FIELD OF THE INVENTION The present invention relates to tomato ringspot virus proteins, DNA molecules encoding these proteins, and their uses.

BACKGROUND OF THE INVENTION Tomato ringspot virus ("TomRSV") is a broad host range nepovirus (Harrison, B. D., et al.,"Nepovirus Groups No. 185,"in Descriptions of Plant Viruses, Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England (1977); Sanfacon, H., "Nepoviruses,"Pathosenesis and Host Specificity in Plant Diseases, Vol. III, Viruses & Viroids, Elsevier Science Ltd., Oxford, pp. 129-141 (1995)) infecting numerous berry and fruit crops including apples, peaches, cherries, plums, raspberries, strawberries, and grapes. Serious diseases have been associated with this virus, including peach stem pitting, peach yellow bud mosaic, apple union necrosis and decline, prune brown line, prune constriction and decline, raspberry crumbly berry, and grapevine decline (Stace-Smith, R.,"Tomato Ringspot Virus,"in Descriptions of Plant Viruses, Commonw. Mycol. Inst./Assoc. Appl. Biol., Kew, England, No. 290 (1984)). Symptoms of these diseases usually appear several years after the plants have been established in the field. TomRSV is transmitted by nematode species of the Xiphinema americanum Cobb group, which are endemic to the Great Lakes region of the United States and Canada, the Northeast and Pacific coast of the United States, and British Columbia, Canada (Brown, D. J. F., et al.,"Transmission of Three North American Nepoviruses by Populations of Four Distinct Species of the Xiphinema americanum Group,"Phvtopathology, 84: 646-649 (1994)).

The genome of TomRSV is bipartite, consisting of two single- stranded, positive-sense, RNA molecules separately encapsidated into isometric particles of about 28 nm in diameter (Schneider, I. R.,"Two Nucleic Acid-Containing

Components of Tomato Ringspot Virus,"Virology, 57: 139-146 (1974)). Both RNAs, RNA 1 of 8,214 nucleotides ("nt") (Rott, M. E., et al.,"Nucleotide Sequence of Tomato Ringspot Virus RNA 1,"J. Gen. Virol., 76: 465-473 (1995)) and RNA 2 of 7,273 nt (Rott, M. E., et al.,"Nucleotide Sequence of Tomato Ringspot Virus RNA-2,"J. Gen. Virol., 72: 1505-1514 (1991)), are translated as two large polyprotein precursors that are cleaved to release functional proteins (Hans, F., et al.,"Tomato Ringspot Nepovirus Protease: Characterization and Cleavage Site Specificity,"J.

Gen. Virol., 76: 917-927 (1995)). Unlike most nepoviruses, TomRSV RNA 1 and RNA 2 have a large (1.5 kbp) 3'end untranslated common region (Rott, M. E., et al., "Comparison of the 5'and 3'End Termini of Tomato Ringspot Virus RNA 1 and RNA 2: Evidence for RNA Recombination,"Virology, 185: 468-472 (1991)). The coat protein ("cp") gene has been localized near the 3'end of RNA 2 for the following nepoviruses: arabis mosaic ("ArMV") (Bertioli, D. J., et al.,"Transgenic Plants and Insect and Cells Expressing the Coat Protein of Arabis Mosaic Virus Produce Empty Virus-Like Particles,"J. Gen. Virol., 72: 1801-1809 (1991)), blueberry leaf mottle ("BBLMV") (Bacher, J., et al.,"Sequence Analysis of the 3'Termini of RNA 1 and RNA 2 of Blueberry Leaf Mottle Virus,"J. Gen. Virol., 75: 2133-2138 (1994)), grapevine fanleaf ("GFLV") (Serghini, M. A.,"RNA 2 of Grapevine Fanleaf Virus: Sequence Analysis and Coat Protein Cistron Location,"J. Gen. Viro., 71: 1433-1441 (1990)), cherry leaf roll ("CLRV") (Scott, N. W., et al.,"The Identification, Cloning, and Sequence Analysis of the Coat Protein Region of a Birch Isolate (I2) of Cherry Leaf Roll Nepovirus,"Arch. Virol., 131: 309-215 (1993)), grapevine chrome mosaic ("GCMV") (Brault, V., et al.,"Nucleotide Sequence and Genetic Organization of Hungarian Grapevine Chrome Mosaic Nepovirus RNA 2," Nucleic Acids Res., 17: 7809-7819 (1989)), raspberry ringspot (Blok, V. C., et al., "The Nucleotide Sequence of RNA 2 of Raspberry Ringspot Nepovirus,"J. Gen.

Virol., 73: 2189-2194 (1992)), strawberry latent ringspot ("SLRV") (Everett, K. R., et al.,"Nucleotide Sequence of the Coat Protein Genes of Strawberry Latent Ringspot Virus: Lack of Homology to the Nepoviruses and Comoviruses,"J. Gen. Virol., 75: 1821-1825 (1994), Kreiah, S., et al.,"Sequence Analysis and Location of Capsid Proteins Within RNA 2 of Strawberry Latent Ringspot Virus,"J. Gen. Virol., 75: 2527-2532 (1994)) tobacco ringspot (Buckley, B.,"Nucleotide Sequence and In

Vitro Expression of the Capsid Protein Gene of Tobacco Ringspot Virus,"Virus Res., 30: 335-349 (1993)), and tomato black ring (Meyer, M., et al.,"The Nucleotide Sequence of Tomato Black Ring Virus RNA-2,"J. Gen. Virol., 67: 1257-1271 (1986)). Rott et al.,"Nucleotide Sequence of Tomato Ringspot Virus RNA-2,"J.

Gen. Virol., 72: 1505-1514 (1991) suggested that the putative location of the cp gene of a raspberry isolate of TomRSV was at the 3'end of RNA 2. Microsequencing of the N-terminal region of purified TomRSV cp allowed the identification of a Q-G cleavage site that was not previously reported for nepoviruses (Hans, F., et al., "Tomato Ringspot Nepovirus Protease: Characterization and Cleavage Site Specificity,"J. Gen. Virol., 76: 917-927 (1995)). However, none of these references have reported a method of imparting high levels of resistance to TomRSV.

In view of the serious risk tomato ringspot virus poses to fruits and the absence of an effective treatment of it, the need to prevent this affliction continues to exist. Thus, methods of imparting resistance to this virus to plants need to be developed. The present invention is directed to overcoming this deficiency in the art.

SUMMARY OF INVENTION The present invention relates to an isolated coat protein or polypeptide for a peach isolate of a tomato ringspot virus. The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, or a transgenic plant, are also disclosed.

Another aspect of the present invention relates to a method of imparting tomato ringspot virus resistance to a plant which includes transforming the plant with a DNA molecule of the present invention.

The present invention also relates to an antibody or binding portion thereof or probe which recognizes the protein or polypeptide.

The present invention provides for a virus resistant transgenic plant line for a group III nepovirus. In addition, the present invention provides for a transgenic plant line which, while having little or no detectable levels of coat protein expression, is still resistant to a nepovirus. Further, the transgenic plant lines of the present invention are virtually immune to infection by the nepovirus.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an illustration of the plant binary vector pGA482GG and expression cassette pNYS containing the TomRSV coat protein ("cp") gene construct of the present invention.

