-3 and 1557-7), demonstrated to have the BWYV CP geneintegrated into the plant genome, did not reveal significant levels of resistance whenchallenged with the homologous virus using the aphid vector, Myzus persicae (Sulz.).111Table of ContentsAbstract ^  iiTable of Contents ^  ivList of Figures  viiiList of Tables ^  xList of Abbreviations ^  xiAcknowledgements  xvIntroduction ^  11.1^Research Objectives  ^11.2^Epiphytology of Beet Western Yellows Virus (BWYV) ^ 31.3^Genomic Organization of Beet Western Yellows Virus  31.4^Genetically Engineered Resistance to Viral Infection ^ 81.5^Agrobacterium tumefaciens Mediated Gene Transfer into Plant Cells ^ 10Materials and Methods ^  142.1^BWYV Propagation and Purification ^  142.2 BWYV Virion RNA Extraction  162.3^Amplification of the BWYV Coat Protein Gene by Reverse Transcriptionand the Polymerase Chain Reaction ^  17iv2.4 DNA Sequencing of the BWYV Coat Protein Gene ^ 192.5^In Vitro Translation Analysis of the BWYV Coat Protein Gene ^ 202.5.1 In Vitro Transcription ^  202.5.2 Translation In Vitro using Wheat Germ Extracts ^ 212.6^Tr-Parental Mating Procedure^  222.7^Transformation and Regeneration of Brassica napus cv. Westar ^ 242.8 DNA Extraction from Plants Using the CTAB Method ^ 252.9 Synthesis of Random-Primed cDNA Probes ^  262.10 Southern Blot Analysis ^  272.11 Leaf RNA Extraction  282.12 Northern Blot Analysis ^  292.13 Production of Monoclonal Antibodies and Polyclonal Antisera ^ 302.14 Evaluation of Existing Monoclonal Antibodies in Western Blot and DotBlot Analysis ^  31Results ^  333.1^Amplification of the BWYV Coat Protein Gene by Reverse Transcription -Polymerase Chain Reaction ^  333.2^Cloning of the Double-Stranded cDNA Copies of the BWYV Coat ProteinGene into the pRT103 Plant Expression Vector ^  363.2.1 DNA Sequencing of the BWYV Coat Protein Gene ^ 38v3.3^In Vitro Translation Analysis of Synthetic Transcripts of the Cloned BWYVCoat Protein Gene ^  503.3.1 Cloning of the BWYV Coat Protein Gene into a Transcription Vectorand the In Vitro Transcription of the BWYV Coat Protein Gene usingthe Bacteriophage T3 RNA Polymerase ^  503.3.2 In Vitro Translation of Synthetic BWYV Coat Protein RNA inWheat Germ Extracts ^  513.4^Construction of the BWYV Coat Protein Gene Binary Plasmids Using thepCGN15 Vector Series (Calgene) ^  513.5^^Mobilization of the BWYV Coat Protein Gene Binary Plasmids intoAgrobacterium tumefaciens EHA101 by the Tr-Parental MatingProcedure ^  573.6^Transformation and Regeneration of Brassica napus cv. Westar ^ 593.7^Evaluation of Regenerated Plants ^  643.7.1 Southern Blot Analysis of DNA Extracted from Regenerated Plants 643.7.2 Northern Blot Analysis of RNA Extracted from Regenerated Plants 713.7.3 PCR Analysis of DNA Extracted from Regenerated Plants   713.7.4 Evaluation of Seeds from Regenerated Plants for KanamycinResistance ^  773.8^Production and Screening of Monoclonal Antibodies and PolyclonalAntisera for Reactivity Against Disrupted BWYV Particles ^ 793.8.1 Evaluation of Antibodies in Western Blot and Dot Blot Analysis . ^ 79vi3.8.2 Production of Monoclonal Antibodies and Polyclonal AntiseraUsing Disrupted BWYV Particles ^  813.9^Preliminary Evaluation of BWYV Resistance in TransgenicBrassica napus cv.Westar ^  82Discussion ^  844.1^Transformation of Brassica napus cv. Westar ^  874.2^Integration Analysis of Regenerated Brassica napus cv. Westar ^ 894.3^Expression Analysis of Regenerated Brassica napus cv. Westar ^ 914.4^Future Outlook ^  93Bibliography ^  95viiList of Figures1.1^Genomic organization of beet western yellows virus ^ 51.2^Agrobacterium tumefaciens Ti plasmid mediated gene transfer ^ 133.1^Amplification of the BWYV coat protein gene by RT-PCR ^ 353.2^Diagram showing the construction of the pRT103/BWYV coat proteingene clones ^  373.3^Strategy used to sequence the BWYV coat protein coding regionof clone BW102D ^  393.4^Nucleotide sequence and amino acid sequence of the BWYV coatprotein coding region ^  403.5^^Nucleotide sequence comparison between the BWYV coat proteincoding region (BWYV1002 meld) and the coat protein sequence ofthe sugarbeet isolate BWYV (GB1) (Veidt et al., 1988) ^ 443.6^Nucleotide sequence comparison between the BWYV coat proteincoding region (BWYV1002 meld) and the coat protein sequence ofthe lettuce isolate BWYV (FL1) (Veidt et al., 1988) ^ 463.7^Amino acid sequence comparison between the BWYV coat protein(derived from BWYV1002 meld) and the coat protein of the sugarbeetisolate BWYV (GB 1) (Veidt et al., 1988) ^  48viii3.8^Amino acid sequence comparison between the BWYV coat protein(derived from BWYV1002 meld) and the coat protein of the lettuceisolate BWYV (FL!) (Veidt et al., 1988) ^  493.9^In vitro translation analysis of synthetic transcripts from the clonedBWYV coat protein gene ^  523.10 Diagram showing the construction of the pCGN1548/BWYV coatprotein gene clones ^  533.11 Diagram showing the construction of the pCGN1557/BWYV coatprotein gene clones ^  553.12 Restriction enzyme analysis of pCGN1548- and pCGN1557-derivedBWYV coat protein gene constructs ^  583.13 Southern blot analysis of DNA extracted from regeneratedBrassica napus cv. Westar ^  663.14 Southern blot analysis of DNA extracted from regeneratedBrassica napus cv. Westar ^  673.15 Southern blot analysis of DNA extracted from regeneratedBrassica napus cv. Westar ^  693.16 Southern blot analysis of the PCR products obtained using DNAextracted from regenerated plants as template ^  733.17 PCR analysis of DNA extracted from regenerated plants ^ 75ixList of Tables3.1^Transformation of Brassica napus cv. Westarwith Agrobacterium tumefaciens EHA101 containingthe indicated binary plasmid construct ^  613.2^Transformation of Brassica juncea cv. Forgewith Agrobacterium tumefaciens EHA101 containingthe indicated binary plasmid construct ^  623.3^Regeneration efficiency of Brassica napus cv. Westarcotyledons after co-cultivation of the cotyledonaryexplants with Agrobacterium tumefaciens EHA101containing the appropriate binary plasmid construct ^ 633.4^Evaluation of seeds from regenerated plantsfor kanamycin resistance ^  784.1^Summary of integration and expression analysis ofregenerated Brassica napus cv. Westar after treatmentof cotyledonary explants using the plant transformationprocedure (Moloney et al., 1989) ^  85xList of AbbreviationsA260 - absorbance at 260 nmAlMV - alfalfa mosaic virusATP (dATP) - adenosine triphosphate (deoxyadenosine triphosphate)bp - base pairBE - 40 mM boric acid / 1 mM EDTA (pH 8.2) bufferBRL - Bethesda Research LaboratoriesBSA - bovine serum albuminBWYV - beet western yellows virusBYDV - barley yellow dwarf virusC - Celsiusca. - aboutCaMV - cauliflower mosaic virusCAT - chloramphenicol acetyl transferasecDNA - complementary DNACi (uCi) - Curie (microCurie)CIP - calf intestinal alkaline phosphataseCMV - cucumber mosaic virusCNV - cucumber necrosis virusCTAB - hexadecyltrimethylammoniumbromidexiCTP (dCTP) - cytidine triphosphate (deoxycytidine triphosphate)cv. - cultivarDAS-ELISA - double antibody sandwich ELISADNA - deoxyribonucleic acidds - double strandedD'TT - dithiothreitolE (uE) - Einstein (microEinstein)EDTA - ethylenediaminetetraacetic acidELISA - enzyme-linked immunosorbent assayg (mg, ug, pg) - gram (milligram, microgram, picogram)g - gravitational constantGTP (dGTP) - guanosine triphosphate (deoxyguanosine triphosphate)HC1 - hydrochloric acid or hydrochloridehr - hourkb - Idlobasekbp - ldlobase pairKC1 - potassium chloridekDa - kiloDaltonL (mL, uL) - litre (millilitre, microlitre)LB - Luria-BertaniMr - relative molecular weightM (mM) - molar (millimolar)xiim - metremas 5' - mannopine synthase promotermas 3' - mannopine synthase terminatorMeHg0H - methylmercuric hydroxideM-MLV - Moloney murine leukaemia virusMgC12 - magnesium chlorideMgSO4 - magnesium sulfatemin - minutemob - mobilization genesmRNA - messenger RNANaC1 - sodium chlorideNaOH - sodium hydroxideNEN - New England NuclearnptlI - neomycin phosphorransferasent - nucleotideORF - open reading framePAGE - polyacrylamide gel electrophoresisPCR - polymerase chain reactionPEG - polyethylene glycolPLRV - potato leafroll viruspoly (A) - polyadenylatedPVX - potato virus XPVY - potato virus YRNA - ribonucleic acidrpm - revolutions per minuteRT - reverse transcriptiontra - transfer geness - secondSDS - sodium dodecyl sulfatess - single strandedSSC - 20X SSC is 3M sodium chloride / 0.3M trisodium citrate (pH 7.5) bufferT-DNA - transfer DNATE - 10 mM Tris-HC1 (pH 7.4) / 1 mM EDTA bufferTEV - tobacco etch virusTi - tumor-inducingtin/ 3' - tm/ terminator from pTiA6TMV - tobacco mosaic virusTNE - 100 mM Tris-HC1 (pH 7.5) / 100 mM NaC1 / 10 mM EDTATris - trishydroxymethylaminomethaneTSWV - tomato spotted wilt virusTSV - tobacco streak virusdTTP - deoxythymidine triphosphateVPg - viral genome-linked proteinxivAcknowledgementsI wish to thank Dr. D'Ann Rochon for her supervision, financial assistance andthe opportunity to work in her lab. Thanks are also extended to the members of mycommittee, Drs. Brian Ellis, Peter Ellis, James Kronstad and Frank Tufaro for theirhelpful advice and suggestions on my thesis.I wish to thank the staff of the Agriculture Canada Vancouver ReasearchStation and Dr. Dean Struble for their help and support, and for providing thereasearch facilities for my work.I am grateful to Carol Riviere and Dr. Donald MacKenzie for their technicalassistance and advice throughout my thesis.A special thanks to Dr. Brian Ellis for "lending me your ear" and for trying tokeep me along the "straight and narrow." Despite your advice, I still learned many ofmy lessons from the school of "hard knocks."I wish to thank Dr. Timmy Sit for his relentless editing of my thesis. Mythesis still isn't the way you would like it but I will remember many of yoursuggestions and comments during my Ph.D. thesis. ..then again maybe not!Many thanks to the gang of three on the first floor, Drs. Peter Ellis, RobertMartin and Dick Stace-Smith, for their encouragement and support. My outlook onscientific research and my sense of humour will never be quite the same again. Avery special thanks to Peter Ellis, who continually told me "it" was going to work. Ican't say, "you never did anything for me."Last, but not least, I wish to thank Grace for her patience and tolerance withmy lack of humour and absent-mindedness during the final stages of my thesis.XVIntroduction1.1^Research ObjectivesBrassica napus, apart from being a major crop (over $1 billion annually inCanada), has proven to be amenable to a variety of methods which lead to theproduction of transgenic plants. The transformation methods include the use ofAgrobacterium vectors (Radke et al., 1988), microprojectile bombardment (Neuhaus etal., 1987) and electroporation (Guerche et al., 1987). Whole plants have beenregenerated from a variety of tissues, such as stem explants (Stringham, 1979), leafand root protoplasts (Newell et al., 1984; Xu et al., 1982) and microspores (Chuong etal., 1988). Nevertheless, these methods are usually labour-intensive and thetransformation efficiency low.Brassica cultivars are known to be susceptible to beet western yellows virus(BWYV) disease (Gilligan et al., 1980; Smith and Hinckles, 1985). Severalpreliminary studies suggest that the occurrence of BWYV in both Brassica cultivarsand in a number of common weeds is significant (Ellis, 1992; Ellis and Stace-Smith,1990; Hampton et al., 1990; Thomas et al., 1993) but the yield losses caused byBWYV have not been examined. Transgenic plants containing the coat protein geneof a virus have been shown to exhibit a delay or even complete inhibition of infectionp.1Introduction p. 2when inoculated with the homologous virus. Such resistance was first demonstrated intobacco for tobacco mosaic virus (TMV; Powell-Abel et al., 1986) and since then forseveral different virus groups including the luteovirus group (Kawchuk et al., 1990;van der Wilk et al., 1990) with potato leafroll virus. The main objective of this studyhas been to attempt to confer genetically engineered resistance in Brassica napus cv.Westar to BWYV through the incorporation of the BWYV coat protein (CP) gene viaAgrobacterium tumefaciens Ti plasmid mediated gene transfer. To this end, doublestranded (ds) complementary DNA (cDNA) copies of the BWYV CP gene weresynthesized from BWYV RNA (extracted from a sugarbeet isolate of BWYV) usingreverse transcription (RT) followed by the polymerase chain reaction (PCR). TheBWYV CP cDNA was cloned into the pRT103 plant expression vector (TOpfer et al.,1987), under the regulatory control of the cauliflower mosaic virus (CaMV) 35Spromoter and polyadenylation signal. The CaMV 35S promoter-BWYV CP gene-polyadenylation signal cassette was used to make binary Ti plasmid constructs in thepCGN15 series of vectors (McBride and Summerfelt, 1990). After mobilization ofthese binary plasmid constructs into A.tumefaciens EHA101 (Hood et al., 1986), the A.tumefaciens were used in the plant transformation procedure described by Moloney etal. (1989) to transform Brassica napus cv. Westar. The regenerated plants were thenevaluated.Introduction^p.31.2^Epiphytology of Beet Western Yellows Virus (BWYV)Beet western yellows virus (BWYV), a member of the luteovirus group(Waterhouse et al., 1988), is a very widespread and economically important plantvirus. Unlike most luteoviruses, BWYV has a very wide host range including manydicotyledonous and at least one monocotyledonous plant species (Rochow and Duffus,1981; Casper, 1988). Some commercially important crop plants susceptible to BWYVdisease include sugarbeet, red beet, lettuce, broccoli, cauliflower, radish, turnip, flaxand oilseed rape (Duffus, 1972; Gilligan et al., 1980). BWYV occurs naturally in anumber of common weed species and often in overwintering hosts of vector aphids(Ellis, 1992). Stunting and chlorosis (interveinal yellowing of the older orintermediate leaves) are typical symptoms of BWYV disease. BWYV is transmitted inthe persistent circulative manner by several species of aphids, the most important ofwhich is Myzus persicae (Sulz.). BWYV is not transmissible by sap inoculation.Furthermore, BWYV is phloem-limited and present in only very low concentrations inplant tissue extracts.1.3^Genomic Organization of Beet Western Yellows VirusThe genome of BWYV consists of a monopartite single stranded (ss) plus-sense RNA, 5641 nucleotides in length (Veidt et al., 1988; Falk et al., 1989). Thegenome is encapsidated in an isometric particle ca. 26 nm in diameter. A genomeIntroduction p. 4linked protein (VPg) is covalently attached to the 5'-end of the RNA and the 3'-endhas no poly(A) tail. The nucleotide sequence of the genomic RNA of a lettuce isolateof BWYV has been determined (Veidt et al., 1988). The BWYV genome iscomprised of six large open reading frames (ORFs) (see Fig. 1.1). ORFs 1 and 2 arelikely both expressed from genomic-length RNA and ORFs 4 and 5 from a single 3'co-terminal subgenomic RNA. Two other luteoviruses, barley yellow dwarf virus(BYDV) and potato leafroll virus (PLRV) employ a similar strategy for the expressionof their corresponding ORFs (Dinesh-Kumar et al., 1992; Tacke et al., 1990; Lamband Hay, 1990). ORF 1 encodes a polypeptide of 29 000 Mr (29 kDa), the function ofwhich is not known. Overlapping extensively with ORF 1 but in a different readingframe is ORF 2 which encodes a polypeptide of 66 000 Mr (66 kDa). ORF 1 beginsat the first AUG codon in the BWYV RNA sequence and ORF 2 at the second. Manyviral RNAs are multicistronic but nevertheless are expressed in a monocistronicfashion; that is, ribosomes scan the RNA from the 5'-end until an AUG is encounteredand only the 5' proximal ORF is translated. ORF 2 of BWYV is likely expressed bya 'leaky' scanning mechanism in which some of the ribosomes bypass the first AUG,which is in a sub-optimal context for translational initiation (Veidt et a/.,1988), andinitiate at the next AUG codon which is in a more favorable context (Kozak, 1989).The 3' terminus of ORF 2 shares a 474 nucleotide overlap with the beginning ofORF 3.Introduction^p. 51 kbpFig. 1.1^Genomic organization of beet western yellows virus. The genome of BWYVconsists of a monopartite single-stranded plus-sense RNA 5641 nucleotides in length. Aviral genome-linked protein (VPg) is covalently attached to the 5' -end of the RNA and the3' -end has no poly(A) tail. The BWYV genome is comprised of six large open readingframes (ORFs). Each ORF is represented by an open box with the predicted size of theprotein product shown above or below the box. The shaded area corresponds to the BWYVcoat protein and putative readthrough product.Introduction^p. 6Expression of ORF 3 involves a -1 translational frameshift somewhere in the regionof overlap permitting translation as an ORF 2/3 frameshift fusion protein ofapproximately 115 kDa (Garcia et al., 1993). Such a frameshift mechanism has beendemonstrated for BYDV and PLRV (Miller et al., 1988; Brault and Miller, 1992;Priifer et al., 1992; Mayo et al., 1989; Kujawa et al., 1993). The BWYV 67 kDaORF (ORF 3) contains the amino acid sequence motif GXXXDOCXNX18.50GDD,where X is any amino acid, present in nearly all known and putative viral RNA-dependent RNA polymerases (Goldbach, 1986). ORF 4 begins approximately 202nucleotides after the stop codon for ORF 3 and encodes a polypeptide of 22 500 Mr(22.5 kDa). Comparison of the amino acid sequence coded for by this ORF with thatof the coat protein ORF of BYDV has identified this BWYV ORF as the BWYV coatprotein gene. Nested entirely within ORF 4, but in another open reading frame, isORF 5, which encodes a polypeptide of 19 500 Mr (19.5 kDa). Comparison of theamino acid sequence coded for by ORF 4 with that of the similarly located 17 kDaORF of BYDV suggests that the 19.5 kDa OEF may correspond to the VPg. Similarto ORF 2, ORF 5 is likely expressed by a 'leaky' scanning mechanism in which someribosomes bypass the first AUG of ORF 4, which is in a suboptimal context fortranslational initiation, and initiate at the next AUG of ORF 5 which is in a morefavorable context. Such a leaky scanning mechanism has been suggested for thecorresponding 17 kDa ORF of PLRV (Tacke et al., 1990). ORF 4 and 5 areexpressed from a single 3' co-terminal subgenomic RNA (Falk et al., 1989). ORF 6immediately follows ORF 4 but ORF 4 terminates with an amber (UAG) terminationIntroduction^p. 7codon. It is possible that the UAG terminator of ORF 4 is read through bytranslational suppression of the ORF 4 amber termination codon to produce an ORF4/6 fusion polypeptide of 74 000 A11. Such translational suppression has been shownfor the corresponding ORFs of BYDV and PLRV (Dinesh-Kumar et al., 1992; Bahneret al., 1990). A 5' region of the polypeptide encoded by ORF 6 appears to be animportant determinant for aphid transmissibility (Bahner et al., 1990). Thecorresponding ORF in PLRV (ORF 5) can encode a 53 lcDa polypeptide which isprobably derived from the rapid proteolysis of the 80 kDa/90 kDa proteins. The 80kDa/90 kDa polypeptide resulting from the readthrough of the coat protein terminationcodon have been detected in extracts of partially purified particles; whereas only the23 kDa coat protein subunit and the 53 lcDa polypeptide could be detected in purifiedprotein extracts (Bahner et al., 1990). Indeed, a small number of protrusions havebeen detected on the surfaces of PLRV particles in favourably stained preparations(Harrison, 1984). Comparisons between aphid-transmissible isolates of PLRV and anisolate that is very poorly aphid-transmissible suggest that the determinant for aphidtransmissibility is located in a region of the readthrough protein that is conserved atthe amino acid sequence level among the three luteoviruses PLRV, BYDV andBWYV. The differences seen in aphid-transmissiblity between the PLRV isolates area result of an alteration in this region as opposed to a deletion (Massalski andHarrison, 1987).Introduction^p. 81.4^Genetically Engineered Resistance to Viral InfectionHamilton (1980) first suggested that virus-induced resistance might be obtainedby transferring a portion of a viral genome into a plant genome. The correspondinggene product(s) might provide protection against infection, either directly or indirectly,in a manner similar to that seen in cross-protection studies. If only a portion of theviral genome were used, the RNA transcript produced would not be infective. Inaddition, the protection provided by the transferred cDNA portion of the viral genomewould be inherited from generation to generation.Subsequent to this suggestion, several developments occurred which facilitatedthe introduction of foreign genes into plant genomes. Fraley (1983) reported theexpression of bacterial genes in plants cells and Chilton et al. (1980) recognized thenatural engineering ability of A. tumefaciens, to transfer the T-DNA (transfer DNA)portion of the tumour-inducing (Ti) plasmid into a wounded plant cell and have the T-DNA stably integrated into the plant genome. The first successful demonstration ofgenetically engineered resistance to a plant virus was reported by Powell-Abel et al.