POTYVIRUS COAT PROTEIN GENES AND PLANTS TRANSFORMED THEREWITH

Abstract: The present invention relates to the coat protein genes of Papaya Ringspot Virus Strain papaya ringspot (PRV-p), Watermelon Mosaic Virus II (WMVII), and Zucchini Yellow Mosaic Virus (ZYMV); to expression vectors which contain a coat protein gene for PRV-p, WMVII or ZYMV, and, additionally, the necessary genetic regulatory sequences needed for expression of a gene transferred into a plant; to bacterial or plant cells which are transformed with an expression vector containing the PRV-p, WMVII or ZYMV coat protein genes; to transgenic plants which are produced from plant cells transformed with an expression vector containing the coat protein gene from PRV-p, WMVII or ZYMV; and to a process of producing transgenic plants which have increased resistance to viral infection.

Description: POTYVIRUS COAT PROTEIN GENES AND PLANTS TRANSFORMED THEREWITH FIELD OF INVENTION The present invention relates to the coat protein genes o potyviruses. More specifically the invention relates to a process fo preparing a coat protein gene from a potyvirus as well as it incorporation into a transfer vector, and its use in produci transformed plant cells and transformed plants which are resistant t viral infections by the particular potyvirus and related viruses fro which the gene is derived. BACKGROUND OF THE INVENTION Potyviruses are a distinct group of plant viruses which ar pathogenic to various crops. Potyviruses include watermelon mosai virus II (WMVII) ; papaya ringspot virus strains papaya ringspot a watermelon mosaic I (PRV-p and PRV-w) , two closely related members o the plant potyvirus group which were at one time classified a distinct virus types, but are presently classified as differen strains of the same virus; zucchini yellow mosaic virus (ZYMV); an many others. These viruses consist of flexous, filamentous particle of dimensions approximately 780 X 12 nanometers. The viral particle contain a single-stranded RNA genome containing about 10,00 nucleotides of positive (+, coding, or sense) polarity. Translatio of the RNA genome of potyviruses shows that the RNA encodes a singl large polyprotein of about 330 kD. This polyprotein contains severa proteins, one of which is a 49kD protease that is specific for th cleavage of the polyprotein into at least six (6) other peptides One of the proteins contained within this polyprotein is a 35 capsid or coat protein which coats and protects the viral RNA fro degradation. The genome organization of several viruses belonging to th potyvirus family group has been studied in detail, in particula tobacco etch virus, tobacco vein mottling virus and 'pepper mottl virus. In each case, the location of the coat protein gene has bee at the 3' -end of the RNA, just prior to a stretch of (200 to 30 bases) terminal adenine nucleotides residues. The location of the 4 kD protease gene appears to be conserved in these viruses. In th tobacco etch virus, the protease cleavage site has been determined t be the dipeptide Gln-Ser, Gln-Gly or Gin-Ala. Conservation of thes dipeptides as the cleavage sites in these viral polyproteins i apparent from the sequences of the above-listed potyviruses. Expression of the coat protein genes from tobacco mosaic virus, alf lfa mosaic virus, cucumber mosaic virus, and potato virus X in transgenic plants has resulted in plants which are resistant to in ection by the respective virus. In order to produce such transgenic plants, the coat protein gene must be inserted into the genome of the plant. Furthermore, the coat protein gene must contain all the genetic control sequences necessary for the expression of the gene after it has been incorporated into the plant genome. Since the coat protein of a potyvirus is produced by the post translational processing of a polyprotein, the coat protein gene isolated from viral RNA does not contain the genetic regulatory sequences needed for gene expression. The coat protein gene does not contain the transcription and translation signals necessary for its expression once transferred and integrated into a plant genome. It must, therefore, be engineered to contain a plant expressible promoter, a translation initiation codon (ATG) and a plant functional poly(A) addition signal (AATAAA) 3' of its translation terminatio codon. In the present invention, the nucleotide sequences of the coa protein genes for WMV-II, PRV-p and ZYMV have been determined, an the genes have been inserted into expression vectors to supply the with the necessary genetic regulatory sequences so that the genes ca be expressed when incorporated into a plant genome. Plant cells ar transformed with the vector construct and the plant cells are induce to regenerate. The resulting plants contain the coat protein gene and produce the coat protein. The production of the protein confer upon the plant an increased resistance to infection by the virus fro which the coat protein gene was derived. INFORMATION DISCLOSURE European patent application EP 0 223 452 describes plants tha are resistant to viral diseases and methods for producing them. Th process described comprises the steps of transforming a plant with DNA insert comprising a promoter, a DNA sequence derived from th virus, and a poly(A) addition sequence. PCT patent application PCT/US86/00514 refers generally to method of conferring resistance to a parasite to a host of th parasite. Allison et al. (1985) "Biochemical Analysis of the Capsi Protein Gene and Capsid Protein of Tobacco Etch Virus: N-Termina Amino Acids Are Located on the Virion's Surface", Virolog 147:309-316, describe the nucleotide sequence at the 3' end of th tobacco etch virus genome encoding the capsid protein. Homology t the sequence encoding the capsid protein of Pepper mottle virus i reported. Allison et al, (1986) "The Nucleotide Sequence of the Codin Region of Tobacco Etch Virus Genomic RNA: Evidence for the Synthesi of a Single Polyprotein", Virology 154:9-20 describe the genom organization of the tobacco etch virus. Carrington, J.C. and Dougherty, W.G. (1987) "Small nuclea inclusion protein encoded by a plant potyvirus genome is a protease" , J. Virology 61:2540-2548, disclose that the viral RNA of tobacco etc virus encodes the 49K protease responsible for cleavage of th polyprotein produced when the viral RNA is translated. Dodds et al. (1985) "Cross Protection between strains o cucumber mosaic virus: effect of host and type of inoculum o accumulation of virions and double-stranded RNA of the challeng strain", Virology 144:301-309, describe increased resistance t challenge by virus conferred to a plant by infection of a differen strain of virus. Dougherty, W.G. et al. (1985) "Nucleotide Sequence at the 3' Terminus .of Pepper Mottle Virus Genomic RNA: Evidence for an Alterna- tive Mode of Potyvirus Capsid Protein Gene Organization" , Virolog 146:282-291, report the nucleotide sequence of the 3' terminus of th viral RNA genome of pepper mottle virus. Dougherty, W.G. et al. (1988) "Biochemical and mutationa analysis of plant virus polyprotein cleavage site", EMBO J. 7:1281-1287, describe the conservation of the proteolytic cleavag site among geographically distinct isolates of tobacco etch virus. Dougherty, W. G. and Carrington, J. C. (1988) "Expression an function of potyviral gene products", Ann. Rev. Phytopathol. 26:123-143, describe potyviruses and some of the similarities th members of the group have with each another. Eggenberger, A. L. et al. (1989) "The nucleotide sequence of Soybean Mosaic Virus Coat Protein region and its expression i Escherichia coli, Agrobacterium tumefaciens, and tobacco callus", Virology, In press, disclose the nucleotide sequence of the coat protein gene for soy bean mosaic virus. Hinchee, M. A. W. et al (1988) "Production of transgenic soybean plants using Agrobacterium-mediated DNA transfer", Bio/tech. 6:915-921, disclose the production of transgenic soybean plants which were transformed with A. tumerfacien plasmids that conferred either Kanamycin resistance/^-glucuronidase activity or Kanamycin resistance/glyphosphate tolerance. Kozak, M. (1986) "Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribo- so es", Cell 44:283-292, discloses the optimal sequence around the ATG initiator codon of the preproinsulin gene for initiation by eukaryotic ribosomes. Loesch-Fries et al. (1987) "Expression of alfalfa mosaic virus RNA 4 in transgenic plants confers virus resistance" , EMBO 6:1845-1851, disclose that expression of the coat protein gene o alfalfa mosaic virus in transgenic plants confers resistance t infection by the virus. Pietrzak et al. (1986) "Expression in plants of two bacteria antibiotic resistant genes after protoplast transformation with a ne plant expression vector", Nucleic Acids Research 14:5857-5868, dis close expression in plants of foreign genes introduced Into the plan using an expression vector containing a movable expression cassett consisting of the Cauliflower mosaic virus 35S promoter an transcription terminator seperated by a polylinker containing severa unique restriction sites. Powell-Abel et al. (1986) "Delay of disease development i transgenic plants that express the tobacco mosaic virus coat protei gene", Science 232:738-743, disclose increased resistance towar infection by tobacco mosaic virus In transgenic plants containing th coat protein gene from tobacco mosaic virus. Quemada, H. D. et al. (1989) "The nucleotide sequences of cDN clones from RNA3 of Cucumber Mosaic Virus strains C and WL", J. Gen Virol. 