PRODUCTION OF VIRAL RESISTANT PLANTS VIA INTRODUCTION OF UNTRANSLATABLE PLUS SENSE VIRAL RNA

Abstract: Plants, such as tobacco, are made resistant to potyvirus infection by transformation with vectors which include a gene, derived from a potyvirus, mutated to encode an untranslatable plus sense RNA molecule. Mutagenized potyvirus genes and plant transformation vectors containing these genes are also disclosed.

Description: "Production of viral resistant plants via introduction of untranslatable plus sense viral RNA FIELD OF THE INVENTION 5 This invention is directed to the production of plants with a reduced susceptibility to virus infection. BACKGROUND OF THE INVENTION Plant viruses are responsible for major losses in worldwide crop production. Much effort is directed 10 towards the development of new plant varieties which exhibit increased resistance to viral infection. Until recently such efforts were primarily based on the traditional plant breeding approach, however this approach is often limited by a lack of sources of 15 resistance within the crop species. The advent of modern molecular biology techniques has facilitated the development of new methods of rendering plant varieties resistant to virus attack that are not limited by a requirement for preexisting resistance genes within a 20 species. Molecular Approaches Many of these molecular approaches are based on the theory of pathogen derived resistance (Sanford and Johnston, 1985) . This theory predicts that a "normal" 25 host (plant) - pathogen (virus) relationship can be disrupted if the host organism expresses essential pathogen derived genes. It has been proposed that host organisms expressing pathogen gene products in excess amounts, at an inappropriate developmental stage, or in 30 a dysfunctional form may disrupt the normal replicative cycle of the pathogen and result in an attenuated or aborted infection of the host. Two approaches typify this pathogen derived f* resistance: coat protein mediated resistance and 35 antisense RNA expression. It has been demonstrated that transgenic plants expressing a plant virus coat protein can be resistant to infection by the homologous virus. This coat protein mediated resistance has been demonstrated for several virus groups. While the mechanism of this resistance is not yet fully understood, it has been suggested that the presence of the plant synthesized coat protein prevents the removal of the protein coat (uncoating) of an invading virus and/or virus movement within the infected plant, leading to resistance. Plants which express an RNA molecule which is complementary to plus sense RNA species encoded by the virus may show a decreased susceptibility to infection by that virus. Such a complementary RNA molecule is termed antisense RNA. It is thought that the plant encoded antisense RNA binds to the viral RNA and thus inhibits its function. Potyviruses The Potato virus Y, or potyvirus, family represents a large number of plant viral pathogens which collectively can infect most crop species including both monocotyledonous and dicotyledonous plants. Potyvirus infection can induce a variety of symptoms including leaf mottling, seed and fruit distortion and can severely compromise crop yield and/or quality (Hollings and Brunt, 1981) . Potyviruses have a single-strand plus sense RNA of circa 10,000 nucleotides which has a viral encoded protein linked to the 5' end and a 3' polyadenylate region. A single open reading frame codes for a 351 kDa polyprotein which is proteolytically processed into mature viral gene products. The RNA is encapsidated by approximately 2,000 copies of a coat protein monomer to form a virion. This capsid protein is encoded by the sequence present at the 3' end of the large open reading frame. Potyviruses can be transmitted by aphids and other sap feeding insects and in some instances can also be transmitted in the seeds of infected plants. Replication of the viral RNA is thought to occur in the cytoplasm of infected plant cells after uncoating. The replication mechanism involves both translation of the plus sense RNA to yield viral gene products (which include a replicase and a proteinase) and also the synthesis of a minus sense RNA strand. This minus sense strand then acts as a template for the synthesis of many plus sense genomes which are subsequently encapsidated in coat protein to yield infectious mature "virions," thus completing the replicative cycle of the virus. Experiments have been reported in which transgenic plants expressing the coat protein gene of a potyvirus show a reduced susceptibility to virus infection (Lawson et al. 1990; Ling et al. 1991; Stark and Beachy 1989) . SUMMARY OF THE INVENTION The disclosed invention concerns a method of producing plants with a decreased susceptibility to virus infection. This is achieved by transforming plants with a DNA molecule which includes a gene derived in part from the genome of a plant virus. This gene is specifically constructed to produce an untranslatable version of a plus sense RNA molecule required for viral replication. Thus, expression of the gene within the plant causes the production of this non-functional molecule which then inhibits viral replication within the plant, rendering the plant resistant to viral infection. In particular, invention provides an alternative and novel approach to rendering plants resistant to potyvirus infection. Plants are transformed with a gene construct engineered to express an untranslatable form of the plus sense RNA which encodes the coat protein of a potyvirus. In the case of Tobacco Etch Virus (TEV) , it is demonstrated that tobacco plants transformed with such a gene construct accumulate the untranslatable plus sense RNA but do not produce detectable levels of the coat protein. It is further shown that these plants are resistant to TEV infection. It is also shown that tobacco cells expressing this untranslatable plus sense RNA do not support TEV replication, unlike control tobacco cells and also unlike tobacco cells which are engineered to express the plus sense translatable RNA and which, as a result, accumulate TEV coat protein. Although the exact mechanism is unknown, it is proposed that the untranslatable plus sense RNA inhibits viral replication by binding to the minus sense RNA and preventing the minus sense RNA from functioning in the replication cycle. It is believed that this approach will be applicable to other potyviruses, to genes other than the coat protein gene and to other plus sense RNA virus families. It is also believed that this, means of inhibiting gene function is applicable to other biological systems, including mammalian viruses. DESCRIPTION OF DRAWINGS Fig. l represents the nucleotide sequence of the Tobacco Etch Virus genome and its deduced amino acid sequence, according to Allison et al. (1986) . The nucleotide sequence of the plus sense strand of the DNA inserts is given. The first nucleotide (N) could not be determined unequivocally. The predicted amino acid sequence of the large ORF of reading frame three of the viron sense RNA is presented in the nucleotide sequence. This sequence is also set forth in SEQ ID No. 1 of the enclosed sequence listing. The termination codon at the end of the large ORF is marked with a *. The putative cleavage site between the large (54,000 Mw) nuclear inclusion protein and the capsid protein is indicated by the arrow. Oligonucleotide primer binding sites are underlined and labeled. Fig. 2 is a schematic representation of the construction of pTC:FL, utilized in construction of transformation vectors for the invention. Restriction endonuclease sites were introduced into pTL 37/8595 at positions A, B and C in the diagram. Following these nucleotide changes the mutated pTL 37/8595 was digested with the restriction enzyme Ncol , the DNA fragment delineated by the restriction enzyme sites at B and C was removed, and the plasmid religated to generate pTC:FL. pTC:FL contains the Tobacco Etch Virus (TEV) coat protein nucleotide sequence flanked by BamHI restriction sites and the TEV 5' and 3' untranslated sequences (UTS) . T7 and SP6 promoters are also shown. Abbreviations used in this diagram are as follows: T7, T7 RNA polymerase promoter sequence; SP6, SP6 RNA polymerase promoter sequence; ori, origin of replication; M13 ori, bacteriophage M13 single-stranded origin of replication; amp r , 3-lactamase gene. Lightly stippled areas are TEV 5' and 3' untranslated sequences; solid black area, TEV genome cDNA nucleotides 144 to 200; striped area, a portion of the TEV Nib gene (TEV nt 8462-8517); heavily stippled areas, cDNA of TEV CP nucleotide sequence (TEV nt 8518-9309) . Fig. 3 is a schematic representation of the forms of the Tobacco Etch Virus coat protein gene inserted into tobacco in the invention. All constructs contained the enhanced CaMV 35S (Enh 35S) promoter, CaMV 35S 5' untranslated sequence (UTS) of 50 bp and the CaMV 35S 3 ' UTS/polyadenylation site of 110 bp. The nomenclature used to describe the transgenic plant lines is presented along with the gene products produced in those plant lines (far right column) . Abbreviations are as follows: 35S, transgenic plants containing the CaMV 35S promoter and 5' and 3' UTS only; FL, transgenic plants containing the transgene coding for full-length, AS and RC transgenic plants contain the transgene expressed as an antisense form of the TEV CP gene, or an untranslated sense form of the TEV CP gene, respectively. Stippled areas represent various forms of the TEV CP nucleotide sequence. Fig. 4 is a graphic representation of the appearance of systemic symptoms in plants infected with Tobacco Etch Virus showing responses of control plants and transformed plants generated as described in the invention. Ten B49 (wild type) plants and ten R2 plants of transgenic plant lines 35S #4, FL #3, FL #24, homozygous for the inserted TEV gene, were mechanically inoculated with 50 μl of 1:10 dilution of infected plant sap (A) . Twenty B49 plants and 20 Rl plants of lines AS #3 and RC #5 were mechanically inoculated with 50 μl of 5 μg/ml TEV (B) . Plants were examined daily for the appearance of systemic symptoms. Plants were evaluated daily, and any plant displaying systemic symptoms (attenuated or wild-type) were recorded as symptomatic. SEQUENCE LISTING The attached sequence listing sets forth nucleotide sequences relevant to the present invention. SEQ ID No. l is the complementary DNA sequence corresponding to the Tobacco Etch Virus Genome. SEQ ID No. 2 is the nucleotide sequence of the modified Tobacco Etch Virus coat protein gene present in pTC:FL. SEQ ID No. 3 is the nucleotide sequence of the modified Tobacco Etch Virus coat protein gene present in pTC:RC. SEQ ID No. 4 is the nucleotide sequence of the modi ied Tobacco Etch Virus coat protein gene present in pTC:AS. It is the inverse complement of SEQ ID No. 2. DETAILED DESCRIPTION The present invention relates to genetically engineered plants which are transformed with a DNA molecule encoding an untranslatable plus sense RNA molecule. Definition of Terms Susceptible plant: A plant that supports viral replication and displays virus-induced symptoms. Resistant plant: A plant wherein virus-induced, symptoms are attenuated and virus replication is