(1986) who found that transgenic tobacco plants expressing the coat protein gene fromTMV displayed a significant delay in symptom development following inoculationwith TMV. Since then, coat protein-mediated resistance has been demonstrated inseveral transgenic plants expressing the viral coat protein for a number of differentviruses including alfalfa mosaic virus (A1MV; Van Dun et al., 1987; Tumer et al.,Introduction^p. 91987; Loesch-Fries et al., 1987), cucumber mosaic virus (CMV; Cuozzo et al., 1988),tobacco streak virus (TSV; Van Dun et al., 1988), potato virus X (PVX; Hemenway etal., 1988; Hoekema et al., 1989), potato virus Y (PVY; Lawson et al., 1990), potatoleafroll virus (PLRV; Kawchuck et al., 1990; van der Wilk et al., 1991), tobacco etchvirus (TEV; Lindbo and Dougherty, 1992) and tomato spotted wilt virus (TSWV;MacKenzie and Ellis, 1992; Goldbach et al., 1992). While in some cases viralresistance was correlated with coat protein accumulation in the plant tissue (A1MV,TMV and CMV), in other cases, viral resistance was correlated with coat proteintranscript accumulation (PVX, PVY, TEV, PLRV, TSWV). High levels of resistanceto PLRV infection in transgenic potatoes in which the PLRV coat protein gene hadbeen introduced, have been observed (Kawchuk et al., 1990). Levels of PLRV coatprotein transcripts were relatively high in the transgenic plants but coat proteinrepresented less than 0.01% of the total leaf protein. Furthermore, resistance wasfound in transgenic plants containing either plus- or anti-sense transcripts of the PLRVcoat protein gene.It is clear that coat protein mediated resistance can reduce virus infection anddisease development for a number of different host-virus systems but the molecularbasis of resistance remains unknown and appears to differ between systems. Severalmechanisms have been proposed for coat protein mediated resistance in transgenicplants including (reviewed by Beachy et al., 1990): (1) interference with disassemblyof the infecting virus or re-encapsidation of the infecting viral RNA, (2) interferenceIntroduction p. 10with the replication of the infecting viral RNA or (3) interference with the systemicspread of the infecting viral RNA.1.5 Agrobacterium tumefaciens Mediated Gene Transfer into Plant CellsA. tumefaciens is an opportunistic soilborne phytopathogen that can causecrown gall tumours in wounded gymnosperms and dicotyledonous angiosperms. ThisGram-negative, rod-shaped bacterum is able to transfer a portion of its large (150-250kb) Ti plasmid, the T-DNA, into the wounded plant cell and have the T-DNA stablyintegrated into the plant genome. The genes encoded on the T-DNA, while ofbacterial origin, contain plant regulatory signals enabling their expression in infectedplant cells. The expression of these genes has two consequences: (1) the synthesis ofphytohormones necessary for the neoplastic transformation of the infected tissue toproduce the characteristic tumorous gall, and (2) the synthesis of opines which serveas a carbon source for Agrobacteria harbouring a Ti plasmid and which induce the traoperon that allows the conjugal transfer of the Ti plasmid to other Agrobacteria.Garfinkel et al. (1981) observed that only two 25 bp border regions (imperfect repeats)of the T-DNA were essential for T-DNA transfer and that the remainder of theplasmid could be deleted and replaced with recombinant DNA. Using this approach,Bevan et al. (1983) and Herrera-Estrella et al. (1983) were the first to successfullyintroduce expressed foreign genes into plants. Herrera-Estrella et al. (1983) obtainedexpression of the octopine synthase and chloramphenicol acetyl transferase (CAT)Introduction^p. 11genes (regulated by the nopaline synthase promoter) in transgenic tumour tissue whileBevan et al. (1983) reported expression of neomycin phosphotransferase (npt II), adominant selectable marker, in transgenic tumours. Since the Ti plasmids were toolarge (150-250 kb) for direct genetic manipulation, smaller vectors were designed thatcontained a selectable marker for introduction into Agrobacterium, with a selectablemarker functional in plants between borders for T-DNA transfer to plants. Thesesmaller vectors were more amenable to the manipulation of recombinant DNA inEscherichia coli. Garfinkel and Nester (1980) also discovered that the vir region ofthe Ti plasmid, containing the genes whose products are necessary for gene transfer,can act in trans on T-DNA carried on another plasmid. This observation led todevelopment of two types of Ti-based vectors: cointegrate vectors and binary vectors.The cointegrate vectors have a region of homology between themselves and theaccepting Ti plasmid. The cointegrate vectors contain pBR322 DNA (into whichforeign genes for expression in plants can be cloned), the left and right borders of theT-DNA, a selectable marker for introduction into Agrobacterium (such as antibioticresistance) and a selectable marker functional in plants (such as npt II for kanamycinresistance). These plasmids, having a ColE1 origin of replication, cannot bemaintained in Agrobacterium. Instead, the cointegrate vectors are mobilized intoAgrobacterium by triparental mating with a plasmid that supplies the mob and tragenes in trans (pRK2013; Ditta et al., 1980), but only the Ti plasmid will bemaintained in Agrobacterium; the antibiotic resistance gene from the cointegrate vectorwill allow for the selection of a single crossover event in which the cointegrate vectorIntroduction^p. 12becomes integrated between the borders of the Ti plasmid. Binary vectors, on theother hand, contain a broad host range origin of replication and a bacterial antibioticresistance gene, for selection and maintenance in Agrobacterium. They also have T-DNA borders between which they typically carry a selectable marker expressed inplants and a polylinker region (containing multiple, unique restriction sites for cloninggenes of interest). The pCGN15-derived plasmids are typical binary vectors (McBrideand Summerfelt, 1990). The pCGN15 series of binary vectors contain the left andright borders of the T-DNA (from pTiA6), a ColE1 origin of replication and transferorigin (from pBR322) expressed in E. coli, the pRiHRI origin of replication from theA. rhizo genes root inducing plasmid for high stability in Agrobacterium, a lac Z' genepolylinker segment from pUC18 incorporated into the T-DNA (blue/white screeningfor recombinant cloned gene plasmids of interest in E. coil), a gentamycin bacterialresistance marker compatible for selection in both E. coli and disarmed A. tumefaciensstrains and a choice between two types of chimeric npt II genes (plant selectablemarker) under the regulatory control of either the CaMV 355 promoter and the tm/terminator (tm/ 3', from pTiA6) or the mannopine synthase promoter (mas 5') and themannopine synthase termination signals (mas 3'). These binary vectors can bemobilized into Agrobacterium by triparental mating or by direct electroporation, butare maintained independently of the disarmed Ti plasmid in Agrobacterium.Following triparental mating, the Agrobacterium harbouring the gene of interest withina modified T-DNA region can be used to infect plant tissue, which must then beregenerated into a whole plant (see Fig. 1.2).BVVYV coatprotein gene cpolyadenylationsignalpCGN154802(15.3 kb)on pillvir regionon pTimas 3'npt IImas 5'Introduction p. 13plant cell wallAgrobacterium tumefaciensFig. 1.2 Agrobacterium tumefaciensTi plasmid mediated gene transfer. Agrobacterium movealong a plant wound exudate concentration gradient and attach to the wounded plant cells. Woundexudate (A) stimulates the vir region of the disarmed Ti plasmid (pEHA101) to (B) act on theT-region and initiate (C) T- strand transfer and integration. The polarity of T-DNA transfer occursfrom right border (RB) to the left border (LB). The example above shows the mobilized binaryplasmid pCGN1548 in the Agrobacterium tumefaciens EHA101. The BWYV coat protein gene,under the control of the CaMV 35S promoter, was cloned in between the RB and LB of the Tiplasmid, pCGN1548. Kan= kanamycin resistance marker; Gm= gentamycin resistance marker;RB/LB= right/left border regions from pTiA6; on pRI= origin of replication of the A. rhizo genesroot inducing plasimd pRiHRI; mas5'/mas3'= mannopine synthase promoter /terminator signalsfrom pTiA6; on Col El= origin of replication and transfer origin from pBR322; npt II= Tn5neomycin phosphotransferase gene; on pTi=origin of replication for the co-resident Ti plasmid.The shaded area on the pCGN154802 binary plasmid construct is the region that is transferred andintegrated into the genome of the plant.p.14Materials and MethodsProcedures commonly used in molecular biology and which are used in thisstudy are essentially as described by Sambrook et al. (1989). Restriction enzymes andDNA modifying enzymes were used according to the manufacturers' specificationsunless otherwise stated.2.1^BWYV Propagation and PurificationBeet western yellows virus was propagated in Brassica napus cv. Westar.Plants were inoculated by viruliferous Myzus persicae Sulz. in a growth chamber(21°C, 16 hr daylight/8 hr night, 60-80 uEm-2s-1 fluorescent and incandescent light) fora 2 to 3 day inoculation access period. The original BWYV, a sugarbeet isolate, waskindly provided by Peter Ellis, Agriculture Canada Vancouver Research Station.Plants were sprayed with PirimorTM to kill the aphids and then placed in a greenhouse(18-23°C). Plants were harvested 4-6 weeks after inoculation. Virus-infected tissuewas frozen in liquid nitrogen in an ice bucket, broken into coarse pieces with awooden pestle and stored at -20°C.The purification method used was a modification of the procedure described byD'Arcy et al. (1989). Approximately 250 g of frozen virus-infected tissue, wasMaterials and Methods p. 15ground until finely powdered in a Waring blender. Sodium phosphate buffer (0.1 M,pH 7.0) was added at a ratio of 2 mL per gram of tissue. Macerating enzyme (0.1%Ultrazym 100, Schweizerische Ferment AG, Basel, Switzerland), 2-mercaptoethanol(0.1% final concentration) and sodium azide (0.02% final concentration) were addedand the slurry stirred until homogeneous. The slurry was left overnight at roomtemperature without stirring. The preparation was made to 1% Triton X-100 andclarified by vigorous stirring for 3 hr at room temperature followed by the addition of1/6 volume chloroform:n-butanol (1:1) with continued vigorous stirring for 10 min atroom temperature. After low speed centrifugation for 20 min at 8 500 rpm in aSorvall GSA rotor, polyethylene glycol (PEG) 6000 and NaC1 were added to theaqueous phase to a final concentration of 8% and 1%, respectively and stirred at 4°Covernight. The preparation was centrifuged as above and the pellets were resuspendedin 0.1 M sodium phosphate buffer (pH 7.0) at a ratio of 10 mL per 50 g originaltissue. The preparation was layered on a 5 mL pad of 20% sucrose in 0.1 M sodiumphosphate buffer (pH 7.0) and centrifuged at 60 000 rpm for 1 hr in a Beckman 70.1rotor. The pellets were resuspended in 20 mL 0.1 M sodium phosphate buffer (pH7.0) and centrifuged for 20 min at 10 000 rpm in a Sorvall SS34 rotor. Thesupernatant was layered onto another 5 mL pad of 20% sucrose and centrifuged at60 000 rpm for 1 hr in a Beckman 70.1 rotor. The pellets were resuspended in 0.5mL 0.1 M sodium phosphate buffer (pH 7.0) with a glass rod, ground in a groundglass homogenizer and layered on top of a 10-40% linear sucrose density gradientprepared by the freeze-thaw method (Davis et al., 1978). After centrifugation atMaterials and Methods p. 1638 000 rpm for 1.5 hr in a Beckman SW41 rotor, the gradients were scanned at 254nm using an ISCO density gradient fractionator. The fractions containing virus werecollected and pelleted by centrifugation at 60 000 rpm for 1 hr in a Beckman 70.1rotor. The final high speed pellet was resuspended with 0.5 mL of 0.1 M sodiumphosphate buffer (pH 7.0). The ultraviolet absorption spectrum (220 to 320 nm) of thepreparation was recorded on a Hewlett-Packard Model 8451 A spectrophotometer. Ayield of 100 ug of purified BWYV was typically obtained from 250 g of virus-infectedtissue.2.2 BWYV Virion RNA ExtractionVirion RNA was isolated from purified virus by extraction withphenol/chloroform in the presence of sodium dodecyl sulfate (SDS). To 300 uL ofpurified virus suspension (0.15 - 0.3 mg virus) was added 500 uL phenol (saturatedwith Tris-HC1 buffer pH 8.0), 500 uL chloroform/octanol (24:1), 200 uL 0.5 M Tris-HC1 (pH 8.9), 10 uL 100 mM EDTA (pH 8.0) and 50 uL 20% SDS. The mixturewas vortexed and the aqueous and organic phases separated by centrifugation for 2minutes in an Eppendorf centrifuge at 14, 000 x g. The aqueous phase was drawn offand the organic phase re-extracted with 500 uL sterile, deionized H20. The resultingaqueous phases were pooled and extracted first with an equal volume ofphenol/chloroform/octanol (25:24:1) and then with an equal volume ofchloroform/octanol (24:1). To the final aqueous phase was added 1/10 volume of 2 MMaterials and Methods p. 17sodium acetate (pH 5.8) and 2 volumes of absolute ethanol. The RNA wasprecipitated at -70°C for 30 minutes and then centrifuged for 15 minutes at 4°C. Thepellet was washed with 70% ethanol, dried and resuspended in autoclaved deionizedH20. The quality of the RNA was assessed by electrophoresis through denaturingagarose gels and was quantified spectrophotometrically (a 1 mg/mL solution of RNAhas an A260 of 25). RNA samples were stored at -70°C.2.3^Amplification of the BWYV Coat Protein Gene by Reverse Transcriptionand the Polymerase Chain ReactionDouble stranded (ds) complementary copies of the BWYV coat protein genewere synthesized from BWYV RNA using reverse transcriptase (RT) followed by thepolymerase chain reaction (PCR), essentially as described by Sambrook et al. (1989).BWYV RNA (100 - 500 ng) was reverse transcribed in a 20 uL reaction volume in thepresence of Taq DNA polymerase buffer (Promega) [50 mM KC1, 50 mM Tris-HC1(pH 9.0), 50 mM NaC1, 10 mM MgC12, 12.5 ug activated calf thymus DNA, 0.1 %Triton X-100]; 1.25 mM dATP, dCTP, dGTP, dTTP; 50 pmol of BWYVoligonucleotide primer #2 (5'-AGA AGG CCA TGG OCT AGO GC-3',complementary to a region just downstream from the coat protein gene at nucleotides4108-4127 of the BWYV RNA); 20 units of RNase inhibitor (Promega); 2.5 mMMgC12 and 200 units of M-MLV reverse transcriptase (BRL). The reaction mixturewas incubated at 37°C for 30 minutes and the M-MLV reverse transcriptase wasMaterials and Methods p. 18inactivated by heating the reaction mixture at 95°C for 5 minutes. To amplify theDNA generated by the reverse transcription, the reaction mixture was amended with50 pmol of BWYV oligonucleotide primer #1 (5'-CGG CAC CAT GGA TAC GGTCGT GGG TAG G-3', identical to the sequence beginning at the coat protein initiatorcodon at nucleotides 3482-3503 of the BWYV RNA), 2.5 units of Taq DNApolymerase (Promega) and Taq DNA polymerase buffer to a final volume of 100 uL.The reaction mixture was overlaid with 100 uL of light mineral oil and theamplification reactions carried out in a thermocycler (EriComp). The first cycle of thePCR included denaturation at 94°C for 5 minutes, annealing at 55°C for 2 minutes andpolymerization at 72°C for 2 minutes. This was followed with 30 cycles ofdenaturation at 94°C for 1 minute, annealing at 55°C for 2 minutes and polymerizationat 72°C for 2 minutes. The final cycle of the PCR involved denaturation at 94°C for1 minute, annealing at 55°C for 2 minutes and polymerization at 72°C for 10 minutes.The mineral oil overlay was removed by the addition of 100 uL of chloroform/octanol(24:1) followed by brief centrifugation. The aqueous phase, which contained theamplified DNA, was drawn off and a sample was analysed by electrophoresis througha 1% agarose gel and visualized by staining with ethidium bromide. The amplifiedDNA from the PCR reaction was prepared for subcloning by digestion with Nco Irestriction enzyme and the Double GeneCleanTM manufacturer's procedure (Bio101Inc.). DNA amplified in this manner was cloned into the Nco I site of the pRT103plasmid. This strategy is described more thoroughly in the Results section 3.2.Materials and Methods p. 192.4 DNA sequencing of the BWYV Coat Protein GeneThe dideoxynucleotide chain termination method of Sanger et al. (1977) wasused to sequence two double stranded plasmid DNA templates (see Section 3.2.1)using a modified T7 DNA polymerase (SequenaseTM, US Biochemicals). Thefollowing protocol was provided by US Biochemicals. Two micrograms of plasmidDNA and 10 ng of sequencing primer [BWYV oligonucleotide primer #1 or #2 orpRT103 oligonucleotide primer #1 (5'-CIT CCT CTA TAT AAG G-3') or #2 (5'-CTA C'TC ACA CAT TAT TC-3')] were denatured in a 40 uL volume containing 200mM NaOH and 0.4 mM EDTA by heating at 95°C for 5 minutes. The reactions werequickly cooled on ice and 1/10 volume of 2 M ammonium acetate and 2 volumes ofabsolute ethanol were added. The DNA was precipitated at -70°C for 30 minutes andthen centrifuged for 15 minutes at 14, 000 x g in an Eppendorf microcentrifuge. Thepellet was washed in 70% ethanol, dried and resuspended in 6 uL H20 and 1.5 uL 5XSequenaseTM buffer [5X buffer is 200 mM Tris-HC1 (pH 7.5), 100 mM MgCl2 and 250mM NaC1]. The DNA was incubated at 37°C for 15 minutes to allow the primer toanneal to the denatured DNA template. To this solution was added 1 uL 100 mMDTT, 2 uL lx labeling mix (1.5 uM each of dCTP, dGTP and dTTP), 2 to 5 uCi of a-[32PNATP (3000 Ci/mmol) and 3 units of SequenaseTM in a total volume of 13 uL.The reactions stood at room temperature for 2 to 5 minutes and 3.3 uL aliquots of thisreaction mixture were added to tubes containing 2.5 uL 50 mM NaC1, 80 uM each ofdGTP, del?, dTTP, dATP and 8 uM of either ddGTP, ddCTP, dd'ITP or ddATP.Materials and Methods p. 20The reactions were incubated at 37°C for a further 20 minutes and to the reactions wasadded 4 uL of a mixture containing 95% formamide, 20 mM EDTA, 0.05%bromophenol blue and 0.05% xylene cyanol FR The sequencing reactions weredenatured in this mixture by heating at 95°C for 3 minutes before loading onto thesequencing gel. Sequencing reactions were electrophoresed through 6%polyacrylamide gels (using wedged spacers with 0.2 to 0.6 mm gradation) containing6.7 M urea for 3-6 hours at constant power (48 watts). Following electrophoresis, thegels were transferred onto Whatman 3MM filter paper, dried under