Figure 2 illustrates the nucleotide sequence of the 3'terminal region of RNA 2 of a peach isolate of TomRSV and deduced amino acid sequence for the polyprotein open reading frame. The sequence of the 3'end untranslatable region (1,552 nucleotides) is given following the polyprotein stop codon (TAA). The'^' between the second and third amino acid residues of the polyprotein denotes the cleavage site (Q-G) between the putative movement protein and the coat protein. The underlined amino acid sequences represent the first 22 amino acids at the N-terminus of the cp and amino acids 133 to 155 obtained from microsequencing the purified cp and a protease-generated polypeptide, respectively. The 19 amino acid residues printed in bold (scattered throughout the cp sequence) represent differences between the peach and raspberry isolates of TomRSV.

Figure 3A illustrates a polymerase chain reaction ("PCR") analysis of Nicotiana benthamiana transgenic lines. Agarose gel representing the amplified products for the negative control (Nb-) (Lanes 1 and 2), the plasmid control (P) (lanes 3 to 5 ; amplified from 1.0,0.1, or 0.01 pg of DNA), and 14 transgenic Ro lines (Nb/TomRSV) (Lanes 6 to 19). From left to right, the lines tested were seven sense (S) lines (S 1 to S7) and seven antisense (AS) lines (AS1 to AS7). The arrow indicates the location of the TomRSV coat protein (cp) PCR-amplified product (1.7 kbp). Lane 20 corresponds to the molecular size standards k-HindIll and f-HueIII, B, Northern blot analysis. All lanes were loaded with 5 llg of total plant RNA. Lane 1 corresponds to a nontransgenic control plant infected with the peach isolate of TomRSV. The probe used contained the cp gene and part of the untranslated 3'end common region for RNA 1 and RNA 2, so it hybridized with both viral RNAs. Lane 2 corresponds to a healthy, nontransformed control plant, and lanes 3 to 7 correspond to five different noninoculated Rs transgenic lines. Note that the transgenic lines expressed either low levels of the cp transcript (lanes 5 to 7 = lines S 1, S 16, and AS3, respectively) or undetectable cp transcripts (lanes 3 and 4 = lines AS4 and S5) even

after long exposure times (5 to 7 days). The location of the cp transcript (1.7 kbp) is indicated in the figure.

Figure 4 illustrates screening for TomRSV resistance of Rl transgenic N. benthamiana lines. Rs progeny were from self-pollinated Ro plants that were resistant to Tom RSV (AS-antisense line, S = sense line). Twenty plants were tested per line. Plants were mechanically inoculated with a 1: 50 inoculum dilution.

Figure 5 shows the reactions of RI transgenic lines of N. tabacum to inoculation with TomRSV. The number of local lesions per leaf varied from susceptible transgenic lines (L33 and L 11) that develop similar numbers of local lesions as the control (nontransformed and vector-transformed) plants to resistant transgenic lines with none (line L38) or significantly reduced number of local lesions (L8, LI, and L41). Five plants were tested per transgenic line. Vertical lines represent standard errors.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an isolated coat protein or polypeptide for a peach isolate of a tomato ringspot virus. The encoding RNA and DNA molecules, in either isolated form or incorporated in an expression system, a host cell, or a transgenic plant, are also disclosed.

As used herein, the term"isolated"when used in conjunction with a nucleic acid molecule or amino acid refers to: 1) a nucleic acid molecule or amino acid which has been separated from an organism in a substantially purified form (i. e. substantially free of other substances originating from that organism), or 2) a nucleic acid molecule or amino acid having the same nucleotide sequence but not necessarily separated from the organism (i. e. synthesized nucleic acid molecules or amino acids).

The present invention relates to an isolated DNA molecule encoding the coat proteins or polypeptides for a peach isolate of a tomato ringspot virus.

As shown in Figure 2, the nucleotide sequence of the 3'terminal region of a RNA 2 of a peach isolate of TomRSV corresponds to SEQ. ID. NO. 1 as follows: GTTCAGGGCG GGTCCTGGCA AGAAGGTACT GAAGCCGCTT TTCTAGGCAA AGTTACCTGT 60 GCGAAGGACG CCAAGGGTGG AACTTTATTG CACACTTTGG ATATTATAAA AGAGTGCAAA 120

TCCCAAAATT TATTAAGGTA TAAAGAATGG CAACGTCAAG GCTTTCTTCA TGGAAAGCTT 180 AGATTGCGCT GCTTCATACC CACTAACATT TTTTGTGGGC ATTCCATGAT GTGTTCTTTG 240 GACGCGTTTG GTCGTTATGA TTCGAACGTG CTAGGTGCTA GTTTTCCAGT GAAGTTGGCA 300 AGTTTATTGC CAACGGAGGT GATTAGTCTA GCTGATGGAC CCGTGGTCAC GTGGACGTTT 360 GATATTGGAC GTCTGTGTGG CCATGGTCTC TATTATTCCG AGGGCGCTTA TGCGAGGCCC 420 AAAATTTATT TTTTAATTCT TTCTGATAAT GATGTTCCTG CAGAAGCAGA TTGGCAATTT 480 ACCTATCAGC TTTTGTTTGA GGATCATACG TTTTCGAATT CCTTTGGGGC GGTTCCTTTT 540 ATTACCTTAC CCCATATTTT TAATAGATTA GATATAGGTT ATTGGCGCGG GCCAACAGAG 600 ATAGATTTAA CATCAACTCC CGCACCAAAC GCCTATCGTT TACTTTTCGG CTTGTCCACT 660 GCTATTAGTG GTAACATGTC GACTTTGAAT GCCAATCAAG CCCTATTGCG TTTTTTTCAG 720 GGCTCGAATG GCACTTTACA TGGGCGCATT AAAAAGATAG GGACAGCACT TACAACTTGT 780 TCCCTTTTAT TATCGTTGCG CCACAAAGAT GCGAGTCTCA CATTGGAGAC CGCATATCAA 840 AGGCCCCATT ACATTTTGGC TGATGGACAA GGGGCTTTTT CACTACCAAT TTCTACCCCC 900 CATGAAGCAA CCTCCTTTGT GGAGGACATG TTGCGCCTGG AGATTTTTGC TATTGCTGGG 960 CCTTTTAGTC CCAAAGATAA TAAAGCAACA TACCAATTCA TGTGTTATTT CGATCACATA 1020 GAATCGGTTG AGGGGGTACC TAGAACTATA GCAGGCGAGC AGCAGTTCAA CTGGTGTAGT 1080 TTAACAAATT CCACAATCGA TGACTGGAGG TTTGAGTGGC CGGCTCGCCT ACCAGATATA 1140 CTTGATGATA AGTCAGAAGT GCTTTTAAGG CAACATCCTT TATCTCTGCT TATCTCATCT 1200 ACCGGTTTTT TTACGGGTAG AGCCATTTTT GTTTTCCAGT GGGGTGTGAA TACTACTGCT 1260 GGGAATATGA AAGGCTCATT TTCTGCGCGC CTGGCCTTTG GCAAGGGCGT GGAGGAAATT 1320 GACCAGACGT CAACAGTGCA ACCACTTGTT GGCGCTTGTG AAGCCCGCAT ACCCGTGGAG 1380 TTTAAGACTT ACACGGGTTA TACTACTTCG GGTCCTCCTG GATCCATGGA ACCATACATT 1440 TACGTGAGGC TTACGCAACC TACGCTTGTG GATAGGCTTT CTGTGAATGT TATTTTACAG 1500 GAGGGATTTT CTTTCTATGG ACCTAGCGTC AAACATTTTA AGAAAGAAGT CGGCACGCCT 1560 AGTGCCACCC TAGAGACAAA TAACCCCGTT GGGCGCCCAC CTGAGAATAT CGATACAGGG 1620 GGTCCCGGCG GCCAGTATGC AGCTGCCTTA CAAGCAGCTC AGCAAGCTGG GAGAAATCCT 1680 TTTGGGCGTG GCTAAGTTGG CTTCCTGAAA GGCGAGTAGC TGCCGTTAGC AGCTTCCAAA 1740 AGGTGGCCTC TTAATTAGCT TTTAATAGGG GTTATCCAGC CTTAAGCAAG CTGGCACCGG 1800 TCCTGATGGA CTACCAGGAA AGCACCTGGT TTGGAAGAAT TCGAGTAAAA TTCTTAAATC 1860 TTGTTTACTC GTGACTTATA GTACATTCAA GAGGAATGAC TCATGTTTTG TCCATTTACA 1920 TGATGGCATA AAGAGTTAAC GGCTCATATG GTGCTCATTA CGTTCAAGTG TTGAAGGATC 1980