attenuated. Plus sense RNA (and sense RNA) : That form of an RNA which can serve as messenger RNA. Minus sense RNA: That form of RNA used as a template for plus sense RNA production. Antisense RNA: RNA complementary to plus sense RNA form. R Q generation: Primary transformants. R x generation: Progeny of primary transformants. R 2 generation: Second generation progeny of RQ generation (i.e., progeny of R x generation). A gene derived in part from a plant virus RNA molecule: At least the portion of the gene encoding the untranslatable RNA molecule is derived from a plant virus RNA molecule. GENERAL DESCRIPTION An untranslatable plus sense RNA molecule is encoded by a gene located on the DNA molecule. The gene comprises DNA derived from a plant virus RNA genome and also DNA from heterologous sources. The DNA from heterologous sources includes elements controlling the expression of the virus-derived DNA sequences. The DNA sequence of the gene is specifically altered so as to render the RNA molecule transcribed from the gene untranslatable. The presence of this untranslatable plus sense RNA within the cells of the transformed plant reduces the susceptibility of the plant to viral infection. More particularly, the portion of the gene which comprises DNA from a plant virus has been derived from a potyvirus. Plants transformed with the DNA molecule containing the gene are less susceptible to infection by potyviruses. Most specifically, the DNA from the potyvirus source has been derived from the coat protein gene of Tobacco Etch Virus and transformed plants are resistant to infection by Tobacco Etch Virus. Plants which can be made resistant to potyvirus infection include, but are not limited to, tobacco. Accordingly, the present invention provides a method for genetically engineering plants by insertion, into the plant genome, a DNA construct containing a recombinant gene derived from a potyvirus genome such that the engineered plants display resistance to the potyvirus. In accordance with one aspect of the present invention, genetically transformed plants which are resistant to infection by a plant potyvirus are produced by inserting into the genome of the plant a DNA sequence which causes the production of an untranslatable coat protein RNA of the potyvirus. In accordance with another aspect of the present invention, a DNA sequence is provided to function in plant cells to cause the production of an untranslatable plus sense RNA molecule. There has also been provided, in accordance with yet another aspect of the present invention, bacterial and transformed plant cells that contain the above-described DNA. In accordance with yet another aspect of the present invention, a differentiated tobacco plant has been provided that comprises transformed tobacco cells which express the untranslatable coat protein RNA of Tobacco Etch Virus and which plants exhibit resistance to infection by Tobacco Etch Virus. Other features and advantages of the present invention will become apparent from the following description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. A mechanism by which an untranslatable plus sense RNA molecule, such as described in the current invention can function to inhibit the normal biological function of a minus sense RNA molecule is proposed. One skilled in the art will recognize that the novel approach described herein is not limited to the specific experimental example given and will appreciate the wider potential utility of the invention. The expression of a plant gene which exists in double-stranded DNA form involves transcription of messenger RNA (mRNA) from one strand of the DNA by RNA polymerase enzyme, and the subsequent processing of the mRNA primary transcript inside the nucleus. This processing involves a 3' nontranslated region which causes polyadenylate nucleotides to be added to the 3' end of the viral RNA. Transcription of DNA into mRNA is regulated by a region of DNA usually referred to as the "promoter." The promoter region contains a sequence of bases that signals RNA polymerase to associate with the DNA and to initiate the transcription of mRNA using one of the DNA strands as a template to make a corresponding strand of RNA. A number of promoters which are active in plant cells have been described in the literature. Promoters which are known or are found to cause transcription of viral RNA in plant cells can be used in the present invention. Such promoters may be obtained from plants or viruses and include, but are not limited to, the CaMV 35S promoter. As described below, it is preferred that the particular promoter selected should be capable of causing sufficient expression to result in the production of an effective amount of untranslatable plus sense RNA to render the plant substantially resistant to virus infection. The amount of untranslatable plus sense RNA needed to induce resistance may vary with the plant type. Accordingly, while the 35S promoter is preferred, it should be understood that this promoter may not be the optimal one for all embodiments of the present invention. Furthermore, the promoters used in the DNA constructs of the invention may be modified, if desired, to affect their control characteristics. DNA sequences have been identified which confer regulatory specificity on promoter regions. For example, the small subunit of the ribulose bis-phosphate carboxylase (ss RUBISCO) gene is expressed in plant leaves but not in root tissues. A sequence motif that represses the expression of the ss RUBISCO gene in the absence of light, to create a promoter which is active in leaves but not in root tissue, has been identified. This and/or other regulatory sequence motifs may be ligated to promoters such as the CaMV 35S promoter to modify the expression patterns of a gene. Chimeric promoters so constructed may be used as described herein. For purposes of this description, the phrase "CaMV 35S promoter" will therefore include all promoters derived by means of ligation with operator regions, random or controlled mutagenesis, as well as tandem or multiple copies of enhancer elements, and the like. The 3 ' nontranslated region of genes which are known or are found to function as polyadenylation sites for viral RNA in plant cells can be used in the present invention. Such 3' nontranslated regions include, but are not limited to, the 3' transcribed, nontranslated region of the CaMV 35S gene and the 3' transcribed, nontranslated regions containing the polyadenylation signals of the tumor-inducing (TI) genes of Agrobacterium , such as the tumor morphology large (tml) gene. For purposes of this description, the phrase "CaMV 35S 3 ' nontranslated region" will therefore include all such appropriate 3 Λ nontranslated regions. The DNA constructs of the disclosed embodiment contain, in double-stranded DNA form, a portion of a cDNA version of the single-stranded RNA genome of TEV. In potyviruses, including TEV, the viral genome includes genes encoding the coat protein, a replicase enzyme and a proteinase. The disclosed embodiment utilizes the region of the genome encoding the coat protein gene. In considering the present invention and the evidence for the proposed mechanism by which an untranslatable plus sense RNA molecule can inhibit viral replication, those skilled in the art will recognize that other portions of a potyvirus genome could be substituted for the coat protein gene. Furthermore, it will be apparent that suitable genomic portions are not limited to complete gene sequences. A disclosed embodiment of the invention utilizes a double-stranded complementary DNA (cDNA) derived from the region of the TEV genome encoding the coat protein gene. To the 5' end of this cDNA is ligated the CaMV 35S promoter and CaMV 35S RNA 5' nontranslated region. To the 3' end is ligated the CaMV 35S 3' nontranslated region. These 5' and 3' sequences are present to cause transcription of the gene in plant cells by the cellular enzyme RNA polymerase to produce an RNA molecule of sequence corresponding to the sequence of the coat protein cDNA sequence. Ordinarily, such an RNA would then be translated by ribosomes which would synthesize a protein of amino acid sequence specified by the nucleotide sequence of the RNA molecule. Particular amino acids are specified by nucleotide triplets termed codons. Codons which stipulate translation initiation and termination are also present in DNA and RNA sequences. The current invention relates to RNA molecules which are untranslatable by ribosomes. In the preferred embodiment the sequence of the TEV cDNA encoding the coat protein is mutated by a standard in vitro mutagenesis technique to produce a frameshift mutation early in the coat protein structural gene immediately followed by three translation termination signal codons. These mutations do not affect the ability of RNA polymerase to transcribe an RNA molecule from the cDNA but prevent translation of the transcribed RNA by ribosomes. Those skilled in the art will recognize that for the disclosed gene and for other genes, DNA sequences can be altered in other ways to cause the DNA to encode an untranslatable plus sense RNA molecule. Thus the disclosed invention is not limited to the mutations disclosed. A disclosed embodiment utilizes a cDNA encoding the coat protein gene of TEV, mutated so as to encode an untranslatable plus sense RNA. It will be obvious to one skilled in the art that further sequence alteration of the cDNA molecule could be used to confer additional features on the untranslatable plus sense RNA molecule. Additional features include those which would result in increased viral resistance of plants transformed with the cDNA molecule encoding an untranslatable plus sense RNA. The inclusion of a ribozyme sequence which causes the RNA catalyzed destruction of the target RNA molecule would constitute one such additional feature. Suitable ribozyme sequences are known, as discussed in Tabler and Tsagris (1991) . A DNA construct in accordance with the present invention is introduced, via a suitable vector and transformation method as described below, into plant cells and plants transformed with the introduced DNA are regenerated. Various methods exist for transforming plant cells and thereby generating transgenic plants. Methods which are known or are found to be suitable for creating stably transformed plants can be used in this invention. The choice of method will vary with the type of plant to be transformed; those skilled in the art will recognize the suitability of particular methods for given plant types. Suitable methods may include, but are not limited to: electroporation of plant protoplasts; liposome mediated transformation; polyethylene mediated transformation; transformation using viruses; microinjection of plant cells; microprojectile bombardment of plant cells and AgroJacterium tumefaciens (AT) mediated transformation. The latter technique is the method of choice for the disclosed preferred embodiment of the present invention. In an embodiment of the current invention, the DNA sequences comprising the CaMV 35S promoter and CaMV 35S nontranslated 3' region and the mutated cDNA encoding an untranslatable plus sense RNA derived from the TEV coat protein gene are combined in a single cloning