vacuum at 80°Cfor 1 hour and then exposed to Kodak XOmatTM film overnight at room temperature.The sequences of the 5' and 3' ends (250 bp) of the BWYV coat protein cDNA weredetermined using the BW102D or BW137D clones and one of the primers, BWYVoligonucleotide #1 or #2 or pRT103 oligonucleotide #1 or #2. The sequence of theinternal portion (200 bp) of the BWYV coat protein cDNA was determined using adeletion clone BW102DASma I and the pRT103 oligonucleotide primer #2.2.5 In Vitro Translation Analysis of the BWYV Coat Protein Gene2.5.1 In Vitro TranscriptionSynthetic transcripts corresponding to the BWYV coat protein region wereproduced in vitro using the bacteriophage T3 RNA polymerase promoter and thepBluescriptil KS+ plasmid (Stratagene) containing the BWYV coat protein cDNAMaterials and Methods p. 21insert from clone BW102D (see Section 3.3.1). The plasmid construct was linearizedwith Sst I restriction enzyme and used for run-off transcription. In vitro transcriptionreactions were carried out in the absence of cap analogue in a 50 uL volumecontaining 40 mM Tris-HC1 (pH 8.0), 10 mM NaC1, 6 mM MgC12, 2 mM spermidine,2.5 mM each of ATP, CTP, GTP and UTP, 10 mM MT, 10 units RNasin, 50 unitsT3 RNA polymerase (BRL) and 2 ug linearized plasmid DNA. The reaction mixturewas incubated at 37°C for 1 hour after which 0.2 units of DNase I was added andincubated for a further 15 minutes at 37°C. The reaction mixture was extracted withphenol/chloroform/octanol (25:24:1), the aqueous phase drawn off and 1/10 volume of2M sodium acetate, pH 5.8 and 2 volumes of absolute ethanol added. The RNA wasprecipitated at -70°C for 1 hour, centrifuged for 15 minutes and the pellet resuspendedin autoclaved deionized H20. The quality and amount of RNA transcript producedwas determined by comparison with known quantities of cucumber necrosis virusRNA after agarose gel electrophoresis and ethidium bromide staining.2.5.2 Translation In Vitro using Wheat Germ ExtractsSynthetic RNA transcripts, prepared as described above, were translated inwheat germ extracts (NEN) in the presence of [35SJ-methionine (NEN; specific activity1200 Ci/mmol) according to the manufacturer's instructions. The 25 uL translationreaction, which included 50-100 ng synthetic RNA transcript, 50 uCi [35S]-methionine,12.5 uL wheat germ extract, 80 uM minus-methionine amino acid mixture and 75Materials and Methods p. 22mM potassium acetate, was carried out at 25°C for 1 hour. Translation products wereanalyzed following electrophoresis through sodium dodecyl sulfate containingpolyacrylamide gels (SDS-PAGE, 15% gel, 0.75 mm thick) using the discontinuousLaemmli buffer system (Laemmli, 1970). Gels were then fixed in three changes of30% methanol/10% acetic acid for 30 minutes each with continuous agitationaccording to the manufacturer's instructions. Gels were dried for 1 hour at 80°C undervacuum on Whatman 3MM paper and then fluorographed using Entensify (NEN) andexposed to X-ray film at -70°C overnight . The sizes of the translation products wereestimated by comparison with the published sizes of the in vitro translation products ofCNV (Rochon et al., 1991).2.6^Tr-Parental Mating ProcedureThe triparental mating procedure was used to introduce the binary plasmidconstructs (154802, 154837, 1548, 155702, 155737 and 1557) into A. tumefaciensEHA101 (Rogers et al., 1986). The three bacteria involved in conjugation were (1)E. coli MM294 containing the binary plasmid constructs, (2) E. coli MM294containing the mobilization plasmid, pRK2013 and (3) A. tumefaciens EHA101containing a disarmed octopine type plasmid. In the triparental mating, the pRK2013plasmid mobilized into the E. coli containing the binary plasmid construct, providedRK2 transfer proteins and the ColE1 mob protein which acts on the born site of thebinary plasmid and thereby mobilized the binary plasmid into A. tumefaciens. TheMaterials and Methods p. 23pCGN15-derived binary plasmids contained the ColE1 and pRiHRI origins forreplication in both E. coli and A. tumefaciens, respectively. Overnight cultures of theE. coli and A. tumefaciens were started: E. coli containing the binary plasmidconstructs of interest were grown in 2 mL of Luria-Bertani (LB) broth plus 10 ug/mLgentamycin, while the E. co/i/pRK2013 and A. tumefaciens were grown in 10 mL LBplus 50 ug/mL kanamycin (the Agrobacterium was grown at 28°C to avoid loss of theTi plasmid). One millilitre (mL) from each of the cultures was mixed together in asterile tube, centrifuged and the pellet resuspended in 2 mL 10 mM MgSO4. Themixture was filtered through a syringe filter (NalgeneTM 25mm, 0.45 um), the filterdisc removed from the casing and transferred sterilely onto a fresh, nondried LB agarplate. The plate was incubated at 28°C overnight and the following day, the filter discwas removed aseptically and placed into a sterile tube containing 2 mL 10 mMMgSO4. The tube was vortexed to remove cells from the filter disc and a 0.1 mLaliquot of the suspension was spread onto a freshly prepared LB agar selection platecontaining 50 ug/mL gentamycin and 100 ug/mL kanamycin. The plates wereincubated at 28°C for 3-4 days after which several hundred colonies appeareddepending on the efficiency of the mating. Several colonies were inoculated into LBcontaining gentamycin and kanamycin and grown at 28°C for 16-24 hours. DNA fromthe Agrobacterium was extracted by the small nucleic acid preparation procedure (Anet al., 1988) and analyzed by agarose gel electrophoresis followed by ethidiumbromide staining and by Southern blot analysis using a random primed, 13211-labeledcDNA BIA/YV coat protein probe (see Section 3.5). Glycerated bacterial cultures,Materials and Methods p. 24consisting of 0.15 mL sterile glycerol and 0.85 mL of the overnight culture, werestored at -70°C. Agrobacterium cells were also streaked onto LB plates containinggentamycin and kanamycin and stored at 4°C for 2 weeks; colonies were restreakedonto fresh plates every 2 weeks.2.7^Transformation and Regeneration of Brassica napus cv. WestarThe plant transformation procedure used was a modification of the proceduredescribed by Moloney et al. (1989) for transformation of Brassica napus cv. Westarutilizing cotyledonary explants. Four days after germination of seeds [on Murashige-Skoog minimal organics medium (MS) with 3% sucrose and 0.7% phytagar, pH 5.8;24°C in a 16 hours light/8 hours dark photoperiod, 60-80 uEm2s-1], cotyledons wereexcised and immersed into a bacterial suspension of A. tumefaciens EHA101containing the binary plasmid of interest. The cotyledonary explants were co-cultivated with the A. tumefaciens for 72 hours (on MS medium, 3% sucrose and 0.7%phytagar, pH 5.8 enriched with 20 uM benzyladenine). The cut ends of B. napuscotyledons have been reported to be highly susceptible to Agrobacterium-mediatedgene transfer and also to display very high regeneration rates (Moloney et al., 1989).After 72 hours, the cotyledons were set out on regeneration medium (MS medium, 3%sucrose and 0.7% phytagar, pH 5.8 enriched with 20 uM benzyladenine) supplementedwith selective antibiotics (15 mg/L kanamycin and 500 mg/L carbenicillin). Shootsappeared on the explants after 3-5 weeks; some shoots that were not transformed withMaterials and Methods p. 25the npt 1:1 gene appeared bleached by the fourth week of culture on kanamycin. Theshoots which remained green were sub-cultured first onto shoot elongation medium(regeneration medium without benzyladenine) for 4-6 weeks and then onto 'rooting'medium (MS medium, 3% sucrose, 4 mg/L indole butyric acid, 0.7% phytagar and500 mg/L carbenicillin) for 4-6 weeks. No kanarnycin was used during this stagesince it was found that more rapid root establishment occurred without the selectionagent. Plantlets were transferred to potting mix and placed in a misting chamber for2-3 weeks after which the plants were transferred to the greenhouse and allowed toflower and set seed.2.8 DNA Extraction from Plants using the CTAB MethodPlant genomic DNA was extracted from the regenerated B. napus cv. Westarby the procedure that utilizes hexadecyltrimethylammonium bromide (CTAB) aspreviously described by Doyle et al. (1990). One gram of fresh, leaf tissue wasground in liquid nitrogen in a chilled mortar and pestle. The powder was gentlymixed into preheated (60°C) CTAB isolation buffer [2% (w/v) CTAB (Sigma), 1.4 MNaCl, 0.2% (v/v) 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris-HC1, (pH 8.0)) in a30 mL centrifuge tube. The sample was incubated at 60°C for 30 minutes withoccasional gentle swirling and afterwards, extracted with chloroform/isoamyl alcohol(24:1;v/v). After centrifugation at 1600 x g in a HB-4 rotor at room temperature, theaqueous phase was drawn off with a wide-bore pipet and 2/3 volume of coldMaterials and Methods p. 26isopropanol was added to precipitate the nucleic acids. The sample was left at roomtemperature overnight. The sample was centrifuged at 500 x g in a SS34 rotor for2 minutes, the supernatant poured off and 20 mL of wash buffer [76% (v/v) ethanol,10 mM ammonium acetate] added. After 20 minutes of washing, the sample wascentrifuged at 1600 x g for 10 min in a SS34 rotor and the pellet resuspended in 1 mLTE [10 mM Tris-HC1 (pH 7.4), 1 mM EDTA]. RNase A was added to a finalconcentration of 10 ug/mL and incubated at 37°C for 30 minutes. The sample wasdiluted with 2 volumes of TE and the DNA precipitated with 7.5 M ammoniumacetate (pH 7.7) added to a final concentration of 2.5 M and 2 volumes of coldethanol. After centrifugation at 10,000 x g for 10 min in a SS34 rotor, the pellet wasair dried and resuspended in 100 uL of TE. The final DNA sample was highlyviscous. The yields, determined spectrophotometrically, ranged from 100 ug to 800 uggenomic DNA per two grams of tissue.2.9 Synthesis of Random-Primed cDNA ProbesRandom primed, [321]-labeled probes were prepared with the Random PrimersDNA Labeling System (BRL) according to the manufacturer's instructions. Twenty-five nanograms of gel purified ds cDNA, which corresponded to the Nco I fragment ofclone BW102D containing the BWYV coat protein gene (essentially the entire BWYVcoat protein gene), was used as template. Specific activity of the probes wasapproximately 1.5 x 109 cpm/ug DNA.Materials and Methods p. 272.10 Southern Blot AnalysisPlant genomic DNA samples (50 - 150 ug) were digested with Hind III orEco R1 restriction enzyme (2.5 - 5 times recommended units) at 37°C for 1.5 - 2.0hours. The DNA samples were electrophoresed through a 1% agarose gel and stainedwith ethidium bromide. The gel was photographed under UV light (320 nm) andmolecular size markers (Lambda DNA digested with Eco RI/Hind III) referenced to afluorescent ruler. The DNA was blotted onto Zeta-probe GT membrane (BioRad)under alkaline conditions (0.4 M NaOH) at room temperature for 8-16 hours accordingto the manufacturer's instructions. The following hybridization method was alsoaccording to the manufacturer's instructions (BioRad; Reed and Mann, 1985). Themembranes were prehybridized in hybridization buffer (7% SDS, 0.25 M Na2HPO4,pH 7.2) at 65°C for 0.5 to 2 hours. Hybridization with random primed, [3211-labeledprobe, corresponding to the BWYV coat protein gene or npt II gene, was carried outin a Hybaid oven (BioCan Scientific) for 16-24 hours at 65°C using approximately 1.9x 106 cpm of probe (25 ng) per mililiter of hybridization buffer. After hybridization,the membranes were washed successively in 5% SDS, 20 mM Na2HPO4 (pH 7.2)and 1% SDS, 20 mM Na2HPO4 (pH 7.2). All washes were at 65°C for 30 minuteseach. Excess moisture was removed from the membranes and they were wrapped inclear, plastic wrap. The membranes were exposed to X-ray film (X-Omat K, Kodak)at -70°C for 6 hours-7 days, depending on the experiment, with the aid of twoMaterials and Methods p. 28Lightening Plus intensifying screens (DuPont).2.11 Leaf RNA extractionApproximately 1-2 g leaf material was ground to a fine powder in liquidnitrogen, mixed vigorously with 3-5 volumes 10x TNE (100 mM Tris-HC1 pH 7.5,100 mM NaC1, 10 mM EDTA), an equal volume of phenol/chloroform/octanol(25:24:1) containing 0.1% SDS and 5% 2-mercaptoethanol and centrifuged at 8 000rpm for 5 minutes at 4°C (SS34 rotor). The aqueous phase was collected, re-extractedwith an equal volume of phenol/chloroform/octanol (25:24:1), and then extracted withchloroform/octanol (24:1). The aqueous phase was combined with 1/10 volume of2 M sodium acetate (pH 5.8) and 2 volumes of absolute ethanol, precipitated at-70°C for 1 hour and then centrifuged as above. The pellet was resuspended in 3 mLof TE, to which 1 mL 8 M LiC1 was added. Samples were left on ice for 1-3 hours,then centrifuged at 8 000 rpm for 10 minutes at 4°C (SS34 rotor). The pellet, whichcontained high molecular weight single stranded RNA, was resuspended in 375 uLwater, then re-precipitated with 1/10 volume 2 M sodium acetate (pH 5.8) and 2volumes absolute ethanol. Samples were placed at -70°C for 1 hour, then centrifugedfor 10 min at 4°C. Pellets were washed with 70% ethanol and the pellet resuspendedin 50 uL of sterile, deionized water. Nucleic acid concentration was determinedspectrophotometrically (one A260 unit of RNA= 40 ug/mL).Materials and Methods p. 292.12 Northern blot analysisPlant RNA , obtained as described above, was denatured with either 5 mMmethylmercuric hydroxide (MeHg0H) and electrophoresed through 1% agarose gelsprepared with BE buffer (40 mM boric acid, 1mM EDTA, pH 8.2) or with glyoxal anddimethyl sulfoxide and electrophoresed through 1% agarose gels prepared with 0.01 Msodium phosphate buffer, pH 7.0 (Sambrook et al., 1989) . After electrophoresis,RNA was blotted onto Zeta-probe GT membranes (BioRad) under alkaline conditions(10 mM NaOH) at room temperature for 6-16 hours. The membranes wereprehybridized in hybridization buffer (50% formamide, 7% SDS, 0.25 M NaC1, 0.12M Na2HPO4, pH 7.2) for 1 hour at 42°C. Hybridization with a random primed, [32P]-labeled probe, corresponding to the BWYV coat protein gene or the npt II gene, wascarried out at 42°C for 6-16 hours. After hybridization, the membranes were washedsuccessively in 2X SSC/0.1% SDS (20 X SSC is 3 M NaC1, 0.3 M trisodium citrate),0.5X SSC/0.1% SDS and finally, in 0.1X SSC/0.1% SDS. All washes were conductedat 60°C for 15 min each. The membranes were wrapped in clear, plastic wrap andexposed to X-ray film at -70°C for 4 hours to 14 days with the aid of two intensifyingscreens (DuPont).Materials and Methods p. 302.13 Production of Monoclonal Antibodies and Polyclonal AntiseraThe production of monoclonal antibodies and polyclonal antisera which reactagainst BWYV coat protein subunits or disrupted BWYV particles is essentially thesame as that described for the production of monoclonal antibodies and polyclonalantisera which react against intact BWYV particles (Ellis and Wieczorek, 1992). Forthe production of monoclonal antibodies, BALB/c mice were immunized with at leastthree injections of virus (50 ug virus each injection). Prior to injection, the purifiedvirus sample was treated in 1% SDS, heated at 95°C for 5 minutes, and dialyzedagainst 0.05 M phosphate buffer, pH 7.0. The first injection was 50 ug of purifieddisrupted virus particles emulsified with Freund's incomplete adjuvant and givensubcutaneously. The second and third injections consisted of 50 ug each of purifieddisrupted virus particles in 0.05 M phosphate buffer given intraperitoneally. Theinjections were given at 3-4 week intervals. Three days after the third injection, themice were sacrificed and the spleens harvested for use in the fusion protocol forhybridoma production. The hybridomas were screened for anti-BWYV (disruptedparticles) antibody production by an indirect enzyme-linked immunosorbent assay(ELISA). Similarly, five purified BWYV preparations (disrupted by treatment in 1%SDS and heating at 95°C for 5 minutes followed by dialysis against 0.05 M phosphatebuffer) were used to immunize a young, New Zealand White rabbit. The firstinjection, 0.1-0.5 mg of purified virus was disrupted and the subunits emulsified withan equal volume of Freund's complete adjuvant, was administered intramuscularly in aMaterials and Methods p. 31hind leg. The subsequent injections of purified disrupted virus particles emulsifiedwith Freund's incomplete adjuvant were given at 3-4 week intervals or longer, whenpurified virus samples were limited. IgG was purified from a test bleed after thefourth injection. The antiserum was evaluated in a double antibody sandwich-ELISA(DAS-ELISA) for reactivity with disrupted BWYV particles (treated as above or withcarbonate buffer, pH 9.2).2.14 Evaluation of Existing Monoclonal Antibodies in Western Blot and DotBlot AnalysisSeveral monoclonal antibodies (510H 1 gG2a a BWYV, 43BC IgM a BWYV &PLRV, 4G12 IgM a BWYV and 26BE IgG1 a PLRV) and polyclonal antisera (rabbita BWYV IgG and rabbit a PLRV IgG), kindly provided by Peter Ellis (AgricultureCanada Research Station), were evaluated for reactivity with disrupted BWYVparticles in Western blot and dot immunobinding assays. Varying amounts of purifiedBWYV samples (100-500 ng) were resuspended with an equal volume of SDS-PAGEsample buffer (4% SDS, 125 mM Tris-HC1 pH 6.8, 10% 2-mercaptoethanol, 0.4%bromophenol blue and 20% glycerol) and then incubated at 85°C for 10 min. Sampleswere centrifuged (13 000 x g) for 5 min and aliquots were electrophoresed through a12% polyacrylamide gel using the buffer system of Laemmli (1970). Separatedproteins were electroblotted (100V, 0.25A, 4°C, 1 hour) onto PVDF membrane (0.2micron, BioRad) in a buffer composed of 25 mM Tris, 192 mM glycine, 20%Materials and Methods p. 32methanol (pH 8.3). The transfer blots were treated with blocking buffer (5% skimmilk powder in 10 mM phosphate buffer, 150 mM NaC1) for 1 hour and incubated(4°C, overnight) first with the diluted monoclonal antibody of interest (in 10 mM TrispH 7.5, 0.15 M NaC1, 1% BSA, 0.1% sodium azide, 0.05% Tween 20) or thepolyclonal antisera and then VINabeled goat anti-mouse IgG or the VINabeled goatanti-rabbit IgG, respectively. The goat IgGs were radioiodinated by the chloramine Tmethod (Sambrook et al., 1989). After washing in PBS-Tween (10 mM phosphatebuffer pH 7.5, 0.05% Tween 20) the membranes were exposed to X-ray film at -70°C overnight with intensifying screens. The specific activity of the radioiodinatedIgG was approximately 5 x 107 cpm/mg protein.Similarly, varying amounts of purified BWYV samples (50-600 ng) werespotted onto PVDF membrane. Untreated samples (intact BWYV particles) andtreated samples (1% SDS, heating at 95°C for 5 min) were spotted onto themembranes and incubated with the monoclonal antibody or polyclonal antisera asabove.p. 33Results3.1^Amplification of the BWYV Coat Protein Gene by Reverse Transcription -Polymerase Chain ReactionDouble-stranded (ds) complementary DNA (cDNA) copies of the BWYV coatprotein gene were synthesized from BWYV RNA using reverse transcription (RT)followed by polymerase chain reaction (PCR). The BWYV RNA was extracted frompurified virus, originally isolated from sugarbeet (provided by Peter Ellis, AgricultureCanada Vancouver Research Station). The first strand of the cDNA was synthesizedutilizing reverse transcriptase and a 20-mer oligonucleotide (BWYV oligonucleotide#2, 5'-AGA AGG CCA TOG GCT AGG GC-3') primer. The sequence of the primerwas based on the published genomic RNA sequence of BWYV (Veidt et al., 1988).The BWYV oligonucleotide #2 is complementary to a region just downstream fromthe coat protein gene at nucleotides 4108-4127 of BWYV RNA. A nucleotidesubstitution in the primer sequence from a C to an A at position 4119 permitted theincorporation of a Nco I restriction enzyme site at the 3' end of the PCR productcorresponding to the 3' terminus of the BWYV coat