CAATAGCCTT GAACTGTGGT GCCATGTGAG GAGATCCACG TTATCTCTGA TTGTCAAAAT 2040 AGACTAGTCT AGGAGACGAT AAATCCTATG TGGGTGAGTC CCATTCTGGC GAGACACGCA 2100 ATGCCTTTTA TTTGTTTGAG GTTATCAAAC ATCATATCTT GAGTCTGCAT TTAAATTCCA 2160 ATAATGTAGT TGTCATAGCC TACCGATGAG CCTGCGAGAA AGGTTCCATG AGGACTAGGG 2220 TTGGCTAACC CTCACTTAAT CTCTCTATTG GTCATTCGAC AGTGCGTCGA GAATTCATGG 2280 GTTTCATCAC CCACATTGAA GCGAGTGTCT CGTAAGAAAC CCACTCGGAT TGATGTACTT 2340 ACCATGCATC CTTTCGAGTA AAGCATCGAT TCCGTCGTTG TGGTTCTTCA ACTGTGGTTT 2400 TAGATGAGCG ATGAGTTGCG CTGCCCGCGT ATGAAGCGTG GAAAAGTAGT CTGAAACGAA 2460 CTTAGTACCA GAGGTAGGAC GCCATTGTTC CAGGCGTTTT TTATGGACAT AAGCTGTAAA 2520 CTTGGTTTCG CAAGCCATGC AGCACCTCCC TTTATTCGTG TACTATCCAG GGGCTCCCGG 2580 TTCTTTCTTA CCGGTACAAT ACCTGGTGAA GCGAATACTT GCGTCGAGGG ATGAGAGTAG 2640 CATGTTCCTA CTCATTGAAG GAATATGTCG TGTTTTCCAC ACGTTAGTGT TAAATGCAGT 2700 ACCCAGCGCC ATAGTGCAAG AATGGTTCCC AGCCACTTTT TCTGGGATTC TAATCGTACG 2760 ACACAATTGC ATGTGTATCG TTGACGGAGG AGTAGCGATC CTCTACCACG CGAGCCTGGA 2820 AGTAATTGCC GGGGCCGAAG AAGGCCAGCA TGCGGTACGA TTAACTTTAG CTGTAATGTA 2880 GTGGTATGTT AAGTTGAGAC TAACTTACCG TACGAGTCAA ACTCCTTGGG TGGATGTGTG 2940 TTCTGCCACC TTGGAGGAAG TAGATGTGAT TTTACCAGTC TGAGACGAGC CATTAATTTG 3000 GTGCTTTTAT TCATTGATGA TAATACTCGT GCAGTTGCAG CTGCACGAGT ATGTTGGTAC 3060 GCACAGTCTA CTCGGATACG GCCGAGTTGC CCTCACAACA GGGATTATCT CTCAATCTTA 3120 ACTACTGCCA GGACGTTGTT TTCGCAGGGT TTTGTTGGTC CGTTTGTGTT TCAAAACGCT 3180 GCTTTGCAAT TTTCTATTTT GTTTTATTGC TTTCGGAGTG TCGAACTTTG TCCAAGTTCA 3240 TAAAAGC 3247 Of particular interest is the portion of SEQ. ID. NO. 1 identified as nucleotide 7 through 1695. As shown in Figure 2, the cleavage site (Q-G) is identified between the putative movement protein and the coat protein of the present invention. The DNA molecule encoding the coat protein of the present invention comprises the nucleotide sequence corresponding to SEQ. ID. NO. 2 as follows: GGCGGGTCCT GGCAAGAAGG TACTGAAGCC GCTTTTCTAG GCAAAGTTAC CTGTGCGAAG 60 GACGCCAAGG GTGGAACTTT ATTGCACACT TTGGATATTA TAAAAGAGTG CAAATCCCAA 120