vector. This vector is subsequently transformed into AT cells and the resultant cells are used to transform cultured tobacco cells. Vectors suitable for the AT mediated transformation of plants with the DNA of the invention are disclosed. It will be obvious to one skilled in the art that a range of suitable vectors is available, including those disclosed by Bevan (1983) , Herrera-Estrella (1983), Klee (1985) and EPO publication 12,516 (Schilperoort et al.). Suitable vectors are available on a commercial basis from Clontech (Palo Alto, CA) and Pharmacia LKB (Pleasant Hill, CA) and other sources. Following the transformation of plant cells and regeneration of transformed plants with the DNA molecules as described, regenerated plants are tested for increased virus resistance. Plants are preferably exposed to the virus at a concentration within a range where the rate of disease development correlates linearly with virus concentration. Methods for virus inoculation are well known to those skilled in the art and are reviewed by Kado and Agrawai (1972) . One such method includes abrading a leaf surface with an aqueous suspension containing an abrasive material such as carborundrum and virus or dusting leaves with such an abrasive material and subsequently applying the virus onto the leaf surface. A virus suspension can be directly inoculated into leaf veins or alternatively plants can be inoculated using insect vectors. The virus suspension may comprise purified virus particles, or alternatively, sap from virus infected plants may be utilized. Transformed plants are then assessed for resistance to the virus. The assessment of resistance or reduced susceptibility may be manifest in different ways dependant on the particular virus type and plant type. Those skilled in the art will realize that a comparison of symptom development on a number of inoculated tintransformed plants with symptom development on similarly inoculated transformed plants will provide a preferred method of determining the effects of transformation with the specified DNA molecule on plant resistance. Symptoms of infection include, but are not limited to leaf mottling, chlorosis and etching. Plants showing increased viral resistance may be recognized by delay in appearance of such symptoms or attenuation or total lack of such symptoms. Example Work with tobacco plants and the Tobacco Etch Virus (TEV) is illustrative of the invention. Construction of crene encoding untranslatable plus sense RNA molecule. The Highly Aphid Transmissible (HAT) isolate of Tobacco Etch Virus (TEV) was obtained from Dr. Tom Pirone (University of Kentucky) and maintained in Nicotiana tabacum (Burley 21) . The virus was purified from Nicotiana tabacum (Burley 21) 20 to 30 days following inoculation. Viral purification and RNA isolation procedures have been described (Dougherty and Hiebert (1980a) . Complementary DNA (cDNA) was synthesized, made double-stranded and inserted into the bacterial plasmid pBR322 as described by Allison et al. (1985a, 1985b, 1986) , herein incorporated by reference. cDNA synthesis was accomplished as follows: Purified viral RNA primed with oligo(dT 12 -ι 8 ) served as a template for single-strand cDNA synthesis by reverse transcriptase. Following the addition of homopolymeric tracts of deoxycytidine 5' monophosphate, second-strand synthesis, primed with oligo(dG 12 -ι 3 ) , was completed with DNA polymerase I. Sail and .EcoRI linkers were ligated to the double-stranded cDNA and inserted into the bacterial plasmid pBR322 (Kurtz and Nicodemus 1981) . The resulting cDNA clones were screened by colony hybridization (Hanahan and Meselson 1980) with oligo(dT 12 -ι 8 ) primed, 32 P-labeled single-stranded TEV cDNA. Plasmid DNA was isolated from colonies which hybridized with the probe, and the Sall/EcoRI cDNA inserts were sized by electrophoresis in a 0.8% (w/v) agarose gel using a horizontal water-cooled gel apparatus. The Sall/EcoRI inserts from the recombinant molecules were isolated from an agarose gel with NA45 membrane (Schleicher & Schuell, Keene, NH) according to the manufacturer's protocol. The following restriction enzymes were used either alone or in combination to digest the isolated cDNA insert: Hindlll , Xhol , Alul , Haelll , Rsal , Sau3k, and Taql . Restriction enzyme digestion products were inserted into the DNA of an appropriate M13 bacteriophage (Messing 1983) selected for the presence of corresponding polylinker restriction sites, and their nucleotide sequences were determined by dideoxy chain termination. Plasmid pTL 37/8595 (Carrington and Dougherty 1987; Carrington et al. 1987, herein incorporated by reference) contains a cDNA copy of the genomic sequence of HAT TEV corresponding to nucleotides (nt) 1-200 and nt 8462-9495 (Fig. 2) . (Numbering of the TEV genome nucleotides is according to that presented in Allison et al. 1986) . The nucleotide sequence and deduced amino acid sequence of the Tobacco Etch Virus genome and the numbering system utilized by Allison et al. (1986) and herein is shown in Fig. 1 and SEQ ID No. 1 in the attached sequence listing. The first and last codons of the coat protein (CP) coding region in the TEV genome are nt 8518-8520 (encoding the amino acid serine) and 9307-9309 (opal stop codon) respectively. pTL 37/8595 was subject to in vitro site-directed mutagenesis as described by Taylor et al. (1985a, 1985b) herein incorporated by reference. In all cases, nucleotide changes were confirmed by dideoxy-nucleotide sequencing (Sanger et al. 1977) . TEV nt 9312-9317 were first mutated (Fig. 2) to generate a BamEI restriction site (GGATCC) . TEV nt 8516-8521 were then altered to generate an Ncol site (CCATGG) , changing the first codon of the TEV CP coding region from AGT . Ser) , to ATG (Met) . A single oligonucleotide was then used to mutate TEV nt 133-138 to a BamEX restriction site (GGATCC) , nt 143-148 to an NcoX restriction site (CCATGG) and nt 142 to a deoxyadenylate residue. These mutations generated an NcoX site centered on the first codon of the TEV ORF and in a good translational start context as described by Kozak (1984) . Digestion of the resulting plasmid with the restriction enzyme NcoX ; removing TEV nt # 143-200/8462-8516, and religation generated plasmid pTC:FL. pTC:FL contained only the TEV CP gene flanked by BamHI restriction sites and TEV 5' and 3 r untranslated sequences (see Fig. 2) . The nucleotide sequence of the TEV CP gene in pTC:FL produced by this mutagenesis scheme is shown in SEQ ID No. 2 in the attached sequence listing. Plasmid pTC:RC (RNA Control, producing untranslatable plus sense RNA) was generated by insertion of a single deoxythymidylate residue after TEV nt 8529, and point mutations of TEV nt 8522 (G to C) , 8534 (C to A) , 8542 (G to A) , and 8543 (A to G) to create a frameshift mutation immediately followed by three stop codons. An NheX restriction site (GCTAGC) was simultaneously generated, for screening purposes, at nt 8539-8544. The nucleotide sequence of the TEV CP gene in pTC:RC produced by this mutagenesis scheme is shown in SEQ ID No. 3 in the attached sequence listing. All plasmids described above were linearized with H±ndlll, transcribed with T7 RNA polymerase (Melton et al. 1984) , and translated in a rabbit reticulocyte lysate containing 35 S Methionine (Dougherty and Hiebert 1980a) . Radiolabeled translation products were analyzed by electrophoretic separation on a 12.5% acrylamide gel containing SDS (Laemmli 1970) and detected by autoradiography. Transcripts of plasmid pTC:RC produced no detectable protein products, while transcripts from pTC:FL produced proteins of the expected sizes. The various forms of the CP nucleotide sequence were then inserted as BamHI cassettes into the plant expression vector pPEV (see below and Fig. 3) . The full length TEV CP open reading frame of pTC:FL was inserted in the reverse orientation to make the antisense (AS) construct pTC:AS. The nucleotide sequence of the TEV CP gene in pTC:AS is shown in SEQ ID No. 4 in the attached sequence listing. Transformation Vector Construction Construction of pPEV. The vector pPEV is part of a binary vector system for Agrobacterium tumefaciens mediated plant cell transformation. Plasmid pPEV was constructed from the plasmids pCGN 2113 (Calgene) , pCIB 710 and pCIB 200 (Ciba Geigy Corp.). pCGN 2113 contains the "enhanced" Cauliflower Mosaic Virus (CaMV) 35S promoter (CaMV sequences -941 to 90/-363 to +2, relative to the transcription start site) in a pUC derived plasmid backbone. pCIB 710 has been described (Rothstein et al. 1987) and pCIB 200 is a derivative of the wide host range plasmid pTJS 75 (Schmidhauser and Helinski 1985) which contains left and right A. tumefaciens T37 DNA borders, the plant selectable NOS/NPT II chimeric gene from the plasmid Bin 6 (Bevan 1984) and part of a pUC polylinker. The small EcoRX-EcoRV DNA fragment of pCIB 710 (Rothstein et al. 1987) was ligated into EcoRX-EcoRV digested pCGN 2113. This regenerated the enhanced CaMV 35S promoter (Kay et al. 1987) of pCGN 2113 and introduced the CaMV 35S 5' and 3' untranslated sequences into pCGN 2113. The CaMV 35S promoterterminator cassette of the resulting plasmid was isolated as an EσoRI-XJal DNA fragment and ligated into .EcoRI-Xjal digested pCIB 200 to generate pPEV. CP nucleotide sequences from PTC:FL, pTC:RC, and pTC:AS were cloned as BamHX cassettes into BamΕLX digested pPEV and orientation of inserts confirmed by digestion with appropriate restriction endonucleases. Transformation and Regeneration of Tobacco pPEV plasmids containing TEV CP ORFs were mobilized from E. coli HB101 into A. tumefaciens A136 containing plasmid pCIB 542 (Ciba Geigy) , using the helper plasmid pRK 2013 in E. coli HB101 and the tri-parental mating system of Ditta et al. (1980) . Plasmid pCIB 42 supplied vir functions necessary for T-DNA transfer. Leaf discs of Nicotiana tabacum cv Burley 49 were transformed and whole plants regenerated according to Horsch et al. (1985) . Transformed tissue was selected by culturing callus on MS plates (Murashige and Skoog 1962) containing 1 μg/ml 6-benzylaminopurine (Sigma Corp.), 01 μg/ml α-naphthaleneacetic acid (Sigma Corp.), 500 μg/ml carbenicillin and 100 μg/ml Kanamycin sulfate (Sigma Corp.). Shoots were rooted on MS plates containing 500 μg/ml carbenicillin and 100 μg/ml kanamycin sulfate, and plantlets were transplanted into soil and transferred directly into the greenhouse approximately 2-3 weeks after rooting. R0, Rl and R2 generation plants were screened by western and/or northern blot analyses. R2 seed (ca. 100 seeds per R2 plant) was screened for the kanamycin- resistant phenotype (kan r ) by surface sterilizing seed in 10% bleach for 5 min. , washing twice in sterile water and germinating on MS plates containing 100 μg/ml kanamycin sulfate. R2 seed lines which were 100% kanamycin resistant were screened by western blot analysis for expression of TEV coat protein. Those transgenic plant lines generated and their nomenclature are presented in Fig. 3. Molecular Analyses of Transgenic Plants Transgenic tobacco plants were analyzed by western and northern blot analyses to determine the nature of protein and RNA products produced respectively. Total RNA samples isolated from the various transgenic lines