protein gene. The second strandof the cDNA, and the subsequent cDNA copies generated in the PCR reaction, weresynthesized with a second 28-mer oligonucleotide (BWYV oligonucleotide #1, 5'-CGGCAC CAT GGA TAC GGT COT GGG TAG G-3') primer. The BWYVResults p. 34oligonucleotide #1 is identical to the sequence beginning at the coat protein initiatorcodon at nucleotides 3482-3503 of BWYV RNA except at position 3485. Anucleotide substitution in the primer sequence from an A to a G (highlighted inboldface) and the addition of seven nucleotides at the 5' end of the BWYVoligonucleotide #1 primer (underlined) permitted the incorporation of a Nco Irestriction enzyme site at the 5' end of the expected PCR product. The resulting dscDNA copy of the BWYV coat protein gene was 652 bp in size as determined byagarose gel electrophoresis (see Fig. 3.1, lane 5).Initial attempts to synthesize double stranded DNA copies of the BWYV coatprotein gene by RT-PCR from RNA extracts of BWYV-infected Brassica napus,collected from the U.B.C. field station (Ellis and Stace-Smith, 1990) wereunsuccessful. PCR products were obtained from RNA extracts of both healthy andinfected plants and were determined by agarose gel electrophoresis to be smaller insize than that expected for the cDNA of the BWYV coat protein gene (compare lanes3 and 4 with lane 5, Fig. 3.1). Subsequent restriction enzyme analysis and partialnucleotide sequencing of the cloned products confirmed that they did not correspondto the BWYV coat protein gene nor to any other part of the BWYV genome (resultsnot shown).5.24 5.054.213.411234 5621.82== 1.901.980.680.590.250.131.55ammo^147aim* 1.320.930.840.58Results p. 35Fig. 3.1 Amplification of the BWYV coat protein gene by RT-PCR. The RNA templatesused in the RT-PCR reactions were as follows: lane (2) contained no RNA template, lane (3)contained RNA from uninfected Brassica napus, lane (4) contained RNA fromBWYV-infected Brassica napus and lane (5) contained BWYV RNA extracted from purifiedvirus. The PCR products obtained were analyzed by electrophoresis through a 1% agarose geland stained with ethidium bromide. Lanes (1) and (6) contained molecular size markers:pBluescribe digested with Pvu II and Rsa I; Lambda DNA digested with Eco RI and Hind III(sizes in kbp).Results^p. 363.2 Cloning of the Double-Stranded cDNA Copies of the BWYV Coat ProteinGene into the pRT103 Plant Expression VectorThe ds cDNA copy of the BWYV coat protein gene obtained by PCRamplification was digested with Nco I restriction enzyme and the compatible endswere ligated into the similarly digested pRT103 plasmid. The pRT103 plasmid carriesthe cauliflower mosaic virus (CaMV) 35S promoter and polyadenylation signal (TOpferet al., 1987; see Fig. 3.2). As well, the AUG codon of the Nco I restriction enzymesite (of the multicloning site) is embedded in the consensus sequence G/AxxAUGGfor optimal ribosome initiation in eukaryotes (Kozak, 1984). The ligation mixture wasused to transform competent Escherichia coil DH5a cells which were then grown onLB-agar containing ampicillin. Several ampicillin-resistant colonies were obtained.Individual colonies were cultured in Luria-Bertani (LB) broth containing ampicillinand their nucleic acid isolated by the small scale mini-preparation procedure. Thepresence and orientation of an insertion was determined by restriction enzymemapping. The BWYV coat protein gene was determined by restriction enzymemapping to be inserted into the Ncol site of the pRT103 plasmid in both the plus-sense orientation (clone BW102D) and the anti-sense orientation (clone BW137D)relative to the CaMV 35S promoter (see Fig. 3.2).RWVV RNAI M-MLV Reverse TranscriptaseBWYV oligonucleotideprimer #2Polymerase Chain ReactionBWYV oligonucleotideprimer #1 & #2I Nco I1 T4 DNA LigaseTransformation of DH5a cellsPlate on LB-agar with AmpicillinSelection of clonesby restriction mappingwith Nco I and Sma I, Hind IIISph IBVVYVmat proteingeneHam HISma IKpn ISot INco IampicillinresistanceCaMVpolyadenylationsignalBVVYVcoat proteingeneXbalBainSma IKpn ISotNcoCaMV 355promoterCaMV 355promoterBW102D3960 bpHine IIPot!Sph IHind IIlinePot ISph IHind HIBW137D3960 bpResults p. 37Fig. 3.2 Diagram showing the construction of the pRT103/13WYV coat protein gene clones. Purified viral RNAwas used as template, together with the BWYV oligonucleotide primer #2, for the first strand cDNA synthesis usingM-MLV reverse transcriptase. The second strand cDNA and subsequent copies, were synthesized by the addition ofthe BWYV oligonucleotide primer #1 in the polymerase chain reaction. Following amplification, the cDNA wasdigested with Nco I restriction enzyme and ligated into the similarly digested pRT103 plasmid using T4 DNAligase. The ligation mixture was used to transform competent DH5acells which were then plated on LB-agarcontaining ampicillin. The DNA from several individual colonies was screened by restriction enzyme mappingusing Nco I and Sma I. The cDNA of the BWYV coat protein was found to be inserted into the NcoI site of thepRT103 plasmid in both the plus-sense (clone BW102D) and anti-sense orientation (clone BW137D) relative to theCaMV 355 promoter.Results p. 383.2.1 DNA Sequencing of the BWYV Coat Protein GeneBoth the pRT103 clones, containing the cDNA of the BWYV coat protein genein the plus-sense orientation (clone BW102D) and in the anti-sense orientation (cloneBW137D) relative to the CaMV 35S promoter, were sequenced using the sameoligonucleotide primers (BWYV oligonucleotide #1, 5' primer and BWYVoligonucleotide #2, 3' primer) previously used in the PCR reactions (see section 3.1).In addition, two pRT103 primers were used to confirm the sequences flanking theNco I site of insertion. The pRT103 oligonucleotide #1 primer is a 16-meroligonucleotide (5'-CTT CCT CTA TAT AAG G-3') whose sequence is identical to aregion of the CaMV 35S promoter upstream (5' end) from the multicloning site. ThepRT103 oligonucleotide #2 primer is a 17-mer oligonucleotide (5'-CTA CTC ACACAT TAT TC-3') whose sequence is complementary to a region downstream (3' end)from the multicloning site. Using these four primers (BWYV oligonucleotide #1 and#2, pRT103 oligonucleotide #1 and #2), the sequences at the 5'- and 3'-ends(approximately 250 bp on each end) of the BW102D and BW137D clones weredetermined (see Fig. 3.3, 3.4). The internal portion of the BWYV coat protein gene(approximately 200 bp) was sequenced using a Sma I deletion clone of BW102D.Clone BW102D, which contains the BWYV coat protein gene inserted in the plus-sense orientation, was digested with Sma I restriction enzyme. The larger portion ofthe plasmid (approximately 3.7 kbp in size) was gel purified and the ends re-ligatal.The Sma I deletion clone (BW102DASma I) contained the BWYV coat protein geneResults p. 39Project: BWYV1, Meld: BWYV10021^ 201^ 401^ 601^ 801Nco I Sma I^Nco I5' IBWYV coat protein gene --->-->BWYV oligo #1^BWYV oligo #2<-->---L026---><---L031---<<---L023--<>^L022----><---L007--<>---L019---><---L029---<> L004^ ><--+2-< L012>^ L009 > <^L010^<>---L028-->^<----L015----<>----L014---->>^L011---->>^L020---->>^L024--->>----L016---->>^L018^>>----L006----><^L001^<<^L002----<<---+1--< L030Fig. 3.3^Strategy used to sequence the BWYV coat protein coding region of cloneBW102D. The 5' and 3' ends (250 bp) of the BWYV coat protein gene were sequencedusing the BWYV oligonucleotide primers #1 and #2 and the pRT103 oligonucleotide primers#1 and #2. The internal portion of the BWYV coat protein gene (200 bp) was sequencedusing a Sma I deletion clone of BW102D and the pRT103 oligonucleotide primer #2. A totalof 23 different sequence readings from the two clones, BW102D and BW102DASma I, wereused to obtain the consensus sequence BWYV1002 (650 bp). The arrows (>---> or <---<)indicate the direction of the sequence. (nn) represents the sequence number in LOnn.3'Results p. 40^10^20^30^40^I I I ICCACCATGGATACGGTCGTGGGTAGGAGAACAATCAATGGAAGAAP P WIRS WVGEQSMEEHHGYGRG—ENNQWKKTMD TVVGRR TINGR50^60^70^80^90I I I I IGACGACCACGCAGGCAAACACGACGCGCTCGGCCGTCTCAGCCAGD DHAGKHDALGRLSQT T TQANT TRS AVS ASRRPRRQTRRARPSQP100^110^120^130I I I ITGGTTGTGGTCCAAACCTCTCGGGCAACACAACGCCGACCTAGACW LWSKPLGQHNADLDGCGPNLSGNT TP T —TVVVVQT SRA TQRRPR140^150^160^170^180I I I I IGACGACGAAGAGGTAATAACCGGACAAGAGGAACTGTTCCTACCAD DEEVI T GQEELF LPT TKR——PDKRNCS YQRRRRGNNR TRG T VP T190^200^210^220I I I IGAGGAGCAGGCTCAAGCGAGACATTTGTTTTCTCGAAAGACAATCE EQAQARHLF SRK TIR SRLK RD ICF LERQSRGAGS SETFVF SKDN230^240^250^260^270I I I I ITCGCGGGAAGTTCCAGCGGACGAATCACGTTCGGGCCGAGTCTATSREVPADESRSGRVYRGKFQR TNHVRAES ILAGS S S GRITF GP SLResults p. 41^280^290^300^310^I I I ICAGACTGCCCAGCATTCTCTAATGGAATACTCAAGGCCTACCATGQ T AQHSLME Y S RP TMRLP S IL —WNTQGLP —SD CPAF^ GILK A YH320^330^340^350^360I I I I IAGTATAAAATCTCGATGGTCATTTTGGAGTTCGTCTCCGAAGCCTS IK SRWSF WS S SPKPV —NL D GHF GVRLR S LE YK I SMVILEF VS E A370^380^390^400I I I ICTTCCCAAAACTCCGGTTCCATCGCTTATGAGCTGGACCCACACTL PK TP VP SLMS W T H TF PK LRFHRL —AGP T LS S QNS GS I AYELDP H410^420^430^440^450I I I I IGTAGACTCGACGCCCTTTCCTCGACCATCAATAAGTTCGGGATCA^ DS TPFPRPSIS  S GS— TRRPF LDHQ—VRDHCRLD AL S STINK  F GI460^470^480^490I I I ICAAAGCCCGGGAGGAGGGCGTTTACAGCGTCTTACATCAACGGGAQ SP GGGRLQRL T ST GK AREE GV Y S VLHQRDTKP GRRAF T AS YING500^510^520^530^540I I I I ICGGATTGGCACGACGTTGCCAAGGACCAATTCAGGATCCTCTACARIGT TLPR TNS GS S TGL ARR CQ GP I QDP L QTDWHDVAKDQFR IL YResults p. 42^550^560^570^580^I I I IAAGGCAATGGTTCTTCATCGATAGCCGGTTCTTTTAGAATCACTAK AMVLHR—PVLLESLRQWFF IDSRFF—NHYK GNGS S S IAGSFR I T590^600^610^620^630I I I I ITAAAGTGTCAATTCCACAACCCCAAATAGGTAGACGAGGAACCCG—SVNS T TPNR— TRNPK VS IP QP QIGRRG TRIKCQFHNPK —VDEEP640^650GCCCTAGCCCATGGGCGAGCTCALAHGR AP—PMGELGP SP WASFig. 3.4 Nucleotide sequence and amino acid sequence of the BWYV coat proteincoding region. The sequence of the BWYV coat protein cDNA was constructed froma meld of 23 different sequence readings of clone BW102D in the ASSEMGELprogram of PCGENE (IntelliGenetics). The first and second initiation codons,ATG,correspond to the BWYV 22.5 kDa coat protein and the 19.5 kDa putativegenome-linked protein (VPg), respectively, and are highlighted in boldface. Alsoshown is the predicted amino acid sequence in the three different open reading frames(ORE). The dashes in the amino acid sequence correspond to stop codons in thenucleotide sequence. The terminator codon, TAG, for each ORE is also shown inboldface. Single letter codes were used for the various amino acids.Results p. 43with approximately 200 bp deleted from the 3' end of the coat protein gene. Since theNco I site at the 3' end of the coat protein gene was also deleted, it was necessary touse the pRT103 oligonucleotide #2 primer to sequence the internal portion of the coatprotein (see Fig. 3.3). The sequences obtained from the clones corresponded closelyto the published coat protein gene sequence from a BWYV isolate collected fromsugarbeet (Veidt et al., 1988) and confirmed that no reading frame errors had beenintroduced during the PCR reaction. The nucleotide sequence of the BWYV coatprotein cDNA (BWYV1002) shared 94.2% and 93.5% sequence identity with thesugarbeet isolate, BWYV (GB 1) and a lettuce isolate, BWYV (FL1), respectively (seeFigs. 3.5, 3.6). A comparison of the protein sequences revealed 94.1% and 92.6%identity with the same sugarbeet isolate and lettuce isolate, respectively (see Figs. 3.7,3.8). Furthermore, the AUG codon of the BWYV coat protein gene was confirmed tobe embedded in the consensus sequence for optimal ribosome initiation, G/AxxAUGGwhere x represents any nucleotide (Kozak, 1984).BWYV (GB 1)BWYV1002BWYV (GB1 )BWYV1002BWYV (GB1 )BWYV1002BWYV (GB 1)BWYV1002BWYV (GB1)BWYV1002BWYV (GB1)BWYV1002BWYV (GB1 )BWYV1002BWYV (GB 1)BWYV1002BWYV (GB1 )BWYV1002BWYV (GB1)BWYV1002BWYV (GB1)BWYV1002 —Results p. 44— CGTTAATGAATACGGTCGTGGGTAGGAGAACAATCAATGG —401^*^III*1111111111111111111111111HIIII— CCACCATGGATACGGTCGTGGGTAGGAGAACAATCAATGG —40— AAGAAGACGACCACGCAGGCA.AACACGACGCGCTCAGCGC —801111111111111111111111111111H11111^11— AAGAAGACGACCACGCAGGCAAACACGACGCGCTCGGCCG —80— TCTCAGCCAGTGGTTGTGGTCCAAACCTCTCGGGCAACAC —1201111111111111111111111111111111111111111— TCTCAGCCAGTGGTTGTGGTCCAAACCTCTCGGGCAACAC —120— AAC GC C GAC C TAGAC GAC GACGAAGAGG TAACAAC C GGAC —1601111111111111111111111111111111^11111111— AAC GC C GAC C TAGAC GAC GAC GAAGAGG TAATAAC C GGAC —160— AAGAGGAACTGTTCCTACCAGAGGAGCAGGCTCGAGCGAG —200111111111111111111111111111111111^111111— AAGAGGAACTGTTCCTACCAGAGGAGCAGGCTCAAGCGAG —200— ACATTTGTTTTCTCAAAAGACAATCTCGCGGGAAGTTCCA —24011111111111111^1111111111111111111111111— ACATTTGTTTTCTCGAAAGACAATCTCGCGGGAAGTTCCA —240— GCGGACGAATCACGTTCGGGCCGAGTCTATCAGACTGCCC —2801111111111111111111111111111111111111111— GCGGACGAATCACGTTCGGGCCGAGTCTATCAGACTGCCC —280— GGCATTCTCTAATGGAATGCTCAAGGCCTACCATGAGTAT —320H11111111111111^111111111111111111111— AGCATTCTCTAATGGAATACTCAAGGCCTACCATGAGTAT —320— AAAATCTCAATGGTCATTTTGGAGTTCGTCTCCGAAGCCT —36011111111^1111111111111111111111111111111— AAAATCTCGATGGTCATTTTGGAGTTCGTCTCCGAAGCCT —360— CT TCCCAAAACTCCGGTTCCATCGCTTACGAGCTGGACCC —40011111^1111111111111111111111^11111111111— CT TCCCAAAACTCCGGT TCCATCGCTTATGAGCTGGACCC —400— ACACTGTAAACTCAACTCCCTTTCCTCAACTATCAACAAG —44011111111^1111^11^1111111111^11^11111^IIIACACTGTAGACTCGACGCCCTTTCCTCGACCATCAATAAG —440BWYV (GB1) —BWYV1002 —BWYV (GB1 ) —BWYV1002 —TTCGGGATCACAAAGCCCGGGAAAGCGGCGTTTACAGCGT —4801111111111111111111111^11111111111111TTCGGGATCACAAAGCCCGGGAGGAGGGCGTTTACAGCGT —480CTTACATCAATGGAAAGGAATGGCACGACGTTGCCGAGGA —5201111111111^11^1^111^111111111111111^1111CTTACATCAACGGGACGGATTGGCACGACGTTGCCAAGGA —520Results^p. 45BWYV (GB1) - CCAATTCAGGATCCTCTACAAAGGCAATGGTTCTTCATCG -5601111111111111111111111111111111111111111BWYV1002 - CCAATTCAGGATCCTCTACAAAGGCAATGGTTCTTCATCG -560BWYV (GB1 ) - ATAGCTGGTTCTTTTAGAATCACCATCAAGTGCCAATTCC -60011111^11111111111111111^11^11111^1111111BWYV1002 - ATAGCCGGTTCTTTTAGAATCACTATAAAGTGTCAATTCC -600BWYV (GB1) - ACAATCCCAAATAGGTAGACGAGGAACCCGGCCCTAGCCC -6401111^11111111111111111111111111111111111BWYV1002 - ACAACCCCAAATAGGTAGACGAGGAACCCGGCCCTAGCCC -640BWYV (GB1) - A-GGGCCTTCT -6501*1111^11BWYV1002 - ATGGGCGAGCTC -652Fig. 3.5^Nucleotide sequence comparison between the BWYV coat protein codingregion (BWYV1002 meld) and the coat protein sequence of the sugarbeet isolate(Veidt et al., 1988), BWYV (GB 1). The first and second ATG codons correspond tothe initiation codon for the BWYV 22.5 IcDa coat protein and 19.5 kDa ORFs,respectively. The character to show that two aligned residues are identical is 'I'.Nucleotide changes introduced via the BWYV oligonucleotide primers are indicated bya '*'. The terminator codon, TAG, for each ORF is shown in boldface.Results^p. 46BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV(FL1)BWYV1002BWYV(FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002— CGTTAATGAATACGGTCGTGGGTAGGAGAATTATCAATGG —401*^111*111111111111111111111^11111111— CCACCATGGATACGGTCGTGGGTAGGAGAACAATCAATGG —40— AAGAAGACGACCACGCAGGCAAACACGACGCGCTCAGCGC —8011111111111111111111111111111111111^11— AAGAAGACGACCACGCAGGCAAACACGACGCGCTCGGCCG —80— CCTCAGCCAGTGGTTGTGGTCCAAACCTCTCGGGCAACAC —120111111111111111111111111111111111111111— TCTCAGCCAGTGGTTGTGGTCCAAACCTCTCGGGCAACAC —120— AAC GC C GAC C TAGAC GACGACGAAGAGG TAACAAC C GGAC —1601111111111111111111111111111111^11111111— AAC GC C GAC C TAGAC GACGACGAAGAGGTAATAACCGGAC —160— AGGAAGAACTGTTCCTACCAGAGGAGCAGGTTCGAGCGAG —2001^11^1111111111111111111111111^11^111111— AAGAGGAACTGTTCCTACCAGAGGAGCAGGCTCAAGCGAG —200— ACATTTGTTTTCTCAAAAGACAATCTCGCGGGAAGTTCCA —24011111111111111^1111111111111111111111111— ACATTTGTTTTCTCGAAAGACAATCTCGCGGGAAGTTCCA —240— GCGGAGCAATCACGTTCGGGCCGAGTCTATCAGACTGCCC —28011111^111111111111111111111111111111111— GCGGACGAATCACGTTCGGGCCGAGTCTATCAGACTGCCC —280— GGCATTCTCTAATGGAATGCTCAAGGCCTACCATGAGTAT —32011111111111111111^111111111111111111111— AGCATTCTCTAATGGAATACTCAAGGCCTACCATGAGTAT —320— AAAATCTCAATGGTCATTTTGGAGTTCGTCTCCGAAGCCT —36011111111^1111111111111111111111111111111— AAAATCTCGATGGTCATTTTGGAGTTCGTCTCCGAAGCCT —360— CTTCCCAAAATTCCGGTTCCATCGCTTACGAGCTGGACCC —4001111111111^11111111111111111^11111111111— CTTCCCAAAACTCCGGTTCCATCGCTTATGAGCTGGACCC —400— ACAC T GTAAAC TCAAC T CCC T T T CC TCAAC TAT CAACAAG —44011111111^1111^11^1111111111^11^11111^111— ACACTGTAGACTCGACGCCCTTTCCTCGACCATCAATAAG —440BWYV (FL1) —BWYV1002 —BWYV (FL1 ) —BWYV1002 —TTCGGGATCACAAAGCCCGGGAAAAGGGCGTTTACAGCGT —4801111111111111111111111^1111111111111111TTCGGGATCACAAAGCCCGGGAGGAGGGCGTTTACAGCGT —480CTTACATCAACGGAACGGAATGGCACGACGTTGCCGAGGA —5201111111111111^11111^111111111111111^1111CTTACATCAACGGGACGGATTGGCACGACGTTGCCAAGGA —520Results p. 47BWYV (FL 1) - CCAATTCAGGATCCTCTACAAAGGCAATGGTTCTTCATCG -5601111111111111111111111111111111111111111BWYV1002 - CCAATTCAGGATCCTCTACAAAGGCAATGGTTCTTCATCG -5 60BWYV (FL1) - ATAGCTGGTTCTTTCAGAATCACCATTAAGTGTCAATTCC -60011111^11111111^11111111^11^1111111111111BWYV1002 - ATAGCCGGTTCTTTTAGAATCACTATAAAGTGTCAATTCC -600BWYV (FL1 ) - ACAACCCCAAATAGGTAGACGAGGAACCCGGCCCTAGCCC -6401111111111111111111111111111111111111111BWYV1002 - ACAACCCCAAATAGGTAGACGAGGAACCCGGCCCTAGCCC -640BWYV(FL1) - A-GGGCCTTCT -6501*1111^11BWYV1002 - ATGGGCGAGCTC -652Fig. 3.6^Nucleotide sequence comparison between the BWYV coat protein codingregion (BWYV1002 meld) and the coat protein sequence of the lettuce isolate (Veidtet al., 1988), BWYV (FL1). The first and second ATG codons correspond to theinitiation codons for the BWYV 22.5 IcDa coat protein and 19.5 lcDa ORF,respectively. The character to show that two aligned residues are identical is 'I'.Nucleotide changes introduced via the BWYV primers are indicated by a '*'. Theterminator codon, TAG, for each ORF is shown in boldface.Results p. 48BWYV (GB1 ) - MNTVVGRRT I NGRRRPRRQTRRAQRS QPVVVVQT S RAT QR -401^111111^111111111111111^111111111111111BWYV1002 - MD TVVGRRT INGRRRPRRQTRRARP SQPVVVVQTSRATQR -40BWYV (GB1) - RPRRRRRGNNRTRGTVPTRGAGSSETFVFSKDNLAGS SSG -801111111111111111111111111111111111111111BWYV1002 - RPRRRRRGNNRTRGTVP TRGAG S S E TFVFSKDNLAGS S SG -80BWYV (GB1) - RI TFGP SLSDCPAFSNGMLKAYHEYKI SMVILEFVSEASS -120IIIIIIIIH1111111^1111111111111111111111BWYV1002 - RI TFGP SLSDCPAFSNGILKAYHEYKI SMVILEFVSEASS -120BWYV (GB1) - QNSGSIAYELDPHCKLNSLSSTINKFGITKPGKAAFTASY -16011111111111111^1^11111111111111^111111BWYV1002 - QNSGSIAYELDPHCRLDALSSTINKFGITKPGRRAFTASY -160BWYV (GB1 ) - INGKEWHDVAEDQFRILYKGNGSSSIAGSFRITIKCQFHN -200111^11111^11111^111111111111111111111111BWYV1002 - INGTDWHDVAKDQFRILYKGNGSSSIAGSFRITIKCQFHN -200BWYV (GB1 ) - PK -20211BWYV1002 - PK -202Fig. 3.7 Amino acid sequence comparison between the BWYV coat protein(derived from BWYV1002 meld) and the coat protein of the sugarbeet isolate (Veidtet al., 1988), BWYV (GB 1). The numbering begins from the methionine whichcorresponds to the first ATG in Fig . The character to show that two alignedresidues are identical is TResults p. 49BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1)BWYV1002BWYV (FL1 )BWYV1002BWYV (FL1)BWYV1002- MNTVVGRRI I NGRRRPRRQTRRAQRP QPVVVVQ T SRATQR -401^111111^11111111111111^11111111111111- MD TVVGRRT I NGRRRPRRQ TRRARP SQPVVVVQTSRATQR -40- RP RRRRRGNNRTGRTVP TRGAG S SETFVFSKDNLAGSSSG -80111111111111^11111111111111111111111111- RP RRRRRGNNRTRGTVP TRGAG S SETFVF SKDNLAGSSSG -80- AI TFGP SLSDCPAF SNGMLKAYHEYKI SMVILEFVSEAS S -1201111111111111111^1111111111111111111111- RI TFGP SLSDCPAFSNGILKAYHEYKI SMVILEFVSEAS S -120- QNSGS IAYELDPHCKLNSLSSTINKFGITKPGKRAFTASY -16011111111111111^1^11111111111111^1111111- QNSGS IAYELDPHCRLDALSSTINKFGITKPGRRAFTASY -160- INGTEWHDVAEDQFRILYKGNGSSS IAGSFRITIKCQFHN -2001111^11111^11111111111111111111111111111- INGTDWHDVAKDQFRILYKGNGSSS IAGSFRITIKCQFHN -200- PK -20211- PK -202Fig. 3.8^Amino acid sequence comparison between the BWYV coat protein(derived from BWYV1002 meld) and the coat protein of the lettuce isolate (Veidt etal., 1988), BWYV (FL1). The numbering begins from the methionine whichcorresponds to the first ATG in Fig. 3.6. The character to show that two alignedresidues are identical is TResults p. 503.3^^In Vitro Translation Analysis of Synthetic Transcripts of the ClonedBWYV Coat Protein Gene3.3.1 Cloning of the BWYV Coat Protein Gene into a Transcription Vector andthe In Vitro Transcription of the BWYV Coat Protein Gene using theBacteriophage T3 PolymeraseThe BWYV coat protein gene was obtained by digestion of BW102D withXho IISst I restriction enzymes. The 654 bp fragment corresponding to the BWYVcoat protein gene was excised from pRT103 and gel purified. The compatible endswere ligated with similarly digested pBluescriptII KS+ plasmid (SITatagene) such thatthe 5' portion of the BWYV coat protein gene was adjacent to the bacteriophage T3RNA polymerase promoter. The ligation mixture was used to transform competentE. coil DH5a cells which were then plated on LB-agar containing ampicillin andBluo-Gal (BRL). Several white ampicillin-resistant colonies were obtained.Individual colonies were cultured in LB broth containing ampicillin and their nucleicacid isolated by the small scale mini-preparation procedure. The presence andorientation of an insertion was determined by restriction enzyme mapping. ThepBluescript/BWYV coat protein gene construct was linearized at the 3' end of theBWYV coat protein gene with Sst I restriction enzyme and used to make syntheticRNA transcripts in vitro with the T3 RNA polymerase.Results p. 513.3.2 In Vitro Translation of Synthetic BWYV Coat Protein RNA in WheatGerm ExtractsSynthetic RNA transcripts were translated in wheat germ extracts in thepresence of [35S1-methionine and the protein products were analyzed by denaturingpolyacrylamide gel electrophoresis (SDS-PAGE) and subsequent autoradiography (seeFig. 