70:1065-1073, reports the nucleotide sequences of cDNA clone from RNA3 of Cucumber Mosaic Virus strains G and WL and compares the to each other and other strains for homology. Shukla, D. D. et al. (1986) "Coat Proteins of Potyviruses" Virology 152:118-125, discloses the amino acid sequence of the potat virus Y coat protein. Shukla, D. D. et al. (1988) "The N and C termini of the Coat Proteins of Potyviruses Are Surface-located and the N Terminus Contains the Major Virus-specific Epitopes", J. Gen. Virol. 69:1497-1508, disclose that the N- and C-termini regions of some potyvirus coat proteins are located at the surface of the viral particles. The viral particles were treated with trypsin and it was observed that the enzyme treatment removed 30-67 amino acids from the N-terminal and 18-20 amino acids from the C-terminal; the variations were dependent on the virus. The remaining portion of the coat protein, the core, was highly conserved among the various viruses. Turner et al. (1987) "Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants", EMBO J. 6:1181-1188, disclose transgenic tobacco and tomato plants transformed with the coat protein gene of alfalfa mosaic virus display increased resistance to infection by alfalfa mosaic virus. Yeh and Gonsalves (1985) "Translation of Papaya Ringspot Virus RNA In vitro: Detection of a Possible Polyprotein That is Processed for Capsid Protein, Cylindrical-Indusion Protein, and Amor¬ phous-Inclusion Protein", Virology 143:260-271, describe the possibility that the RNA genome encodes a single proprotein which undergoes post-translational processing to form the potyvirus protein products. The following scientific publications are of interest but not relevant: An et al. (1985) "New cloning vehicles for transformation of higher plants", EMBO J. 4:277-285 describe the construction of an expression plasmid which may be stably replicated in both E. coli and A. tumerfaciens. An, G. (1986) "Development of plant promoter expression vectors and their use for analysis of differential activity of nopaline synthase promoter in transformed tobacco cells", Plant Physiol. 81:86-91, reports differences in promoter activities of transferred genes within the same cells as well as in independently derived cell lines. Bevan et al. (1983) "Structure and transcription of the nopaline synthase gene region of T-DNA", Nucleic Acids Research 11:3 * 69-385, disclose the DNA sequence and plant-tumor transcription pattern of a portion of DNA from A. tumerfaciens strain T37. Depicker et al. (1982) "Nopaline synthase: transcript mapping and DNA sequence", J. Mol. Appl. Genet. 1:561-573, disclose the DNA sequences 5' and 3' to the nos gene found in A. tumerfaciens. Hepburn, A. et al. (1985) "The use of pNJ5000 as an intermediate vector for genetic manipulation of Agrobacterium Ti-plasmids", J. General Microbio. 131:2961-2969, describe vectors which are used to transfer narrow host range vectors from E. coli to A. turnerf ciens. Klein et al. , (1987) "High-velocity microprojec iles for delivering mucleic acids into living cells", Nature 327:70-73, disclose that nucleic acids may be delivered into living cells using accelerated, small tungsten balls which pierce the cells without killing them. Klein et al. , (1988) "Factors influencing gene delivery into Ze mays cells by high-velocity micropro ectiles" , Bio/tech. 6:559-563, disclose that two days after bombarding plant cells with a plasmi coated microprojectile, expression of an gene encoding an enzym could be detected. Mazur, B. J. and Chui, C.-F. (1985) "Sequence of a genomic DN clone for the small subunit of ribulose bis-phosphat carboxylase-oxygenase from tobacco" , Nucleic Acids Researc 13:2373-2386, disclose the DNA sequence of the small subunit o ribulose bis-phosphate carboxylase-oxygenase from tobacco. McCabe, D. E. , et al. , (1988) "Stable transformation of soybea (Glycine max) by particle acceleration", Bio/tech. 6:923-926 disclose expression in soybean shoots of foreign genes introduce into immature soybean seeds using DNA coated microprojectiles. Olson, M. K. et al (1989) "Enhancement of heterologou polypeptide expression by alterations in the ribosome-binding-sit sequence", J. Biotech. 9:179-190, discloses the increase In gen expression of heterologous genes In E. coli due to the presence of a A -rich 5' untranslated region. Slightom et al. (1983) "Complete nucleotide sequence of a Frenc bean storage protein gene: Phaseolin", Proc. Natl. Acad. Sci. U.S.A 80:1897-1901, disclose the complete nucleotide sequences of the gen and the mRNA coding for a specific phaselin type French bean majo storage protein. Vilaine, F. and Casse-Delbart, F. (1987) "Independent inductio of transformed roots by the TL and TR regions of the Ri plasmid o agropine type Agrobacterium rhizogenes", Mol. Gen. Genet. 206:17-23 disclose the respective role of Tl- and TR-DNA in root induction b agropine type Agrobacterium rhizogenes Ri plasmids. None of these documents, either alone or taken together, teache or suggests the instant invention which relates to potyvirus coa protein genes and plants transformed therewith. SUMMARY OF THE INVENTION The present invention relates to the coat protein genes o Papaya Ringspot Virus Strain papaya ringspot (PRV-p) , Watermelo Mosaic Virus II (WMVII) , and Zucchini Yellow Mosaic Virus (ZYMV) . The present invention relates to a recombinant DNA molecul which encodes a potyvirus coat protein. The present inventio relates to a recombinant DNA molecule comprising a potyvirus coa protein gene operably linked to genetic regulatory sequence necessary for gene expression. The present invention relates to expression vectors whic contain a coat protein gene for potyviruses, and, additionally, th necessary genetic regulatory sequences needed for expression of gene transferred into a plant. The present invention also relates t bacterial or plant cells which are transformed with an expressio vector containing the coat protein genes. Furthermore, the presen invention relates to transgenic plants which are.produced from plan cells transformed with an expression vector containing the coa protein gene from potyviruses. In addition, the present inventio relates to a process of producing transgenic plants which hav increased resistance to viral infection. DETAILED DESCRIPTION OF THE INVENTION Charts 1, 2 and 3 contain DNA nucleotide sequences of the coa protein genes of PRV-p, WMVII and ZYMV, respectively. Charts 4 and compare the nucleotide sequences of various coat protein genes Charts 6-14 are set forth to illustrate the constructions of thi invention. Certain conventions are used to illustrate plasmids an DNA fragments as follows: (1) The single line figures represent both circular and linea double-stranded DNA. (2) Asterisks (*) indicate that the molecule represented i circular. Lack of an asterisk indicates the molecule i linear, (3) Junctions between natural boundaries of functional com ponents are indicated by vertical lines along th horizontal lines. (4) Genes or functional components are indicated below th horizontal lines. (5) Restriction sites are indicated above the horizontal lines (6) Distances between genes and restriction sites are not t scale. The figures show the relative positions only unles indicated otherwise. (7) The following abbreviations are used to denote function an components: a) P ca ■**■* ■ CaMV35S promoter; b) I c — CMV intergenie region, the intergenie regio comprising the Initiation codon and AT rich 5 untranslated region; c) S ca *•*■* - * , CaMV35S poly(A) addition signal; and d) Nos = Nos nptll gene. Most of the recombinant DNA methods employed in practicing th present invention are standard procedures, well known to thos skilled in the art, and described in detail in, for example, Europea Patent Application Publication Number 223452, published November 29 1986, which is incorporated herein by reference. Enzymes ar obtained from commercial sources and are used according to th vendor's recommendations or other variations known in the art General references containing such standard techniques include th following: R. Wu, ed. (1979) Methods in Enzymology, Vol. 68; J. Miller (1972) Experiments in Molecular Genetics; T. Maniatis et al (1982) Molecular Cloning: A Laboratory Manual; D. M. Glover, e (1985) DNA Cloning Vol. II; H.G. Polites ; and K.R. Marotti (1987) " step-wise protocol for cDNA synthesis".; Biotechniques 4:514-520 S.B. Gelvin and R.A. Schilperoort, eds. Introduction, Expressio and Analysis of Gene Products in Plants, all of which a incorporated by reference. For the purposes of the present disclosure the followi definitions apply. "Promoter" means a promoter which is functional in the ho plant. "Initiation region" includes the initiation codon an nucleotides flanking the initiation codon. "Operably linked" refers to the linking of nucleotide region encoding specific genetic information such that the nucleotid regions are contiguous, the functionality of the region is preserve and will perform its function relative the the other regions as par of a