were analyzed in northern blot hybridization studies. Total nucleic acids were isolated from tissue and RNA precipitated with LiCl as described by Verwoerd et al. (1989) . RNAs were electrophoretically separated on 1.2% agarose gels containing 6% (v/v) formaldehyde and transferred to nitrocellulose. Prehybridization and hybridization conditions were as described in Sambrook et al. (1989) . Strand specific riboprobes were generated from SP6 ^ or T7 DNA dependent RNA polymerase transcription reactions of pTL 37/8595 linearized with the restriction enzymes Asp718 (Boehringer Mannheim, Indianapolis, IN) or Hindlll , respectively, using α-labelled 32 p-CTP ribonucleotide and suggested procedures (Promega, Madison, WI) . An RNA transcript of approximately 1,000 nt was expected with all transgenic plant lines. Such a TEV CP transcript was detected in CP expressing plant lines by using a minus sense riboprobe containing the TEV CP sequence. A similar transcript was detected in AS plants by using a plus sense riboprobe containing the TEV CP sequence. The transcript in the RC line, while detected with a minus sense riboprobe, may have migrated as a slightly larger (ca 1,100-1,200 nt) RNA species, possibly due to termination at an alternately selected site and/or a longer poly-A tail on the transcript. Differing levels of CP transcript accumulation were observed among different transgenic plant lines. Transgenic plant lines expressing the coat protein of TEV were identified by western blot analysis using polyclonal antisera to TEV CP. Tissue samples of regenerated plants were ground in 10 volumes of 2X Laemmli (Tris-glicine) runner buffer (Laemmli 1970) and clarified by centrifugation in a microcentrifuge for 10 min. at 10,000xg. Protein concentration was estimated by the dye binding procedure of Bradford (1976) using BSA as a standard. Protein samples (50 μg total protein) were separated on a 12.5% polyacrylamide gel containing SDS and subjected to the i munoblot transfer procedures described by Towbin et al. (1979) . Anti-TEV coat protein polyclonal primary antibodies, alkaline phosphatase conjugated secondary antibodies and the chromogenic substrates NBT (para-nitro blue tetrazolium chloride) and BCIP (5-bromo-4-chloro-3-indoyl phosphate para-toluidine salt) were used to detect bound antigen. Coat protein products produced in FL plants were stable and accumulated to different levels in individual transgenic plant lines. It was estimated by western blot analysis that between 0.01% to 0.001% of total extracted protein was TEV CP. Assessment of Resistance to TEV Eight-week-old (circa 15 cm tall) Rl and R2 plants were inoculated with either purified virus preparations or infected plant sap. Inoculum was applied with sterile, premoistened cotton swabs. Infected plant sap inoculum was prepared by grinding TEV-infected N. tabacum Burley 21 leaf tissue (2 weeks postinoculation) in carborundum and 50 mM sodium phosphate buffer (pH 7.8) at a ratio of lgm:02gm:10mls, respectively, and filtering the homogenate through cheesecloth. TEV virons were purified as described by Dougherty and Hiebert (1980b) . One leaf per plant was dusted lightly with carborundum (320 grit) and inoculated at two interveinal locations with 50 μl (total) of inoculum. Inoculated plants were examined daily and the appearance and severity of systemic symptoms recorded. Symptoms on any leaf above the inoculated leaf were considered to be systemic. Typically, inoculation of Burley 49 plants with TEV (either purified virus or plant sap) resulted in severe chlorosis and mosaic and mottle on systemically infected leaves approximately 6-7 days after inoculation. Severe etching of the leaf followed within a few days. It was observed that transgenic plants containing only the CaMV promoter and untranslated sequences (i.e., 35S plant line) responded to challenge inoculation in a manner similar to wild type Burley 49, developing extensive chlorosis and etching at the same rate (Fig. 4A) . Plant lines which expressed FL TEV CP showed little or no delay in the appearance of symptoms when inoculated with infected plant sap. However, FL transgenic plants did show a slight attenuation of symptoms and eventually (2-4 weeks after initial appearance of symptoms) , younger leaf tissue emerged devoid of symptoms and virus as demonstrated by bacjc inoculation experiments. Typically chlorosis and etching on older systemic leaves was limited. Ten independently transformed RC lines and seven independently transformed AS lines were obtained. Progeny from three of the RC lines, including line RC #5 and from one of the AS lines, including AS #3, showed an altered response to viral infection relative to control plants. All of these lines were verified to be transformed and were producing expected RNA products. A possible explanation for the variation in observed phenotype is the previously noted "position effect" whereby the expression of genes from identical DNA sequences integrated at different locations within the genome show varying patterns of tissue specificity. Ten R2 expressing plants of the FL expressing line were inoculated with infected plant sap, and 20 Rl plants of lines AS #3 and RC #5 were inoculated with 50 μl of a 5 μg/ml solution of purified TEV. Identical results to those obtained by purified TEV inoculation were obtained when AS #3 and RC #5 Rl plants were inoculated with TEV-infected plant sap, as described above. Transgenic Burley 49 plant lines AS #3 and RC #5, expressing only TEV CP related RNA sequences, showed a delay in the appearance of symptoms and a modification of symptoms when inoculated with TEV (Fig. 4B) . Since the 20 Rl plants were not screened for expression of CP RNA prior to inoculation, some of the symptomatic plants represented non-expressing plants in which the gene of interest had been lost during Mendelian segregation. Modified symptoms on AS #3 plants appeared as small chlorotic lesions often associated with a vein. Most of the leaves were devoid of symptoms and virus (determined by back inoculation experiments) . Approximately 15% of RC #5 plants showed symptoms which were identical to those of infected Burley 49. However, the remaining RC #5 plants were entirely asymptomatic, and virus was not detected in back inoculation studies. Plants from TEV resistant AS and RC lines showed no increased resistance, relative to untransformed controls, to infection by two other members of the potyvirus family, namely Tobacco Vein Mottling Virus and Potato Virus Y. R 2 generation plants derived from TEV-resistant RC plants showed the expected Mendelian pattern of inheritance of the TEV-resistant phenotype. Analysis of TEV Replication in Protoplasts Derived from Transgenic Plant Lines In an attempt to explain the results obtained when AS and RC transgenic plants were challenged with TEV, it was sought to determine if all of the transgenic plant lines would support virus replication at a level comparable to Burley 49. Accumulation of viral encoded proteins was used as an indirect indicator of viral replication. Protoplasts were derived from leaf tissue of homozygous CP expressing plants and electroporated according to the procedure of Luciano et al. (1987) with TEV RNA. Protoplasts were prepared from transgenic plants and electroporated according to the procedure of Luciano et al. (1987) . Protoplasts (1 X 10 6 ) were resuspended in 450 μl electroporation buffer (330 mM mannitol, 1 mM KP0 < , pH 7.0, 150 M KC1) and electroporated using a BTX Transfector 300 (BTX San Diego, CA) (950 micro Farads, 130-volt pulse amplitude, 3.5 mm electrode gap) in the presence or absence of 6 μg of purified TEV RNA. After electroporation, protoplasts were incubated for 96 hours in incubation medium as described in Luciano et al. (1987) . Protoplasts were extracted in 2X Laemmli (Trisglycine) running buffer, and 5 x 10* extracted protoplasts were then subjected to western blot analysis as described above. Protoplast viability was measured by dye exclusion as described in Luciano et al. (1987). All electroporated protoplast samples had equivalent viability counts. The results indicated that protoplasts from all FL plant lines supported virus replication at levels comparable to wild type Burley 49 protoplasts. Rl transgenic plants from lines AS #3 and RC #5 were initially screened by northern analysis, and leaves from positive expressors were used in the production of protoplasts. Transfected protoplasts derived from AS #3 plants supported TEV replication, albeit at a reduced level. Protoplasts derived from RC #5 transgenic plant leaf tissue did not support TEV replication at a detectable level. These results, and those presented in the whole plant inoculation series, suggested AS and RC plants interfere with TEV replication. Discussion of Data The above example indicates that varying degrees of protection from TEV infection can be achieved by overexpression of coat protein and by expression of an antisense RNA. The current invention which comprises the expression of an untranslatable plus sense RNA molecule provides protection against TEV infection that is more effective than either of these two methods. Plants of line RC #5, transformed with the disclosed DNA molecule encoding an untranslatable plus sense RNA derived from the TEV coat protein gene, were asymptomatic and appear to be completely protected from virus infection. The disclosed invention therefore represents a new and effective way of generating potyvirus resistant germplasm. Tobacco protoplasts derived from plants expressing the antisense RNA supported a reduced level of TEV replication compared to control cells derived from untransformed plants. In contrast, tobacco protoplasts derived from plants of line RC ' #5, expressing the untranslatable plus sense RNA did not support detectable TEV replication. This suggests that the untranslatable plus sense RNA was more effective at blocking TEV replication in the cells of those transformed plants tested. It is proposed that the untranslatable plus sense RNA inhibits viral replication by hybridizing to the minus sense RNA replicative template of TEV. The finding that plants expressing untranslatable plus sense RNA derived from the TEV coat protein gene are not protected from infection by Potato Virus Y or Tobacco Vein Mottling Virus is therefore explained by the circa 40-50% amino acid sequence divergence between the coat proteins of these viruses and TEV (Allison et al. 1986; Robaglia et al. 1989; Domier et al. 1986). From the above-described findings, it would be reasonable and entirely predictable that if plants were transformed with a gene encoding an untranslatable plus sense RNA derived from a gene which was highly conserved between viruses of the potyvirus family, that these plants would be protected from infection by a wide range of viruses. Regions of the potyvirus genome which are sufficiently conserved between potyvirus types to be potentially useful in such an approach may be readily determined by one skilled in the art. Highly conserved regions may be determined by reference to published sequence data (Allison et al. 1986; Robaglia et al. 1989; Domier et al. 1986; Lain et al. 1989; Maiss et al. 1989) . The utility of the identified regions could be readily