3.9). The size of the protein product corresponded to the expected size of theBWYV coat protein (22.5 kDa) and not to the product encoded by ORF 5 (19.5 kDa).3.4^Construction of the BWYV Coat Protein Gene Binary Plasmids Using thepCGN15 Vector Series (Calgene)Clones BW102D and BW137D were digested with Hind III restriction enzyme (seeFig. 3.10 and 3.11). The appropriate fragments of approximately 1.3 kbp in size weregel purified and the compatible ends re-ligated into the Hind III digested pCGN1548and pCGN1557 binary vectors (McBride and Summerfelt, 1990). The 1.3 kbpfragments excised from either clone BW102D or clone BW137D contained the CaMV355 promoter, the BWYV coat protein gene (in the plus-sense orientation or the anti-sense orientation, respectively) and the CaMV polyadenylation signal. The pCGN1548and pCGN1557 binary vectors contain the npt II (neomycin phosphotransferase) geneas a plant selectable marker regulated by the mannopine synthase (mas 5') promoter /termination signal (mas 3') and by the CaMV 35S promoter / tm/Results^p. 521^2^3^4Fig. 3.9 In vitro translation analysis of the synthetic transcripts of the cloned BWYV coatprotein gene. The BWYV coat protein cDNA was cloned next to the T3 promoter ofpBluescriptII KS+ plasmid. The Bluescript/BWYV coat protein gene construct waslinearized with Sst I restriction enzyme and used to make synthetic RNA transcriptsin an in vitro transcription run off reaction. Synthetic RNA transcripts were translated inwheat germ extracts in the presence of [35S]-methionine and the protein products wereanalyzed by denaturing polyacrylamide gel electrophoresis (15% gel) and subsequentautoradiography. The following templates were used in the in vitro translation reactions:lanes (1) cucumber necrosis virus (CNV) RNA (50 ng) used as a protein molecular weightstandard, (2) BWYV RNA extracted from purified virus (100 ng), (3) BWYV coat proteinsynthetic transcript (50 ng) and (4) no RNA. The arrow points to the BWYV coat protein.The high molecular weight band in lane 2 likely corresponds to the BWYV 66 kDa, encodedby ORF 2. The numbers on the left correspond to the sizes of CNV in vitro translationproducts.Results p. 53Fig. 3.10^Diagram showing the construction of the pCGN1548/BWYV coat protein gene clones.BW102D and BW137D clones were digested with Hind III restriction enzyme in separate reactions. The 1.3kbp Hind III fragment was gel purified from both of the clones and ligated into the Hind III site of thebinary plasmid pCGN1548 (Calgene) using T4 DNA ligase. The ligation mixture was used to transformcompetent DH5oc cells which were then plated on LB-agar containing ampicillin and Bluo-gal (BRL). TheDNA from several individual white colonies was screened by restriction enzyme mapping using Sma I andXho I. The Hind III fragment containing the CaMV 35S promoter/polyadenylation signal and the BWYVcoat protein gene in the plus-sense orientation and the anti-sense orientation were successfully inserted intothe Hind III site of pCGN1548; making the pCGN154802 and pCGN154837 binary plasmid constructs,respectively. The restriction enzyme sites for Eco RI (E), Hind III (H), Sma I (S) and Xho I (X) are shown.See section 1.5 for a description of the components of the pCGN15 binary plasmids. (CIP= calf intestinalphosphatase; npt II= Tn5 neomycin phosphotransferase gene; Gm= gentamycin resistance marker; on pRI=origin of replication of the A. rhizogenes root inducing plasmid pRiHRI; RB/LB= right /left border regionsfrom pTiA6; mas 5',--  mannopine synthase promoter from pTiA6; mar 3'= mannopine synthase terminationsignals from pTiA6; tm/ 3= tm/ terminator from pTiA6; on Col El= origin of replication and transfer originfrom pBR322; lac 1= E. coli lac alpha polylinker from pUC18)Nco IBWYV coatprotein genemasopt 11mas 5'LBpCGN154837(15.3 kb)oil PR!on pRIResults^p. 54Hind HISph IPatCaMVpolyadenylatio)signalBWYVgeneCaM:3SPTinpoiromoterHindi!Pst ISph IHind IllBW102D3960 bpXbalHam HISma Is.n.i KB.1co IBollXho IXbaIBan, HISma IKpnSat INco I--Sma ICO iXho I3960 bpHine IIPI ISph IHind IIIHind IIISph IPot IHind IIIgel purify1.3 kbp fragmentT4 DNA LigaseTransformationof DH5 a cellsPlate on LB-agarwith GentamycinSelection of clonesby restriction mappingHind III, CIPKpn IBain HIXba IPat!HindlIIonHind III, CIP!Hind IIIgel purify1.3 kbp fragmentSelection of clonesby restriction mappingT4 DNA Ligase1 Transformationof DHSOC cellsPlate on LB-agarwith Gentamycinpolyadenylation ssignal^ERBonCol El ■X CaMV 35SpromotermasN\°Pt IIpCGN154802 mas 5'(15.3 kb)LBpolyadenylationsignal^ERBonCol ElBWYV coatprotein geneX CaMV 35SpromoterHxResults p. 55Fig. 3.11^Diagram showing the construction of the pCGN1557/BWYV coat protein gene clones.BW102D and BW137D clones were digested with Hind III restriction enzyme in separate reactions. The 1.3kbp Hind III fragment was gel purified from both of the clones and ligated into the Hind III site of thebinary plasmid pCGN1557 (Calgene) using T4 DNA ligase. The ligation mixture was used to transformcompetent DH5cc cells which were then plated on LB-agar containing ampicillin and Bluo-gal (BRL). TheDNA from several individual white colonies was screened by restriction enzyme mapping using Sma I andXho I. The Hind III fragment containing the CaMV 35S promoter/polyadenylation signal and the BWYVcoat protein gene in the plus-sense orientation and the anti-sense orientation were successfully inserted intothe Hind III site of pCGN1557; making the pCGN155702 and pCGN155737 binary plasmid constructs,respectively. The restriction enzyme sites for Eco RI (E), Hind III (H), Sma I (S) and Xho I (X) are shown.(CIP= calf intestinal phosphatase; npt II= Tn5 neomycin phosphotransferase gene; Gm= gentamycinresistance marker; on pRI= origin of replication of the A. rhizogenes root inducing plasmid pRiHRI;RB/LB= right /left border regions from pTiA6; mas 5'= mannopine synthase promoter from pTiA6;mas 3= mannopine synthase termination signals from pTiA6; tm/ 3'= tm/ terminator from pTiA6; on ColEl= origin of replication and transfer origin from pBR322; lac Z'= E. coli lac alpha polylinker from pUC18)T4 DNA LigaseTransformation1of DH5 a cellsPlate on L-agarwith Gentamycinand Bluo-galSelection of clonesby restriction mappingCaMV 35SromoterH Xpolyadenylationsignalon—Sma ICalVIV 355promoterHine IIPot ISph IHind III!Hind IIIgel purify1.3 kbp fragmentHind HIPst IXba IBarn HIKpn Ico Ial IXho ICaMV 35SpromoterHRBpolyadenylationX S^signaloilHind IIISph IPot IXCaMV^balpolyadenylation^Bam HIsignal Sm. IKpn IBWYV 1 . I Icoat protein I 1 Igeneampicillinresistance Nco IBW137D3960 bp3960 bpHine HPst ISph IHind HIXbalHam HISees Ipo ISot INco IHind III, CH'■11110.Hind IIIgel purify1.3 kbp fragmentHind III, CIP.4—..—T4 DNA LigaseTransformationof DH5a cellsPlate on LB-agarwith Gentamycinand Bluo-galSelection of clonesby restriction mapping 1Results^p. 56Results p. 57terminator (tm/ 3'), respectively. The ligation mixture was used to transformcompetent E. coli DH5cc cells which were then plated on LB-agar containing theantibiotic gentamycin and the chromogenic substrate Bluo-Gal (BRL). Several whitegentamycin-resistant colonies were obtained. Individual colonies were cultured inLuria-Bertani (LB) broth containing gentamycin and their nucleic acid isolated by thesmall scale mini-preparation procedure. The presence and orientation of an insertionwas determined by restriction enzyme mapping. Several clones were obtained (seeFig. 3.12): pCGN154802 and pCGN155702 containing the BWYV coat protein gene inthe plus-sense orientation relative to the CaMV 35S promoter; pCGN154837 andpCGN155737 containing the BWYV coat protein gene in the anti-sense orientationrelative to the CalVIV 35S promoter.3.5^Mobilization of the BWYV Coat Protein Gene Binary Plasmids intoAgrobacterium tumefaciens EHA101 by the Tr-Parental Mating ProcedureThe binary plasmid constructs pCGN154802, pCGN154837, pCGN155702 andpCGN155737 were used to transform E. coli MM294 cells which were then plated onLB-agar containing gentamycin. The binary plasmids pCGN1548 and pCGN1557were also used to transform E. coli MM294 cells as controls. Several gentamycin-resistant colonies were obtained. Individual colonies of each construct were culturedin LB broth containing gentamycin. Cultures containing the appropriate constructwere co-cultured with E. coli MM294 containing the pRK2013 helper plasmid andResults^p. 58A 1 2 3 4 5 6 7 8 9 10 11B 1 234  5 6 7 8 9 10 11Fig. 3.12 Restriction enzyme analysis of pCGN1548- and pCGN1557-derived BWYV coatprotein gene constructs. Plasmid constructs, containing the BWYV coat protein gene in theplus-sense orientation (relative to the CaMV 35S promoter) - pCGN154802 (A,lanes 3,6,9) andpCGN155702 (B, lanes 2,5,8) - and in the anti-sense orientation (relative to the CaMV 35Spromoter) - pCGN154837 (A, lanes 4, 7, 10) and pCGN155737 (B, lanes 3, 6, 9) - were digestedwith Hin d III (lanes 2-4),Sma I (lanes 5-7) and Xho I (lanes 8-10) restriction enzymes. Plasmidsnot containing the BWYV coat protein gene were similarly digested as controls - pCGN1548 (A,lanes 2, 5, 8) and pCGN1557 (B, lanes 4, 7, 10). The resulting products were analyzed byelectrophoresis through a 1% agarose gel and stained with ethidium bromide. Lanes 1 and 11contain molecular size markers: Lambda DNA digested with Eco RI and Hind III andpBluescribe digested with Pvu II and Rsa I (sizes shown in kbp), respectively.Results p. 59Agrobacterium tumefaciens EHA101 (Hood et al., 1986) and grown on LB-agarcontaining both antibiotics, gentamycin and kanamycin. Several gentamycin andkanamycin-resistant A. tumefaciens colonies were obtained. The presence of theappropriate binary plasmid construct could not be determined by small scale nucleicacid preparation of the A. tumefaciens and subsequent restriction enzyme mappingsince the yield of plasmid was too low. But the presence of the appropriate binaryplasmid was confirmed by digestion of the nucleic acid preparation with Hind IIIrestriction enzyme, size fractionated by agarose gel electrophoresis and subsequentSouthern blot analysis using a random-primed, [32P]-labeled cDNA BWYV coatprotein gene probe (results not shown). Nucleic acid preparations from the A.tumefaciens containing the appropriate binary plasmid construct were also used totransform competent E. coli DH5a cells. The cells were then plated onto LB-agarcontaining gentamycin and Bluo-gal (BRL). Several white gentamycin-resistantcolonies were obtained, cultured and their nucleic acid isolated by the small scalemini-preparation procedure. The presence of the appropriate binary plasmid constructwas confirmed by restriction enzyme mapping.3.6^Transformation and Regeneration of Brassica napus cv. WestarBrassica napus cv. Westar was transformed with the appropriate binary plasmidconstructs according to the plant transformation procedure utilizing cotyledonaryexplants (Moloney et al., 1989). The A. tumefaciens EHA101 used in theResults p. 60transformation procedure contained one of the following binary plasmid constructs:pCGN154802, pCGN154837, pCGN155702 and pCGN155737. As a control, somecotyledons were not treated with A. tumefaciens in order to determine the base level ofregeneration in untreated B. napus cv. Westar under the plant tranformation conditions.In all, 5280 cotyledonary explants were used in four separate attempts to transform B.napus cv. Westar (see Table 3.1). Several regenerated, kanamycin-resistant shootswere subcultured and eventually placed in the greenhouse where they were allowed toflower and set seed. Shoots that had not been transformed with the npt II geneappeared bleached by the fourth week of culture. The regeneration efficiency of thecotyledonary explants after treatment with the A. tumefaciens ranged from 0.8 to 3.6%as compared to 17 to 80% in the untreated cotyledonary explants (see Table 3.3).Thirty-eight regenerated plants were recovered from the first two transformationattempts: five which had been treated with the A. tumefaciens containing thepCGN154802 binary plasmid construct, one with the pCGN154837 binary plasmidconstruct, three with the pCGN1548 binary plasmid, fifteen with the pCGN155702binary plasmid construct, six with the pCGN155737 binary plasmid construct and eighttreated with the A. tumefaciens containing the pCGN1557 binary plasmid. All theregenerated plants appeared morphologically identical to untreated B. napus cv.Westar except for plant 155702-9, which was dwarfish and sterile. Severalmodifications implemented in the third (July 1992) and fourth (September 1992)attempts could not be evaluated since A. tumefaciens used in the plant transformationprocedure reappeared and contaminated all the regenerated shoots. The reappearanceResults^p. 61Table 3.1^Transformation of Brassica napus cv. Westar with Agrobacterium tumefaciens EHA101 containingthe indicated binary plasmid construct. Four separate attempts were made following the plant transformationprocedure using cotyledonary explants (Moloney et al., 1989). (---) indicates that no cotyledons were treated onthis date.Number of Brassica napus cv. Westar cotyledons treated in the planttransformation procedureAgrobacterium/constructsJan.24192 Apr.5/92 Apr.6/92 Jul.9/92 Sept.23/92 TotalsEHA101/pCGN155702170 110 140 360 400 1180EHA101/pCGN155737170 110 130 360 400 1170EHA101/pCGN1557170 110 --- 50 --- 330EHA101/pCGN154802--- --- 140 360 400 900EHA101/pCGN154837--- --- 140 360 400 900EHA101/pCGN1548--- --- 140 50 --- 190noAgrobacterium190 110 130 120 60 610Totals 700 440 820 1540 1660 5280Results^p. 62Table 3.2 Transformation of Brassica juncea cv. Forge with Agrobacterium tumefaciens EHA101 containingthe indicated binary plasmid construct. An attempt was made to transform Brassica juncea cv. Forge, using theplant transformation procedure (Moloney et cd., 1989), for a comparison of the regeneration efficiency for adifferent species. (---) indicates that no cotyledons were treated on this date.Agrobacterium IconstructsNumber of Brassica juncea cv. Forge cotyledons treated intransformation procedureJan.24/92 Apr.6/92 Totalsnumberofexplantstreatedregenerationefficiency %numberofexplantstreatedregenerationefficiency %EHA101 / pCGN155702 20 0.0 100 0.0 120EHA101 / pCGN155737 20 0.0 --- --- 20EHA101 / pCGN1557 20 0.0 --- --- 20no Agrobacterium 28 0.0 90 0.0 118Totals 88 0.0 190 0.0 278Results p. 63Table 3.3^Regeneration efficiency of Brassica napus cv. Westar cotyledons after co-cultivation of thecotyledonary explants with Agrobacterium tumefaciens EHA101 containing the appropriate binary plasmidconstruct. (---) indicates that no cotyledons were treated on this date.Agrobacterium/constructsDate regenerated Brassica napus cv. Westar placed in greenhouseApril to June July Octobernumberregeneratedregenerationefficiency %numberregeneratedregenerationefficiency %numberregeneratedregenerationefficency %EHA 101 /pCGN1557026 / 170* 3.5 9 / 250 3.6 2 / 360 0.6EHA 101 /pCGN1557374 / 170 2.4 2 / 240 0.8 0 / 360 0.0EHA 101 /pCGN15574 / 170 2.4 4 / 110 3.6 0 / 50 0.0EHA 101 /pCGN154802--- --- 5 / 140 3.6 0 / 360 0.0EHA101 /pCGN154837--- --- 1 / 140 0.7 0 / 360 0.0EHA 101 /pCGN1548--- --- 3 / 140 2.1 1 / 50 2.0Totals 14 / 510 2.7 24 / 1020 2.4 3 / 1560 0.2noAgrobacterium152 / 190 80 156 / 240 65 20/ 120 17* number of plants regenerated / number of cotyledons treated.Results^p. 64was likely due to a reduction in the concentration of antibiotics used during the shootrooting stage or to an excessive initial inoculum of A. tumefaciens. Modifications(suggested by Sharon E. Radke, Calgene and Gijs H. van Rooijen, University ofCalgary) to the plant transformation procedure included: (1) co-cultivation of the A.tumefaciens (reduced inoculum) with excised cotyledons for 24 hours instead of 72hours, (2) use of kanamycin throughout the transformation procedure in order toreduce the number of possible "escapes" (kanamycin sensitive shoots) and (3) use ofcefotaxime instead of or in combination with carbenicillin to inhibit the reappearanceof the A. tumefaciens after the regenerated shoots have been subcultured. Brassicajuncea cv. Forge was also used in the plant transformation procedure but noregenerated shoots were recovered. This confirms that the plant transformationprocedure used (Moloney et al., 1989) is genotype specific for Brassica napus (seeTable 3.2).3.7^Evaluation of Regenerated Plants3.7.1 Southern Blot Analysis of DNA Extracted from Regenerated PlantsPlant DNA was isolated from regenerated plants by the CTAB extractionmethod. Approximately 50 to 150 ug of plant DNA sample (the amount depended onthe final concentration of DNA obtained in the extraction) was digested with eitherHind III or Eco RI restriction enzyme and size fractionated by agarose gelResults p. 65electrophoresis. The DNA was transferred onto Zeta-Probe GT membrane (BioRad)by alkaline capillary blotting. The Southern blots were probed with either randomprimed, [32P]-labeled DNA corresponding to the npt II gene or to the BWYV coatprotein gene.Probing of the Southern blots, of Hind III digested DNA, with [32P]-labeledBWYV coat protein cDNA revealed a 1.3 kbp fragment in the plant DNA samplesfrom three plants (154802-1, 154802-3 and 1557-7; see Fig. 3.13, lanes 5, 7 and 4,respectively). Hybridization to a 1.3 kbp fragment containing the CaMV 35Spromoter/BWYV coat protein gene (plus-sense and anti-sense orientation)/CaMVpolyadenylation signal occurred as predicted to Hind III digested DNA from theappropriate binary plasmid constructs (pCGN154802, pCGN154837, pCGN155702 andpCGN155737; Fig. 3.13, lanes 16, 17, 13 and 14, respectively) but did not occur asexpected to Hind III digested binary plasmids used as controls (pCGN1548 andpCGN1557; Fig. 3.13, lanes 18 and 15, respectively). Hybridization of the BWYVcoat protein cDNA probe to Hind III digested DNA from plant 1557-7 (supposedly abinary plasmid not containing the BWYV coat protein gene) suggested that the planthad been, in fact, transformed with a BWYV coat protein gene binary plasmidconstruct. Plant 1557-7 was likely labeled incorrectly. Probing of Eco RI digestedplant DNA with random primed, [32P]-labeled DNA corresponding to the npt II generevealed a 1.0 kbp fragment in the DNA sample from one plant (1557-7) and a 2.5kbp fragment in the DNA sample from another plant (155737-5; see Fig. 3.14 A, lanes—21.85.05=4.21 3.41—1.98—0.93—0.84CRResults^p. 661 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fig. 3.13 Southern blot analysis of DNA extracted from regenerated Brassica nai9uscv. Westar. Approximately 50-150 ug of genomic DNA extracted from regeneratedplants was digested with Hind III restriction enzyme and electrophoresed through a1% agarose gel. The size fractionated Hin d HI digested genomic DNA was transferredonto Zeta-Probe GT membrane (BioRad) by alkaline capillary blotting. The membraneswere probed with random primed, [3211-labeled BWYV coat protein cDNA andautoradiographed. Lanes 1-2 contained Hind III digested genomic DNA correspondingto plants 155737-5 to -6 (ie. plants transformed with construct 155737), lanes 3-4 toplants 1557-5 and -7, lanes 5-9 to plants 154802-1 to -5, lane 10 to plant 154837-1 andlanes 11-12 to plants 1548-1 and -2. In addition, lane 20 contained Hind HI digestedgenomic DNA extracted from an untransformed plant. The controls included: Hinddigested pCGN155702, pCGN155737, pCGN1557, pCGN154802, pCGN154837, andpCGN1548 plasmid DNA (approx. 