AATTTATTAA GGTATAAAGA ATGGCAACGT CAAGGCTTTC TTCATGGAAA GCTTAGATTG 180 CGCTGCTTCA TACCCACTAA CATTTTTTGT GGGCATTCCA TGATGTGTTC TTTGGACGCG 240 TTTGGTCGTT ATGATTCGAA CGTGCTAGGT GCTAGTTTTC CAGTGAAGTT GGCAAGTTTA 300 TTGCCAACGG AGGTGATTAG TCTAGCTGAT GGACCCGTGG TCACGTGGAC GTTTGATATT 360 GGACGTCTGT GTGGCCATGG TCTCTATTAT TCCGAGGGCG CTTATGCGAG GCCCAAAATT 420 TATTTTTTAA TTCTTTCTGA TAATGATGTT CCTGCAGAAG CAGATTGGCA ATTTACCTAT 480 CAGCTTTTGT TTGAGGATCA TACGTTTTCG AATTCCTTTG GGGCGGTTCC TTTTATTACC 540 TTACCCCATA TTTTTAATAG ATTAGATATA GGTTATTGGC GCGGGCCAAC AGAGATAGAT 600 TTAACATCAA CTCCCGCACC AAACGCCTAT CGTTTACTTT TCGGCTTGTC CACTGCTATT 660 AGTGGTAACA TGTCGACTTT GAATGCCAAT CAAGCCCTAT TGCGTTTTTT TCAGGGCTCG 720 AATGGCACTT TACATGGGCG CATTAAAAAG ATAGGGACAG CACTTACAAC TTGTTCCCTT 780 TTATTATCGT TGCGCCACAA AGATGCGAGT CTCACATTGG AGACCGCATA TCAAAGGCCC 840 CATTACATTT TGGCTGATGG ACAAGGGGCT TTTTCACTAC CAATTTCTAC CCCCCATGAA 900 GCAACCTCCT TTGTGGAGGA CATGTTGCGC CTGGAGATTT TTGCTATTGC TGGGCCTTTT 960 AGTCCCAAAG ATAATAAAGC AACATACCAA TTCATGTGTT ATTTCGATCA CATAGAATCG 1020 GTTGAGGGGG TACCTAGAAC TATAGCAGGC GAGCAGCAGT TCAACTGGTG TAGTTTAACA 1080 AATTCCACAA TCGATGACTG GAGGTTTGAG TGGCCGGCTC GCCTACCAGA TATACTTGAT 1140 GATAAGTCAG AAGTGCTTTT AAGGCAACAT CCTTTATCTC TGCTTATCTC ATCTACCGGT 1200 TTTTTTACGG GTAGAGCCAT TTTTGTTTTC CAGTGGGGTG TGAATACTAC TGCTGGGAAT 1260 ATGAAAGGCT CATTTTCTGC GCGCCTGGCC TTTGGCAAGG GCGTGGAGGA AATTGACCAG 1320 ACGTCAACAG TGCAACCACT TGTTGGCGCT TGTGAAGCCC GCATACCCGT GGAGTTTAAG 1380 ACTTACACGG GTTATACTAC TTCGGGTCCT CCTGGATCCA TGGAACCATA CATTTACGTG 1440 AGGCTTACGC AACCTACGCT TGTGGATAGG CTTTCTGTGA ATGTTATTTT ACAGGAGGGA 1500 TTTTCTTTCT ATGGACCTAG CGTCAAACAT TTTAAGAAAG AAGTCGGCAC GCCTAGTGCC 1560 ACCCTAGAGA CAAATAACCC CGTTGGGCGC CCACCTGAGA ATATCGATAC AGGGGGTCCC 1620 GGCGGCCAGT ATGCAGCTGC CTTACAAGCA GCTCAGCAAG CTGGGAGAAA TCCTTTTGGG 1680 CGTGGCTAA 1689 The coat protein of the tomato ringspot virus has an amino acid sequence corresponding to SEQ. ID. NO. 3 as follows :

Gly Gly Ser Trp Gln Glu Gly Thr Glu Ala Ala Phe Leu Gly Lys Val 1 5 10 15 Thr Cys Ala Lys Asp Ala Lys Gly Gly Thr Leu Leu His Thr Leu Asp 20 25 30 Ile Ile Lys Glu Cys Lys Ser Gln Asn Leu Leu Arg Tyr Lys Glu Trp 35 40 45 Gln Arg Gln Gly Phe Leu His Gly Lys Leu Arg Leu Arg Cys Phe Ile 50 55 60 Pro Thr Asn Ile Phe Cys Gly His Ser Met Met Cys Ser Leu Asp Ala 65 70 75 80 Phe Gly Arg Tyr Asp Ser Asn Val Leu Gly Ala Ser Phe Pro Val Lys 85 90 95 Leu Ala Ser Leu Leu Pro Thr Glu Val Ile Ser Leu Ala Asp Gly Pro 100 105 110 Val Val Thr Trp Thr Phe Asp Ile Gly Arg Leu Cys Gly His Gly Leu 115 120 125 Tyr Tyr Ser Glu Gly Ala Tyr Ala Arg Pro Lys Ile Tyr Phe Leu Ile 130 135 140 Leu Ser Asp Asn Asp Val Pro Ala Glu Ala Asp Trp Gln Phe Thr Tyr 145 150 155 160 Gln Leu Leu Phe Glu Asp His Thr Phe Ser Asn Ser Phe Gly Ala Val 165 170 175 Pro Phe Ile Thr Leu Pro His Ile Phe Asn Arg Leu Asp Ile Gly Tyr 180 185 190 Trp Arg Gly Pro Thr Glu Ile Asp Leu Thr Ser Thr Pro Ala Pro Asn 195 200 205 Ala Tyr Arg Leu Leu Phe Gly Leu Ser Thr Ala Ile Ser Gly Asn Met 210 215 220 Ser Thr Leu Asn Ala Asn Gln Ala Leu Leu Arg Phe Phe Gln Gly Ser 225 230 235 240 Asn Gly Thr Leu His Gly Arg Ile Lys Lys Ile Gly Thr Ala Leu Thr 245 250 255 Thr Cys Ser Leu Leu Leu Ser Leu Arg His Lys Asp Ala Ser Leu Thr 260 265 270 Leu Glu Thr Ala Tyr Gln Arg Pro His Tyr Ile Leu Ala Asp Gly Gln 275 280 285

Gly Ala Phe Ser Leu Pro Ile Ser Thr Pro His Glu Ala Thr Ser Phe 290 295 300 Val Glu Asp Met Leu Arg Leu Glu Ile Phe Ala Ile Ala Gly Pro Phe 305 310 315 320 Ser Pro Lys Asp Asn Lys Ala Thr Tyr Gln Phe Met Cys Tyr Phe Asp 325 330 335 His Ile Glu Ser Val Glu Gly Val Pro Arg Thr Ile Ala Gly Glu Gln 340 345 350 Gln Phe Asn Trp Cys Ser Leu Thr Asn Ser Thr Ile Asp Asp Trp Arg 355 360 365 Phe Glu Trp Pro Ala Arg Leu Pro Asp Ile Leu Asp Asp Lys Ser Glu 370 375 380 Val Leu Leu Arg Gln His Pro Leu Ser Leu Leu Ile Ser Ser Thr Gly 385 390 395 400 Phe Phe Thr Gly Arg Ala Ile Phe Val Phe Gln Trp Gly Val Asn Thr 405 410 415 Thr Ala Gly Asn Met Lys Gly Ser Phe Ser Ala Arg Leu Ala Phe Gly 420 425 430 Lys Gly Val Glu Glu Ile Asp Gln Thr Ser Thr Val Gln Pro Leu Val 435 440 445 Gly Ala Cys Glu Ala Arg Ile Pro Val Glu Phe Lys Thr Tyr Thr Gly 450 455 460 Tyr Thr Thr Ser Gly Pro Pro Gly Ser Met Glu Pro Tyr Ile Tyr Val 465 470 475 480 Arg Leu Thr Gln Pro Thr Leu Val Asp Arg Leu Ser Val Asn Val Ile 485 490 495 Leu Gln Glu Gly Phe Ser Phe Tyr Gly Pro Ser Val Lys His Phe Lys 500 505 510 Lys Glu Val Gly Thr Pro Ser Ala Thr Leu Glu Thr Asn Asn Pro Val 515 520 525 Gly Arg Pro Pro Glu Asn Ile Asp Thr Gly Gly Pro Gly Gly Gln Tyr 530 535 540 Ala Ala Ala Leu Gln Ala Ala Gln Gln Ala Gly Arg Asn Pro Phe Gly 545 550 555 560 Arg Gly

The molecular weight was calculated from the amino acid sequence and was 60 to 64 KDa, preferably about 62 KDa.