determined using the methodologies described above and substituting the defined region for the TEV coat protein gene. Regions of the potyvirus genome potentially suitable include, but are not limited to the genes encoding the viral replicase and the viral proteinase. Furthermore, it will be apparent to one skilled in the art that highly conserved portions of a particular gene may also serve in this role. It will also be apparent to one skilled in the art that the described invention may also be used to produce plants resistant to viruses outside of the potyvirus family in instances where these viruses also produce a minus sense RNA replicative template. BIBLIOGRAPHY Allison et al. 1985a. Virology 147:309-316. Allison et al. 1985b. Proc. Natl. Acad. Sci. USA £2:3969-3972. Allison et al. 1986. Virology 154:9-20. Bevan 1984. Nucl. Acids Res. 11:8711-8721. Bradford 1976. Nucl. Acids Res. 12:8711-8721. Carrington and Dougherty 1987. J. Virol. :2540-2548. Carrington et al. 1987. Nucl. Acids Res. 15:10066. Ditta et al. 1980. Proc. Natl. Acad. Sci. USA 77:7347-7351. Domier et al. 1986. Nucl. Acids Res. 14.:5417-5430. Dougherty and Hiebert 1980a. Virology 101:466-474. Dougherty and Hiebert 1980b. Virology 104.:183-194. Hanahan and Meselson 1980. Gene 10:63-67. Herrera-Estrella et al. 1983. Nature (London) 303:209-213. Hollings and Brunt 1981. Commonwealth Mycological Institute/Association of Applied Biologists Descrip. Plant Viruses. No. 245. Horsch et al. 1985. Science 227:1229-1231. Kado and Agrawai 1972. Principles and Techniques in Plant Virology. Kay et al. 1987. Science 236:1299-1302. Kozak 1984. Nature (London) 308:241-246. Kurtz and Nicodemus 1981. Gene 13:145-152. Laemmli 1970. Nature (London) 227:680-685. Lain et al. 1989. Virus Res. 13:157-172. Lawson et al. 1990. Bio/Technology 8.:127-134. Ling et al. 1991. Bio/Technology 9:752-758. Luciano et al. 1987. Plant Science 51:295-303. Maiss et al. 1989. J. Gen. Virol. 70:513-524. Melton et al. 1984. Nucl.■ Acids Res. 12.:7145-7156. Messing 1983. "Methods in Enzymology" (R. Wu, L. Grossman, and K. Moldave, eds.). Vol. 101c, pp. 20-78. Academic Press, New York. Murashige and Skoog 1962. Plant Physiol. 15:473-497. Robaglia et al. 1989. J. Gen. Virol. 20:935-947. Rothstein et al. 1987. Gene 53:153-161. Sambrook et al. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratories, Cold Spring Harbor, New York. Sanford and Johnston 1985. J. Theor. Biol. 113:395-405. Sanger et al. 1977. Proc. Natl. Acad. Sci. USA 24:5463-5467. Schmidhauser and Helinski 1985. J. Bact. 164:446-455. Stark and Beachy 1989. Bio/Technology i1257-1262. Tabler and Tsagris 1991. Gene 100:175-183. Taylor et al. 1985a. Nucl. Acids Res. 13.:8749-8764. Taylor et al. 1985b. Nucl. Acids Res. 13.:8765-8785. Towbin et al. 1979. Proc. Natl. Acad. Sci. USA 6:4350-4354. Verwoerd et al. 1989. Nucl. Acids Res. 12=2372. SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: William G. Dougherty and John A. Lindbo (ii) TITLE OF INVENTION: Production of Plants Showing Immunity to Viral Infection via Introduction of Genes Encoding Untranslatable Plus Sense RNA Molecules (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Richard J. Polley (B) STREET: One World Trade Center 121 S.W. Salmon Street, Suite 1600 (C) CITY: Portland (D) STATE: Oregon (E) COUNTRY: United States of America (F) ZIP: 97204 (V) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette, 5.25 inch (B) COMPUTER: IBM PC Compatible (C) OPERATING SYSTEM: MS DOS (D) SOFTWARE: WordPerfect 5.1 (Vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: 07/838,509 (B) FILING DATE: February 19, 1992 (C) CLASSIFICATION: 435 (vi) PRIOR APPLICATION DATA: None (Vii) ATTORNEY/AGENT INFORMATION (A) NAME: Richard J. Polley, Esq. (B) REGISTRATION NUMBER: 28,107 (C) REFERENCE/DOCKET NUMBER: 245-35829/RJP (viii) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (503) 226-7391 (B) TELEFAX: (503) 228-9446 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 9495 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (ii) MOLECULE TYPE: (A) DESCRIPTION: cDNA to genomic RNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: No (V) FRAGMENT TYPE: N/A (Vi) ORIGINAL SOURCE: (A) ORGANISM: Tobacco Etch Virus (TEV) (B) STRAIN: Highly Aphid Transmitted (HAT) (vii) IMMEDIATE SOURCE: TEV propagated in N. tabacum N/A Coat protein gene Genomic nucleotides 4ATION: SEQ. ID No. 1 is the cDNA corresponding to the Tobacco Etch Virus Genome. (X) PUBLICATION INFORMATION: (A) AUTHORS: Allison et al. (B) TITLE: The nucleotide sequence of the coding region of Tobacco Etch Virus Genomic RNA: Evidence for the Synthesis of a Single Polyprotein (C) JOURNAL: Virology (D) VOLUME: 154 (E) ISSUE: — (F) PAGES : 9-20 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1: NAAATAACAA ATCTCAACAC AACATATACA AAACAAACGA ATCTCAAGCA ATCAAGCATT 60 CTACTTCTAT TGCAGCAATT TAAATCATTT CTTTTAAAGC AAAAGCAATT TTCTGAAAAT 120 TTTCACCATT TACGAACGAT AGCA ATG GCA CTG ATC TTT GGC ACA GTC AAC GCT 174 Met Ala Leu lie Phe Gly Thr Val Asn Ala 1 5 10 AAC ATC CTG AAG GAA GTG TTC GGT GGA GCT CGT ATG GCT TGC GTT ACC 222 Asn He Leu Lys Glu Val Phe Gly Gly Ala Arg Met Ala Cys Val Thr 15 20 25 AGC GCA CAT ATG GCT GGA GCG AAT GGA AGC ATT TTG AAG AAG GCA GAA 270 Ser Ala His Met Ala Gly Ala Asn Gly Ser He Leu Lya Lys Ala Glu 30 35 40 GAG ACC TCT CGT GCA ATC ATG CAC AAA CCA GTG ATC TTC GGA GAA GAC 318 Glu Thr Ser Arg Ala He Met His Lys Pro Val He Phe Gly Glu Asp 45 50 55 TAC ATT ACC GAG GCA GAC TTG CCT TAC ACA CCA CTC CAT TTA GAG GTC 366 Tyr He Thr Glu Ala Asp Leu Pro Tyr Thr Pro Leu His Leu Glu Val 60 65 70 GAT GCT GAA ATG GAG CGG ATG TAT TAT CTT GGT CGT CGC GCG CTC ACC 414 Asp Ala Glu Met Glu Arg Met Tyr Tyr Leu Gly Arg Arg Ala Leu Thr 75 80 85 90 CAT GGC AAG AGA CGC AAA GTT TCT GTG AAT AAC AAG AGG AAC AGG AGA 462 His Gly Lys Arg Arg Lys Val Ser Val Asn Asn Lys Arg Asn Arg Arg 95 100 105 AGG AAA GTG GCC AAA ACG TAC GTG GGG CGT GAT TCC ATT GTT GAG AAG 510 Arg Lys Val Ala Lys Thr Tyr Val Gly Arg Asp Ser He Val Glu Lys 110 115 120 ATT GTA GTG CCC CAC ACC GAG AGA AAG GTT GAT ACC ACA GCA GCA GTG 558 He Val Val Pro His Thr Glu Arg Lys Val Asp Thr Thr Ala Ala Val 125 130 135 GAA GAC ATT TGC AAT GAA GCT ACC ACT CAA CTT GTG CAT AAT AGT ATG 606 Glu Asp He Cys Asn Glu Ala Thr Thr Gin Leu Val His Asn Ser Met 140 145 150 CCA AAG CGT AAG AAG CAG AAA AAC TTC TTG CCC GCC ACT TCA CTA AGT 654 Pro Lys Arg Lys Lys Gin Lys Asn Phe Leu Pro Ala Thr Ser Leu Ser 155 160 165 170 AAC GTG TAT GCC CAA ACT TGG AGC ATA GTG CGC AAA CGC CAT ATG CAG 702 Asn Val Tyr Ala Gin Thr Trp Ser He Val Arg Lys Arg His Met Gin 175 180 185 GTG GAG ATC ATT AGC AAG AAG AGC GTC CGA GCG AGG GTC AAG AGA TTT 750 Val Glu He He Ser Lys Lys Ser Val Arg Ala Arg Val Lys Arg Phe 190 195 200 GAG GGC TCG GTG CAA TTG TTC GCA AGT GTG CGT CAC ATG TAT GGC GAG 798 Glu Gly Ser Val Gin Leu Phe Ala Ser Val Arg His Met Tyr Gly Glu 205 210 215 AGG AAA AGG GTG GAC TTA CGT ATT GAC AAC TGG CAG CAA GAG ACA CTT 846 Arg Lys Arg Val Asp Leu Arg He Asp Asn Trp Gin Glή Glu Thr Leu 220 225 230 CTA GAC CTT GCT AAA AGA TTT AAG AAT GAG AGA GTG GAT CAA TCG AAG 894 Leu Asp Leu Ala Lys Arg Phe Lys Asn Glu Arg Val Asp Gin Ser Lys 235 240 245 250 CTC ACT TTT GGT TCA AGT GGC CTA GTT TTG AGG CAA GGC TCG TAC GGA 942 Leu Thr Phe Gly Ser Ser Gly Leu Val Leu Arg Gin Gly Ser Tyr Gly 255 260 265 CCT GCG CAT TGG TAT CGA CAT GGT ATG TTC ATT GTA CGC GGT CGG TCG 990 Pro Ala His Trp Tyr Arg His Gly Met Phe He Val Arg Gly Arg Ser 270 275 280 GAT GGG ATG TTG GTG GAT GCT CGT GCG AAG GTA ACG TTC GCT GTT TGT 1038 Asp Gly Met Leu Val Asp Ala Arg Ala Lys Val Thr Phe Ala Val Cys 285 290 295 CAC TCA ATG ACA CAT TAT AGC GAC AAA TCA ATC TCT GAG GCA TTC TTC 1086 His Ser Met Thr His Tyr Ser Asp Lys Ser He Ser Glu Ala Phe Phe 300 305 310 ATA CCA TAC TCT AAG AAA TTC TTG GAG TTG AGA CCA GAT GGA ATC TCC 1134 He Pro Tyr Ser Lys Lys Phe Leu Glu Leu Arg Pro Asp Gly He Ser 315 320 325 330 CAT GAG TGT ACA AGA GGA GTA TCA GTT GAG CGG TGC GGT GAG GTG GCT 1182 His Glu Cys Thr Arg Gly Val Ser Val Glu Arg Cys Gly Glu Val Ala 335 340 345 GCA ATC CTG ACA CAA GCA CTT TCA CCG TGT GGT AAG ATC ACA TGC AAA 1230 Ala He Leu Thr Gin Ala Leu Ser Pro Cys Gly Lys He Thr Cys Lys x 350 355 360 CGT TGC ATG GTT GAA ACA CCT GAC ATT GTT GAG GGT GAG TCG GGA GAA 1278 Arg Cys Met Val Glu Thr Pro Asp He Val Glu Gly Glu Ser Gly Glu 365 370 375 AGT GTC ACC AAC CAA GGT AAG CTC CTA GCA ATG CTG AAA GAA CAG TAT 1326 Ser Val Thr Asn Gin Gly Lys Leu Leu Ala Met Leu Lys Glu Gin Tyr 380 385 390 CCA GAT TTC CCA ATG GCC GAG AAA CTA CTC ACA AGG TTT TTG CAA CAG 1374 Pro Asp Phe Pro Met Ala Glu Lys Leu Leu Thr Arg Phe Leu Gin Gin 395 400 405 410 AAA TCA CTA GTA AAT ACA AAT TTG ACA GCC TGC GTG AGC GTC AAA CAA 1422 Lys Ser Leu Val Asn Thr Asn Leu Thr Ala Cys Val Ser Val Lys Gin 415 420 425 CTC ATT GGT GAC CGC AAA CAA GCT CCA TTC ACA CAC GTA CTG GCT GTC 1470 Leu He Gly Asp Arg Lys Gin Ala Pro Phe Thr His Val Leu Ala Val 430 435 440 AGC GAA ATT CTG TTT AAA GGC AAT AAA CTA ACA GGG GCT GAT CTC GAA 1518 Ser Glu He Leu Phe Lys Gly Asn Lys Leu Thr Gly Ala Asp Leu Glu 445 450 455 GAG GCA AGC ACA CAT ATG CTT GAA ATA GCA AGG TTC TTG AAC AAT CGC 1566 Glu Ala Ser Thr His Met Leu Glu He Ala Arg Phe Leu Asn Asn Arg 460 465 470 ACT GAA AAT ATG CGC ATT GGC CAC CTT GGT TCT TTC AGA AAT AAA ATC 1614 Thr Glu Asn Met Arg He Gly His Leu Gly Ser Phe Arg Asn Lys He 475 480 485 490 TCA TCG AAG GCC CAT GTG AAT AAC GCA CTC ATG TGT GAT AAT CAA CTT 1662 Ser Ser Lys Ala His Val Asn Asn Ala Leu Met Cys Asp Asn Gin Leu 495 500 505 GAT CAG AAT GGG AAT TTT ATT TGG GGA CTA AGG GGT GCA CAC GCA AAG 1710 Asp Gin Asn Gly Asn Phe He Trp Gly Leu Arg Gly Ala His Ala Lys 510 515 520 AGG TTT CTT AAA GGA TTT TTC ACT GAG ATT GAC CCA AAT GAA GGA TAC 1758 Arg Phe Leu Lys Gly Phe Phe Thr Glu He Asp Pro Asn Glu Gly Tyr 525 530 535 GAT AAG TAT GTT ATC AGG AAA CAT ATC AGG GGT AGC AGA AAG CTA GCA 1806 Asp Lys Tyr Val He Arg Lys His He Arg Gly Ser Arg Lys Leu Ala 540 545 550 ATT GGC AAT TTG ATA ATG TCA ACT GAC TTC CAG ACG CTC AGG CAA CAA 1854 He Gly Asn Leu He Met Ser Thr Asp Phe Gin Thr Leu Arg Gin Gin 555 560 565 570 ATT CAA GGC GAA ACT ATT GAG CGT AAA GAA ATT GGG AAT CAC TGC ATT 1902 He Gin Gly Glu Thr He Glu Arg Lys Glu He Gly Asn His Cys He 575 580 585 TCA ATG CGG AAT GGT AAT TAC GTG TAC CCA TGT TGT TGT GTT ACT CTT 1950 Ser Met Arg Asn Gly Asn Tyr Val Tyr Pro Cys Cys Cys Val Thr Leu 590 595 600 GAA GAT GGT AAG GCT CAA TAT TCG GAT CTA AAG CAC CCA ACG AAG AGA 1998 Glu Asp Gly Lys Ala Gin Tyr Ser Asp Leu Lys His Pro Thr Lys Arg ' 605 610 615 CAT CTG GTC ATT GGC AAC TCT GGC GAT TCA AAG TAC CTA GAC CTT CCA 2046 His Leu Val He Gly Asn Ser Gly Asp Ser Lys Tyr Leu Asp Leu Pro 620 625 630 GTT CTC AAT GAA GAG AAA ATG TAT ATA GCT AAT GAA GGT TAT TGC TAC 2094 Val Leu Asn Glu Glu Lys Met Tyr He Ala Asn Glu Gly Tyr Cys Tyr 635 640 645 650 ATG AAC ATT TTC TTT GCT CTA CTA GTG AAT GTC AAG GAA GAG GAT GCA 2142 Met Asn He Phe Phe Ala Leu Leu Val Asn Val Lys Glu Glu Asp Ala 655 660 665 AAG GAC TTC ACC AAG TTT ATA AGG GAC ACA ATT GTT CCA AAG CTT GGA 2190 Lys Asp Phe Thr Lys Phe He Arg Asp Thr He Val Pro Lys Leu Gly 670 675 680 GCG TGG CCA ACA ATG CAA GAT GTT GCA ACT GCA TGC TAC TTA CTT TCC 2238 Ala Trp Pro Thr Met Gin Asp Val Ala Thr Ala Cys Tyr Leu Leu Ser 685 690 695 ATT CTT TAC CCA GAT GTC CTG AGA GCT GAA CTA CCC AGA ATT TTG GTT 2286 He Leu Tyr Pro Asp Val Leu Arg Ala Glu Leu Pro Arg He Leu Val 700 705 710 GAT CAT GAC AAC AAA ACA ATG CAT GTT TTG GAT TCG TAT GGG TCT AGA 2334 Asp His Asp Asn Lys Thr Met His Val Leu Asp Ser Tyr Gly Ser Arg 715 720 725 730 ACG ACA GGA TAC CAC ATG TTG AAA ATG AAC ACA ACA TCC CAG CTA ATT 2382 Thr Thr Gly Tyr His Met Leu Lys Met Asn Thr Thr Ser Gin Leu He 735 740 745 GAA TTC GTT CAT TCA GGT TTG GAA TCC GAA ATG AAA ACT TAC AAT GTT 2430 Glu Phe Val His Ser Gly Leu Glu Ser Glu Met Lys Thr Tyr Asn Val 750 755 760 GGA GGG ATG AAC CGA GAT GTG GTC ACA CAA GGT GCA ATT GAG ATG TTG 2478 Gly Gly Met Asn Arg Asp Val Val Thr Gin Gly Ala He Glu Met Leu 765 770 775 ATC AAG TCT ATA TAC AAA CCA CAT CTC ATG AAG CAG TTA CTT GAG GAA 2526 He Lys Ser He Tyr Lys Pro His Leu Met Lys Gin Leu Leu Glu Glu 780 785 790 GAG CCA TAC ATA ATT GTC CTG GCA ATA GTC TCC CCT TCA ATT TTA ATT 2574 Glu Pro Tyr He He Val Leu Ala He Val Ser Pro Ser He Leu He 795 800 805 810 GCC ATG TAC AAC TCT GGA ACT TTT GAG CAG GCG TTA CAA ATG TGG TTG 2622 Ala Met Tyr Asn Ser Gly Thr Phe Glu Gin Ala Leu Gin Met Trp Leu 815 820 825 CCA AAT ACA ATG AGG TTA GCT AAC CTC GCT GCC ATC TTG TCA GCC TTA 2670 Pro Asn Thr Met Arg Leu Ala Asn Leu Ala Ala He Leu Ser Ala Leu 830 835 840 GCG CAA AAG TTA ACT TTG GCA GAT TTG TTC GTC CAG CAG CGT AAT TTG 2718 Ala Gin Lys Leu Thr Leu Ala Asp Leu Phe Val Gin Gin Arg Asn Leu 845 850 855 ATT AAT GAG TAT GCG CAG GTA ATT TTG GAC AAT CTG ATT GAC GGT GTC 2766 He Asn Glu Tyr Ala Gin Val He Leu Asp Asn Leu He Asp Gly Val 860 . 