25 pg, lanes 13-18, respectively). Lane 19 containedLambda DNA digested with Eco RI and Hind III used as a molecular size marker (sizesshown in kbp on right).21.8 —5.05 5.24 _-4.21 —3.41 —1.98 — 1.57 --1320.93 —0.84 —0.58 —Results^p. 671 2 3 4 5 6 7 8 9 10 11 12 13 14 1510 11 12 13 14 1521.85.05 5.244.213.411.981191.320.930.840.58Fig. 3.14 Southern blot analysis of DNA extracted from regenerated Brassica napuscv. Westar. Approximately 50-150 ug of genomic DNA extracted from regeneratedplants was digested with EcoRI restriction enzyme and electrophoresed through a1% agarose gel. The size fractionated digested genomic DNA was transferred ontoZeta-Probe GT membrane (BioRad) by alkaline capillary blotting. The membranes wereprobed with random primed, [ 32 P[-labeled nPt II cDNA (blot A) or BWYV coatprotein cDNA (blot B) and autoradiographed. Lanes 6-9 contained Eco RI digestedgenomic DNA corresponding to plants 155702-12 to -15, lanes 10-12 to plants155737-4 to -6 and lanes 13-15 to plants 1557-5 to -7. The controls included: EcoRIdigested plasmid DNA pCGN155737 and pCGN1557 (lanes 3,4), EcoRI digestedgenomic DNA extracted from an untransformed plant (lane 5) and 25 pg each of nplcDNA and BWYV coat protein cDNA mixed in EcoRI digested genomic DNA (lane2). Lane 1 contained Lambda DNA digested with EcoRI and Hind III used asmolecular size marker (sizes shown in kbp).Results p. 6815 and 11, respectively). A 1.0 kbp fragment containing the npt II gene occurred aspredicted from Eco RI digestion of all binary plasmid constructs including the binaryplasmids used as controls. The 2.5 kbp fragment (DNA sample from plant 155737-5;see Fig. 3.14A, lane 11) is likely to be the result of incomplete digestion of the Eco RIsite between the npt II gene and the tm/ terminator (tin! 3' region). Probing of thesame Southern blots, after stripping of the npt II cDNA probe from the blot, withrandom primed [32131-labeled DNA corresponding to the BWYV coat protein geneproduced a fragment (> 5.0 kbp) in the DNA sample from plant 1557-7 and a similarfragment (but weaker signal) in the DNA sample from plant 154802-3 (see Fig. 3.14B,lane 15; Fig. 3.15B, lane 8, respectively). A fragment no smaller than 2.3 kbp waspredicted from Eco RI digestion of the binary plasmid constructs (pCGN154802,pCGN154837, pCGN155702and pCGN155737). The BWYV coat protein gene inpCGN155702 and pCGN155737 is flanked by only one Eco RI restriction site andtherefore, the size of the fragment revealed in the Southern blot analysis would dependon the left border integrating next to a plant Eco RI restriction site. Genomic DNAfrom plant 154802-3 was also digested with Hind III and hybridization of the probecorresponding to the BWYV coat protein gene revealed a 1.3 kbp fragment (see Fig.3.15B, lane 9), as predicted from digestion of all the binary plasmid constructs. Sincehybridization was found to occur with Hind III digested DNA from plant 154802-1using the probe corresponding to the BWYV coat protein gene (see Fig. 3.13, lane 5),it was surprising to find that no hybridization occurred with Eco RI digested DNAfrom plant 154802-1 (see Fig. 3.15B, lane 6) using the BWYV coat protein probe .21.85.05 5.244.213.411.981.901.571.32Results^p. 691 2  3 4 5 6 7 8 9 10 11 12 13 14 1521.85.053.411.51.321 2 3 4^5^6 7 8 9 10 11 12 13 14 15Fig. 3.15 Southern blot analysis of DNA extracted from regenerated Brassica napuscv. Westar. Approximately 50-150 ug of genomic DNA extracted from regeneratedplants was digested with Eco RI restriction enzyme and electrophoresed through a1% agarose gel. The size fractionated EcoRI digested genomic DNA was transferredonto Zeta-Probe GT membrane (BioRad) by alkaline capillary blotting. The membraneswere probed with random primed, 32P]- labeled npt II cDNA (blot A) or BWYV coatprotein cDNA (blot B) and autoradiographed. Lane 5 contained EccRI digested genomicDNA corresponding to plant 1557-8, lanes 6-8,10,11 to plants 154802-1 to -5, lane 12 toplant 154837-1 and lane 13-15 to plants 1548-1 to -3. In addition, lane 9 contained Hind111 digested genomic DNA corresponding to plant 154802-3. The controls included: EcoRI digested plasrnid DNA of pCGN154802, pCGN154837, and pCGN1548 (lanes 2-4,respectively). Lane 1 contained Lambda DNA digested with EcoRI and Hind Ill usedas molecular size marker (sizes shown in kbp).Results p. 70Similarly, hybridization was found to occur with Eco RI digested DNA from plant155737-5 using the probe corresponding to the npt II gene (see Fig. 3.14A, lane 11)but no hybridization occurred with Eco RI digested DNA from plant 155737-5 (seeFig. 3.14B, lane 11) using the BWYV coat protein probe. No hybridization ofBVVYV coat protein cDNA probe was expected nor occurred for the binary plasmids(pCGN1548 and pCGN1557) used as controls.The results from Southern blot analysis of Hind III digested plant genomicDNA corresponding to the regenerated plants suggests, although not conclusively, thatthree plants have been successfully transformed with the BWYV coat protein gene(plants 154802-1, 154802-3 and 1557-7). Plant 1557-7, which supposedly had beentransformed with the binary plasmid 1557 containing no BWYV coat protein gene,was likely incorrectly labeled. Furthermore, Southern blot analysis of Eco RIdigested genomic DNA indicated that two plants had been successfully co-transformedwith the npt II gene (plants 155737-5 and 1557-7). Surprisingly, the npt II gene couldnot be detected in Southern blot analysis of the genomic DNA from plants 154802-1and 154802-3, for which the presence of the BWYV coat protein gene had beenindicated in Hind III digests (Eco RI digested plant DNA produced a signal only forthe plant 154802-3). Similarly, the BWYV coat protein gene could not be detected inSouthern blot analysis of the genomic DNA from plant 155737-5, for which thepresence of the npt H gene had been indicated and presumed to be present since theplant was kanamycin resistant (see Discussion).Results p. 713.7.2 Northern Blot Analysis of RNA Extracted from Regenerated PlantsTotal leaf RNA was extracted from all regenerated plants except for plant155702-9, which was stunted and sterile. Approximately 10 to 40 ug of RNA wassize fractionated by electrophoresis through glyoxal containing or MeHg0H-containingagarose gels and transferred onto Zeta-Probe GT membrane by alkaline capillaryblotting. The Northern blots were probed with either random primed, [3211-labeledDNA corresponding to the npt II gene or to the BWYV coat protein gene. In threeseparate attempts, RNA transcripts of the npt II gene and BWYV coat protein genecould not be detected in the RNA samples extracted from any of the regeneratedplants (results not shown). Both controls were detected in Northern blot analysis; theyincluded 100 pg of BWYV RNA extracted from purified virus and 50 pg of syntheticBWYV coat protein transcript.3.7.3 PCR Analysis of DNA Extracted from Regenerated PlantsThe polymerase chain reaction (PCR) was used for detection of the BWYVcoat protein gene in DNA samples extracted from regenerated plants. A PCR primerbased on the CaMV 35S promoter sequence, 5'-CAC TAT CCT TCG CAA GAC CCTTCC TC-3' (35S-1 primer kindly supplied by Robert R. Martin, Agriculture CanadaVancouver Research Station) was used in combination with either of the original PCRprimers (BWYV oligonucleotide #1 or #2) used to synthesize the BWYV coat proteinResults p. 72gene cDNA. In the DNA samples from plants expected to contain the BWYV coatprotein gene in the plus-sense orientation relative to the CaMV 35S promoter, the 35S-1 primer was used with BWYV oligonucleotide primer #2 in the PCR reaction. In theDNA samples from plants expected to contain the BWYV coat protein gene in theanti-sense orientation, the 35S-1 primer was used with BWYV oligonucleotide primer#1 in the PCR reaction. Finally, in the DNA samples from untransformed plants orplants that contained the control binary plasmid, the 35S-1 primer was used with bothBWYV oligonucleotide primers #1 and #2. The expected size of the PCR productusing the 35S-1 primer in combination with either the BWYV oligonucleotide primer#1 or #2 is approximately 720 bp. The PCR products were analyzed by agarose gelelectrophoresis followed by Southern blot analysis using a random primed, [32P]-labeled DNA probe corresponding to the BWYV coat protein gene.The Southern blot analysis was used to confirm that the PCR productscorresponded to the BWYV coat protein gene (as opposed to an unrelated productfound previously with RT-PCR from both healthy and BWYV-infected plants, seeSection 3.1). Southern blot analysis of the PCR products revealed a 720 bp fragmentin several plant DNA samples (see Fig. 3.16). When the PCR products wereelectrophoresed through a 1% agarose gel and visualized by staining with ethidiumbromide it was found that in some lanes the most prominent band was about 500 bp in21.85.05 5.244.213.411.90 1.981.32 1.570.930.840.5821.85.05 5.4.3.411:481.570.720.50Results p. 731 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40•Fig. 3.16 Southern blot analysis of the PCR products obtained using DNA extracted fromregenerated plants as template. Template DNA used in the PCR reactions was extracted from thefollowing regenerated plants: 155702-1 to -15 (lanes 2-16), 155737-1 to -6 (lanes 17-22),1557-7 (lane 23), 154802-1 to -5 (lanes 24-28), 154837-1 (lane 29) and 1548-2 to -3 (lanes30,31). The PCR products obtained were analyzed by electrophoresis through a 1% agarose geland stained with ethidium bromide. The PCR products were transferred onto Zeta-Probe GTmembrane (BioRad) by alkaline capillary blotting. The membranes were probed with randomprimed, [3211-labeled BWYV coat protein cDNA and autoradiographed. Templates used in thePCR reactions as controls included the binary plasmids pCGN155702, pCGN155737,pCGN1557, pCGN154802, pCGN154837 and pCGN1548 (lanes 32-37, respectively). PlantDNA extracted from uninfected Brassica napus cv. Westar (lane 39) and water (lane 40) werealso included as controls. Lanes 1 and 38 contain molecular size markers (in kbp): LambdaDNA digested with Eco RI and Hind III. The numbers on the right indicate the expected sizeof the coat protein PCR product, 720 bp and of the 500 bp non-specific band, likely a result ofmis-priming using the CalVIV 35S-1 primer.Results p. 74size (see Fig. 3.17, lanes 3-7). This band likely resulted from mis-priming using the35S-1 primer. Previously, an attempt to amplify the BWYV coat protein gene and thenpt II gene as a single fragment in constructs containing two CaMV 35S promoters(see Fig. 3.11, pCGN155702) of approximately 5 kbp in size using only the 35S-1primer in the PCR reaction resulted in more than one band, one of which was about500 bp in size. Southern blot analysis of the PCR products demonstrated that thesebands did not correspond to the BWYV coat protein gene or the npt II gene (resultsnot shown). Southern blot analysis of the PCR products using the binary plasmidconstructs as templates produced a 720 bp fragment for the pCGN154802,pCGN155702 (see Fig. 3.16, lanes 24-28, lanes 2-9, 11-15) constructs but no fragmentfor the pCGN1548 and pCGN1557 binary plasmids, as predicted (see Fig. 3.16, lane37 and 34, respectively). Surprisingly, the BWYV coat protein gene could not bedetected in any of the regenerated plants transformed with the anti-sense binaryplasmid constructs (pCGN154837 and pCGN155737). However, a PCR product of720 bp in size was obtained using the anti-sense binary plasmid constructs,pCGN154837 and pCGN155737 as controls. A 720 bp fragment was detected bySouthern blot analysis of the PCR products in several plant DNA samples whichincluded: thirteen plants treated with the A. tumefaciens harbouring the 155702construct, five with the 154802 construct (including plants 154802-1 and 154802-3which were shown previously by Southern blot analysis to possibly contain the coatprotein gene) and plant 1557-7. A 630 bp fragment was revealed in the Southern blotanalysis of the PCR products from the plant 1557-7 (see Fig. 3.16, lane 23), whichResults p. 75 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 2021.85.05 5.244.213.411.90 1.981.571.320.930.840.580.720.5021 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 4021.85.05 5.244.213.411.901.981.570.720.50Fig. 3.17 PCR analysis of DNA extracted from regenerated plants. A PCR primerbased on the CaMV 35S promoter sequence was used in combination with either of thePCR primers (BWYV oligonucleotide #1 and #2) for detection by the polymerasereaction of the BWYV coat protein gene (anti-sense orientation and plus-senseorientation, respectively) in DNA samples extracted from regenerated plants. TemplateDNA used in the PCR reactions was extracted from the following regenerated plants:155702-1 to -15 (lanes 2-16), 155737-1 to -6 (lanes 17-22), 1557-7 (lane 23), 154802-1to -5 (lanes 24-28), 154837-1 (lane 29) and 1548-2 to -3 (lanes 30,31). The PCRproducts obtained were analyzed by electrophoresis through a 1% agarose gel andstained with ethidium bromide. Templates used as controls included the binaryplasmids pCGN155702, pCGN155737, pCGN1557, pCGN154802, pCGN154837 andpCGN1548 (lanes 32-37, respectively). Plant DNA extracted fromuninfected Brassica napus cv. Westar (lane 39) and water (lane 40) were alsoincluded as the controls. In the DNA samples where the BWYV coat protein gene wasnot expected to be detected by PCR, all three PCR primers were used (lanes 23, 30, 31,34, 37, 39 and 40). Lanes 1 and 38 contain molecular size markers (in kbp): LambdaDNA digested with Eco RI and Hind III. The numbers on the right indicate theexpected size of the coat protein PCR product, 720 bp and of the 500 bp non-specificband, likely a result of mis-priming using the CaMV 35S-1 primer.Results p. 76was not expected to contain the BWYV coat protein gene but nevertheless hybridizedto a BWYV coat protein probe when the plant DNA extracts were analyzed bySouthern blotting (see Section 3.7.1). In the PCR reactions in which the DNAtemplate was extracted from plants that were presumed to have been transformed witha binary plasmid (pCGN1548 and pCGN1557) not containing the BWYV coat protein,all three primers (BWYV oligonucleotide primer #1 and #2, and CaMV 35S-1oligonucleotide primer) were included in the reactions. The 630 bp fragment seen inthe Southern blot analysis of the PCR products from the plant 1557-7 likely resultedfrom the annealing of the BWYV oligonucleotide primers #1 and #2 and subsequentamplification of the BWYV coat protein. Alternatively, the annealing of the CaMV35S-1 oligonucleotide primer and BWYV oligonucleotide primer #2 would haveresulted in the amplification of a portion of the CaMV 35S promoter upstream of theBWYV coat protein gene as a 720 bp fragment. The results from the PCR analysis ofthe plant genomic DNA followed by Southern blot analysis suggests that several otherplants were successfully transformed with the BWYV coat protein gene constructs:plants 155702-1 to -8, -10 to -14, 1557-7 and 154802-1 to -5. The PCR analysis didnot detect the BWYV coat protein gene in any of the plants treated with the A.tumefaciens harbouring the construct containing the gene in the anti-sense orientation.Primers were not obtained for use in PCR to amplify the npt II gene from the plantDNA samples. Attempts to detect the BWYV coat protein gene by RT-PCR in RNAsamples extracted from the regenerated plants were unsuccessful.Results p. 773.7.4 Evaluation of Seeds from Regenerated Plants for Kanamycin ResistanceThe seeds (R1) of the primary transformants of B. napus cv. Westar weregerminated in the presence of 50 ug/mL kanamycin (see Table 3.4). The transformedplants containing the plant selectable marker npt II, which confers kanamycinresistance, had green first true leaves whereas the first true leaves of plants that werenot resistant to kanamycin became bleached within two weeks after germination. Twoplants showed no resistance to kanamycin (untransformed B. napus and plant154802-5) as indicated by the bleaching of both the first true leaves and cotyledons.Several plants showed partial resistance to kanamycin (indicated by bleaching of firsttrue leaves but partially green cotyledons). A few plants showed only slightly betterresistance as indicated by the pale green color of their first true leaves. The plantswere scored for their level of resistance to kanamycin (no resistance, partial resistanceand good resistance) and the segregation ratio (resistant: non-resistant plants)compared to the theoretical ratio of 3: 1; partial resistance was counted as resistance inthe calculation of the segregation ratio. Of seventeen plants tested, ten plantsproduced seeds displaying ratios greater than 3:1, and seven had ratios less than 3:1(see Table 3.4). Plants 154802-1 and 154802-3, which were shown previously bySouthern blot analysis to possibly contain the coat protein gene but not the npt II gene,had ratios of 9:1 and 5:1, respectively. Surprisingly, plant 1557-7, which was shownpreviously by Southern blot analysis to contain not only the coat protein gene but alsothe npt II gene, had a segregation ratio of 2.2:1. Plant 154802-5, which was shownResults^p. 78Table 3.4^Evaluation of seeds from regenerated plants for kanamycin resistance. Seeds (R1) from theprimary transformants of Brassica napus cv. Westar were germinated on MS media supplemented with50 g/mL kanamycin (see Section 3.7.4). RI seed from untransformed regenerated B. napus cv. Westar was alsoincluded. The plants were scored for kanamycin resistance two weeks after germination; (--) no lcanamycinresistance, bleached cotyledons and first true leaves; (-+) partial kanamycin resistance, green (or partial)cotyledons and bleached first true leaves; (++) kanamycin resistance, green cotyledons and pale green first trueleaves. The ratio of lcanamycin resistant to non-resistant plants was calculated; a 3:1 ratio is the theoretical ratioexpected. Partial kanamycin resistance was scored as resistance in determining the final ratio.PrimaryTransformantNumberGerminatedKanamycin Resistance-- -+ ++ ration:1R1 13/20 13 0 0 0.0155702-1 17/20 4 7 6 3.2155702-4 17/20 3 3 11 4.7155702-5 13/20 2 9 2 5.5155702-6 20/20 6 11 3 2.3155702-10 12/20 5 6 1 1.4155702-11 14/20 2 3 9 6.0155702-12 20/20 3 13 4 5.7155702-13 19/20 2 5 12 8.5155737-1 19/20 