functional unit. "AT rich 5' untranslated region" is a nucleotide sequenc composed of at least 60% adenine or thymine nucleotides. "Untranslated flanking region" refers to nucleotide sequence which are 3' of the termination codon and end at the poly(A) additio signal. These sequences enhance production of the peptide encoded b the upstream gene. "Vector" is a vehicle by means of which DNA fragments can b introduced into host organisms. "Expression vector" is a vehicle by means of which DNA fragment that contain sufficient genetic information and can, therefore, b expressed by the host, can be introduced into host organisms. "Antipathogen gene" is a gene which encodes a DNA sequence whic is either the antisense sequence of a pathogenic gene or th antipathogenic gene encodes a peptide whose presence in an organis confers an increased resistence to a pathogen. To .practice the present invention, the coat protein gene of virus must be isolated from the viral genome and inserted into vector containing the genetic regulatory sequences necessary t express the inserted gene. Accordingly, a vector must be constructe to provide the regulatory sequences such that they will be functiona upon inserting a desired gene. When the expression vector/inser construct is assembled, it is used to transform plant cells which ar then used to regenerate plants. These transgenic plants carry th viral gene in the expression vector/insert construct. The gene i expressed in the plant and increased resistance to viral infection i conferred thereby. Several different courses exist to isolate the coat protei gene. To do so, one having ordinary skill in the art can us information about the genome organization of potyviruses to locat and isolate the coat protein gene. The coat protein gene is locate at the 3' end of the RNA, just prior to a stretch of about 200-30 adenine nucleotide residues. Additionally, the Information relate to proteolytic cleavage sites is used to determine the N- erminus o the potyvirus coat protein gene. The protease recognition sites ar conserved in the potyviruses and have been determined to be eithe the dipeptide Gln-Ser, Gln-Gly or Gin-Ala. The nucleotide sequence which encode these dipeptides can be determined. Using methods well known in the art, a quantity of virus i grown up and harvested. The viral RNA is then seperated and the coa protein gene can be isolated using a number of known procedures. cDNA library is created using the viral RNA. The methods followed t do this are well known in the art. The viral RNA is treated wit reverse transcriptase and a complementary DNA molecule is produced A DNA complement of the complementary DNA molecule is produced an that sequence represents a DNA copy of the original viral RN molecule. Thus, a double stranded DNA molecule is generated whic contains the sequence information of the viral RNA. These DN molecules can be cloned in E. coli plasmid vectors after th additions of restriction enzyme linker molecules by DNA ligase. Th various fragments are inserted into cloning vectors which are the used to transform E. coli and create a cDNA library. Since the coat protein gene is located just 5' to the poly region, oligonucleotides that can hybridize to the polyA region ca be used as hybridization probes to screen the cDNA library an determine if any of the transformed bacteria contain DNA fragment with sequences coding for the polyA region. The cDNA inserts in a bacterial colonies which contain this region can be sequenced. T coat protein gene Is present in its entirety in colonies which ha sequences that extend 5' to the sequence which encodes the proteo lytic cleavage site described above. Alternatively, cDNA fragments may be inserted into expressio vectors. Antibodies against the coat protein may be used to scree the cDNA expression library and the gene can be isolated fro colonies which express the protein. Using the sequences disclosed in Charts 1, 2 and 3, the co protein genes for the respective viruses may be synthesiz chemically by methods well known in the art. Alternatively, t information in Charts 1, 2 and 3 may be" used to synthesi oligonucleotides which can be used as probes to .screen a cD library. The nucleotide sequences of the coat protein genes for WMV-I PRV-p and ZYMV have been determined and the genes have been insert into expression vectors. The expression vectors contain t necessary genetic regulatory sequences for expression of an insert gene. The coat protein gene is inserted such that those regulato sequences are functional so that the genes can be expressed wh incorporated into a plant genome. In order to express the viral gene, the necessary genet regulatory sequences must be provided. Since the coat protein. of potyvirus is produced by the post translational processing of polyprotein, the coat protein gene isolated from viral RNA does n contain the genetic regulatory sequences needed for gene expressio The coat protein gene does not contain the transcription a translation signals necessary for its expression once transferred a integrated into a plant genome. It must, therefore, be engineered contain a plant expressible promoter, a translation initiation cod (ATG) and a plant functional poly(A) addition signal (AATAAA) 3' its translation termination codon. In the present invention, t coat protein is inserted into a vector which contains a cloning si for insertion 3' of the initiation codon and 5' of the poly( signal. The promoter is 5' of the initiation codon such that when structural gene is inserted at the cloning site, a functional unit formed in which the inserted gene is expressed under the control the various genetic regulatory sequences. In the preferred embodiment of the present invention, addition genetic regulatory sequences are provided. As described above, expression vector must contain a promoter, an intiation codon and poly(A) addition signal. In order to get a higher level expression, untranslated regions 5' and 3' to the inserted genes a provided. Furthermore, certain sequences flanking the initiati codon optimize expression. The promoter used is one that is chos for high level expression. A 5' untranslated region which results in higher level expre sion of an inserted gene is provided downstream from the promoter a upstream from the initiation codon. This region contains at lea 60% of the sequence a Adenine and Thymine. There is a statistic bias for expression when such an AT rich region is located betwee the promoter and intiation codon. This preference is utilized in th preferred embodiment of the present invention by inclusion of an A rich 5' -untranslated region intermediate of the promoter and initi ation codon. The preferred embodiment of the present invention also contain specific nucleotide sequence flanking the initiation codon. Thi preferred sequence, termed Kozak's element, is AAXXATGG wherein represents any of the four nucleotides. The presence of th initiation codon following Kozak's rule results in higher leve expression when used in an expression vector. In the preferre embodiment of the present invention, the small subunit from the S RUBISCO contains an initiation codon in which Kozak's element i used. Furthermore, the preferred embodiment of the present inventio contains a 3' untranslated region downstream from the cloning sit where the coat protein gene is inserted and upstream from the poly(A addition signal. The sequence of this 3' untranslated region result in a statistical bias for protein production. The sequence promote high level expression. The poly(A) addition signal is found directl downstream from the 3' untranslated region and can be derived fro the same source. In the preferred embodiment of the prese invention, the 3' untranslated region and poly(A) addition signal a derived from CaMV 35S gene or the phaseolin seed storage prote gene. The poly(A) addition signal from CaMV, nopaline synthas octopine synthase, bean storage protein, and SS RUBISCO genes a also suitable for this construction. Several promoters whi function in plants are available, but the best promoters are t constitutive promoter from cauliflower mosaic virus (CaMV, a pla DNA virus) and the small subunit of ribulose bis-phospha carboxylase-oxygenase (SS RUBISCO) gene. Using methods well known to those skilled in the art, pla cells are transformed with the vector construct and the plant cel are induced to regenerate. The resulting plants contain the co protein genes and produce the coat protein. The production of t protein confers upon the plant an increased resistance to infecti by the virus from which the coat protein gene was derived. DESCRIPTION OF THE PREFERRED EMBODIMENTS Example 1 Isolation of WMVII RNA Watermelon mosaic virus II (WMV II) was propagated in zucchin squash (Cucurbita pepo L) plants and RNA was isolated by the metho described by Yeh and Gonsalves (Virology 143:260, 1985). Example 2 Isolation of PRV-p RNA Papaya ringspot virus strain prv (PRV-p) was propagated in jell melon, Cucumis metuliferus (Nand.) Mey. Ace. 2549 plants and RNA wa isolated by the method described by Yeh and Gonsalves (Virolog 143:260, 1985). Example 3 Isolation of ZYMV RNA Zucchini yellow mosaic virus (ZYMV) was propagated in zucchin squash (Cucurbita pepo L) plants and RNA was isolated by the metho described by Yeh