865 870 AGG GTT AAT CAT TCG CTA TCC CTA GCA ATG GAA ATT GTT ACT ATT AAG 2814 Arg Val Asn His Ser Leu Ser Leu Ala Met Glu He Val Thr He Lys 875 880 885 890 CTG GCC ACC CAA GAG ATG GAC ATG GCG TTG AGG GAA GGT GGC TAT GCT 2862 Leu Ala Thr Gin Glu Met Asp Met Ala Leu Arg Glu Gly Gly Tyr Ala 895 900 905 GTG ACC TCT GAA AAG GTG CAT GAA ATG TTG GAA AAA AAC TAT GTA AAG 2910 Val Thr Ser Glu Lys Val His Glu Met Leu Glu Lys Asn Tyr Val Lys 910 915 920 GCT TTG AAG GAT GCA TGG GAC GAA TTA ACT TGG TTG GAA AAA TTC TCC 2958 Ala Leu Lys Asp Ala Trp Asp Glu Leu Thr Trp Leu Glu Lys Phe Ser 925 930 935 GCA ATC AGG CAT TCA AGA AAG CTC TTG AAA TTT GGG CGA AAG CCT TTA 3006 Ala He Arg His Ser Arg Lys Leu Leu Lys Phe Gly Arg Lys Pro Leu 940 945 950 ATC ATG AAA AAC ACC GTA GAT TGC GGC GGA CAT ATA GAC TTG TCT GTG 3054 He Met Lys Asn Thr Val Asp Cys Gly Gly His He Asp Leu Ser Val 955 960 965 970 AAA TCG CTT TTC AAG TTC CAC TTG GAA CTC CTG AAG GGA ACC ATC TCA 3102 Lys Ser Leu Phe Lys Phe His Leu Glu Leu Leu Lys Gly Thr He Ser 975 980 985 AGA GCC GTA AAT GGT GGC GCA AGA AAG GTA AGA GTA GCG AAG AAT GCC 3150 Arg Ala Val Asn Gly Gly Ala Arg Lys Val Arg Val Ala Lys Asn Ala 990 995 1000 ATG ACA AAA GGG GTT TTT CTC AAA ATC TAC AGC ATG CTT CCT GAC GTC 3198 Met Thr Lys Gly Val Phe Leu Lys He Tyr Ser Met Leu Pro Asp Val 1005 1010 1015 TAC AAG TTT ATC ACA GTC TCG AGT GTC CTT TCC TTG TTG TTG ACA TTC 3246 Tyr Lys Phe He Thr Val Ser Ser Val Leu Ser Leu Leu Leu Thr Phe 1020 1025 1030 TTA TTT CAA ATT GAC TGC ATG ATA AGG GCA CAC CGA GAG GCG AAG GTT 3294 Leu Phe Gin He Asp Cys Met He Arg Ala His Arg Glu Ala Lys Val 1035 1040 1045 1050 GCT GCA CAG TTG CAG AAA GAG AGC GAG TGG GAC AAT ATC ATC AAT AGA 3342 Ala Ala Gin Leu Gin Lys Glu Ser Glu Trp Asp Asn He He Asn Arg 1055 1060 1065 ACT TTC CAG TAT TCT AAG CTT GAA AAT CCT ATT GGC TAT CGC TCT ACA 3390 Thr Phe Gin Tyr Ser Lys Leu Glu Asn Pro He Gly Tyr Arg Ser Thr 1070 1075 1080 GCG GAG GAA AGA CTC CAA TCA GAA CAC CCC GAG GCT TTC GAG TAC TAC 3438 Ala Glu Glu Arg Leu Gin Ser Glu His Pro Glu Ala Phe Glu Tyr Tyr 1085 1090 1095 AAG TTT TGC ATT GGA AAG GAA GAC CTC GTT GAA CAG GCA AAA CAA CCG 3486 Lys Phe Cys He Gly Lys Glu Asp Leu Val Glu Gin Ala Lys Gin Pro 1100 1105 1110 GAG ATA GCA TAC TTT GAA AAG ATT ATA GCT TTC ATC ACA CTT GTA TTA 3534 Glu He Ala Tyr Phe Glu Lys He He Ala Phe He Thr Leu Val Leu 1115 1120 1125 1130 ATG GCT TTT GAC GCT GAG CGG AGT GAT GGA GTG TTC AAG ATA CTC AAT 3582 Met Ala Phe Asp Ala Glu Arg Ser Asp Gly Val Phe Lys He Leu Asn 1135 1140 1145 AAG TTC AAA GGA ATA CTG AGC TCA ACG GAG AGG GAG ATC ATC TAC ACG 3630 Lys Phe Lys Gly He Leu Ser Ser Thr Glu Arg Glu He He Tyr Thr 1150 1155 1160 CAG AGT TTG GAT GAT TAC GTT ACA ACC TTT GAT GAC AAT ATG ACA ATC 3678 Gin Ser Leu Asp Asp Tyr Val Thr Thr Phe Asp Asp Asn Met Thr He 1165 1170 1175 AAC CTC GAG TTG AAT ATG GAT GAA CTC CAC AAG ACG AGC CTT CCT GGA 3726 Asn Leu Glu Leu Asn Met Asp Glu Leu His Lys Thr Ser Leu Pro Gly 1180 1185 1190 GTC ACT TTT AAG CAA TGG TGG AAC AAC CAA ATC AGC CGA GGC AAC GTG 3774 Val Thr Phe Lys Gin Trp Trp Asn Asn Gin He Ser Arg Gly Asn Val 1195 1200 1205 1210 AAG CCA CAT TAT AGA ACT GAG GGG CAC TTC ATG GAG TTT ACC AGA GAT 3822 Lys Pro His Tyr Arg Thr Glu Gly His Phe Met Glu Phe Thr Arg Asp 1215 1220 1225 ACT GCG GCA TCG GTT GCC AGC GAG ATA TCA CAC TCA CCC GCA AGA GAT 3870 Thr Ala Ala Ser Val Ala Ser Glu He Ser His Ser Pro Ala Arg Asp 1230 1235 1240 TTT CTT GTG AGA GGT GCT GTT GGA TCT GGA AAA TCC ACA GGA CTT CCA 3918 Phe Leu Val Arg Gly Ala Val Gly Ser Gly Lys Ser Thr Gly Leu Pro 1245 1250 1255 TAC CAT TTA TCA AAG AGA GGG AGA GTG TTA ATG CTT GAG CCT ACC AGA 3966 Tyr His Leu Ser Lys Arg Gly Arg Val Leu Met Leu Glu Pro Thr Arg 1260 1265 1270 CCA CTC ACA GAT AAC ATG CAC AAG CAA CTG AGA AGT GAA CCA TTT AAC 4014 Pro Leu Thr Asp Asn Met His Lys Gin Leu Arg Ser Glu Pro Phe Asn 1275 1280 1285 1290 TGC TTC CCA ACT TTG AGG ATG AGA GGG AAG TCA ACT TTT GGG TCA TCA 4062 Cys Phe Pro Thr Leu Arg Met Arg Gly Lys Ser Thr Phe Gly Ser Ser 1295 1300 1305 CCG ATC ACA GTC ATG ACT AGT GGA TTC GCT TTA CAC CAC TTT GCA CGA 4110 Pro He Thr Val Met Thr Ser Gly Phe Ala Leu His His Phe Ala Arg 1310 1315 1320 AAC ATA GCT GAG GTA AAA ACA TAC GAT TTT GTC ATA ATT GAT GAA TGT 4158 Asn He Ala Glu Val Lys Thr Tyr Asp Phe Val He He Asp Glu Cys 1325 1330 1335 CAT GTG AAT GAT GCT TCT GCT ATA GCG TTT AGG AAT CTA CTG TTT GAA 4206 His Val Asn Asp Ala Ser Ala He Ala Phe Arg Asn Leu Leu Phe Glu 1340 1345 1350 CAT GAA TTT GAA GGA AAA GTC CTC AAA GTG TCA GCC ACA CCA CCA GGT 4254 His Glu Phe Glu Gly Lys Val Leu Lys Val Ser Ala Thr Pro Pro Gly 1355 1360 1365 1370 AGA GAA GTT GAA TTT ACA ACT CAG TTT CCC GTG AAA CTC AAG ATA GAA 4302 Arg Glu Val Glu Phe Thr Thr Gin Phe Pro Val Lys Leu Lys He Glu 1375 1380 1385 GAG GCT CTT AGC TTT CAG GAA TTT GTA AGT TTA CAA GGG ACA GGT GCC 4350 Glu Ala Leu Ser Phe Gin Glu Phe Val Ser Leu Gin Gly Thr Gly Ala 1390 1395 1400 AAC GCC GAT GTG ATT AGT TGT GGC GAC AAC ATA CTA GTA TAT GTT GCT 4398 Asn Ala Asp Val He Ser Cys Gly Asp Asn He Leu Val Tyr Val Ala 1405 1410 1415 AGC TAC AAT GAT GTT GAT AGT CTT GGC AAG CTC CTT GTG CAA AAG GGA 4446 Ser Tyr Asn Asp Val Asp Ser Leu Gly Lys Leu Leu Val Gin Lys Gly 1420 1425 1430 TAC AAA GTG TCG AAG ATT GAT GGA AGA ACA ATG AAG AGT GGA GGA ACT 4494 Tyr Lys Val Ser Lys He Asp Gly Arg Thr Met Lys Ser Gly Gly Thr 1435 1440 1445 1450 GAA ATA ATC ACT GAA GGT ACT TCA GTG AAA AAG CAT TTC ATA GTC GCA 4542 Glu He He Thr Glu Gly Thr Ser Val Lys Lys His Phe He Val Ala 1455 1460 1465 ACT AAC ATT ATT GAG AAT GGT GTA ACC ATT GAC ATT GAT GTA GTT GTG 4590 Thr Asn He He Glu Asn Gly Val Thr He Asp He Asp Val Val Val 1470 1475 1480 GAT TTT GGG ACT AAG GTT GTA CCA GTT TTG GAT GTG GAC AAT AGA GCG 4638 Asp Phe Gly Thr Lys Val Val Pro Val Leu Asp Val Asp Asn Arg Ala 1481 1490 1495 GTG CAG TAC AAC AAA ACT GTG GTG AGT TAT GGG GAG CGC ATC CAA AAA 4686 Val Gin Tyr Asn Lys Thr Val Val Ser Tyr Gly Glu Arg He Gin Lys 1500 1505 1510 CTC GGT AGA GTT GGG CGA CAC AAG GAA GGA GTA GCA CTT CGA ATT GGC 4734 Leu Gly Arg Val Gly Arg His Lys Glu Gly Val Ala Leu Arg He Gly 1515 1520 1525 1530 CAA ACA AAT AAA ACA CTG GTT GAA ATT CCA GAA ATG GTT GCC ACT GAA 4782 Gin Thr Asn Lys Thr Leu Val Glu He Pro Glu Met Val Ala Thr Glu 1535 1540 1545 GCT GCC TTT CTA TGC TTC ATG TAC AAT TTG CCA GTG ACA ACA CAG AGT 4830 Ala Ala Phe Leu Cys Phe Met Tyr Asn Leu Pro Val Thr Thr Gin Ser 1550 1555 1560 GTT TCA ACC ACA CTG CTG GAA AAT GCC ACA TTA TTA CAA GCT AGA ACT 4878 Val Ser Thr Thr Leu Leu Glu Asn Ala Thr Leu Leu Gin Ala Arg Thr 1565 1570 1575 ATG GCA CAG TTT GAG CTA TCA TAT TTT TAC ACA ATT AAT TTT GTG CGA 4926 Met Ala Gin Phe Glu Leu Ser Tyr Phe Tyr Thr He Asn Phe Val Arg 1580 1585 1590 TTT GAT GGT AGT ATG CAT CCA GTC ATA CAT GAC AAG CTG AAG CGC TTT 4974 Phe Asp Gly Ser Met His Pro Val He His Asp Lys Leu Lys Arg Phe 1595 1600 1605 1610 AAG CTA CAC ACT TGT GAG ACA TTC CTC AAT AAG TTG GCG ATC CCA AAT 5022 Lys Leu His Thr Cys Glu Thr Phe Leu Asn Lys Leu Ala He Pro Asn 1615 1620 1625 AAA GGC TTA TCC TCT TGG CTT ACG AGT GGA GAG TAT AAG CGA CTT GGT 5070 Lys Gly Leu Ser Ser Trp Leu Thr Ser Gly Glu Tyr Lys Arg Leu Gly 1630 1635 1640 TAC ATA GCA GAG GAT GCT GGC ATA AGA ATC CCA TTC GTG TGC AAA GAA 5118 Tyr He Ala Glu Asp Ala Gly He Arg He Pro Phe Val Cys Lys Glu 1645 1650 1655 ATT CCA GAC TCC TTG CAT GAG GAA ATT TGG CAC ATT GTA GTC GCC CAT 5166 He Pro Asp Ser Leu His Glu Glu He Trp His He Val Val Ala His 1660 1665 1670 AAA GGT GAC TCG GGT ATT GGG AGG CTC ACT AGC GTA CAG GCA GCA AAG 5214 Lys Gly Asp Ser Gly He Gly Arg Leu Thr Ser Val Gin Ala Ala Lys 1675 1680 1685 1690 GTT GTT TAT ACT CTG CAA ACG GAT GTG CAC TCA ATT GCG AGG ACT CTA 5262 Val Val Tyr Thr Leu Gin Thr Asp Val His Ser He Ala Arg Thr Leu 1695 1700 1705 GCA TGC ATC AAT AGA CGC ATA GCA GAT GAA CAA ATG AAG CAG AGT CAT 5310 Ala Cys He Asn Arg Arg He Ala Asp Glu Gin Met Lys Gin Ser His 1710 1715 1720 TTT GAA GCC GCA ACT GGG AGA GCA TTT TCC TTC ACA AAT TAC TCA ATA 5358 Phe Glu Ala Ala Thr Gly Arg Ala Phe Ser Phe Thr Asn Tyr Ser He 1725 1730 1735 CAA AGC ATA TTT GAC ACG CTG AAA GCA AAT TAT GCT ACA AAG CAT ACG 5406 Gin Ser He Phe Asp Thr Leu Lys Ala Asn Tyr Ala Thr Lys His Thr 1740 1745 1750 AAA GAA AAT ATT GCA GTG CTT CAG CAG GCA AAA GAT CAA TTG CTA GAG 5454 Lys Glu Asn He Ala Val Leu Gin Gin Ala Lys Asp Gin Leu Leu Glu 1755 1760 1765 1770 TTT TCG AAC CTA GCA AAG GAT CAA GAT GTC ACG GGT ATC ATC CAA GAC 5502 Phe Ser Asn Leu Ala Lys Asp Gin Asp Val Thr Gly He He Gin Asp 1775 1780 1785 TTC AAT CAC CTG GAA ACT ATC TAT CTC CAA TCA GAT AGC GAA GTG GCT 5550 Phe Asn His Leu Glu Thr He Tyr Leu Gin Ser Asp Ser Glu Val Ala 1790 1795 1800 AAG CAT CTG AAG CTT AAA AGT CAC TGG AAT AAA AGC CAA ATC ACT AGG 5598 Lys His Leu Lys Leu Lys Ser His Trp Asn Lys Ser Gin He Thr Arg 1805 1810 1815 GAC ATC ATA ATA GCT TTG TCT GTG TTA ATT GGT GGT GGA TGG ATG CTT 5646 Asp He He He Ala Leu Ser Val Leu He Gly Gly Gly Trp Met Leu 1820 1825 1830 GCA ACG TAC TTC AAG GAC AAG TTC AAT GAA CCA GTC TAT TTC CAA GGG 5694 Ala Thr Tyr Phe Lys Asp Lys Phe Asn Glu Pro Val Tyr Phe Gin Gly 1835 1840 1845 1850 AAG AAG AAT CAG AAG CAC AAG CTT AAG ATG AGA GAG GCG CGT GGG GCT 5742 Lys Lys Asn Gin Lys His Lys Leu Lys Met Arg Glu Ala Arg Gly Ala 1855 1860 1865 AGA GGG CAA TAT GAG GTT GCA GCG GAG CCA GAG GCG CTA GAA CAT TAC 5790 Arg Gly Gin Tyr Glu Val Ala Ala Glu Pro Glu Ala Leu Glu His Tyr 1870 1875 1880 TTT GGA AGC GCA TAT AAT AAC AAA GGA AAG CGC AAG GGC ACC ACG AGA 5838 Phe Gly Ser Ala Tyr Asn Asn Lys Gly Lys Arg Lys Gly Thr Thr Arg 1885 1890 1895 GGA ATG GGT GCA AAG TCT CGG AAA TTC ATA AAC ATG TAT GGG TTT GAT 5886 Gly Met Gly Ala Lys Ser Arg Lys Phe He Asn Met Tyr Gly Phe Asp 1900 1905 1910 CCA ACT GAT TTT TCA TAC ATT AGG TTT GTG GAT CCA TTG ACA GGT CAC 5934 Pro Thr Asp Phe Ser Tyr He Arg Phe Val Asp Pro. Leu Thr Gly His 1915 1920 1925 1930 ACT ATT GAT GAG TCC ACA AAC GCA CCT ATT GAT TTA GTG CAG CAT GAG 5982 Thr He Asp Glu Ser