10 6 3 0.9155737-6 19/20 4 15 0 3.81557-7 19/20 6 3 10 2.2154802-1 20/20 2 8 10 9.0154802-2 19/20 8 11 0 1.4154802-3 18/20 3 10 5 5.0154802-4 15/20 8 5 2 0.9154802-5 16/20 16 0 0 0.0154837-1 19/20 2 2 15 8.5Results p. 79only by PCR analysis to contain the BWYV coat protein gene, demonstrated noresistance to kanamycin. Similarly, the progeny from untransformed B. napus, asexpected, displayed no resistance to kanamycin. The level of resistance to kanamycinlikely is a function of the number of functioning npt II gene integrations into thegenome of the plant. Although the germination of twenty plants (seeds) is insufficientfor statistical analysis, the ratios suggest that many of the plants had likely been co-transformed with the npt II gene. None of these plants could be recovered for furthertesting as they became stunted in their growth, even after being transferred tokanamycin-free medium.3.8^Production and Screening of Monoclonal Antibodies and PolyclonalAntisera for Reactivity Against Disrupted BWYV Particles3.8.1 Evaluation of Antibodies in Western Blot and Dot Blot AnalysisBWYV infection of B. napus was monitored by using a polyclonal antiserumand a monoclonal antibody in an enzyme linked immunosorbent assay (ELISA).BWYV coat protein expression in transgenic plants is expected to eventually result inthe formation of coat protein subunits. Ideally, it would have been useful to screen fortransgenic plants expressing BWYV coat protein subunits by ELISA. Severalmonoclonal antibodies (510H 1 gG2a a BWYV, 43BC IgM a BWYV & PLRV, 4012IgM a BWYV and 26BE IgG1 a PLRV) and polyclonal antisera (rabbit a BWYV IgGResults p. 80and rabbit a PLRV IgG), kindly provided by Peter Ellis (Agriculture Canada ResearchStation), were evaluated for the ability to react with disrupted BWYV particles. Theseantibodies were originally produced by injection of intact purified BWYV virions intomice and rabbits, for monoclonal antibodies and polyclonal antisera, respectively. Themonoclonal antibody 510H (mouse a BWYV), the polyclonal antiserum (rabbit aBWYV) and goat IgG (goat a mouse) were used in triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) to monitor BWYV infection of B. napuscv. Westar (Ellis and Wieczorek, 1992).Initially, the monoclonal antibodies were evaluated for the ability to bindBWYV coat protein under the conditions of a Western blot analysis. Various amounts(100-500 ng) of purified disrupted BWYV were electrophoresed through a 10% SDS-polyacrylamide gel and electro-blotted onto PVDF membrane (0.2 micron, BioRad).The trans-blots were incubated with the appropriate monoclonal antibody / [125I]labeled goat anti-mouse IgG, washed and autoradiographed. None of the monoclonalantibodies appeared to react with the BWYV coat protein under the denaturingconditions of the Western blot analysis. Furthermore, incubation of the polyclonalantiserum (a BWYV) / [125INabeled goat anti-rabbit with the same trans-blotsdemonstrated that the polyclonal antiserum did not react with the BWYV coat proteinunder these conditions (results not shown).Various amounts (100-500 ng) of intact purified BWYV virion and disruptedResults^p. 81BWYV particles (1% SDS, 95°C for 5 min) were spotted onto PVDF membrane,incubated with the appropriate monoclonal antibody / ['I]-labeled goat anti-mouseIgG, washed and autoradiographed. All the monoclonal antibodies tested reacted withthe intact purified BWYV particles on the membrane except for 26BE (a PLRV).However, none of the monoclonal antibodies reacted with the disrupted BWYVparticles spotted onto the membrane. Furthermore, the polyclonal antiserum specificfor BWYV also reacted with the intact purified BWYV particles spotted on themembrane but not with the disrupted BWYV particles. As expected, the polyclonalantisera specific for PLRV did not react with either intact or disrupted BWYVparticles on the membrane.Based on this analysis, the above monoclonal antibodies and polyclonal antiserawould not have been useful for screening transgenic B. napus expressing BWYVcoat protein subunits.3.8.2 Production of Monoclonal Antibodies and Polyclonal Antisera usingDisrupted BWYV ParticlesPurified BWYV particle preparations were disrupted by treatment with 1%SDS and subsequent heating at 95°C for 5 min followed by dialysis against 0.1 Mphosphate buffer, pH 7.0. The denatured particles were used to immunize BALB/cmice and a rabbit for the production of monoclonal antibodies and polyclonal antisera,Results p. 82respectively. BALB/c mice were immunized with three injections of virus (50 ug perinjection) before their spleens were harvested and used in the fusion protocol to makehybridomas (Ellis and Wieczorek, 1992). The hybridomas were screened by anindirect TAS-ELISA. Hybridoma clones secreting BWYV-specific antibodies againstdisrupted virus particles, but not intact virus particles could not be found. Similarly, arabbit was immunized with five injections of virus (0.5 mg per injection). Test bleeds(30 mL) were taken after the third and fourth injections. Immunoglobulins (IgG)purified from a portion of the test bleeds were evaluated in double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) for the ability to react with intactand disrupted BWYV particles as well as possible BWYV coat protein subunits intransgenic plants. Preliminary results indicated that the purified IgGs did not reactagainst intact BWYV particles but did react against disrupted BWYV particles(treatment with SDS or carbonate buffer, pH 9.2). The purified IgG was used inDAS-ELISA to screen for any regenerated plants that might be expressing BWYV coatprotein subunits. None of the plants appeared to be expressing the BWYV coatprotein at a level detectable by ELISA (results not shown).3.9^Preliminary Evaluation of BWYV Resistance in Transgenic Brassicanapus cv.WestarThe seeds (R1) from the primary transformants of B. napus cv. Westar (18plants from 154802-3, 14 plants from 155737-6, 14 plants from 1557-7) and the seedsResults p. 83(R1) from untransformed regenerated plants were germinated and placed in pots inthe greenhouse (16 hr light, 21°C / 8 hr darkness, 16°C). Limited space dictated that alimited number of transgenic lines be evaluated. The progeny of plants 154802-3 and1557-7 were evaluated because they were previously shown by Southern blot and PCRanalysis to contain the BWYV coat protein gene. Thus, plants 154802-3 and 1557-7were expected to show the best levels of resistance to infection by BWYV. Plant155737-6 could not be demonstrated in Southern blot analysis or PCR analysis tocontain the BWYV coat protein. However, germination of the progeny from this plantshowed that they possessed good resistance to kanamycin. Thus, plant 155737-6 wasnot expected to show any resistance to infection by BWYV. After 2-3 weeks, whenthe first true leaves were 4-5 cm in length, 20-40 viruliferous green peach aphids(Myzus persicae) were placed on the plants for an inoculation access period of 72hours. Prior to the inoculation, the aphids were allowed at least 48 hr access toBWYV-infected plants. Physalis pubescens L., an indicator plant, was also inoculatedat the same time. After 5-6 weeks, leaf samples were taken from each plant andtested by TAS-ELISA for BWYV infection. Neither the inoculated transgenic nor theuntransformed B. napus cv. Westar nor the indicator plants showed any of the typicalsymptoms of BWYV infection. However, all plants were infected with a high titre ofBWYV as determined by TAS-ELISA (results not shown).p. 84DiscussionThe beet western yellows virus (BWYV) coat protein gene was successfullyintroduced into the genome of Brassica napus cv. Westar via the Agrobacteriumtumefaciens Ti plasmid mediated gene transfer. A summary of the integration andexpression analysis of the regenerated plants is provided in Table 4.1. Southern blotanalysis of Hind III digested plant DNA indicated that the BWYV coat protein genewas present in the genome of three plants (154802-1, 154802-3 and 1557-7).However, Southern blot analysis of Eco RI digested plant DNA showed that the npt IIgene was present only in plants 1557-7 and 155737-5 but not 154802-1 and 154802-3.Furthermore, the blots of the same Eco RI digested plant DNA showed that theBWYV coat protein gene was present in the genome of plants 1557-7 and 154802-3.PCR analysis of the same plant genomic DNA followed by Southern blot analysis ofthe PCR products indicated that several plants contained the BWYV coat protein genein the plant genome (plants 155702-1 to -8, -10 to -14, 1557-7 and 154802-1 to -5).No RNA transcripts of either the BWYV coat protein or of the npt II gene productcould be detected in Northern blots but germination of the seeds from the primarytransformants on kanamycin indicated that the npt II gene product, which conferskanamycin resistance, is functional in many of the plants. However, preliminaryevaluation of the progeny of three promising lines, demonstrated to have the BWYVcoat protein gene integrated into their genomes, did not find significant levels ofDiscussion^p. 85Table 4.1^Summary of integration and expression analysis of regenerated Brassica napus cv. Westar aftertreatment of cotyledonary explants using the plant transformation procedure (Moloney et al., 1989). (n/s) Nosignal was detected for the indicated gene, (???) the plant is highly suspect for the presence of the indicatedgene, (nit) the sample was not tested, (n/a) not applicable.Date plantplaced intogreenhouseConstruct Southern blot analysis Northern blotanalysisPCR GerminationanalysisEco RI digestedplant DNAHind IIIdigestedkanamycinresitancenptIIgeneBWYVCP geneBWYVCPnpt IIgeneBWYVCPBWYVCPgene# ration:1April 1/92 155737-1 n/s n/s n/s n/s n/s n/s 9/19 0.9April 30/92 155702-1 n/s n/s n/s n/s n/s Yes 13/17 3.2155737-2 n/s n/s n/s n/s n/s n/s n/t n/t155737-3 n/s n/s n/s n/s n/s n/s n/t n/t1557-1 n/s n/s n/s n/s n/s n/s n/t nit1557-2 n/s n/s n/s n/s n/s n/t n/t1557-3 n/s n/s his n/s n/s n/s n/t nit1557-4 n/s n/s n/s n/s n/s n/s nit n/tMay 1/92 155702-2 n/s n/s n/s n/s n/s Yes n/t nitJune 2/92 155702-3 n/s n/s n/s n/s n/s Yes n/t n/t155702-4 n/s n/s n/s n/s n/s Yes 14/17 4.7155702-5 n/s n/s n/s n/s n/s Yes 11/13 5.5155702-6 n/s n/s n/s n/s n/s Yes 14/20 2.3155737-4 n/s n/s n/s n/s n/s n/s n/t n/tJuly 2/92 155702-7 n/s n/s n/s n/s n/s Yes n/t155702-8 n/s n/s n/s n/s n/s Yes n/t n/t155702-9 n/s n/s n/s n/a n/a n/s n/a n/a155702-10 n/s n/s n/s n/s Ws Yes 7/12 1.4155702-11 n/s n/s n/s n/s n/s Yes 12/14 6.0155702-12 n/s n/s n/s n/s n/s Yes 12/14 6.0155702-13 n/s n/s n/s n/s n/s Yes 17/19 8.5Discussion^p. 86Table 4.1^(coned)Date plantplaced intogreenhouseConstruct Southern blot analysis Northern blotanalysisPCR GerminationanalysisEco RI digestedplant DNAHind IIIdigestedIcanamycinresitancenptrfgeneBWYVCP geneBWYVCPnpt IIgeneBWYVCPBWYVCPgene# ration:1July 2/92 155702-14 n/s n/s lils n/s n/s Yes n/t155702-15 n/s n/s n/s n/s n/s n/s nit n/t155737-6 n/s n/s n/s n/s n/s n/s 15/19 3.81557-5 n/s n/s n/s n/s n/s n/s nit n/t1557-6 n/s n/s n/s n/s n/s n/s n/t nA1557-7 Yes Yes Yes n/s n/s Yes 13/19 3.21557-8 n/s n/s n/s n/s n/s n/s tilt n/t154802-1 n/s n/s Yes n/s n/s Yes 18/20 9.0154802-2 n/s n/s n/s n/s n/s Yes 11/19 1.4154802-3 ??? ??? Yes n/s n/s Yes 15/18 5.0154802-4 n/s n/s n/s n/s n/s Yes 7/15 0.9154802-5 n/s n/s n/s n/s n/s Yes 0/16 0.0154837-1 ??? n/s n/s n/s n/s n/s 17/20 5.71548-1 n/s n/s n/s n/s n/s n/s n/t n/t1548-2 n/s n/s n/s n/s n/s n/s n/t n/t1548-3 n/s n/s n/s n/s n/s n/s n/t n/tuntreated n/s n/s n/s n/s n/s n/s 0/13 0.0Discussion p. 87resistance when these plants were inoculated with the homologous virus using theaphid vector, Myzus persicae (Sulz.).4.1^Transformation of Brassica napus cv. WestarThe plant transformation procedure used was that described by Moloney et al.(1989) for the transformation of Brassica napus cv. Westar utilizing cotyledonaryexplants. The regeneration efficiency of the cotyledonary explants, after inoculationwith the A. tumefaciens, ranged from 0.8-3.6% as compared to 17-80% in theuninoculated cotyledonary explants. Thirty-eight regenerated plants were recoveredfrom 4670 cotyledonary explants which had been inoculated with A. tumefaciens. Theregeneration efficiency observed was considerably less than that reported (55%) byMoloney et al. (1989) but compared favorably with previous work described usingfloral stem 'thin cell layer' (2% efficiency, Charest a al., 1988), hypocotyls (2.5%efficiency, Radke a al., 1988) and longitudinal stem sections (10% efficiency, Pua aal., 1987). Two other groups have observed low regeneration efficiency (Calgene Inc.,California and Plant Biotechnology Institue, Saskatoon; personal communication) usingthis same plant transformation procedure. Additional difficulties with the planttransformation procedure included: (1) contamination of explants when using highinocula of A. tumefaciens, even under the selection of the antibiotic, carbenicillin and(2) the apparently high number of regenerated plants which escaped selection bykanamycin. The contamination was greatest in the third (July 1992) and fourthDiscussion p. 88(September 1992) attempts to transform B. napus cv. Westar. Although theregeneration efficiency of shoots remained low for the third and fourth attempts, theplants that did regenerate were soon overcome by the rapidly growing A. tumefaciensand died. Of the thirty eight plants regenerated during the first two planttransformation attempts, it became apparent from germination studies that 33-40% ofthe putative transgenic plants possessed little or no resistance to kanamycin; eventhough the regenerated plants were grown in the selection of kanamycin. Severalmodifications to the plant transformation procedure (kindly suggested by S. Radke,Calgene and G.H. van Rooijen, University of Calgary) may address the difficultiesobserved with this procedure. (1) Reducing the initial inoculum of A. tumefaciens,reducing the co-cultivation period of the A. tumefaciens with the cotyledonary explantsfrom 72 hours to 24 hours and using cefotaxime in combination with the carbenicillinmight reduce the amount of tissue necrosis and hopefully increase the regenerationefficiency of the plant transformation procedure. The B. napus cv. Westarcotyledonary explants appeared to be sensitive to high levels of A. turnefaciensinoculum. A large inoculum resulted in early tissue necrosis, especially along theexposed cut ends of the explants. Eventually, the high concentrations of A.tumefaciens overcame the antibiotic selection with carbenicillin and killed any shootsthat managed to regenerate into plants. (2) Although exposure to kanamycin wasfound to increase the time required for the formation of roots (Moloney et al., 1989),the use of kanamycin throughout the procedure should reduce the possibility thatplants that are not kanamycin resistant, and therefore not transformed, will survive.Discussion p. 89The plant transformation procedure described by Moloney et al. (1989) useskanamycin only up to the shoot stage. Since the polarity of T-DNA gene transferoccurs from the right border to the left border in the Ti plasmid (Rubin, 1986), plantstransformed with the npt II gene presumably would have been co-transformed with theBWYV coat protein using the binary plasmid constructs described in Section 3.4.4.2^Integration Analysis of Regenerated Brassica napus cv. WestarThe genomic DNA extracted from the regenerated plants was analyzed bySouthern blot analysis and PCR analysis (see Table 4.1). Southern blot analysis ofHind III digested genomic DNA, using a random primed, [3211-labeled probecorresponding to the BWYV coat protein gene, indicated that the BWYV coat proteingene was present in the genome of three plants (154802-1, 154802-3 and 1557-7).However, Southern blot analysis of Eco RI digested genomic DNA, using a randomprimed, [3211-labeled probe corresponding to the npt II gene, showed that the npt IIgene was present only in the genome of plants 1557-7 and 155737-5. Surprisingly,the npt II gene was not detected in the genome of plants 154802-1 and 154802-3.Probing of the same Southern blots, after stripping the npt II cDNA probe from theblots, with random primed, [3211-labeled DNA corresponding to the BWYV coatprotein gene indicated that the BWYV coat protein gene was present in the genome ofplant 1557-7 and 154802-3. But the BWYV coat protein gene could not be detectedin the genome of plants 154802-1 and 155737-5 after digestion of their DNA withDiscussion p. 90Eco RI. The reason that the plant transformed with construct 154802-1 hybridized tothe coat protein probe when the plant DNA was digested with Hind III but not withEco RI is not understood at this time. It is possible that the coat protein gene isindeed present but could not be detected in the Southern blot analysis due to problemswith the particular plant DNA sample (possibly degradation); especially since the PCRanalysis indicated the presence of the coat protein gene. The lack of hybridization tothe npt II probe using the same Eco RI digested DNA could be explained similarly.Indeed the result from the germination analysis of R1 seed would support the findingthat the original plant was transformed at least with the npt II gene since thesegregation ratio was 9:1. Similarly, germination analysis of R1 seeds from plantstransformed with 154802-3 would suggest the presence of the npt II gene (see Table4.1).The genome size of Brassica napus has been reported to be 2.3-2.56 pg/2C or1129-1235 Mbp/C (Arumuganathan and Earle, 1991; where 2C represents the diploidgenome and 1C the haploid genome). Assuming that only a single copy of theBWYV coat protein gene is present in each genome, 100 ug of plant genomic DNAwould contain ca. 25 pg of BWYV coat protein DNA (Rogers and Bendich, 1988).Twenty-five picograms of plasmid DNA gave very strong signals in the Southernblots, so the lack of a hybridization signal in the plant DNA samples should mean thatthese plants were not transformed. However, these conclusions are difficult to draw inview of the variable intensity of signal obtained with control plasmid DNA which wasDiscussion p. 91intended to be 25 pg / lane (see Fig. 3.13, lanes 13, 14, 16 and 17). The BWYV coatprotein gene was detected in several other plants (plants 155702-1 to -8, -10 to -14,1557-7 and 154802-1 to -5) by PCR analysis of the genomic DNA followed bySouthern blot analysis. PCR primers for the detection of the npt II gene were notavailable. Since the polarity of T-DNA transfer is from the right border to the leftborder of the Ti plasmid (Rubin, 1986), the BWYV coat protein gene would have beentransferred before the npt II gene in the binary plasmid constructs was used (seeSection 3.4). The possibility arises that a plant can be transformed with the BWYVcoat protein gene but not the npt II gene (see Section 3.4). This may be the case forseveral plants (1548002-2, -4,-5 and 155702-10) which were shown by PCR analysisto contain the BWYV coat protein but showed little or no resistance to kanamycin ingermination studies, as evidenced by the low segregation ratios (see Table 4.1).Truncated or rearranged integration has been previously reported in Brassica napustransformants (Radke et al., 1988) and in Nicotiana tabacum (Van Lijsbettens et al.,1986).4.3^Expression analysis of regenerated Brassica napus cv. WestarThe expression of the BWYV coat protein and the npt II gene product wereevaluated in Northern blots, by a functional bioassay for kanamycin resistance and byELISA. Northern blot analysis of the RNA extracted from the regenerated plantscould not detect either BWYV coat protein transcripts or npt II transcripts. PreviousDiscussion p. 92work with transgenic potato transformed with the PLRV coat protein gene indicatedthat PLRV coat protein RNA transcripts could be detected in a total