and Gonsalves (Virology 143:260, 1985). Example 4 Synthesis of double-stranded cDNA The procedure used to make double stranded cDNA from isolate viral RNA is the same for all viral RNA isolated above. The purifie RNA was subjected to the cDNA synthesis protocol described by Polite and Marotti (Biotechniques 4:514, 1986) and because this RNA contain an A-rich region at its 3'-end (similar to that found for man eukaryotic mRNAs) the procedure was straight-forward. The synthesi of double stranded cDNA was also done as described by Polites an Marotti. After double-stranded cDNA was synthesized, it was purifie by passage through a G-100 Sephadex column, precipitated wit ethanol, and suspended in 20 μl of 10X EcoRI methylase buffer (100 m NaCl, 100 mM Tris-HCl, pH 8.0, 1 mM EDTA, 80 μM S-adenosy methionine, and 100 μg/ml bovine serum albumin) . An additiona amount of S-adenosyl methionine (1 μl of a 32 mM solution) was adde to the reaction mixture, followed by the addition of 1 μl (20 units) EcoRI methylase. The reaction was incubated at 37°C for 30 minute and stopped by incubation at 70°C for 10 minutes. Then 1 μl ( units) of E. coli DNA polymerase I Klenow fragment was added an incubated at 37°C for 10 minutes, followed by phenol/chlorofor extraction and ethanol precipitation. The pellet was washed in 70 ethanol, then 70% ethanol/0.3 M sodium acetate. The pellet was drie and resuspended in 8 μl of 0.5 μg/μl phosphorylated EcoRI linkers (Collaborative Research, Inc., 128 Spring St., Lexington, MA 02173). One μl of 10X ligase buffer (800 mM Tris-HCl ph 8.0, 200 mM MgCl 2 . 150 mM DTT, 10 mM ATP) and 1 μl of T4 DNA ligase (4 units) were added, and the reaction was incubated overnight at 15°C. The ligation reaction was stopped by incubation at 65°C for 10 minutes. Sixty μl of H 2 0, 10 μl of 10X EcoRI salts (900 mM Tris-HCl pH 8.0, 100 mM MgCl2, 100 mM NaCl), and 10 μl of EcoRI (10 units/μl) were added, and the reaction was incubated at 37°C for 1 hour. The reaction was stopped by phenol/chloroform and chloroform extractions. The reaction mixture was then size fractionated by passage through a Sephadex G-100 column and the fractions containing the largest double stranded cDNA molecules were concentrated by butanol extractions, precipitated with ethanol, and resuspended in 10 μl of H2O. Five μl of the double stranded cDNAs was added to 0.5 μg of pUC19 DNA (which had been previously treated with phosphatase to remove the 5' phosphates), 1 μl of 10X ligase buffer, and 1 μl of T4 ligase, and the reaction was incubated at 15°C for 16 hours. The resulting ligated pUC19-coat protein gene double stranded cDNA molecules were transformed into E.coli host cells as described by the manufacturer (Bethesda Research Laboratories, Inc., Gaithersburg, MD 20877) and plated on medium containing 50 μg/ml ampicillin, 0.04 mM IPTG, an 0.004% X-Gal. Bacterial colonies showing no blue color were selecte for further analysis. Clones containing the 3' -region and possibl the coat protein gene were identified by hybridization against a 3 p- labeled oligo-dT. Bacterial colonies showing hybridization to this probe should contain at least the poly(A) region of the particula potyvirus genome. Several of the hybridizing bacterial clones wer selected and plasmid DNAs were isolated according to methods known t those skilled in the art. Example 5 Identification of the PRV-p Coat Protein Gene Several of the cloned cDNAs of PVP-p RNA were sequenced by th chemical DNA sequencing method described by Maxam and Gilber (Methods of Enzymology 65:499, 1980). Based on this Information an comparative analysis with other potyviruses clone number pPRV-117 wa found to contain a complete copy of the PRV-p coat protein gene. Th N-terminus of the coat protein was identified by the location of th dipeptide sequence Gln-Ser. The length of the PRV-p coat protei gene coding region is consistent with a gene encoding a protein o about 33 kDal. The sequence of the PRV-p coat protein gene an protein are shown in Chart 1. In addition, comparison of thi sequence with that of the related virus PRV-w described by Nagel an Heibert (Virology 143:435, 1985) shows that the two coat protei genes share 98% nucleotide and amino acid similarities (Chart 4) Because these two viruses share extensive identities in their coa proteins, expression of the coat protein gene from PRV-p is expecte to yield plants resistant to both PRV-p and PRV-w. Example 6 • Construction of a Plant-expressible PRV-p Coat Protei Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal an CMV 5' Untranslated Region and Translation Initiator ATG. Attachment of the necessary plant regulatory signals to the PRV p coat protein gene was accomplished by constructing a translationa fusion with a clone originally designed for the expression of the CM coat protein gene, using clone pUC1813/CPl9. Plasmid pUC1813/CP19 i a vector suitable for agrobacterium mediated gene transfer. A EcoRI-EcoRI fragment was removed from pDH51/CP19 and placed into th EcoRI site of the plasmid, pUC1813 (available from Robert K. Department of Chemistry, Washington State University, Pullman Washington) , creating plasmid pUC1813/CP19. Plasmid pUC1813/CP19 wa described in U.S. patent application Serial Number 07/135,591 file on December 21, 1987 incorporated herein by reference. This trans lational fusion clone was constructed by first identifying tw restriction enzyme sites within clone pUC1813/CP19. One site (Tthll I) is located between amino acids 13 to 17 while the other site (Bst I) is located near the end of the 3' -untranslated region of the CM coat protein gene. Addition of these specific restriction enzyme sites to the PRV- coat protein gene was accomplished by the polymerase chain reactio technique, using an instrument and Taq polymerase purchased fro Perkin Elmer-Cetus, Emeryville, Ca. Specifically, two respective 5' and 3' oligomers (CGACGTCGTCAGTCCAAGAATGAAGCTGTG, containing a Tthll I site and (CCCACGAAAGTGGGGTGAAACAGGGTCGAGTCAG, containing a BstX site) , sharing at least 20 nucleotides with the PRV-p coat protei gene were used to prime synthesis and gene amplification of the coa protein gene. After synthesis, the amplified fragments were digeste with Tthlll I and BstX I to expose the sites. As shown in Chart 6, pUCl813/CP19 is the expression vector whic contains the CMV coat protein gene. Plasmid pUC1813/CPl9 contain Tthlll I and BstX I sites. The digested, amplified fragments are ligated into the respec¬ tive exposed sites of pUC1813/CP19 and the expected new construction was identified using methods known to those skilled in the art. Polymerase chain reaction techniques were used to amplify PRV-P coat protein gene containing the Tthllll and BstXI sites. The plasmid pUC1813/CP19 and PRV-P coat protein gene fragments were digested with Tthllll and BstXI and ligated to each other. The resulting clone, designated pUC1813/CP19-PRVexp, was subjected to nucleotide sequencing to ensure that the cloning and gene amplification did not introduce any detrimental artifacts. The sequence showed no arti- facts, suggesting that this plant expression cassette should be capable of expressing a PRV-p coat protein gene which contains an additional 16 amino acids of CMV coat protein at its N-terminus. Example 7 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible PRV-p Coat Protein Gene Construction. As depicted in Chart 7, the plant expression cassette for the PRV-p coat protein gene was transferred into a suitable micro T-DNA vector which contains the necessary Agrobacterium T-DNA transfer signals for transfer from an Agrobacterium and integration into a plant genome, and a wide host-range origin of replication (for replication in Agrobacterium). Plasmid pUCl813/CP19-PRVexp was digested with Hind III and the resulting 2.2 kb insert fragmen containing the plant-expressible cassette was removed and ligate Into the Hind III site of the modified Agrobacterium-derived micro- vector pGA482 (modification included the addition of the β- glucuronidase gene). The micro T-DNA vector, pGA482, is availabl from G. An, Institute of Biological Chemistry, Washington Stat University, Pullman, WA. The resulting plasmid was designated, pGA482/G/CP19-PRVexp and is shown in Chart 7. This plasmid (o derivatives thereof) was transferred Into virulent or avirulen strains of Agrobacterium tu efaciens or rhizogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRIB278b) , and others. Strain A208 C58, LBA4404, and A4RS are available from American Type Cultur Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD. Bacteri A4RS(pRiB278b)is available from Dr. F. Casse-Delbart, C.N.R.A ' ., Routede Saint Cyr. F78000, Versailles, France. Strain C58Z707 i available from Dr. A.G.Hepburn, Dept. of Agronomy, University o Illinois, Urbana, IL. After transfer of the engineered plasmid pGA482/G/CP19-PRVex into any of the above listed Agrobacterium strains, these Agro bacterium strains can be used to transfer and integrate within plant genome the plant-expressible PRV-p coat protein gene containe within its T-DNA region. This transfer can be accomplished using th standard methods for T-DNA transfers which are known to those skille in the art, or this transfer can be accomplished using the method described in a U.S. patent application Serial Number 07/135,655 