The sequence of the 3'-end untranslatable region is identified as nucleotides 1696 through 3247 of SEQ. ID. NO. 1 and corresponds to SEQ. ID.

NO. 4 as follows: GTTGGCTTCC TGAAAGGCGA GTAGCTGCCG TTAGCAGCTT CCAAAAGGTG GCCTCTTAAT 60 TAGCTTTTAA TAGGGGTTAT CCAGCCTTAA GCAAGCTGGC ACCGGTCCTG ATGGACTACC 120 AGGAAAGCAC CTGGTTTGGA AGAATTCGAG TAAAATTCTT AAATCTTGTT TACTCGTGAC 180 TTATAGTACA TTCAAGAGGA ATGACTCATG TTTTGTCCAT TTACATGATG GCATAAAGAG 240 TTAACGGCTC ATATGGTGCT CATTACGTTC AAGTGTTGAA GGATCCAATA GCCTTGAACT 300 GTGGTGCCAT GTGAGGAGAT CCACGTTATC TCTGATTGTC AAAATAGACT AGTCTAGGAG 360 ACGATAAATC CTATGTGGGT GAGTCCCATT CTGGCGAGAC ACGCAATGCC TTTTATTTGT 420 TTGAGGTTAT CAAACATCAT ATCTTGAGTC TGCATTTAAA TTCCAATAAT GTAGTTGTCA 480 TAGCCTACCG ATGAGCCTGC GAGAAAGGTT CCATGAGGAC TAGGGTTGGC TAACCCTCAC 540 TTAATCTCTC TATTGGTCAT TCGACAGTGC GTCGAGAATT CATGGGTTTC ATCACCCACA 600 TTGAAGCGAG TGTCTCGTAA GAAACCCACT CGGATTGATG TACTTACCAT GCATCCTTTC 660 GAGTAAAGCA TCGATTCCGT CGTTGTGGTT CTTCAACTGT GGTTTTAGAT GAGCGATGAG 720 TTGCGCTGCC CGCGTATGAA GCGTGGAAAA GTAGTCTGAA ACGAACTTAG TACCAGAGGT 780 AGGACGCCAT TGTTCCAGGC GTTTTTTATG GACATAAGCT GTAAACTTGG TTTCGCAAGC 840 CATGCAGCAC CTCCCTTTAT TCGTGTACTA TCCAGGGGCT CCCGGTTCTT TCTTACCGGT 900 ACAATACCTG GTGAAGCGAA TACTTGCGTC GAGGGATGAG AGTAGCATGT TCCTACTCAT 960 TGAAGGAATA TGTCGTGTTT TCCACACGTT AGTGTTAAAT GCAGTACCCA GCGCCATAGT 1020 GCAAGAATGG TTCCCAGCCA CTTTTTCTGG GATTCTAATC GTACGACACA ATTGCATGTG 1080 TATCGTTGAC GGAGGAGTAG CGATCCTCTA CCACGCGAGC CTGGAAGTAA TTGCCGGGGC 1140 CGAAGAAGGC CAGCATGCGG TACGATTAAC TTTAGCTGTA ATGTAGTGGT ATGTTAAGTT 1200 GAGACTAACT TACCGTACGA GTCAAACTCC TTGGGTGGAT GTGTGTTCTG CCACCTTGGA 1260 GGAAGTAGAT GTGATTTTAC CAGTCTGAGA CGAGCCATTA ATTTGGTGCT TTTATTCATT 1320 GATGATAATA CTCGTGCAGT TGCAGCTGCA CGAGTATGTT GGTACGCACA GTCTACTCGG 1380 ATACGGCCGA GTTGCCCTCA CAACAGGGAT TATCTCTCAA TCTTAACTAC TGCCAGGACG 1440 TTGTTTTCGC AGGGTTTTGT TGGTCCGTTT GTGTTTCAAA ACGCTGCTTT GCAATTTTCT 1500 ATTTTGTTTT ATTGCTTTCG GAGTGTCGAA CTTTGTCCAA GTTCATAAAA GC 1552

The protein or polypeptide of the present invention is preferably produced in purified form (preferably, at least about 80%, more preferably 90%, pure) by conventional techniques. Typically, the protein or polypeptide of the present invention is isolated by lysing and sonication. After washing, the lysate pellet is resuspended in buffer containing Tris-HCI. During dialysis, a precipitate forms from this protein solution. The solution is centrifuged, and the pellet is washed and resuspended in the buffer containing Tris-HCl. Proteins are resolved by electrophoresis through an SDS 12% polyacrylamide gel.

The DNA molecule encoding the tomato ringspot virus protein or polypeptide of the present invention can be incorporated in cells using conventional recombinant DNA technology. Generally, this involves inserting the DNA molecule into an expression system to which the DNA molecule is heterologous (i. e. not normally present). The heterologous DNA molecule is inserted into the expression system or vector in proper sense orientation and correct reading frame. The entire DNA molecule or smaller fragments of it can be used to produce transgenic plants. See U. S. Patent Application Serial No. 09/025,635, which is hereby incorporated by reference. Alternatively, it can have an antisense orientation. Antisense RNA technology involves the production of an RNA molecule that is complementary to the messenger RNA molecule of a target gene; the antisense RNA can potentially block all expression of the targeted gene. In the anti-virus context, plants are made to express an antisense RNA molecule corresponding to a viral RNA (that is, the antisense RNA is an RNA molecule which is complementary to a plus sense RNA species encoded by an infecting virus). Such plants may show a decreased susceptibility to infection by that virus. Such a complementary RNA molecule is termed antisense RNA. The vector contains the necessary elements for the transcription and translation of the inserted protein-coding sequences.

U. S. Patent No. 4,237,224 to Cohen and Boyer, which is hereby incorporated by reference, describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of

transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinia virus. Recombinant viruses can be generated by transfection of plasmids into cells infected with virus.

Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES. tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC184, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/- or KS +/- (see"Stratagene Cloning Systems"Catalog (1993) from Stratagene, La Jolla, Calif, which is hereby incorporated by reference), pQE, pIH821, pGEX, pET series (see Studier et. al.,"Use of T7 RNA Polymerase to Direct Expression of Cloned Genes,"Gene Expression Technology, vol. 185 (1990), which is hereby incorporated by reference), and any derivatives thereof. Recombinant molecules can be introduced into cells via transformation, transduction, conjugation, mobilization, or electroporation. The DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Maniatis et al., Molecular Cloning : A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), which is hereby incorporated by reference.

A variety of host-vector systems may be utilized to express the protein-encoding sequence (s). Primarily, the vector system must be compatible with the host cell used. Host-vector systems include but are not limited to the following: bacteria transformed with bacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containing yeast vectors; mammalian cell systems infected with virus (e. g., vaccina virus, adenovirus, etc.); insect cell systems infected with virus (e. g., baculovirus); and plant cells infected by bacteria or transformed via particle bombardment (i. e. biolistics). The expression elements of these vectors vary in their strength and specificities. Depending upon the host- vector system utilized, any one of a number of suitable transcription and translation elements can be used.