leaf RNA extractonly after a poly(A) RNA enrichment had been performed (Kawchuck et al., 1990).Germination of the B. napus R1 seeds from the primary transformants on kanamycin-containing media indicated that the npt II gene product was functional in many of thetransformed plants (see Section 3.7.4) suggesting that npt II transcripts are present inthe plant but were not detected in the blotting experiments described in this thesis. Itis possible that many of the plants which showed kanamycin resistance represent"escapes" however, the germination analysis from seeds derived from the primarytransformants indicated that the kanamycin resistant phenotype segregated as adominant gene (— 3:1 ratio) and therefore these plants likely do not represent kanamycin"escapes". BWYV coat protein subunits were not detected by DAS-ELISA using apolyclonal antisera that was produced using BWYV coat protein subunits. Althoughthe CaMV 35S promoter has been known to confer high levels of expression in avariety of plants cells, the expression levels of the BWYV coat protein and the npt Hgene product in the primary transformants appeared to be low or not detectable. Intransgenic potatoes, containing the PLRV coat protein chimeric gene under the controlof the duplicated CaMV 35S promoter, transcription levels of the PLRV coat proteingene were high but little or no coat protein was detected (Kawchuk et al., 1990).Inoculation of the transgenic potato plants with PLRV resulted in low virus titers thatremained low or decreased indicating sustained resistance. Similar resistance to PLRVinfection was found with plants transformed with the PLRV coat protein gene in anti-Discussion p. 93sense orientation which suggests that resistance of transgenic potatoes against PLRVmay be mediated by the coat protein gene transcript. Preliminary evaluation of theprogeny of two promising lines (154802-3 and 1557-7), which were shown bySouthern blot analysis and PCR analysis to contain the BWYV coat protein gene inthe plant genome, did not find significant levels of resistance when these plants wereinoculated with the homologous virus using the aphid vector, Myzus persicae (Sulz.).4.4^Future outlookThe ability to transform Brassica napus cv. Westar has proven useful foroverall improvement to the quality of the crop and for the study of plant, bacterial andviral genes. Significant improvements in the quality of the oil and protein content ofB. napus seeds have been obtained by the introduction of the stearoyl-acyl carrierprotein desaturase gene in the antisense orientation (Knutzon et al., 1992) and amethionine-rich seed protein from a Brazil nut (Altenbach et al., 1992). The beetwestern yellows virus (BWYV) coat protein gene was successfully introduced into thegenome of Brassica napus cv. Westar via the Agrobacterium tumefaciens Ti plasmidmediated gene transfer. Regeneration efficiency of B. napus cv. Westar using theplant transformation procedure as described by Moloney et al. (1989) was lower thanreported. But Southern blot analysis and PCR analysis indicate, although notconclusively, that the coat protein gene has been integrated into the plant genome. Itis not understood at this time the reason why BWYV coat protein RNA transcripts orDiscussion p. 94BWYV coat protein subunits were not found. But the lack of BWYV coat proteinRNA transcripts and of coat protein subunits could explain why B. napus cv. Westartransformed with the BWYV coat protein gene showed no resistance to infection byBWYV when inoculated with infected Myzus persicae (Sulz.). The usefulness of theplant transformation procedure described by Moloney et al. (1989) will depend onimprovement of the overall regeneration and transformation efficiency anddevelopment of a procedure for other Brassicaceae cultivars such as Brassica juncea.Several other genes besides the coat protein gene have proven useful for conferringviral resistance in transgenic plants; these include a portion of the viral replicase non-structural gene of TMV in tobacco (Golemboski et al., 1990), viral satellite RNA fromCMV in tobacco (Baulcombe et al., 1986), virus specific antibodies (Baulcombe,1986) and ribozymes in tobacco (Haseloff and Gerlach, 1988). The commercialsuccess of pathogen resistant transgenic plants will depend on the identification ofplant resistance genes rather than foreign genes, the development of planttransformation selectable markers found naturally in plants (especially in transgenicplant products that are for human consumption) and the alleviation of fears towardsthe release of genetically altered plants.p. 95BibliographyALTENBACH, S., KUO, C.-C., STARACI, L.C., PEARSON, K.W., WAINWRIGHT, C.,GEORGESCU, A., TOWNSEND, J. (1992) Accumulation of a Brazil nut albuminin seeds of transgenic canola results in enhanced levels of seed proteinmethionine. Plant Molecular Biology 18, 235-245.AN, G., EBERT, P.R., MITRA, A., HA, S.B. (1988) Binary vectors. In Plant MolecularBiology Manual, A3: 1-19. Gelvin, S.B., Schilperoort, R.A., Verma, D.S.(editors), Kluwer Academic Publishers, Boston © 1992.ARUMUGANATHAN, K., EARLE, E.D. (1991) Nuclear DNA content of some importantplant species. Plant Molecular Biology Reporter 9, 208-218.BAHNER, I., LAMB, J., MAYO, M.A., HAY, R.T. (1990) Expression of the genome ofpotato leafroll virus: readthrough of the coat protein termination codon in vivo.Journal of General Virology 71, 2251-2256.BAULCOMBE, D.C., SAUNDERS, G.R., BEVAN, M.W., MAYO, M.A., HARRISON, B.D.(1986) Expression of biologically active viral satellite RNA from the nucleargenome of transformed plants. Nature 321, 446-449.BEACHY, R.N., LOESCH-FRIES, S., TUMER, N.E. (1990) Coat protein-mediatedresistance against virus infection. Annual Review of Phytopathology 28, 451-474.BEVAN, M.W., FLAVELL, R.B., CHILTON, M.D. (1983) A chimaeric antibioticresistance gene as a selectable marker for plant cell transformation. Nature304, 184-187.BRAULT, V., MILLER, W.A. (1992) Translation frameshifting mediated by a viralsequence in plant cells. Proceeding of the National Academy of Sciences USA89, 2262-2266.Bibliography p. 96CASPER, R. (1988) Luteoviruses. In The plant viruses: polyhedral virions withmonopartite RNA genomes. (R. Koenig, Ed.) 3, pp. 235-258. Plenum, NewYork.CHAREST, P.J., HOLBROOK, L.A., GABARD, J., IYER, V.N., MIKI, B.L. (1988)Agrobacterium-mediated transformation of thin cell layer explants fromBrassica napus L. Theoretical and Applied Genetics 75, 438-445.CHILTON, M.D., SAIKI, R.K., YDAV, N., GORDON, M.P., QUETIER (1980) T-DNAfrom Agrobacteriurn Ti plasmid is in the nuclear DNA of crown gall tumourcells. Proceedings of the National Academy of Sciences USA 77, 4060-4064.CHUONG, P.V., PAULS, K.P., BEVERSDORF, W.D. (1988) High-frequencyembryogenesis in male sterile plants of Brassica napus through microsporeculture. Canadian Journal of Botany 66 1676-1680.CUOZZO, M., O'CONNELL, K.M., KANIEWSKI, W., FANG, R.-N., CHUA, N.H., TUMER,N.E. (1988) Viral protection in transgenic tobacco plants expressing thecucumber mosaic virus coat protein or its antisense RNA. Biotechnology 6,549-557.DAVIS, P.B., PEARSON, C.K. (1978) Characterization of density gradients prepared byfreezing and thawing a sucrose solution. Annals of Biochemistry 91, 343-349.D'ARcY, C.J., MARTIN, R.R., SPIEGEL, S. (1989) A comparative study of luteoviruspurification methods. Canadian Journal of Plant Pathology 11, 251-255.DINESH-KUMAR, S.P., BRAULT, V., MILLER, W.A. (1992) Precise mapping and invitro translation of a trifunctional subgenomic RNA of barley yellow dwarfvirus. Virology 187, 711-722.arm, G., STANFIELD, S., CORBIN, D., HELSINKI, D.R. (1980) Broad host range DNAcloning system for gram negative bacteria: construction of a gene bank inRhizobium meliloti. Proceedings of the National Academy of Sciences USA77, 7347-7351.DOYLE, J.J., DOYLE, J.L. (1990) Isolation of plant DNA from fresh tissue. BRLFocus 12, 13-15.Bibliography p. 97DUFFUS, J.E. (1972) Beet western yellows virus. In CMIIAAB Description of PlantViruses, no. 89.ELLIS, P.J. (1992) Weed hosts of beet western yellows virus and potato leafroll virusin British Columbia. Plant Disease 76, 1137-1139.ELLIS, P.J., STACE-SMITH, R. (1990) Occurrence of beet western yellows virus incanola and mustard. Abstract in Canadian Phytopathology Society meetings,1992.Ellis, P.J., Wieczorek, A. (1992) Production of monoclonal antibodies to beet westernyellows virus and potato leafroll virus and their use in luteovirus detection.Plant Disease 76, 75-78.FALK, B.W., CHIN, L.-S., DUFFUS, J.E. (1989) Complementary DNA cloning andhybridization analysis of beet western yellows luteovirus RNAs. Journal ofGeneral Virology 70, 1301-1309.FRALEY, R.T. (1983) Expression of bacterial genes in plant cells. Proceedings of theNational Academy of Sciences USA 80, 4803-4807.GARCIA, A., VAN DUIN, J., PLELT, C.W.A. (1993) Differential response to frameshiftsignals in eukaryotic and prokaryotic translational systems. Nucleic AcidsResearch 21, 401-406.GARFINKEL, D.J., SIMPSON, R.B., REAM, L.W., WHITE, F.F., GORDON, M.P., NESTER,E.W. (1981) Genetic analysis of crown gall; fine structure map of the T-DNAby site directed mutagenesis. Cell 27, 143-154.GARFINKEL, D.J., NESTER, E.W. (1980) Agrobacterium tumefaciens mutants affectedin crown gall tumorigenesis and octopine catabolism. Journal of Bacteriology144, 732-743.GILLIGAN, C.A., PECHAN, P.M., DAY, R., HILL, S.A. (1980) Beet western yellowsvirus on oilseed rape. Plant Pathology 29, 53.GOLDBACH, R.W. (1986) Molecular evolution of plant RNA viruses. Annual Reviewof Phytopathology 24, 289-310.Bibliography p. 98GOLDBACH, R., DE HAAN, P., GIELEN, J.J.L., PRINS, M., WLTKAMP, I.G., VAN SHEPEN,A., PETERS, D., VAN GIUNSVEN, M.Q.J.M. (1992) Characterization of RNA-mediated resistance to tomato spotted wilt virus in transgenic tobacco plants.Biotechnology 10, 1133-1137.GOLEMBOSKI, D.B., LOMONOSSOFF, G.P., ZAITLIN, M. (1990) Plants transformed witha tobacco mosaic nonstructural gene sequence are resistant to the virus.Proceedings of the National Academy of Sciences USA 87, 6311-6315.GUERCHE, P., JOUANIINT, L., TEPFER, D., PELLETIER, G. (1987). Genetic transformationof oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacteriumrhizo genes and analysis of inheritance of the transformed phenotype.Molecular and General Genetics 206, 382-386.HAMILTON, R.I. (1980) Defenses triggered by previous invaders: viruses. In Plantdisease: an advance treatise. (editors I.G. Horsfall and E.B. Cowling) 5, p.279. Academic Press, New York.HAMPTON, R.O., HEMPHILL, D.D., MANSOUR, N.S. (1990) BWYV infects westernOregon. American Vegetable Grower May 1990.HARRISON, B.D. (1984) Potato leafroll virus. CMIIAAB Descriptions of PlantViruses, no. 291.HASELOFF, J., GERLAC'H, W.L. (1988) Simple RNA enzymes with new and highlyspecific endoribonuclease activities. Nature 334, 585-591.HEMENWAY, C., FANG, R.-X., KANIEWSKI, W.K., CHUA, N.-H., TUMER, N.E. (1988)Analysis of the mechanism of protection in transgenic plants expressing thepotato virus X coat protein or its antisense RNA. EMBO Journal 7, 1273-1280.HERRERA-ESTRELLA, L., DE BLOCK, M., MESSENS, E., HERNALSTEENS, J.-P., VANMONTAGU, M., SCHELL, J. (1983) Chimeric genes as dominant selectablemarkers in plant cells. EMBO Journal 2, 987-995.HOEKEMA, A., HUISMAN, M.J., MOLENDYK, L., VAN DEN ELSEN, P.J.M.,CORNELISSEN, B.J.C. (1989) The genetic engineering of two commercialpotato cultivars of resistance to potato virus X. Biotechnology 7, 273-278.Bibliography p. 99HOOD, E.E., HELMER, G.L., FRALEY, R.T., CHILTON, M.D. (1986) The hypervirulenceof Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542outside of T-DNA. Journal of Bacteriology 168, 1291-1301.KAWCHUK, L.M., MARTIN, R.R., MCPHERSON, J. (1990) Resistance in transgenicpotato expressing the potato leafroll virus coat protein gene. Molecular Plant-Microbe Interactions 3, 301-307.KNurzoN, D.S., THOMPSON, G.A., RADKE, S.E., JOHNSON, W.B., KNAuF, V.C. (1992)Modification of Brassica seed oil by antisense expression of a stearoyl-acyl=Tier protein desaturase gene. Proceedings of the National Academy ofSciences USA 89, 2624-2628.KOZAK, M. (1989) The scanning model for translation: an update. Journal of CellBiology 108, 229-241.KUJAWA, A.B., DRUGEON, G., HULANICKA, D., HAENNI, A.-L. (1993) Structuralrequirements for efficient translational frameshifting in the synthesis of theputative viral RNA-dependent RNA polymerase of potato leafroll virus.Nucleic Acids Research 21, 2165-2171.LAEMMLI, U.K. (1970) Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227, 680-685.LAMB, J.W., HAY, R.T. (1990) Ribozymes that cleave potato leafroll virus RNAwithin the coat protein and polymerase genes. Journal of General Virology71, 2257-2264.LAWSON, C., KANIEWSKI, W., HALEY, L., ROZMAN, R., NEWELL, C., SANDERS, P.,TUMER, N.E. (1990) Engineering resistance to mixed infection into acommercial potato cultivar: resistance to potato virus X and potato Y intransgenic Russet Burbank. Biotechnology 8, 127-134.LINDBO, J.A., DOUGHERTY, W.G. (1992) Pathogen-derived resistance to a potyvirus:immune and resistant phenotypes in transgenic tobacco expressing alteredforms of a potyvirus coat protein nucleotide sequence. Molecular Plant-Microbe Interactions 5, 144-153.Bibliography p. 100LOESCH-FRIES, S., MERLO, D., ZLNNER, T., BURHOP, L., HILL, K., KRAHN, K., JERis,N., NELSON, S., STALK, E. (1987) Expression of alfalfa mosaic virus RNA4 intransgenic plants confers virus resistance. EMBO Journal 6, 1845-1851.MACKENZIE, D.J., ELLIS, P.J. (1992) Resistance to tomato spotted wilt virus infectionin transgenic tobacco expressing the viral nucleocapsid gene. Molecular Plant-Microbe Interactions 5, 34-40.MAYO, M.A., ROBINSON, D.J., JOLLY, C.A., HYMAN, L. (1989) Nucleotide sequenceof potato leafroll luteovirus RNA. Journal of General Virology 70, 1037-1051.MASSALSKI, P.R. HARRISON, B.D. (1987) Properties of monoclonal antibodies topotato leafroll luteovirus and their use to distinguish virus isolates differing inaphid transmissibility. Journal of General Virology 68, 1813-1821.McBRIDE, K.E. SUMMERFELT, K.R. (1990) Improved binary vectors forAgrobacterium-mediated plant transformation. Plant Molecular Biology 14,269-276.MILLER, W.A., WATERHOUSE, P.M., GERLACH, W.L. (1988) Sequence andorganization of barley yellow dwarf virus genomic RNA. Nucleic AcidsResearch 16, 6097-6111.MOLONEY, M.M., WALKER, J.M., SHARMA, K.K. (1989) High efficiencytransformation of Brassica napus using Agrobacterium vector. Plant CellReports 8, 238-242.NEUHAUS, G., SPANGENBERG, G., MITTELSTEIN SCHERD, 0., SCHWEIGER, H.-G.(1987). Transgenic rape seed plants obtained by microinjection of DNA intomicrospore-derived embryoids. Theoretical and Applied Genetics 75, 30-36.NEWELL, C.A., RHOADS, M.L., BIDNEY, D.L. (1984) Cytogenetic analysis of plantsregenerated from tissue explants and mesophyll protoplasts of winter rape,Brassica napus L. Canadian Journal of Genetics and Cytology 26, 752-761.POWELL-ABEL, P., NELSON, R.S., HOFFMAN, N., ROGERS, S.G., FRALEY, R.T.,BEACHY, R.N. (1986) Delay of disease development in transgenic plants thatexpress the tobacco mosaic virus coat protein gene. Science 232, 738-743.Bibliography p. 101PROPER, D., TACKE, E., Soimrrz, J., Kuu, B., KAUFMANN, A., ROHDE, W. (1992)Ribosomal frameshifting in plants: a novel signal directs the -1 frameshift inthe synthesis of the putative viral replicase of potato leafroll luteovirus. EMBOJournal 11, 1111-1117.PUA, E.C., MEHRA-PALTA, A., NAGY, F., CHUA, N.H. (1987) Transgenic plants ofBrassica napus L. Biotechnology 5, 815-817.Radke, S.E., Andrews, M., Moloney, M.M., Crouch, M.L., Kridl, J.C., Knauf, V.C.(1988). Transformation of Brassica napus L. using Agrobacteriumtumefaciens:developmentally regulated expression of a reintroduced napin gene.Theoretical and Applied Genetics 75, 685-694.REED, K.C., MANN, D.A. (1985) Rapid transfer of DNA from agarose gels to nylonmembranes. Nucleic Acids Research 13, 7207.ROCHON, D.M., JOHNSTON, J.C., RIVIERE, C.J. (1991) Molecular analysis of thecucumber necrosis virus genome. Canadian Journal of Plant Pathology 13,142-154.ROCHOW, W.F., DUFF'US, J.E. (1981) Luteoviruses and yellows diseases. InHandbook of plant virus infections and comparative diagnosis. pp. 147-170.Edited by E. Kurstak. Amsterdam: Elsevier/North-Holland.ROGERS, S.G., HORSCH, R.B., FRALEY, R.T. (1986) Gene transfer in plants:production of transformed plants using Ti plasmid vectors. MethodsEnzymology 118, 627-640.ROGERS, S.O., BENDICH, A.J. (1988) Extraction of DNA from plant tissues. In PlantMolecular Biology Manual, A6: 1-10. Gelvin, S.B., Schilperoort, R.A.,Verma, D.S. (editors), Kluwer Academic Publishers, Boston © 1992.RUBIN, R.A. (1986) Genetic studies on the role of octopine T-DNA border regions incrown gall tumour formation. Molecular and General Genetics 202, 312-320.SAMBROOK, J., FRITSCH, E.F., MANIATIS, T. (1989) Molecular cloning: a laboratorymanual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NewYork.Bibliography p. 102SANGER, F., NICKLEN, S., COULSON, A.R. (1977) DNA sequencing with chain-terminating inhibitors. Proceedings of the National Academy of Sciences USA74, 5463-5467.Smrm, H.G., HINCKLES, J.A. (1985) Studies on beet western yellows in oilseed rape(Brassica napus ssp. oleifera) and sugarbeet (Beta vulgaris). Annals of AppliedBiology 107, 473-484.STRINGHAM, G.R. (1979) Regeneration in leaf-callus cultures of haploid rapeseed(Brassica napus L.). Journal of Plant Physiology 92, 459-462.TACKE, E., PROPER, D., SALAMINI, F., ROHDE, W. (1990) Characterization of a potatoleafroll luteovirus subgenomic RNA: differential expression by internaltranslation initiation and UAG suppression. Journal of General Virology 71,2265-2272.THOMAS, P.E., HANG, A.N., REED, G., GILLILAND, G.C. (1993) Role of winterrapeseed culture on epidemiologies of potato leafroll diseases. Plant Disease77, 420-423.TOPPER, R., MATzEiT, V., GRONENBORN, B., SCHELL, J., STEINBISS, H. (1987) A setof plant expression vectors for transcriptional and translational fusions. NucleicAcids Research 15, 5890.TUMER, N.E., O'CONNELL, K.M., NELSON, R.S., SANDERS, P.R., BEACHY, R.N. (1987)Expression of alfalfa mosaic virus coat protein gene confers cross-protection intransgenic tobacco and tomato plants. EMBO Journal 6, 1181-1188.VAN DER WILK, F., HUISMAN, M.J., CORNELISSEN, B.J.C., HUTTINGA, H., GOLDBACH,R. (1989) Nucleotide sequence and organization of potato leafroll virusgenomic RNA. FEBS Letters 245, 51-56.VAN DER WILK, F., WILLINK, D.P.-L., HUISMAN, M.J., HUTTINGA, H., GOLDBACH, R.(1991) Expression of the potato leafroll luteovirus coat protein gene intransgenic potato plants inhibits viral infection. Plant Molecular Biology 17,431-439.VAN DUN, C.M.P., BOL, J.F., VAN VLOTEN-DOTING, L. (1987) Expression of alfalfamosaic virus and tobacco rattle virus protein genes in transgenic tobacco plants.Virology 159, 299-305.Bibliography p. 103VAN DUN, C.M.P., VAN VLOTEN-DOTING, BOL, J.F. (1988) Expression of alfalfamosaic virus complementary DNA 1 and 2 in transgenic tobacco plants.Virology 163, 572-578.VAN LUSBETTENS, M., INZE, D., SCHELL, J., VAN MONTAGU, M. (1986) Transformedcell clones as a tool to study T-DNA integration mediated by Agrobacteriumtumefaciens. Journal of Molecular Biology 188, 129-145.VEIDT, I., LOT, H., LEISER, M., SACHEIDECKER, D., GUILLEY, H., RICHARDS, K.E.,JONARD, G. (1988) Nucleotide sequence of beet western yellows virus RNA.Nucleic Acids Research 16, 9917-9932.WATERHOUSE, P.M., GILDOW, F.E., JOHNSTONE, G.R. (1988) Luteovirus group. InCMIIAAB Descriptions of Plant Viruses, no. 339.Xu, Z.H., DAVEY, M.R., COCKING, E.C. (1982) Plant regeneration from rootprotoplasts of Brassica. Plant Science Letters 24, 117-121.

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0086246/manifest

Comment

Related Items