filed on December 21, 1987, entitled "Agrobacterium Mediate Transformation of Germinating Plant Seeds" and incorporated herein b reference. Example 8 Construction of a Plant-expression Cassette fo Expression of Various Genes in Transgenic Plants. In the preferred embodiment of the present invention, th following expression cassette was constructed to provide th necessary plant regulatory signals (which include the addition of promoter, 5' untranslated region, translation initiation codon, and polyadenylation signal) to the gene inserts in order to achiev high level expression of the inserts. The expression cassette may b used to express any genes inserted therein. Accordingly, th applicability- of the expression cassette goes beyond its use i expressing coat protein genes. Rather, the expression cassette ma be used to express any desired protein in transgenic plants. Th expression cassette is the preferred expression system for expressin viral coat protein genes in plants. The expression cassette of the preferred embodiment contains: constitutive promoter; a 5' untranslated region which enhances gen expression; an initiation codon which comprise Kozak's element; cloning site where the gene to be expressed may be inserted t produce a functional expression unit; and a 3' untranslated regio which comprises a poly(A) addition signal and untranslated flankin regions which result in a higher level of expression. More specifically, the expression cassette which is th preferred embodiment of the present invention consists of th cauliflower mosaic virus (CaMV) 35S transcript promoter, the 5' untranslated region of cucumber mosaic virus (CMV) , the CM translation initiation codon, and the CaMV polyadenylation signal The construction of this expression cassette utilized the Polymeras Chain Reaction (PCR) technique to obtain correct position of the plant regulatory signals and the addition of convenient restriction enzyme sites which allow for the easy addition of a coat protein gene and the excision of the completed cassette so it can be transferred 5 to other plasmids. To accomplish the construction of this expression cassette the following oligomers were synthesized: 1. 5'-GAAGCTTCCGGAAACCTCCTCGGATTCC-3', contains a Hindlll site at its 5' -end and contains 21 bases which are identical to 21 bases ' 10 in the 5'-flanking region of CaMV. 2. 5'-GCCATGGTTGACTCGACTCAATTCTACGAC-3', contains a Ncol site at its 5' -end which contains a translation Initiation codon which conforms to Kozak's rules and has 21 bases which are identical to 21 bases in the antisense strand of the CMV 5' -untranslated region. 15 3. 5'-GCCATGGTTGCGCTGAAATCACCAGTCTC-3' , contains a Ncol site at its 5'-end (which contains the same translation initiation codon as oligomer 2) and has 20 bases which are identical to 20 bases in the 3'-untranslated region of CaM . 4. 5'-GAAGCTTGGTACCACTGGATTTTGGTT-3' , contains a Hindlll site 20 at its 3' -end and has a 20 base match with the flanking DNA region 3' of the CaMV polyadenylation site (on the antisense strand) . These oligomers were used to amplify sequences contained within the CMV expression clone referred to as pUC1813/CP19, shown in Chart 6, and referred to above. As depicted in Chart 8, the PCR technique 25 was used to amplify the gene regulatory regions in pUC1813/CP19. Amplification of the 5' -flanking, CMV 5' -untranslated region, and CMV initiation codon (which was modified to conform to Kozak's rule AAXXATGG) resulted in a fragment of about 400 base pairs in lengt and amplification of the CaMV 3-untranslated and flanking regions 30 resulted in a fragment of about 200 base pairs in length. These fragments were digested with Ncol and Hindlll, isolated from polyacrylamide gel, and then ligated into Hindlll digested an phosphatase treated pUC18. The resulting clone is referred to a pl8CaMV/CMV-exp and is shown in Chart 8. 35 Example 9 Identification of the WMVII Coat Protein Gene The cloned WMVII cDNA insert from clone pWMVII-41-3.2 which wa produced as described above, was sequenced by using both the chemica (Maxam and Gilbert, Methods of Enzymology 65:499, 1980) and enzymati (Sanger et al. , Proc. Natl. Acad. Sci. U.S.A. 74:5463, 1977) method Based on this information and comparative analysis with oth potyviruses, the nucleotide sequence of clone pWMVII-41-3.2 was fou to contain a complete copy of the WMVII coat protein gene. The terminus of the coat protein was suggested by the location of t dipeptide sequence Gln-Ser. The length of the WMVII coat prote gene coding region (281 amino acids) is consistent with a ge encoding a protein of about 33 kD. The sequences of this WMVII co protein gene and protein are shown in Chart 2. In additio comparison of this sequence with that obtained from the related vir Soybean Mosaic Virus (SMV) strain N described by Eggenberger et a shows that they share overall about 88% identity and excluding the terminal length differences they share about 92.5% identity, s Chart 5. Because these two virus coat proteins share extensive ami acid identities, expression of the coat protein gene from WMVII expected to yield plants resistant to WMVII infection and could yiel plants resistant to SMV infection. Example 10 Construction of a Plant-expressible WMVII Coat Prote Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal a CMV Intergenic Region and Translation Initiator ATG. As depicted in Chart 9, attachment of the necessary pla regulatory signals to the WMVII coat protein gene was accomplished using the PCR technique to amplify the WMVII coat protein gene usi oligomers which would add the necessary s.ites to its 5' and sequences. Following this amplification the resulting fragment digested with the appropriate restriction enzyme and cloned into t Ncol site of the above described expression cassette containi plasmid, pl8CaMV/CMV-exp. Clones containing the WMVII coat protei gene insert need only be checked to determine correct orientatio with respect with the CaMV promoter. However, to ensure that artifacts have been incorporated during the PCR amplification t entire coat protein gene region is checked by nucleotide sequenc analysis. To obtain the amplified WMVII coat protein gene with Nco restriction enzyme sites on both ends the following two oligomer were synthesized: 1. 5'-ACCATGGTGTCTTTACAATCAGGAAAAG-3' , which adds a Ncol sit to the 5' -end of the WMVII coat protein gene and retains the same AT translatlon start codon which is present in the expression cassette, pl8CaMV/CMV-exp. 2. 5'-ACCATGGCGACCCGAAATGCTAACTGTG-3' , which adds a Ncol site to the 3' -end of the WMVII coat protein gene, this site can be ligated into the expression cassette, pl8CaMV/CMV-exp. The cloning of this PCR WMVII coat protein gene, using these two oligomers, into pl8CaMV/CMV-exp yields a plant expressible WMVII gene (referred to as pl8WMVII-exp) which, following transcription and translation, will generate a WMVII coat protein which is identical to that derived from the WMVII coat protein gene nucleotide sequence, see Chart 2. However, this coat protein will differ, because of necessary genetic engineering to add the ATG initiation codon and by including the last four amino acids of the 54 kD nuclear inclusion protein (which is adjacent to the Glu-Ser protease cleavage site); the amino acids added are Val-Ser-Leu-Glu-N-ter WMVII. The addition of these four amino acid residues should not affect the ability of this coat protein to yield plants which are resistant to WMVII infections, because the N-terminal region of potyvirus coat proteins appear not to be well conserved for either length or amino aci identity. However, if this is found to be a problem its replacement would involve the use of a different oligomer to obtain N-terminal variations of the WMVII coat protein gene. The cloned constructio of the plant expressible WMVII coat protein gene is referred to as p!8WMVII-exp, and is shown in Chart 9. Example 11 Construction of a Micro T-DNA Plasmid Containing the Plant-expressible WMVII Coat Protein Gene Construction. As depicted in Chart 10, the plant expression cassette for th WMVII coat protein gene was transferred into a suitable micro-T-DN vector which contains the necessary Agrobacterium T-DNA transfe signals (to mediated transfer from an Agrobacterium and integratio Into a plant genome) and wide-host range origin of replication (fo replication in Agrobacterium) to form plasmid pGA482/G/CPWMVII-exp. To construct this plasmid, plasmid pl8WMVII-exp was digested wit Hind III (which cuts within the polycloning sites of pUC18, wel outside of the expression cassette), and an 1.8 kb fragmen containing the plant-expressible cassette was removed and ligate into the Hind III site of the modified Agrobacterium-derived micro vector pGA482 (modification included the addition of the β glucuronidase gene). The micro T-DNA vector, pGA482, is shown in Chart 7 and available from G. An, Institute of Biological Chemistry, Washington State University, Pullman, WA. The resulting plasmid was designated, pGA482/G/CPWMVII-exp is shown in Chart 10. This plasmid (or derivatives thereof) was transferred into virulent or avirulent strains of Agrobacterium tumef ciens or rhizogenes, such as A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRiB278b) , and others. Strains A208 