Different genetic signals and processing events control many levels of gene expression (e. g., DNA transcription and messenger RNA ("mRNA") translation).

Transcription of DNA is dependent upon the presence of a promotor which is a DNA sequence that directs the binding of RNA polymerase and thereby promotes mRNA synthesis. The DNA sequences of eucaryotic promotors differ from those of procaryotic promotors. Furthermore, eucaryotic promotors and accompanying genetic signals may not be recognized in or may not function in a procaryotic system, and, further, procaryotic promotors are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presence of the proper procaryotic signals which differ from those of eucaryotes.

Efficient translation of mRNA in procaryotes requires a ribosome binding site called the Shine-Dalgarno ("SD") sequence on the mRNA. This sequence is a short nucleotide sequence of mRNA that is located before the start codon, usually AUG, which encodes the amino-terminal methionine of the protein. The SD sequences are complementary to the 3'-end of the 16S rRNA (ribosomal RNA) and probably promote binding of mRNA to ribosomes by duplexing with the rRNA to allow correct positioning of the ribosome. For a review on maximizing gene expression, see Roberts and Lauer, Methods in Enzymologv, 68: 473 (1979), which is hereby incorporated by reference.

Promotors vary in their"strength" (i. e. their ability to promote transcription). For the purposes of expressing a cloned gene, it is desirable to use strong promotors in order to obtain a high level of transcription and, hence, expression of the gene. Depending upon the host cell system utilized, any one of a number of suitable promotors may be used. For instance, when cloning in E. coli, its bacteriophages, or plasmids, promotors such as the T7 phage promoter, lac promotor, trp promotor, recA promotor, ribosomal RNA promotor, the PR and PL promotors of coliphage lambda and others, including but not limited, to lacUV5, ompF, bla, lpp, and the like, may be used to direct high levels of transcription of adjacent DNA segments. Additionally, a hybrid trp-lacUVS (tac) promotor or other E. coli promotors produced by recombinant DNA or other

synthetic DNA techniques may be used to provide for transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen which inhibit the action of the promotor unless specifically induced. In certain operons, the addition of specific inducers is necessary for efficient transcription of the inserted DNA. For example, the lac operon is induced by the addition of lactose or IPTG (isopropylthio-beta-D-galactoside). A variety of other operons, such as trp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient gene transcription and translation in procaryotic cells. These transcription and translation initiation signals may vary in"strength"as measured by the quantity of gene specific messenger RNA and protein synthesized, respectively. The DNA expression vector, which contains a promotor, may also contain any combination of various"strong"transcription and/or translation initiation signals. For instance, efficient translation in E. coli requires a Shine-Dalgarno ("SD") sequence about 7- 9 bases 5'to the initiation codon ("ATG") to provide a ribosome binding site.

Thus, any SD-ATG combination that can be utilized by host cell ribosomes may be employed. Such combinations include but are not limited to the SD-ATG combination from the cro gene or the N gene of coliphage lambda, or from the E. coli tryptophan E, D, C, B or A genes. Additionally, any SD-ATG combination produced by recombinant DNA or other techniques involving incorporation of synthetic nucleotides may be used.

Once the isolated DNA molecules encoding the various tomato ringspot virus proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system. Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like.

The present invention also relates to RNA molecules which encode the tomato ringspot virus proteins or polypeptides described above. The transcripts can be synthesized using the host cells of the present invention by any

of the conventional techniques. The mRNA can be translated either in vitro or in vivo. Cell-free systems typically include wheat-germ or reticulocyte extracts. In vivo translation can be effected, for example, by microinjection into frog oocytes.

One aspect of the present invention involves using one or more of the above DNA molecules encoding the various proteins or polypeptides of a tomato ringspot virus from peach to transform plants in order to impart tomato ringspot virus resistance to the plants. The mechanism by which resistance is imparted in not known. In one hypothetical mechanism, the transformed plant can express the coat protein or polypeptide, and, when the transformed plant is inoculated by a tomato ringspot virus, the expressed coat protein or polypeptide surrounds the virus, thereby preventing translation of the viral DNA.

In this aspect of the present invention the subject DNA molecule incorporated in the plant can be constitutively expressed. Alternatively, expression can be regulated by a promoter which is activated by the presence of tomato ringspot virus. Suitable promoters for these purposes include those from genes expressed in response to tomato ringspot virus infiltration.

The isolated DNA molecules of the present invention can be utilized to impart tomato ringspot virus resistance for a wide variety of plants.

The DNA molecules are particularly well suited to imparting resistance to fruit plants, such as grapes, apples, peaches, cherries, plums, raspberries, and strawberries.

Plant tissue suitable for transformation include leaf tissue, root tissue, meristems, zygotic and somatic embryos, and anthers. It is particularly preferred to utilize embryos obtained from anther cultures.

The expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Tissue cells transformed in accordance with the present invention can be grown in vitro in a suitable medium to impart tomato ringspot virus resistance. Transformed cells can be regenerated into whole plants such that the protein or polypeptide imparts resistance to tomato ringspot virus in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express that DNA molecule to produce

the above-described tomato ringspot virus proteins or polypeptides and, thus, to impart tomato ringspot resistance.

One technique of transforming plants with the DNA molecules in accordance with the present invention is by contacting the tissue of such plants with an inoculum of a bacteria transformed with a vector comprising a gene in accordance with the present invention which imparts tomato ringspot virus resistance. Generally, this procedure involves inoculating the plant tissue with a suspension of bacteria and incubating the tissue for 48 to 72 hours on regeneration medium without antibiotics at 25-28°C.

Bacteria from the genus Agrobacterium can be utilized to transform plant cells. Suitable species of such bacterium include Agrobacterium tumefaciens and Agrobacterium rhizogenes. Agrobacterium tumefaciens (e. g., strains C58, LBA4404, or EHA105) is particularly useful due to its well-known ability to transform plants.

Heterologous genetic sequences can be introduced into appropriate plant cells, by means of the Ti plasmid of A. tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid is transmitted to plant cells on infection by Agrobacterium and is stably integrated into the plant genome. J. Schell, Science, 237: 1176-83 (1987), which is hereby incorporated by reference.

Another approach to transforming plant cells with a gene which imparts resistance to pathogens is particle bombardment (also known as biolistic transformation) of the host cell. This can be accomplished in one of several ways.

The first involves propelling inert or biologically active particles at cells. This technique is disclosed in U. S. Patent Nos. 4,945,050,5,036,006, and 5,100,792, all to Sanford et al., and in Emerschad et al.,"Somatic Embryogenesis and Plant Development from Immature Zygotic Embryos of Seedless Grapes (Vitis vinifera),"Plant Cell Reports, 14: 6-12 (1995) ("Emerschad (1995)"), which are hereby incorporated by reference. Generally, this procedure involves propelling inert or biologically active particles at the cells under conditions effective to penetrate the outer surface of the cell and to be incorporated within the interior thereof. When inert particles are utilized, the vector can be introduced into the cell by coating the particles with the vector containing the heterologous DNA.