Thr Asn Ala Pro He Asp Leu Val Gin His Glu 1935 1940 1945 TTT GGA AAG GTT AGA ACA CGC ATG TTA ATT GAC GAT GAG ATA GAG CCT 6030 Phe Gly Lys Val Arg Thr Arg Met Leu He Asp Asp Glu He Glu Pro 1950 1955 1960 CAA AGT CTT AGC ACC CAC ACC ACA ATC CAT GCT TAT TTG GTG AAT AGT 6078 Gin Ser Leu Ser Thr His Thr Thr He His Ala Tyr Leu Val Asn Ser 1965 1970 1975 GGC ACG AAG AAA GTT CTT AAG GTT GAT TTA ACA CCA CAC TCG TCG CTA 6126 Gly Thr Lys Lys Val Leu Lys Val Asp Leu Thr Pro His Ser Ser Leu 1980 1985 1990 CGT GCG AGT GAG AAA TCA ACA GCA ATA ATG GGA TTT CCT GAA AGG GAG 6174 Arg Ala Ser Glu Lys Ser Thr Ala He Met Gly Phe Pro Glu Arg Glu 1995 2000 2005 2010 AAT GAA TTG CGT CAA ACC GGC ATG GCA GTG CCA GTG GCT TAT GAT CAA 6222 Asn Glu Leu Arg Gin Thr Gly Met Ala Val Pro Val Ala Tyr Asp Gin 2015 2020 2025 TTG CCA CCA AAG AAT GAG GAC TTG ACG TTT GAA GGA GAA AGC TTG TTT 6270 Leu Pro Pro Lys Asn Glu Asp Leu Thr Phe Glu Gly Glu Ser Leu Phe 2030 2035 2040 AAG GGA CCA CGT GAT TAC AAC CCG ATA TCG AGC ACC ATT TGT CAT TTG 6318 Lys Gly Pro Arg Asp Tyr Asn Pro He Ser Ser Thr He Cys His Leu 2045 2050 2055 ACG AAT GAA TCT GAT GGG CAC ACA ACA TCG TTG TAT GGT ATT GGA TTT 6366 Thr Asn Glu Ser Asp Gly His Thr Thr Ser Leu Tyr Gly He Gly Phe 2060 2065 2070 GGT CCC TTC ATC ATT ACA AAC AAG CAC TTG TTT AGA AGA AAT AAT GGA 6414 Gly Pro Phe He He Thr Asn Lys His Leu Phe Arg Arg Asn Asn Gly 2075 2080 2085 2090 ACA CTG TTG GTC CAA TCA CTA CAT GGT GTA TTC AAG GTC AAG AAC ACC 6462 Thr Leu Leu Val Gin Ser Leu His Gly Val Phe Lys Val Lys Asn Thr 2095 2100 2105 ACG ACT TTG CAA CAA CAC CTC ATT GAT GGG AGG GAC ATG ATA ATT ATT 6510 Thr Thr Leu Gin Gin His Leu He Asp Gly Arg Asp Met He He He 2110 2115 2120 CGC ATG CCT AAG GAT TTC CCA CCA TTT CCT CAA AAG CTG AAA TTT AGA 6558 Arg Met Pro Lys Asp Phe Pro Pro Phe Pro Gin Lys Leu Lys Phe Arg 2125 2130 2135 GAG CCA CAA AGG GAA GAG CGC ATA TGT CTT GTG ACA ACC AAC TTC CAA 6606 Glu Pro Gin Arg Glu Glu Arg He Cys Leu Val Thr Thr Asn Phe Gin 2140 2145 2150 ACT AAG AGC ATG TCT AGC ATG GTG TCA GAC ACT AGT TGC ACA TTC CCT 6654 Thr Lys Ser Met Ser Ser Met Val Ser Asp Thr Ser Cys Thr Phe Pro 2155 2160 2165 2170 TCA TCT GAT GGC ATA TTC TGG AAG CAT TGG ATT CAA ACC AAG GAT GGG 6702 Ser Ser Asp Gly He Phe Trp Lys His Trp He Gin Thr Lys Asp Gly 2175 2180 2185 CAG TGT GGC AGT CCA TTA GTA TCA ACT AGA GAT GGG TTC ATT GTT GGT 6750 Gin Cys Gly Ser Pro Leu Val Ser Thr Arg Asp Gly Phe He Val Gly 2190 2195 2200 ATA CAC TCA GCA TCG AAT TTC ACC AAC ACA AAC AAT TAT TTC ACA AGC 6798 He His Ser Ala Ser Asn Phe Thr Asn Thr Asn Asn Tyr Phe Thr Ser 2205 2210 2215 GTG CCG AAA AAC TTC ATG GAA TTG TTG ACA AAT CAG GAG GCG CAG CAG 6846 Val Pro Lys Asn Phe Met Glu Leu Leu Thr Asn Gin Glu Ala Gin Gin 2220 .2225 2230 TGG GTT AGT GGT TGG CGA TTA AAT GCT GAC TCA GTA TTG TGG GGG GGC 6894 Trp Val Ser Gly Trp Arg Leu Asn Ala Asp Ser Val Leu Trp Gly Gly 2235 2240 2245 2250 CAT AAA GTT TTC ATG AGC AAA CCT GAA GAG CCT TTT CAG CCA GTT AAG 6942 His Lys Val Phe Met Ser Lys Pro Glu Glu Pro Phe Gin Pro Val Lys 2255 2260 2265 GAA GCG ACT CAA CTC ATG AAT GAA TTG GTG TAC TCG CAA GGG GAG AAG 6990 Glu Ala Thr Gin Leu Met Asn Glu Leu Val Tyr Ser Gin Gly Glu Lys 2270 2275 2280 AGG AAA TGG GTC GTG GAA GCA CTG TCA GGG AAC TTG AGG CCA GTG GCT 7038 Arg Lys Trp Val Val Glu Ala Leu Ser Gly Asn Leu Arg Pro Val Ala 2285 2290 2295 GAG TGT CCC AGT CAG TTA GTC ACA AAG CAT GTG GTT AAA GGA AAG TGT 7086 Glu Cys Pro Ser Gin Leu Val Thr Lys His Val Val Lys Gly Lys Cys 2300 2305 2310 CCC CTC TTT GAG CTC TAC TTG CAG TTG AAT CCA GAA AAG GAA GCA TAT 7134 Pro Leu Phe Glu Leu Tyr Leu Gin Leu Asn Pro Glu Lys Glu Ala Tyr 2315 2320 2325 2330 TTT AAA CCG ATG ATG GGA GCA TAT AAG CCA AGT CGA CTT AAT AGA GAG 7182 Phe Lys Pro Met Met Gly Ala Tyr Lys Pro Ser Arg Leu Asn Arg Glu 2335 2340 2345 GCG TTC CTC AAG GAC ATT CTA AAA TAT GCT AGT GAA ATT GAG ATT GGG 7230 Ala Phe Leu Lys Asp He Leu Lys Tyr Ala Ser Glu He Glu He Gly 2350 2355 2360 AAT GTG GAT TGT GAC TTG CTG GAG CTT GCA ATA AGC ATG CTC GTC ACA 7278 Asn Val Asp Cys Asp Leu Leu Glu Leu Ala He Ser Met Leu Val Thr 2365 2370 2375 AAG CTC AAG GCG TTA GGA TTC CCA ACT GTG AAC TAC ATC ACT GAC CCA 7326 Lys Leu Lys Ala Leu Gly Phe Pro Thr Val Asn Tyr He Thr Asp Pro 2380 2385 2390 GAG GAA ATT TTT AGT GCA TTG AAT ATG AAA GCA GCT ATG GGA GCA CTA 7374 Glu Glu He Phe Ser Ala Leu Asn Met Lys Ala Ala Met Gly Ala Leu 2395 2400 2405 2410 TAC AAA GGC AAG AAG AAA GAA GCT CTC AGC GAG CTC ACA CTA GAT GAG 7422 Tyr Lys Gly Lys Lys Lys Glu Ala Leu Ser Glu Leu Thr Leu Asp Glu 2415 2420 2425 CAG GAG GCA ATG CTC AAA GCA AGT TGC CTG CGA CTG TAT ACG GGA AAG 7470 Gin Glu Ala Met Leu Lys Ala Ser Cys Leu Arg Leu Tyr Thr Gly Lys 2430 2435 2440 TTG GGA ATT TGG AAT GGC TCA TTG AAA GCA GAG TTG CGT CCA ATT GAG 7518 Leu Gly He Trp Asn Gly Ser Leu Lys Ala Glu Leu Arg Pro He Glu 2445 2450 2455 AAG GTT GAA AAC AAC AAA ACG CGA ACT TTC ACA GCA GCA CCA ATA GAC 7566 Lys Val Glu Asn Asn Lys Thr Arg Thr Phe Thr Ala Ala Pro He Asp 2460 2465 2470 ACT CTT CTT GCT GGT AAA GTT TGC GTG GAT GAT TTC AAC AAT CAA TTT 7614 Thr Leu Leu Ala Gly Lys Val Cys Val Asp Asp Phe Asn Asn Gin Phe 2475 2480 2485 2490 TAT GAT CTC AAC ATA AAG GCA CCA TGG ACA GTT GGT ATG ACT AAG TTT 7662 Tyr Asp' Leu Asn He Lys Ala Pro Trp Thr Val Gly Met Thr Lys Phe 2495 2500 2505 TAT CAG GGG TGG AAT GAA TTG ATG GAG GCT TTA CCA AGT GGG TGG GTG 7710 Tyr Gin Gly Trp Asn Glu Leu Met Glu Ala Leu Pro Ser Gly Trp Val 2510 2515 2520 TAT TGT GAC GCT GAT GGT TCG CAA TTC GAC AGT TCC TTG ACT CCA TTC 7758 Tyr Cys Asp Ala Asp Gly Ser Gin Phe Asp Ser Ser Leu Thr Pro Phe 2525 2530 2535 CTC ATT AAT GCT GTA TTG AAA GTG CGA CTT GCC TTC ATG GAG GAA TGG 7806 Leu He Asn Ala Val Leu Lys Val Arg Leu Ala Phe Met Glu Glu Trp 2540 2545 2550 GAT ATT GGT GAG CAA ATG CTG CGA AAT TTG TAC ACT GAG ATA GTG TAT 7854 Asp He Gly Glu Gin Met Leu Arg Asn Leu Tyr Thr Glu He Val Tyr 2555 2560 2565 2570 ACA CCA ATC CTC ACA CCG GAT GGT ACT ATC ATT AAG AAG CAT AAA GGC 7902 Thr Pro He Leu Thr Pro Asp Gly Thr He He Lys Lys His Lys Gly 2575 2580 2585 AAC AAT AGC GGG CAA CCT TCA ACA GTG GTG GAC AAC ACA CTC ATG GTC 7950 Asn Asn Ser Gly Gin Pro Ser Thr Val Val Asp Asn Thr Leu Met Val 2590 2595 2600 ATT ATT GCA ATG TTA TAC ACA TGT GAG AAG TGT GGA ATC AAC AAG GAA 7998 He He Ala Met Leu Tyr Thr Cys Glu Lys Cys Gly He Asn Lys Glu 2605 2610 2615 GAG ATT GTG TAT TAC GTC AAT GGC GAT GAC CTA TTG ATT GCC ATT CAC 8046 Glu He Val Tyr Tyr Val Asn Gly Asp Asp Leu Leu He Ala He His 2620 2625 2630 CCA GAT AAA GCT GAG AGG TTG AGT AGA TTC AAA GAA TCT TTC GGA GAG 8094 Pro Asp Lys Ala Glu Arg Leu Ser Arg Phe Lys Glu Ser Phe Gly Glu 2635 2640 2645 2650 TTG GGC CTG AAA TAT GAA TTT GAC TGT ACC ACC AGG GAC AAG ACA CAG 8142 Leu Gly Leu Lys Tyr Glu Phe Asp Cys Thr Thr Arg Asp Lys Thr Gin 2655 2660 2665 TTG TGG TTC ATG TCA CAC AGG GCT TTG GAG AGG GAT GGC ATG TAT ATA 8190 Leu Trp Phe Met Ser His Arg Ala Leu Glu Arg Asp Gly Met Tyr He 2670 2675 2680 CCA AAG CTA GAA GAA GAA AGG ATT GTT TCT ATT TTG GAA TGG GAC AGA 8238 Pro Lys Leu Glu Glu Glu Arg He Val Ser He Leu Glu Trp Asp Arg 2685 2690 2695 TCC AAA GAG CCG TCA CAT AGG CTT GAA GCC ATC TGT GCA TCA ATG ATT 8286 Ser Lys Glu Pro Ser His Arg Leu Glu Ala He Cys Ala Ser Met He 2700 2705 2710 GAA GCA TGG GGT TAT GAC AAG CTG GTT GAA GAA ATC CGC AAT TTC TAT 8334 Glu Ala Trp Gly Tyr Ast> Lys Leu Val Glu Glu He Arg Asn Phe Tyr 2715 2720 2725 2730 GCA TGG GTT TTG GAA CAA GCG CCG TAT TCA CAG CTT GCA GAA GAA GGA 8382 Ala Trp Val Leu Glu Gin Ala Pro Tyr Ser Gin Leu Ala Glu Glu Gly 2735 2740 2745 AAG GCG CCA TAT CTG GCT GAG ACT GCG CTT AAG TTT TTG TAC ACA TCT 8430 Lys Ala Pro Tyr Leu Ala Glu Thr Ala Leu Lys Phe Leu Tyr Thr Ser 2750 2755 2760 CAG CAC GGA ACA AAC TCT GAG ATA GAA GAG TAT TTA AAA GTG TTG TAT 8478 Gin His Gly Thr Asn Ser Glu He Glu Glu Tyr Leu Lys Val Leu Tyr 2765 2770 2775 GAT TAC GAT ATT CCA ACG ACT GAG AAT CTT TAT TTT CAG AGT GGC ACT 8526 Asp Tyr Asp He Pro Thr Thr Glu Asn Leu Tyr Phe Gin Ser Gly Thr 2780 2785 2790 GTG GAT GCT GGT GCT GAC GCT GGT AAG AAG AAA GAT CAA AAG GAT GAT 8574 Val Asp Ala Gly Ala Asp Ala Gly Lys Lys Lys Asp Gin Lys Asp Asp 2795 2800 2805 2810 AAA GTC GCT GAG CAG GCT TCA AAG GAT AGG GAT GTT AAT GCT GGA ACT 8622 Lys Val Ala Glu Gin Ala Ser Lys Asp Arg Asp Val Asn Ala Gly Thr 2815 2820 2825 TCA GGA ACA TTC TCA GTT CCA CGA ATA AAT GCT ATG GCC ACA AAA CTT 8670 Ser Gly Thr Phe Ser Val Pro Arg He Asn Ala Met Ala Thr Lys Leu 2830 2835 2840 CAA TAT CCA AGG ATG AGG GGA GAG GTG GTT GTA AAC TTG AAT CAC CTT 8718 Gin Tyr Pro Arg Met Arg Gly Glu Val Val Val Asn Leu Asn His Leu 2845 2850 2855 TTA GGA TAC AAG CCA CAG CAA ATT GAT TTG TCA AAT GCT CGA GCC ACA 8766 Leu Gly Tyr Lys Pro Gin Gin He Asp Leu Ser Asn Ala Arg Ala Thr 2860 2865 2870 CAT GAG CAG TTT GCC GCG TGG CAT CAG GCA GTG ATG ACA GCC TAT GGA 8814 His Glu Gin Phe Ala Ala Trp His Gin Ala Val Met Thr Ala Tyr Gly 2875 2880 2885 2890 GTG AAT GAA GAG CAA ATG AAA ATA TTG CTA AAT GGA TTT ATG GTG TGG 8862 Val Asn Glu Glu Gin Met Lys He Leu Leu Asn Gly Phe Met Val Trp 2895 2900 2905 TGC ATA GAA AAT GGG ACT TCC CCA AAT TTG AAC GGA ACT TGG GTT ATG 8910 Cys He Glu Asn Gly Thr Ser Pro Asn Leu Asn Gly Thr Trp Val Met 2910 2915 2920 ATG GAT GGT GAG GAT CAA GTT TCA TAC CCG CTG AAA CCA ATG GTT GAA 8958 Met Asp Gly Glu Asp Gin Val Ser Tyr Pro Leu Lys Pro Met Val Glu 2925 2930 2935 AAC GCG CAG CCA ACA CTG AGG CAA ATT ATG ACA CAC TTC AGT GAC CTG 9006 Asn Ala Gin Pro Thr Leu Arg Gin He Met Thr His Phe Ser Asp Leu 2940 2945 2950 GCT GAA GCG TAT ATT GAG ATG AGG AAT AGG GAG CGA CCA TAC ATG CCT 9054 Ala Glu Ala Tyr He Glu Met Arg Asn Arg Glu Arg Pro Tyr Met Pro 2955 2960 2965 2970 AGG TAT GGT CTA CAG AGA AAC ATT ACA GAC ATG AGT TTG TCA CGC TAT 9102 Arg Tyr Gly Leu Gin Arg Asn He Thr Asp Met Ser Leu Ser Arg Tyr 2975 2980 2985 GCG TTC GAC TTC TAT GAG CTA ACT TCA AAA ACA CCT GTT AGA GCG AGG 9150 Ala Phe Asp Phe Tyr Glu Leu Thr Ser Lys Thr Pro Val Arg Ala Arg 2990 2995 3000 GAG GCG CAT ATG CAA ATG AAA GCT GCT GCA GTA CGA AAC . AGT GGA ACT 9198 Glu Ala His Met Gin Met Lys Ala Ala Ala Val Arg Asn Ser Gly Thr 3005 3010 3015 AGG TTA TTT GGT CTT GAT GGC AAC GTG GGT ACT GCA GAG GAA GAC ACT 9246 Arg Leu Phe Gly Leu Asp Gly Asn Val Gly Thr Ala Glu Glu Asp Thr 3020 3025 3030 GAA CGG CAC ACA GCG CAC GAT GTG AAC CGT AAC ATG CAC ACA CTA TTA 9294 Glu Arg His Thr Ala His Asp Val Asn Arg Asn Met His Thr Leu Leu 3035 3040 3045 3050 GGG GTC CGC CAG TGA TAGTTTCTGC GTGTCTTTGC TTTCCGCTTT TAAGCTTATT 9349 Gly Val Arg Gin GTAATATATA TGAATAGCTA TTCACAGTGG GACTTGGTCT TGTGTTGAAT AGTATCTTAT 9409 ATATTTTAAT ATGTCTTATT AGTCTCATTA CTTAGGCGAA CGACAAAGTG AGGTCACCTC 9469 GGTCTAATTC TCCTATGTAG TGCGAG 9495 (3) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 792 (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Double (D) TOPOLOGY: Circular (ii) MOLECULE TYPE: cDNA to genomic RNA (iϋ) HYPOTHETICAL: No (iv) ANTI-SENSE: No (v) FRAGMENT TYPE: N/A (vi) ORIGINAL SOURCE: (A) ORGANISM: Tobacco Etch Virus (B) STRAIN: Highly Aphid Transmitted (C) INDIVIDUAL ISOLATE: N/A (vii) IMMEDIATE SOURCE: (A) LIBRARY: No (B) CLONE: pTC:FL (viϋ) POSITION IN GENOME: N/A (ix) FEATURE: (A) NAME/KEY: Mutations (AGT→ATG) introduced into nucleotides corresponding to genomic nucleotides 8518-8520 of SEQ ID No. 1, to create initiating methionine codon. (B) LOCATION: Nucleotides 1-3 of SEQ ID No. 2 (C) IDENTIFICATION METHOD: — (D) OTHER INFORMATION: SEQ ID NO: 2 is the modified Tobacco Etch