C58, LBA4404, and A4RS are available from American Type Culture Collection (ATCC) , 12301 Parklawn Drive, Rockville, MD. Bacteria A4RS(pRiB278b)is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr. F78000, Versailles, France. Bacteria C58Z707 is available from Dr. A.G.Hepburn, Dept. of Agronomy, University of Illinois, Urbana, IL. After transfer of the engineered plasmid pGA482/G/CPWMVII-exp into any of the above listed Agrobacterium strains, these Agrobacterium strains can be used to transfer and integrate within a plant genome the plant-expressible WMVII coat protein gene contained within its T-DNA region. This transfer can be accomplished using the standard methods for T-DNA transfers which are known to those skilled in the art, or this transfer can be accomplished using the methods described in U.S. Patent application SN 07/135,655 filed December 21, 1987 entitled "Agrobacterium Mediated Transformation of Germinating Plant Seeds". In addition, it has recently been shown that such Agrobacteria are capable of transferring and integrating their T-DNA regions into the genome of soybean plants. Thus these strains could be used to transfer the plant expressible WMVII coat protein gene into the genome of soybean to develop a soybean plant line which is resistant to infection from soybean mosaic virus strains. Example 12 Microprojectile Transfer of pWMVII-exp into Plant Tissues. Recently an alternative approach for the transfer and integration of DNA into a plant genome has been developed. This technique relies on the use of microprojectiles on which the DNA (plasmid form) is attached. These microprojectiles are accelerated to high velocities and their momentum is used to penetrate plant cell walls and membranes. After penetration into a plant cell the attached DNA leaches off the microprojectile and is transferred to the nucleus where DNA repair enzymes integrate the "free" DNA into the plant genome. In Its present form the process is entirely random, but plant tissues which have been successfully transformed b the plasmid DNA (or part of it) can be identified and cultured to homogeneity by the use of selectable marker genes (such as the bacterial neomycin phospho ransferase II gene, NPTII) , or reporter genes (such as the bacterial beta-glucuronidase gene, Gus). Successful use of particle acceleration to transform plants has recently been shown for soybean and the transfer of pl8WMVII-exp int the genome could result in obtaining soybean plants which ar resistant to infections from soybean mosaic virus strains. The use of this process for the transfer of pl8WMVII-exp can b accomplished after the addition of either plant expressible gene NPTII or Gus genes or both. Plasmids that have the nptll and Gu genes to pl8WMVII-exp are shown in Chart 11, and referred to a pl8GWMVII-exp and pl8NGWMVII-exp. In addition, the constructio described in Example 11 can also be used for microprojectile transfe as it already has both the nptll and Gus genes attached to th pWMVII-exp cassette (see Chart 10) . The only difficulty which th use of pGA482GG/cpWMVII-exp may encounter during transfer by th microprojectile process is due to its large size, about 18kb, whic may have a lower efficiency transfer and such larger plasmi generally yield less DNA during propagation. To construct plasmid pl8GWMVii-exp, plasmid pl8WMVii-exp i digested with BamHI and ligated with a 3.0 kilobase Ba HI isolate fragment containing the Gus gene. To construct plasmid pl8NGWMVii exp, the plasmid pl8GWMVii-exp is digested with Smal and ligated wit a 2.4 kb isolated fragment containing the Nos-nptll gene generated b digestion with Dral and Stul. Example 13 Identification of the ZYMV Coat Protein Gene. The cloned ZYMV cDNA insert from clone pZYMV-15, which wa cloned using the method described above, was sequenced by using bot the chemical (Maxam and Gilbert, Methods of Enzymology 65:499, 1980 and enzymatic (Sanger et al., Proc. Natl. Acad. Sci. U.S.A. 74:5463 1977) methods. Based on this information and comparative analysi with other potyviruses the nucleotide sequence of clone pZYMV-15 wa found to contain a complete copy of the ZYMV coat protein gene. Th N-terminus of the coat protein was suggested by the location of th dipeptide sequence Gln-Ser which Is characteristic of cleavage site in potyviruses (see Dougherty et al. EMBO J. 7:1281, 1988). The length of the ZYMV coat protein gene coding region (280 amino acids) is consistent with a gene encoding a protein of about 31.3 kD. The sequences of this ZYMV coat protein gene and protein are shown in Chart 3. Example 14 Construction of a Plant-expressible ZYMV Coat Protein Gene Cassette with CaMV 35S Promoter and Polyadenylation Signal and CMV Intergenic Region and Translation Initiator ATG. As depicted in Chart 12, attachment of the necessary plant regulatory signals to the ZYMV coat protein gene was accomplished by using the PCR technique to amplify the ZYMV coat protein gene using oligomers which would add the necessary sites to its 5' and 3' sequences. Following this amplification the resulting fragment is digested with the appropriate restriction enzyme and cloned into the Ncol site of the above expression cassette containing plasmid, pUC18CP-exp. Clones containing the ZYMV coat protein gene insert need only be checked to determine correct orientation with respect with the CaMV promoter. However, to ensure that no artifacts have been incorporated during • the PCR amplification the entire coat protein gene region is checked by nucleotide sequence analysis. To obtain the amplified ZYMV coat protein gene with Ncol restriction enzyme sites on both ends the following two oligomers were synthesized: 1. 5'-ATCATTCCATGGGCACTCAACCAACTGTGGC-3' , which adds a Ncol site to the 5' -end of the ZYMV coat protein gene and retains the same ATG translation start codon which is present in the expression cassette, pUClδcpexp. 2. 5'-AGCTAACCATGGCTAAAGATATCAAATAAAGCTG-3' , which adds a Ncol site to the 3' -end of the ZYMV coat protein gene, this site can be ligated into the expression cassette, pUC18cpexp. The cloning of this PCR ZYMV coat protein gene, using these two oligomers, into pUClδcpexp yields a plant expressible ZYMV gene (referred to as pUClδcpZYMV) which following transcription and translation will generate a ZYMV coat protein which is identical to that derived from the ZYMV coat protein gene nucleotide sequence, see Chart 3. However, this coat protein will differ, because of neces¬ sary genetic engineering to add the ATG initiation codon followed by Gly, which is the amino acid 3' adjacent to the Ser of the polyprot- ein cleavage site (see Chart 3) . The Gly amino acid residue wa selected for the potential N-terminal amino acid because man potyvirus coat proteins have either an Ser, Gly, or Ala at their N terminal. However, if this is found to be a problem its replacemen would involve the use of a different oligomer to obtain a differen N-terminal amino acid for the ZYMV coat protein. The clone construction of the plant expressible ZYMV coat protein gene i referred to pUC18cpZYMV, and is shown in Chart 12. Example 15 Construction of a Micro T-DNA Plasmid Containing th Plant-expressible ZYMV Coat Protein Gene Construction. Following the teachings of Example 11 with appropriat modifications, the construction of a micro T-DNA plasmid containing plant-expressible ZYMV coat protein was constructed. Plasmi pUClδcpZYMV (Chart 12) was digested with Hind III (which cuts withi the polycloning sites of ρUC18, well outside of the expressio cassette), and a 1.6 kb fragment containing the plant-expressib cassette was removed and ligated into the Hind III site of the micr vecto pGA482 (Chart 7). The resulting plasmid was designate pGA482GG/cpZYMV is shown in Chart 13. After transfer of the engineered plasmid pGA482GG/cpZYMV in Agrobacterium strains, the Agrobacterium strains can be used transfer and integrate within a plant genome the plant-expressib ZYMV coat protein gene contained within its T-DNA region. Example 16 Microprojectile Transfer of pUClβcpZYMV into Pla Tissues. Following the teachings of Example 12, the micropro ecti transfer technique can be used to introduce the ZYMV coat prote gene with appropriate genetic regulatory sequences into pla tissues. The use of this process for the transfer of pUC18cpZYMV can accomplished after the addition of either plant expressible gen NPTII or Gus genes or both. Plasmids that have the nptll and G genes to pUClScpZYMV are shown in Chart 14 and referred to pUC18GcpZYMV and pUC18NGcpZYMV. In addition, the constructi described in Example 15 can also be used for microprojectile transf as it already has both the nptll and Gus genes attached to t pUC18cpZYMV cassette (see Chart 13) . The only difficulty which t use of pGA482GG/cpZYMV may encounter during transfer by t microprojectile process is due to its large size, about 18kb, which may have a lower efficiency transfer and such larger plasmid generally yield less DNA during propagation. To construct plasmid pUClδGCPZYMV, plasmid pUC18CPZYMV is digested with BamHI and ligated to a 3.0 BamHI isolated fragment which contains the Gus gene. To construct plasmid pUC18GCPZYMV, plasmid pUC18GCPZYMV is digested with Smal and ligated with a 2.4 kb isolated fragment containing the Nos nptll gene isolated by digestion with Dral and Stul. CHARTS CHART 1 CAG iTCCAAGAATGAAGCTGTGGATGCTGGTTTGAATGAAAAACTCAAAGAGAAGGAAAAT 1 ■ * ■ » ■ » +-__-. __.