Alternatively, the target cell can be surrounded by the vector so that the vector is carried into the cell by the wake of the particle. Biologically active particles (e. g., dried bacterial cells containing the vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies.

Fraley, et al., Proc. Natl. Acad. Sci. USA, 79: 1859-63 (1982), which is hereby incorporated by reference.

The DNA molecule may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA, 82: 5824 (1985), which is hereby incorporated by reference). In this technique, plant protoplasts are electroporated in the presence of plasmids containing the expression cassette.

Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.

After transformation, the transformed plant cells must be regenerated.

Plant regeneration from cultured protoplasts is described in Evans et al., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co., New York, 1983); and Vasil I. R. (ed.), Cell Culture and Somatic Cell Genetics of Plants, Acad.

Press, Orlando, Vol. I, 1984, and Vol. III (1986), which are hereby incorporated by reference.

It is known that practically all plants can be regenerated from cultured cells or tissues, including but not limited to, all major species of sugarcane, sugar beets, cotton, fruit trees, and legumes.

Means for regeneration vary from species to species of plants, but generally a suspension of transformed protoplasts or a petri plate containing explants is first provided. Callus tissue is formed and shoots may be induced from callus and subsequently rooted. Alternatively, embryo formation can be induced in the callus tissue. These embryos germinate as natural embryos to form plants. The culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as corn and alfalfa. Efficient regeneration will depend on

the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.

Once transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the DNA construct is present in the resulting plants. Alternatively, transgenic seeds or propagules (e. g., cuttings) are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.

The tomato ringspot virus protein or polypeptide can also be used to raise antibodies or binding portions thereof or probes. The antibodies can be monoclonal or polyclonal. It is particularly preferred that the antibodies specifically bind to the tomato ringspot virus protein and the antibodies be purified. A"purified antibody"is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.

Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, antibody. An antibody that"specifically binds"recognizes and binds a tomato ringspot virus protein (e. g., a coat protein) but which does not substantially recognize and bind other molecules in a sample (e. g., a biological sample) which naturally includes that tomato ringspot virus protein. An antibody which"specifically binds"to a tomato ringspot virus protein is sufficient to detect a tomato ringspot virus protein in such a biological sample using one or more standard immunological techniques available to those in the art (for example, Western Blotting or immunoprecipitation).

Monoclonal antibody production may be effected by techniques which are well-known in the art. Basically, the process involves first obtaining immune cells (lymphocytes) from the spleen of a mammal (e. g., mouse) which has been previously immunized with the antigen of interest either in vivo or in vitro. The antibody-secreting lymphocytes are then fused with (mouse) myeloma cells or transformed cells, which are capable of replicating indefinitely in cell

culture, thereby producing an immortal, immunoglobulin-secreting cell line. The resulting fused cells, or hybridomas, are cultured, and the resulting colonies screened for the production of the desired monoclonal antibodies. Colonies producing such antibodies are cloned, and grown either in vivo or in vitro to produce large quantities of antibody. A description of the theoretical basis and practical methodology of fusing such cells is set forth in Kohler and Milstein, Nature, 256: 495 (1975), which is hereby incorporated by reference.

Mammalian lymphocytes are immunized by in vivo immunization of the animal (e. g., a mouse) with the protein or polypeptide of the present invention. Such immunizations are repeated as necessary at intervals of up to several weeks to obtain a sufficient titer of antibodies. Following the last antigen boost, the animals are sacrificed and spleen cells removed.

Fusion with mammalian myeloma cells or other fusion partners capable of replicating indefinitely in cell culture is effected by standard and well-known techniques, for example, by using polyethylene glycol ("PEG") or other fusing agents. (See Milstein and Kohler, Eur. J. Immunol., 6: 511 (1976), which is hereby incorporated by reference.) This immortal cell line, which is preferably murine, but may also be derived from cells of other mammalian species, including but not limited to rats and humans, is selected to be deficient in enzymes necessary for the utilization of certain nutrients, to be capable of rapid growth, and to have good fusion capability. Many such cell lines are known to those skilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.

Typically, such antibodies can be raised by administering the protein or polypeptide of the present invention subcutaneously to New Zealand white rabbits which have first been bled to obtain pre-immune serum. The antigens can be injected at a total volume of 100 1ll per site at six different sites. Each injected material will contain synthetic surfactant adjuvant pluronic polyols, or pulverized acrylamide gel containing the protein or polypeptide after SDS-polyacrylamide gel electrophoresis. The rabbits are then bled two weeks after the first injection and periodically boosted with the same antigen three times every six weeks. A sample of serum is then collected 10 days after each boost. Polyclonal antibodies

are then recovered from the serum by affinity chromatography using the corresponding antigen to capture the antibody. Ultimately, the rabbits are euthenized with pentobarbital 150 mg/Kg IV. This and other procedures for raising polyclonal antibodies are disclosed in Harlow et. al., editors, Antibodies: A Laboratory Manual (1988), which is hereby incorporated by reference.

In addition to utilizing whole antibodies, binding portions of such antibodies can be used. Such binding portions include Fab fragments, F (ab') 2 fragments, and Fv fragments. These antibody fragments can be made by conventional procedures, such as proteolytic fragmentation procedures, as described in Goding, Monoclonal Antibodies: Principles and Practice, New York: Academic Press, pp. 98-118 (1983), which is hereby incorporated by reference.

The present invention also relates to probes found either in nature or prepared synthetically by recombinant DNA procedures or other biological procedures. Suitable probes are molecules which bind to tomato ringspot viral antigens identified by the monoclonal antibodies of the present invention. Such probes can be, for example, proteins, peptides, lectins, or nucleic acid probes.

The antibodies or binding portions thereof or probes can be administered to tomato ringspot virus infected cultivars. Alternatively, at least the binding portions of these antibodies can be sequenced, and the encoding DNA synthesized. The encoding DNA molecule can be used to transform plants together with a promoter which causes expression of the encoded antibody when the plant is infected by tomato ringspot virus. In either case, the antibody or binding portion thereof or probe will bind to the virus and help prevent the usual TomRSV response.

Antibodies raised against the proteins or polypeptides of the present invention or binding portions of these antibodies can be utilized in a method for detection of tomato ringspot virus in a sample of tissue. Any reaction of the sample with the antibody is detected using an assay system which indicates the presence of tomato ringspot virus in the sample. A variety of assay systems can be employed, such as enzyme-linked immunosorbent assays,

radioimmunoassays, gel diffusion precipitin reaction assays, immunodiffusion assays, agglutination assays, fluorescent immunoassays, protein A immunoassays, or immunoelectrophoresis assays.