Virus coat protein gene present in pTC:FL. - - (X) PUBLICATION INFORMATION: (A) AUTHORS: Allison et al. (B) TITLE: The nucleotide sequence of the coding region of Tobacco Etch Virus Genomic RNA: Evidence for the Synthesis of a Single Polyprotein (C) JOURNAL: Virology (D) VOLUME: 154 (E) ISSUE: — (F) PAGES: 9-20 (A) AUTHORS: Lindbo and Dougherty (B) TITLE: Untranslatable Transcripts of the tobacco etch virus coat protein gene sequence can interfere with tobacco etch virus replication in Transgenic Plants and Protoplasts (C) JOURNAL: Virology (D) VOLUME: 189 (E) ISSUE: — (F) PAGES: 725-733 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: ATG GGC ACT 9 Met Gly Thr 1 GTG GAT GCT GGT GCT GAC GCT GGT AAG AAG AAA GAT CAA AAG GAT GAT 57 Val Asp Ala Gly Ala Asp Ala Gly Lys Lys Lys Asp Gin Lys Asp Asp 5 10 15 AAA GTC GCT GAG CAG GCT TCA AAG GAT AGG GAT GTT AAT GCT GGA ACT 105 Lys Val Ala Glu Gin Ala Ser Lys Asp Arg Asp Val Asn Ala Gly Thr 20 25 30 35 TCA GGA ACA TTC TCA GTT CCA CGA ATA AAT GCT ATG GCC ACA AAA CTT 153 Ser Gly Thr Phe Ser Val Pro Arg He Asn Ala Met Ala Thr Lys Leu 40 45 50 CAA TAT CCA AGG ATG AGG GGA GAG GTG GTT GTA AAC TTG AAT CAC CTT 201 Gin Tyr Pro Arg Met Arg Gly Glu Val Val Val Asn Leu Asn His Leu 55 60 65 TTA GGA TAC AAG CCA CAG CAA ATT GAT TTG TCA AAT GCT CGA GCC ACA 249 Leu Gly Tyr Lys Pro Gin Gin He Asp Leu Ser Asn Ala Arg Ala Thr 70 75 80 CAT GAG CAG TTT GCC GCG TGG CAT CAG GCA GTG ATG ACA GCC TAT GGA 297 His Glu Gin Phe Ala Ala Trp His Gin Ala Val Met Thr Ala Tyr Gly 85 90 95 GTG AAT GAA GAG CAA ATG AAA ATA TTG CTA AAT GGA TTT ATG GTG TGG 345 Val Asn Glu Glu Gin.Met Lys He Leu Leu Asn Gly Phe Met Val Trp 100 105 110 115 TGC ATA GAA AAT GGG ACT TCC CCA AAT TTG AAC GGA ACT TGG GTT ATG 393 Cys He Glu Asn Gly Thr Ser Pro Asn Leu Asn Gly Thr Trp Val Met 120 125 130 ATG GAT GGT GAG GAT CAA GTT TCA TAC CCG CTG AAA CCA ATG GTT GAA 441 Met Asp Gly Glu Ast> Gin Val Ser Tyr Pro Leu Lys Pro Met Val Glu 135 " 140 145 AAC GCG CAG CCA ACA CTG AGG CAA ATT ATG ACA CAC TTC AGT GAC CTG 489 Asn Ala Gin Pro Thr Leu Arg Gin He Met Thr His Phe Ser Asp Leu 150 155 160 GCT GAA GCG TAT ATT GAG ATG AGG AAT AGG GAG CGA CCA TAC ATG CCT 537 Ala Glu Ala Tyr He Glu Met Arg Asn Arg Glu Arg Pro Tyr Met Pro 165 170 175 AGG TAT GGT CTA CAG AGA AAC ATT ACA GAC ATG AGT TTG TCA CGC TAT 585 Arg Tyr Gly Leu Gin Arg Asn He Thr Asp Met Ser Leu Ser Arg Tyr 180 185 190 195 GCG TTC GAC TTC TAT GAG CTA ACT TCA AAA ACA CCT GTT AGA GCG AGG 633 Ala Phe Asp Phe Tyr Glu Leu Thr Ser Lys Thr Pro Val Arg Ala Arg 200 205 210 GAG GCG CAT ATG CAA ATG AAA GCT GCT GCA GTA CGA AAC AGT GGA ACT 681 Glu Ala His Met Gin Met Lys Ala Ala Ala Val Arg Asn Ser Gly Thr 215 220 225 AGG TTA TTT GGT CTT GAT GGC AAC GTG GGT ACT GCA GAG GAA GAC ACT 729 Arg Leu Phe Gly Leu Asp Gly Asn Val Gly Thr Ala Glu Glu Asp Thr 230 235 240 GAA CGG CAC ACA GCG CAC GAT GTG AAC CGT AAC ATG CAC ACA CTA TTA 777 Glu Arg His Thr Ala His Asp Val Asn Arg Asn Met His Thr Leu Leu 245 250 255 GGG GTC CGC CAG TGA 792 Gly Val Arg Gin 260 (4) INFORMATION FOR SEQ ID NO: 3 : (i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 793 (B) TYPE : Nucleic Acid (C) STRANDEDNESS : Double (D) TOPOLOGY: Circular ( ii) MOLECULE TYPE : cDNA to genomic RNA ( ϋi) HYPOTHETICAL: No (iv) ANTI-SENSE : No (v) FRAGMENT TYPE: N/A (vi) ORIGINAL SOURCE : (A) ORGANISM: Tobacco Etch Virus (B) STRAIN : Highly Aphid Transmitted (C) INDIVIDUAL ISOLATE : N/A (vii) IMMEDIATE SOURCE : (A) LIBRARY : No (B) CLONE : pTC: RC (viii) POSITION IN GENOME: N/A ( ix) FEATURE : (A) NAME/KEY: Mutation of AGT-GGC (Ser-Gly) to ATG-GCC (Met-Ser) (B) LOCATION: Nucleotides 1-6 of SEQ ID NO. 3 (corresponding to nucleotides 8518-8523 of SEQ ID NO. 1) (A) NAME/KEY: Frameshift mutation (insertion of T) producing stop codon (B) LOCATION: Nucleotide 13 of SEQ ID No. 3 (corresponding to position between nucleotides 8529 and 8530 of SEQ. ID No. 1) (D) OTHER INFORMATION: SEQ ID No: 3 is the modified Tobacco Etch Virus coat protein gene present in pTC:RC. (X) PUBLICATION INFORMATION: (A) AUTHORS: J. A. Lindbo and W. G. Dougherty (B) TITLE: Pathogen-Derived Resistance to a Potyvirus: Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence (C) JOURNAL: Molecular Plant-Microbe Interactions the Tobacco Etch Virus Coat Protein Gene Sequence Can Interfere with Tobacco Etch Virus Replication in Transgenic Plants and Protoplasts (C) JOURNAL: Virology (D) VOLUME: 189 (E) ISSUE: — (F) PAGES: 725-733 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ATG GCC ACT 9 Met Ser Thr GTG TGA TGA TGGTGCTAGC GCTGGTAAGA AGAAAGATCA AAAGGATGAT 58 Val AAAGTCGCTG AGCAGGCTTC AAAGGATAGG GATGTTAATG CTGGAACTTC 108 AGGAACATTC TCAGTTCCAC GAATAAATGC TATGGCCACA AAACTTCAAT 158 ATCCAAGGAT GAGGGGAGAG GTGGTTGTAA ACTTGAATCA CCTTTTAGGA 208 TACAAGCCAC AGCAAATTGA TTTGTCAAAT GCTCGAGCCA CACATGAGCA 258 GTTTGCCGCG TGGCATCAGG CAGTGATGAC AGCCTATGGA GTGAATGAAG 308 AGCAAATGAA AATATTGCTA AATGGATTTA TGGTGTGGTG CATAGAAAAT 358 GGGACTTCCC CAAATTTGAA CGGAACTTGG GTTATGATGG ATGGTGAGGA 408 TCAAGTTTCA TACCCGCTGA AACCAATGGT TGAAAACGCG CAGCCAACAC 458 TGAGGCAAAT TATGACACAC TTCAGTGACC TGGCTGAAGC GTATATTGAG 508 ATGAGGAATA GGGAGCGACC ATACATGCCT AGGTATGGTC TACAGAGAAA 558 CATTACAGAC ATGAGTTTGT CACGCTATGC GTTCGACTTC TATGAGCTAA 608 CTTCAAAAAC ACCTGTTAGA GCGAGGGAGG CGCATATGCA AATGAAAGCT 658 GCTGCAGTAC GAAACAGTGG AACTAGGTTA TTTGGTCTTG ATGGCAACGT 708 GGGTACTGCA GAGGAAGACA CTGAACGGCA CACAGCGCAC GATGTGAACC 758 GTAACATGCA CACACTATTA GGGGTCCGCC AGTGA 793 (5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 792 (B) TYPE: Nucleic acid (C) STRANDEDNESS: Double (D) TOPOLOGY: Circular (ϋ) MOLECULE TYPE: cDNA to genomic RNA (iϋ) HYPOTHETICAL: No (iv) ANTI-SENSE: Yes (v) FRAGMENT TYPE: N/A (vi) ORIGINAL SOURCE: (A) ORGANISM: Tobacco Etch Virus (B) STRAIN: Highly Aphid Transmitted (C) INDIVIDUAL ISOLATE: N/A (vii) IMMEDIATE SOURCE: (A) LIBRARY: No (B) CLONE: pTC:AS (viii) POSITION IN GENOME: N/A (ix) FEATURE: (A) NAME/KEY: — (B) LOCATION: — (C) IDENTIFICATION METHOD: — (D) OTHER INFORMATION: SEQ ID No. 4 is the modified Tobacco Etch Virus Coat protein gene present in pTC:AS. It is the inverse complement of SEQ ID No. 2. (x) PUBLICATION INFORMATION: (A) AUTHORS: J. A. Lindbo and . G. Dougherty (B) TITLE: Untranslatable Transcripts of the Tobacco Etch Virus Coat Protein Gene Sequence Can Interfere with Tobacco Etch Virus Replication in Transgenic Plants and Protoplasts (C) JOURNAL: Virology (D) VOLUME: 189 (E) ISSUE: — (F) PAGES: 725-733 (A) AUTHORS: J. A. Lindbo and W. G. Dougherty (B) TITLE: Pathogen-Derived Resistance to a Potyvirus: Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence (C) JOURNAL: Molecular Plant-Microbe Interactions (D) VOLUME: 5 (E) ISSUE: 2 (F) PAGES: 144-153 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: TCACTGGCGG ACCCCTAATA GTGTGTGCAT GTTACGGTTC ACATCGΪGCG CTGTGTGCCG 60 TTCAGTGTCT TCCTCTGCAG TACCCACGTT GCCATCAAGA CCAAATAACC TAGTTCCACT 120 GTTTCGTACT GCAGCAGCTT TCATTTGCAT ATGCGCCTCC CTCGCTCTAA CAGG7GTTTT 180 TGAAGTTAGC TCATAGAAGT CGAACGCATA GCGTGACAAA CTCATGTCTG TAATGTTTCT 240 CTGTAGACCA TACCTAGGCA TGTATGGTCG CTCCCTATTC CTCATCTCAA TATACGCTTC 300 AGCCAGGTCA CTGAAGTGTG TCATAATTTG CCTCAGTGTT GGCTGCGCGT TTTCAACCAT 360 TGGTTTCAGC GGGTA7GAAA CTTGATCCTC ACCATCCATC ATAACCCAAG TTCCGTTCAA 420 ATTTGGGGAA G7CCCA7TTT CTATGCACCA CACCATAAAT CCATTTAGCA ATATTTTCAT 480 TTGCTCTTCA T7CACTCCA7 AGGC7C7CA7 CACTGCC7CA TGCCACGCGG CAAACTGCTC 540 ATGTGTGGC7 CGAGCATT7G ACAAA7CAAT TTGCTGTGGC TTG7ATCCTA AAAGG7GAT7 600 CAAGTTTACA ACCACC7C7C CCCTCATCCT 7GGATATT-.A AGTTTTGTGG CCATAGCAT 7 660 7ATTCGTGGA ACTGAGAATG T7CCTG--AGT TCCAGCATTA ACATCCCTAT CCTTTGAAG C 720 CTGCTCAGCG ACTTTATCAT CrTTTTGAT C TTTCTTCTTA CCAGCGTCAG CACCAGCATC 780 CACAGTGCCC AT 792

Claims: CLAIMS 1. A plant-transformation vector comprising a DNA molecule that includes a gene derived, in part, from a plant virus RNA molecule, wherein the gene is mutated to encode an untranslatable plus sense RNA molecule. 2. The vector of claim 1 wherein the gene is derived, in part, from potyvirus RNA. 3. The vector of claim 2 wherein the potyvirus is Tobacco Etch Virus. 4. The vector of claim 2 wherein the gene is derived, in part, from a coat protein gene of a potyvirus. 5. The vector of claim 4 wherein t:he gene is derived, in part, from the coat protein gene of Tobacco Etch Virus. 6. A bacterial cell containing the vector of claim 1. 7. The bacterial cell of claim 8 wherein the bacterial cell is an Agrobacterium tumefaciens cell. 8. A transformed plant cell comprising a heterologous DNA chromosomal insert that includes a gene derived from a plant virus RNA molecule, wherein the gene is mutated to encode an untranslatable plus sense RNA molecule. 9. The plant cell of claim 8 wherein the gene is derived from potyvirus RNA. 10. The plant cell of claim 9 wherein the potyvirus is Tobacco Etch Virus. 11. The plant cell of claim 10 wherein the gene is derived from a coat protein gene of a potyvirus. 12. The plant cell of claim 10 wherein the gene is derived from the coat protein gene of Tobacco Etch Virus and the plant cell is a tobacco plant cell. 13. A differentiated plant comprising transformed plant cells of claim 8. 14. A differentiated plant comprising transformed plant cells of claim 9. 15. A differentiated plant comprising transformed plant cells of claim 10. 16. A differentiated plant comprising transformed plant cells of claim 11. 17. A differentiated plant comprising transformed plant cells of claim 12. 18. A recombinant gene comprising: control regions which regulate transcription of the gene; and a region, derived from a plant virus, mutated so as to render the RNA transcribed from the gene untranslatable. 19. The recombinant gene of claim 18 wherein the plant virus is a potyvirus. 20. The recombinant gene of claim 19 wherein the virus-derived region is derived from the region of the viral genome encoding a coat protein. 21. The recombinant gene of claim 20 wherein the potyvirus is Tobacco Etch Virus. 22. A method of producing plants with a reduced susceptibility to viral infection, comprising: forming a recombinant gene derived, in part, from viral RNA wherein the gene is mutated to encode an untranslatable plus sense RNA molecule; and transforming plants with the recombinant gene. 23. The method of claim 22 wherein the method of producing plants comprises: constructing a recombinant gene comprising a region of a viral genome capable of being transcribed in a plant; mutating the recombinant gene to encode an untranslatable plus sense RNA molecule; cloning the recombinant untranslatable gene into a plant transformation vector; transforming plant cells .with the transformation vector; and culturing transformed cells under conditions suitable for regeneration of transformed plants. 24. The method of claim 23 wherein the viral genome is a potyvirus genome. 25. The method of claim 24 wherein the region of the viral genome encodes a coat protein. 26. The method of claim 25 wherein the viral genome is the Tobacco Etch Virus genome. 27. The method of claim 26 wherein the plants are tobacco plants.

Inventors: DOUGHERTY WILLIAM G, LINDBO JOHN A, DOUGHERTY, WILLIAM, G., LINDBO, JOHN, A.
Applicants: OREGON STATE, THE STATE OF OREGON ACTING BY AND THROUGH THE OREG
Application year: 1993
Main classification: C12N15
IPC codes: A01H5, C07H21, C07K14, C12N1, C12N5, C12N15