__.♦ _ ♦ ββ G! nSβrLy»AsnGluAI«V« IA»pAI«GfyLβuA»nGI uLy*L-»υLy»GIuLy*GIuAsn CAGAAAGAAAAAGAAAAAGAAAAACAAAAAGAGAAAGAAAAAGACGGTGCTAGTGACGGA βl * ♦ + + 4 ♦ 120 GlnLy»GIuL aGI uL »C!uL *G1nLy*GIuL sG!uL βAβpGTyλ1aSβrAaoG!y AATGATGTGTCAACTAGCACAAAAACTGGAGAGAGAGATAGAGATGTCAATGTTGGGACC 21 4 ♦ ♦ * ♦ ♦ 180 AsnAspY* IS * BrThrSβrThrLy«ThrGl GluArBAspArgA»pV._ tAsnVa IGI Thr AGTGGAACTTTCACTGTTCCGAGAATTAAATCATTTACTGATAAGATGGTTCTACCGAGA 81 -t- + ♦ + ♦ + 240 SβrGl ThrPhβThrV* IProArgl I•LyβSβrPhβThrA»pLy»MβtV* IL«uProArg ATTAAGGGGAAGACTGTCCTTAATTTAAATCATCTTCTTCAGTACAATCCGCAACAAATT 41 + ♦ •» * 4 - 300 I IβLysGl ysThrVi ILeuAsnLβu snHi sLβuLeuGInTyrAsnProGIπGInlIβ GACATTTCTAACACTCGTGCCACTCATTCACAATTTGAGAAGTGGTATGAGGGAGTGAGG 01 4 4 4 4- 4 4 360 As i IβSerAsnThrArgAlβThrHi βSβrGlnPh*G1uLy«TrpT rG1uGl V» lArg AATGATTATGGCCTTAATGATAATGAAATGCAAGTGATGCTAAATGGTTTGAT-GGTTTGG βl 4 4 * 4 42 AsnAs TyrGl LβuAsnAspAsnGIuU*tGlnV« 1U*tL«uA»nGl L«uMβtV» ITrp TGTATCGAGAATGGTACATCTCCAGACATATCTGGTGTCTGGGTTATGATGGATGGGGAA 21 4 4 4 4 * 4 480 Cy»II«GluAsnGlyThrSβrProAspIlβSβrGl V*ITrpVaIMβtMβtAspGl Glu ACCCAAGTTGATTATCCAATCAAGCCTTTGATTGAGCATGCTACTCCGTCATTTAGGCAA 81 4 4 4 4 4 4 540 ThrGlnYιlAspTyrProI Ly«ProL*uIleClυHlβAI*ThrProSerPh«λrgGln -ATTATGGCTCACTTTAGTAACGCGGCACAAGCATACATTGCGAAGAGAAATGCTACTGAG 41 _. * ♦ + 4- 4 600 II«M*tAI»HlaPh»SβrA»nAI«AlβGluAlβTyrII*AlιLy»ArgA»n lβThrGlu CHART 1 (Continued) ACCTACATGCCGCαGTATGGAATCAAGAGAAATTTGACTGACATTAGCCTCGCTACAT C 601 4 * 4 4 4 ββ ArgTyrMβt roArgtyrClyl ! «Ly »ArgAsnL«uThrA» I l<*S«rL«uAlaArgTyr CCTTTCGACTTCTATGAGGTGAATTCGAAAACACCTGATAGGGCTCGCGAAGCTCACATG 861 4 4 4 4 4 4 720 A I aPhβAspPhβT rGI uV» I AsnSβrLysThrProAspArgA I tArgG luA I aH i sMβt CAGATGAAGGCTGCAGCGCTGCGAAACACCAGTCGCAAAATGTTTGGTATGCACGGCAGT 21 4 4 4 4 4 4 780 G I fiMetLys I a A I a A I aLβuArgAsnThrSβrArgLy βUβtPhβG I yMβtAspG I ySβr GTTAGTAACAAGGAAGAAAACACGGAGAGACACACAGTGGAAGATGTCAATAGAGACATG 81 4 4 4 4 4 4 840 Va ISβrAsnLysGlυGluAsnThrGluArgH'iaThrVa IGIuAapVaIAsnArgAapMβt CACTCTCTCCTGGGTATGCGCAACTAAATACCTGCGCTTGTGTGTTTGTTGAGTCTGACT 841 ♦ * ' f * ♦ + 900 HisSβrLβuLβuGly βtArgAsnEn CGACCCTGTTTCACCTTATGGTACTATATAAGCATTAGAATACAGAGTGGC1GCGCCACC 901 4 4 4 4 4 4 960 GCTTCTATTTTACAGTGAGGGTAGCCCTCCGTGCTTTTAGTATTATTCGAGTTCTCTGAG gβl ♦ ♦ + + ♦ * 1020 TCTCCATACAGTGTGGGTGGCCCACGTGATATTCGAGCCTCTTAGAATGAGAAAAAAAAA 1021 ♦ + +-- •» -> + 1080 AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGAATTCC 1081 * * •» •» 1124 CHART 2 GTGTCTTTACAATCAGGAAAAGAAACAGTTGAAAATTTGGACGCAGGGAAAGAATCAAAG ♦ * -. * 6 ø Va ISerLβuGlnSerGIyLysGI uThrVa IGIuAsnLeuAspAIaGI LysGIuSβrLyS T AAAGATGCCAGTGACAAAGGGAATAAGCCGCAGAACTCGCAAGTTGGTCAGGGTAGCAAG LysAspAlaSβrAspLysGl AsnLysProGlnAsnSβrGlnVa IGlyGInGIySβrLys GAACCAACAAAAACCGGCACAGTCAGCAAGGATGTAAATGTTGGATCGAAAGGAAAAGAA ♦ + + * * 18 GluProThrLysThrGlyThrVa ISβrLysAspVaIAsnVaIGlySβrLysGIyLysGIu GTCCCACGACTACAAAAGATAACAAAGAAAATGAATCTTCCAACAGTTGGTGGGAAAATC VaIProArgLβuGInLysiieThrLysLys etAsnLeuProThrVa IGI GI L siIθ ATTCTTAGCTTAGACCATTTGCTTGAGTACAAACCTAGTCAAGTTGATTTGTTTAACACT 4- 4 4 4 4- 3 11 eLβuSβrLeuAspH i sLβuLβuG I uT rLysProSerG I πVa I AspLβuPhβAsnThr CGAGCAACAAAAACACAATTTGAATCATGGTACAGCGCAGTCAAAGTTGAGTATGATCTT 4 4 4- 4 + 3 ArgA! aThrLysThrG I nPhβG I uSβrTrpTyrSβrA I aVa ILysVa I G I uTyrAspLβu AATGATGAGCAAATGGGTGTGATTATGAATGGTTTTATGGTTTGGTGTATCGATAACGGT 4 "4 + + 4 4 4 AsnAspGIuGInMβtGI Va 111β βtAsnGI PheMβtYa iTrpC siIβAspAsnGI ACATCTCCAGATGTCAATGGAGTGTGGGTAATGATGGATGGGGAAGAGCAAGTTGAGTAC CCACTAAAGCCAATTGTTGAAAATGCAAAGCCAACTTTGAGACAAATCATGCACCATTTC 4- + 4 4 4 5 roLeulysProI1βVa IGIuAsnAIaLysProThrLβuArgGInlIe etHϊsHisPhe TCAGATGCAGCGGAAGCATATATTGAAATGAGAAACTCTGAAAGTCCGTATATGCCTAGA 4- 4 4 4- 4- 4- 6 SerAspAf aA I aG 1 uA I aTyrl IβG I uMetArgAsnSβrG 1 uSβrProTyrMβtProArg TACGGATTACTGAGAAATTTGAGAGACAGGGAATTAGCACGCTATGCTTTTGACTTCTAT 4 --4- 4 4. 4- 4 6 Ty PGI yLβuLβuArgAsnLeuArgAspArgG I uLβuA I aArgT rA I aPhβAspPhβTy r GAGGTTACTTCTAAAACGCCAAATAGGGCAAGAGAAGCAATAGCTCAAATGAAGGCCGCG + -4 + + 4 4 7 GluVa IThrSerLysThrProAsnArgAlaArgGluAIallβAlaGI nMetLysAI aAl a GCTCTCGCGGGAGTTAACAGCAGGTTATTTGGACTTGATGGTAATATCTCGACCAATTCC .- + - - 4 7 A I Leu laGlyVa l snSβrArgLβuPhβG l LβuAspGl Asnl IβSβrThrAsnSer CHART 2 (Continued) GAAAATACTGGGAGGCACACTGCAAGGGACGTAAATCAGAATATGCATACTTTGTTGGGT 781 ♦ * 4 4 840 GluAsπThrGlyArgH.sThrAlaArgAspValAsπGlnAsnMetH.sThrLeuLβuGly ATGGGTCCACCGCAGTAAAGACTAGGTAAACTGGTCACAGTTAGCATTTCGGGTCGTTAT 841 4 4 4 4 4 4 900 MetGlyProProGlnEnd AA rsT T TTCT A TAATATAACAT TCGCACTTTATTTTAGTATAOTGTfti T T T rATrArrTT 901 4 4 4 4 4 960 TGTACTGTTTATGTTAGCGTGGTTTAACCACCTTTGTGTGTGCTTTATATTATAGTTTAT 961 4 4 4 4 4 4 1020 GCGTAGCAGGGAGAACCATTACAATGCCGGAGTTGTTTGTAGTGTGATTTCATCACGGTT 1021 4 4 4 4 4 1080 AATAGCCGAGGTACGGTAATGTTTGTTGCCTAAAAAAAAAAAAAAAAAAAAAAAA 1081 4 4 4 4 + 1135 CHART 3 ATGCTCCAATCAGGCACTCAACCAACTGTGGCAGACGCTAGAGTTACAAAGAAAGATAAA 1 * „ ♦ ., , _, βø Uβ Lβu IπS-βrGIyThrGInProThrVaIAIaAspAIaArgVa IThrLysLysAspL s GAAGATGACAAAGGGGAAAACAAGGATTTCACAGGCTCCGGCTCAGGTGAGAAAACAGTA βl -> 4 4. 4 4 4 120 GIuAspAspLys I GIuAsπL sAspPhβThrGI SβrGIySβrGI GIuLysThrV I GTAGCTGCCAAGAAAGACAAGGATGTGAATGCTGGTTCTCATGGGAAAATTGTGCCGCGT 21 + • 4 4 4 180 ValAlaAlaLysLysAspLysAspValAsnAl GlySβrHisGl LysII-aYalProArg CTTTCGAAGATCACAAAGAAAATGTCATTGCCACGCGTGAAAGGGAATGTGATACTCGAT 81 ♦ -r 4 4 4- ♦ 240 LβuSβrLysIlβThrLysLysMβtSβrLβuProArgVa ILysGlyAsnVaIIIeLβuAsp ATCGATCATTTGCTGGAATATAAGCCGGATCAAATTGAGTTATACAACACACGAGCGTCT 41 4 * 4 4 4 4 300 I l eAspH i sLβuLβuG I uTyrlysProAspG I nl I βG l uLβuTyrAsnThrArgA taSβr CATCAGCAATTTGCCTCTTGGTTCAACCAAGTTAAGACAGAATATGATCTGAATGATCAA 01 * 4 4 4 4 4 360 H I aG I πG I nPhβA I Ser-TrpPheAsnG I nV I LysThrG I uTyrAspLβuAsπAspG I n CAGATGGGAGTTGTGATGAACGGTTTCATGGTTTGGTGTATTGAAAATGGCACCTCACCT 61 4- 4 4 4 4 4 420 GInMβtGIyVa IV IMβfcAsnGIyPheMetVaITrpCysiIeGIuAsnGIyThrSβrPro GACATTAATGGAGTGTGGTTTATGATGGACGGAAATGAACAAGTTGAGTATCCTTTGAAA 21 4- 4 4 4 4 4- 480 AsplI-aAsπGI V ITrpPhβMβ MβtAs GIyAsnGIuGInV IGIuT rProLβuLys CCGATAGTTGAAAATGCAAAGCCAACGCTGCGGCAAATAATGCATCATTTTTCAGATGCA 81 -— -4-—-— ♦ ♦-- 4 ♦ 4 540 Prol I *V I G I uAsnA I aLysProThrLβuArgG I nl I •MetH \ sH i sPhβSβrAspA 1 a GCGGAGGCATATATAGAGATGAGAAATGCAGAGGCACCATACATGCCGAGGTATGGTTTG l * 4 4 4- 4 4 800 AJaGluAlaTyrI GIuMβtΛrgAsnAl GluAl ProTyrM«tProArgTyrGlyL«u CHART 3 (Continued) CTTCGAAACCTACGGGATAGGAGTTTAGCACGATACGCTTTCGATTTCTATGAAGTCAAT 601 4 4 4 4 4 4 660 LβuArgAsnLeuArgAspArgSerLeuAIaArgT rAIaPh#AspPh«T PGIuVaIAsrt TCTAAAACTCCTGAAAGAGCCCATGAAGCTGTTGCGCAGATGAAAGCAGCAGCTCTTAGC 661 4 4 4 4 4 4 720 SerLysThrProGluArgAl HisGluAlaV lAlaGlnMβtLys laAlaAlaLeuS-βr AATGTTTCTTCAAGTGTGTTTGGCCTTAGTGAAATCGTTGCCACCACTAGCGAAGCCACA 721 4 * ♦ 4 ♦ * 780 AsnVaISβrSβrSβrVaIPhβGIyLβuSβrGIulIβVaIAIaThrThrSβrGIuAIaThr CTGAACGGCACACTGCACGTGATGTTAATAGAAACATGCCACACCTTACTAGGTGTGAAT 781 4 4 4 4-- 4 4 840 LβuAsnGlyThrlβuHisValMetLβuIlβGluThrCysHisThrLβuLβuGl ValAsn ACAATGCAGTAAAGGGTAGGCCGCCTACCTAGGTTATCGCTTCGCTGCCGACGTAATTCT 8 1 4 4 4 4 4 4 900 ThrMβtGlnEπd AATATTTACCAGCTTTATTTGATATCTTTAGATTTCCAGAGTGGGCCTCCCACCTTTAAA 901 4 ♦ 4 4 4 960 GCGTAGAGTTTATGCTTAGTTGTCCAGGAGTGCCGTAGTCCTGTCGGAAGCTTTAGTGTG 961 * 4 4 4 ♦ 4 1020 AGCCTCTCACGAATAAGCTCGAGATTAGACTCCGTTTGCAAGCCTAAAAAAAAAAAAAAA 1021 4 4 * 4 4 4 1080 AAAA 1081 1084 CHART 4 PRV-p CAGTOCAAGAATGAAGCTGTGGATGCTGCTTTGAATGAAAAACTCAAAGA 50 IN! ill I I MM I MINIMI 111111111111111 M PR V -w CAGTCCAAAAATGAAGCTGTGGATACTGGTTTGAATGAAAAATTCAAAGA 50 GAAGGAAAATCAGAAAGAAAAAGAAAAAGAAAAACAAAAAGAGAAAGAAA 100 MIIIMI 1111111111 II t i M 1111 iI It M 11 M II 11111111 AAAGGAAAAACAGAAAGAAAAAGAAAAAGAAAAACAAAAAGAGAAAGAAA 100 AAGACGGTGCTAGTGACGGAAATGATGTGTCAACTAGCACAAAAACTGGA 150 IMIII I H 11111111111111111 II 11 M i I II I i 11 M 1111111 AAGACGATGCTAGTGACGGAAATGATGTGTCAACTAGCACAAAAACTGGA 150 GAGAGAGATAGAGATGTCAATGTTGGGACCAGTGGAACTTTCACTGTTCC 200 IMMMMIM MIMMIMMMIIMIIIMMIIMMMIMM GAGAGAGATAGAGATGTCAATGTTGGGACCAGTGGAACTTTCACTGTTCC 200 GAGAATTAAATCATTTACTGATAAGATGGTTCTACCGAGAATTAAGGGGA 250 IIMMMMMMMMMMMMM 1111111111111111111 I GAGAATTAAATCATTTACTGATAAGATGATTOTACCGAGAATTAAGGGAA 250 AGACTGTCCTTAATTTAAATCATCTTCTTCAGTACAATCCGCAACAAATT 300 II MMMIIIMMMMM II MIIIMI MIMMIMIMM AGTCTGTCCTTAATTTAAATCACCTACTTCAGTATAATCCGCAACAAATT 300 GACATTTCTAACACTCGTGCCACTCATTCACAATTTGAGAAGTGGTATGA 350 MMMIMMMMMM 11111111111111111111111 GACATTTCTAACACTCGTGCCACTCAGTCACAATTTGAGAAGTGGTATGA 350 GGGAGTGAGGAATGATTATGGCCTTAATGATAATGAAATGCAAGTGATGC 400 MM IMMIIMMIMMMMMIIIMMMMIMMMMMM GGGAGTGAGGAATGATTATGGCCTTAATGATAATGAAATGCAAGTGATGC 400 TAAATGGTTTGATGGTTTGGTGTATCGAGAATGGTACATCTCCAGACATA 450 111 II 11111111111111 i I M 11 i II I II 1111111111111111111 TAAATGGTTTGATGGTTTGGTGTATCGAGAATGGTACATCTCCAGACATA 450 CHART 4 (Continued) TCTGGTGTCTGGGTTATCATGCATGGGGAAACCCAACTTGATTATCCAAT 50 I M M I I I I I I I I M I M M II M I I M I M I M I I I I M I I M II II M TCTGGTGTCTGGGTTATGATGGATGGGGAAACCCAAGTTGATTATCCAAT 600 CAAGCCTTTGATTGAGCATGCTACTCCGTCATTTAGGCAAATTATGGCTC 550 I I I I I I I I I I I I 11111111111111 I 1111 I I 111111 I I I I 111111 CAAGCCTTTAATTGAGCATGCTACTCCGTCATTTAGGCAAATTATGGCTC 550 ACTTTAGTAACGCGGCAGAAGCATACATTGCGAAGAGAAATGCTACTGAG 600 MIIIIIMMMMMMIMMMMMMII 111111111111111 ACTTTAGTAACGCGGCAGAAGCATACATTGCGAAAAGAAATGCTACTGAG 600 AGGTACATGCCGCGGTATGGAATCAAGAGAAATTTGACTGACATTAGCCT 650 II II MIMMMMIMMM MMMIIMMMMIMMMIMM AGGTACATGCCGCGGTATGGAATCAAGAGAAATTTGACTGACATTAGCCT 650 CGCTAGATACGCTTTCGACTTCTATGAGGTGAATTCGAAAACACCTGATA 700 CGCTAGATACGCTTTCGACTTCTATGAGGTGAATTCGAAAACACCTGATA 700 GGGCTCGCGAAGCTCACATGCAGATGAAGGCTGCAGCGCTGCGAAACACC 750 MMIMMMM IMIMMMMMIMMMM GGGCTCGCGAAGCCCACATGCAGATGAAGGCTGCAGCACTGCGAAACACT 750 AGTCGCAAAATGTTTGGTATGGACGGCAGTGTTAGTAACAAGGAAGAAAA 800 AGTCGCAGAATGTTTGGTATGGACGGCAGTGTTAGTAACAAGGAAGAAAA 800 CACGGAGAGACACACAGTGGAAGATGTCAATAGAGACATGCACTCTCTCC 850 MMMMMMMMMMMM 1111111111111111111111111 CACGGAGAGACACACAGTGGAAGACGTCAATAGAGACATGCACTCTCTCC 850 TGGGTATGCGCAACTAA 867 MMIMMIMMIM TGGGTATGCGCAACTAA 887 CHART 5 SMV SGKEKEGDUDADKDPKKSTSSSKG AGTSSKDVNV 34 MM MINIM I! I M IIIMI WMVII SGKETVENLDAGKESKKDASDKGNKPQNSQVGQGSKEPTKTGTVSKDVNV 50 SMV GSKGKVVPRLQKITRKMNLPMVEGKIILSLDHLLEYKPNQVDLFNTRATR 84 Mill MIIMMMMM I 111111111 II 1111 I IIMIIIIlll WMVII GS GKEVPRLQKIT KMNLPTVGG IILSLDHLLEYKPSQVDLFNTRATK 100 SMV TqFEAWYNAVKDEYELDOEQMGVVMNGFMVWCIDNGTSPDANGYWYMMDG 134 MM II III I II 11 I I 11111111 M 1111 M 1 I I i I WMVII TQFESWYSAVKVEYDLNDEQMGVIMNGFMVWCIDNGTSPOVNGVWVMMDG 150 SMV EEQIEYPLKPIVENA PTLRQIMHHFSDAAEAYIEMRNSESPYMPRYGLL 184 NNNNINNMM MNIIMIIMIMNMINMIIMINM WMVII EEQVEYPLKPIVENAKPTLRQIMHHFSDAAEAYIEMRNSESPYMPRYGLL 200 SMV RNLRDRELARYAFDFYEVTSKTPNRAREAIA MKAAALSGVNNKLFGLDG 234 I II N N III Ml III Nil IN NIMIINMMM III WMVII RNLRDRELARYAFDFYEVTSKTPNRAREAIAQMKAAALAGVNSRLFGLDG 250 SMV NISTNSENTERHTARDVNQNMHTLLGMGPPQ 265 INI I II IIININN IN INNNIIN WMVII NISTNSENTGRHTARDVN NMHTLLGMGPPQ 281 CHART 6 pPRV117 PRV-p Coat Protein Gene pUC1813/Cpl9 Hindlll Ncol Tthllll BstXI Ncol Hindlll + i I * ca I_ I ICMV Coat Protein Gene 'ca pUC1813/Cpl9-PRVexp Hindlll Ncol Tthllll BstXI Ncol Hindlll 4- 4 4 ca CMV I PRV-p Coat 'ca Coat Protein (16AA) gene CHART 7 pGA482 Hindlll Gene pGA482/G/CP19-PRVexp Hindlll Tthllll BstXI Hindlll + 4- 4 Nos cai CMV I PRV-p Coat S ca | ICaMV Coat Protein Gus Protein Gene Gene (16 A) CHART 8 Hindlll Ncol Ncol Hindll pl8CaMV/CMV-exp Hindlll Ncol Hindlll 4- 4 4- ca 'ca CHART 9 pWMVII-41-3.2 WMVII Coat Protein Gene PCR Generated Gene Ncol Ncol 4- 4 WMVII Coat Protein Gene pl8WMVII-exp Hindlll Ncol Ncol Hindlll BamHI Smal 4- 4- 4- 4- 4- 4. * I ca WMV Coat Protein 'ca Gene CHART 10 pGA482/G/CPWMVII-exp Hindlll Ncol Ncol Hindlll 4 4 4 4 B, Nos ■ ca WMVII Coat 'ca CaMV Protein Gus Gene Gene CHART 11 pl8GWMVII-exp Hindlll Ncol Ncol Hindlll BamHI BamHI Smal 4 4 4 4 4 4 4 ca WMV Coat 'ca CaMV Protein Gus Gene Gene pl8NGWMVII-exp Hindlll Ncol Ncol Hindlll BamHI BamHI 4 4 4 4 4 4 ■ ca WMVII Coat 'ca CaMV Nos Protein Gus Gene Gene CHART 12 pZYMV-15 ZYMV Coat Protein Gene ZYMV Coat Protein Gene Ncol Ncol 4 4 ZYMV Coat Protein Gene pUC18CpZYMV Hindlll Ncol Ncol Hindlll BamHI Smal 4 4 4 4 4 4 ca I c [.ZYMV Coat Protein 'ca Gene CHART 13 pGA482/GG/cpZYMV Hindlll Ncol Ncol Hindlll 4 4 4 4 Nos ca ZYMV Coat 'ca CaMV Protein Gus Gene Gene -43 - CHART 14 pUC18GCpZYMV Hindlll Ncol Ncol Hindlll BamHI BamHI Smal 4 4 | 4 4 4 4 P ca |I c |ZYMV Coat 'ca CaMV Protein Gus Gene Gene pUCl8NGCpZYMV Hindlll Ncol Ncol Hindlll BamHI BamHI 4 4 4 4 4 4 ca I c IZYMV Coat 'ca CaMV Nos Protein Gus Gene Gene

Claims: CLAIMS 1. A recombinant DNA molecule which encodes a potyvirus coa protein, said recombinant DNA molecule selected from the grou consisting of Papaya ringspot virus strain papaya ringspot PRV-p coa protein gene, Watermelon mosaic virus II WMVII coat protein gene, an Zucchini yellow mosaic virus ZYMV coat protein gene. 2. A recombinant DNA molecule according to Claim 1 wherein sai recombinant DNA molecule encodes Papaya ringspot virus strain papay ringspot PRV-p coat protein, said recombinant DNA molecule having th nucleotide sequence shown In Chart 1. 3. A recombinant DNA molecule according to Claim 1 wherein sai recombinant DNA molecule encodes Watermelon mosaic virus II WMVI coat protein, said recombinant DNA molecule having the nucleoti sequence shown in Chart 2. 4. A recombinant DNA molecule according to Claim 1 wherein sa recombinant DNA molecule encodes Zucchini yellow mosaic virus ZY coat protein, said recombinant DNA molecule having the nucleoti sequence shown in Chart 3. 5. A recombinant DNA molecule according to Claim 1 furth comprising: a) a promoter; b) an initiation region; and, c) a poly(A) addition signal; wherein said promoter is upstream and operably linked to sa Initiation region, said initiation region is upstream and operab linked to said recombinant DNA molecule encoding a coat protein, a said recombinant DNA molecule encoding a coat protein is upstream a operably linked to said poly(A) addition signal. 6. A recombinant DNA molecule according to Claim 5 wherein sa promoter is Cauliflower mosaic virus CaMV 35S promoter. 7. A recombinant DNA molecule according to Claim 5 wherein sa initiation region is selected from the group consisting of initiation region derived from the 5' untranslated region of Cucumbe mosaic virus CMV coat protein gene and an initiation region derive from the 5' untranslated region of SS RUBISCO gene. 8. A recombinant DNA molecule according to Claim 5 wherein sai initiation region comprises the sequence AAXXATGG. 9. A recombinant DNA molecule according to Claim 5 wherein sai poly(A) addition signal is selected from the group consisting of: th pol (A) signal derived from Cauliflower mosaic virus CaMV 35S gene the pol (A) signal derived from phaseolin storage protein gene; th pol (A) signal derived from nopaline synthase gene; the poly(A signal derived from octopine synthase gene; the poly(A) signa derived from bean storage protein gene; and, the poly(A) signa derived from SS RUBISCO. 10. A recombinant DNA molecule according to Claim 6 wherein sai initiation region is derived from the 5' untranslated region o Cucumber mosaic virus CMV coat protein gene and said poly(A) additio signal is derived from Cauliflower mosaic virus CaMV 35S gene. 11. A recombinant DNA molecule according to Claim 5 furthe comprising an AT rich 5' untranslated region wherein: a) said AT rich region is downstream from said promoter an upstream from said initiation region; b) said initiation region comprises the sequence AAXXATGG and, c) said poly(A) addition signal contains untranslate flanking sequences. 12. A recombinant DNA molecule according to Claim 14 wherein sai promoter is Cauliflower mosaic virus CaMV 35S promoter. 13. A recombinant DNA molecule according to Claim 11 wherein sai AT-rich 5' untranslated region is derived from the 5' untranslate region of a gene selected from the group consisting of Cucumbe mosaic virus CMV coat protein gene and SS RUBISCO gene. 14. A recombinant DNA molecule according to Claim 11 wherein said initiation region is derived from the 5' untranslated region of a gene selected from the group consisting of Cucumber mosaic virus CMV coat protein gene and SS RUBISCO gene. 15. A recombinant DNA molecule according to Claim 11 wherein said initiation region comprises the sequence AAXXATGG. 16. A recombinant DNA molecule according to Claim 11 wherein said poly(A) addition signal is selected from the group consisting of: the poly(A) signal derived from Cauliflower mosaic virus CaMV 35S gene; the poly(A) signal derived from phaseolin storage protein gene; the poly(A) signal derived from nopalinesynthase gene; the poly(A) signal derived from octopine synthase gene; the poly(A) signal derived from bean storage protein gene; and, the poly(A) signal derived from SS RUBISCO. 17. A recombinant DNA molecule according to Claim 12 wherein; a) said AT rich 5' untranslated region and said initiation region are derived from the 5' untranslated region of Cucumber mosaic virus CMV coat protein gene; b) said initiation region comprises the sequence AAXXATGG; and, c) and said poly(A) addition signal is derived from Cauli- flower mosaic virus CaMV 35S gene. 18. A transformed plant cell containing a recombinant DNA molecule according to Claim 5. 19. A transformed plant cell according to Claim 18 containing a recombinant DNA molecule according to Claim 10. 20. A transformed plant cell according to Claim 18 containing recombinant DNA molecule according to Claim 11. 21. A transformed plant cell according to Claim 20 containing recombinant DNA molecule according to Claim 17. 22. A transgenic plant selected from the group consisting of the families Cucuribitaceae, Caricaceae, Solanaceae, and Leguminoseae comprising transformed plant cells according to Claim 18. 23. A transgenic plant according to Claim 22 comprising transformed plant cells according to Claim 19. 24. A transgenic plant according to Claim 22 comprising transformed plant cells according to Claim 20. 25. A transgenic plant according to Claim 24 comprising transformed plant cells according to Claim 21. 26. A process for producing a transgenic plant which is resistant to viral infection comprising the steps of: .a) constructing a recombinant DNA molecule according to Claim 5; b) transforming plant cells with said recombinant DNA; and c) regenerating plants from said transformed plant cells.

Inventors: GONSALVES DENNIS, L HOSTIS BRIGITTE, QUEMADA HECTOR D, SLIGHTOM JERRY L, GONSALVES, DENNIS, L'HOSTIS, BRIGITTE, QUEMADA, HECTOR, D., SLIGHTOM, JERRY, L.
Applicants: CORNELL RES FOUNDATION INC, UPJOHN CO, CORNELL RESEARCH FOUNDATION, INC., THE UPJOHN COMPANY
Application year: 1989
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N5, C12N15, C12R1