Alternatively, tomato ringspot virus can be detected in such a sample using a nucleotide sequence of the DNA molecule, or a fragment thereof, encoding for a protein or polypeptide of the present invention. The nucleotide sequence is provided as a probe in a nucleic acid hybridization assay known in the art, including, but not limited to, Southern blots (Southern, J. Mol. Biol., 98: 503- 17 (1975)); Northern blots (Thomas et al., Proc. Natl. Acad. Sci. USA, 77: 5201- 05 (1980)); Colony blots (Grunstein et al., Proc. Natl. Acad. Sci. USA, 72: 3961- 65 (1975); which are hereby incorporated by reference). Alternatively, the isolated DNA molecules of the present invention can be used in a gene amplification detection procedure (e. g., a polymerase chain reaction). See H. A.

Erlich et al.,"Recent Advances in the Polymerase Chain Reaction,"Science, 252: 1643-51 (1991), which is hereby incorporated by reference. Any reaction with the probe is detected so that the presence of tomato ringspot virus in the sample is indicated.

The following examples are provided to illustrate embodiments of the present invention but are by no means intended to limit its scope.

EXAMPLES The coat protein ("cp") gene and the 3'end untranslated region of a peach isolate of tomato ringspot virus ("TomRSV") were cloned from purified total viral RNA and sequenced. Reverse transcription and polymerase chain reaction ("RT-PCR") were used to engineer the TomRSV cp gene so that it could be cloned into plasmid vectors designed for either in vitro transcription or plant expression. The cloned TomRSV cp gene was used to transform Nicotiana benthamiana and N. tabacum plants, a systemic and a local lesion host, respectively. After challenge innoculation with the TomRSV peach isolate, several Ro and Rl and R2 resistant transgenic lines containing sense and antisense cp constructs exhibited different levels of protection, ranging from complete resistance, to delay in symptom appearance, or reduction in symptom severity.

Interestingly, cp gene expression levels were undectable by enzyme-linked immunosorbent assay ("ELISA") in the resistant lines containing cp sense constructs, and levels of cp transcripts were low or undetectable by Northern blot on resistant sense and antisense lines. The high level of resistance obtained in Nicotiana spp offers important prospects for the engineering of TomRSV resistance into several economically important fruit and berry crops susceptible to this nepovirus.

Material and Methods Virus purification and RNA isolation. A peach isolate of TomRSV originally obtained from California was used. This isolate induces peach yellow bud mosaic, and was propagated in cowpea and purified as previously described (Bitterlin, M. W., et al.,"Serological Grouping of Tomato Ringspot Virus Isolates: Implications for Diagnosis and Cross-Protection,"Phytopathology, 78: 278-285 (1988), which is hereby incorporated by reference). Total viral RNA was extracted from purified virions (Serghini, M. A.,"RNA 2 of Grapevine Fanleaf Virus: Sequence Analysis and Coat Protein Cistron Location,"J. Gen. Viro., 71: 1433-1441 (1990), which is hereby incorporated by reference) and fractionated by sucrose density gradiant centrifugation. However, because of their similar molecular mass, RNA 1 and RNA 2 could not be separated. Therefore, total viral RNA was used as a template for oligo (dT)-primed cDNA synthesis.

Reverse transcription and polymerase chain reaction (RT-PCR), cloning, and sequencing of the 3'end of RNA 2. cDNA clones containing the 3' end sequences of RNA 1 and RNA 2 were constructed by cDNA synthesis using oligo (dT) primers and murine leukemia virus ("MLV")-reverse transcriptase (Promega Corp., Madison, WI). The second cDNA strand was synthesized according to the method of Gubler, U., et al.,"A Simple and Very Efficient Method for Generating cDNA Libraries,"Gene, 25: 263-269 (1983), which is hereby incorporated by reference, extremities were filled with T4 DNA polymerase (Promega Corp., Madison, WI), and EcoRI linkers were added using T4 DNA ligase (Promega Corp., Madison, WI). The cDNA molecules were digested with EcoRI and ligated into the EcoRI site of the plasmid pUC 18 (Pharmacia LKB Biotechnology Inc., Piscataway,

NJ). Recombinant plasmids were electroporated into Escherichla coli strain DH5a.

The RNA species from which each oligo (dT)-primed clone was transcribed was determined by probing Northern blots of purified TomRSV RNA with labeled cloned cDNA. The cDNA clones corresponding to the 3'end untranslated region of RNA 2 and the C-terminus of the cp gene were characterized by restriction digestion and sequenced using cesium chloride purified plasmid DNA and the dideoxynucleotide chain termination method (Sanger, F., et al.,"DNA Sequencing with Chain- Terminating Inhibitors,: Proc. Natl. Acad. Sci. USA, 74: 5463-5467 (1977), which is hereby incorporated by reference). Sequence information was used to design primers for RT-PCR cloning.

Three cDNA clones were produced from total viral RNA by RT-PCR using MLV-reverse transcriptase (Promega Corp., Madison, WI), Taq DNA polymerase (Perkin-Elmer Corp., Norwalk, CT), and appropriate primers. A 1.5-kbp RT-PRC cDNA product was generated using primers 91-32 (5'-AGCACCATGGTCTGTCGAAAACAAAACTTGC-3') (SEQ. ID. No: 5) and 91-34 (5'-AGCTGACCATGGCTTGGACAAAGTTCGACACTACG-3') (SEQ. ID.

NO. 6), designed to prime the amplification of the untranslated 3'end region of TomRSV. A 2.2-kbp RT-PCR product that was obtained using primers 91-33 (5'-AGCTGACCATGGAAGCTTCCATTAGAGCTTATC-3') (SEQ. ID. NO. 7) and 91-34 corresponded to the 3'end noncoding region and the sequence coding for part of the C-terminal region of the cp. A third 1.7-kbp RT-PCR product was amplified corresponding to the full-length cp gene using primer 91-76 (5'-AGCTAGTCTAGACCATGGTTCAGGGCGGGTCCTGGCAAG-3') (SEQ. ID.

NO. 8), designed at the start of the cp gene (based on the microsequencing data of the cp), and primer 91-77 (5'-GCATGATCTAGACCATGGTAAAAGCTAATTAAGAGGCCACC-3') (SEQ.

ID. NO. 9), located 43 nt downstream of the end of the RNA 2 open reading frame as deduced from sequencing the RNA 2 cDNA clones.

The cloning primer contained an Xbal site (TCTAGA), followed by a NcoI site (CCATGG) with the ATG initiation codon in the context of the plant translation consensus sequence (Lutcke, H. A., et al.,"Selection of the AUG Initiation Codon Differs in Plants and Animals,"Eur. Mol. Biol. Organ. J., 6: 43-48 (1987),

which is hereby incorporated by reference). The XbaI site was used to facilitate cloning into the in vitro transcription vector and the in vivo plant expression vector.

The RT-PCR products were ligated into the plasmid vector pGMM (a pBluescript derivative engineered as described below) used for sequencing and in vitro translation experiments. Manual 35S-sequencing was done using the Sequenase T7 polymerase sequencing kit (United States Biochemical Corp., Cleveland OH) and automated sequencing was done using the Taq DyeDeoxyterminator cycle sequencing kit (Applied Biosystems, Inc., Foster City, CA). All clones were sequenced completely in both directions using primers designed at 300-to 350-nt intervals. Sequence data analysis was done using the Genetics Computer Group., Inc. sequence analysis software package (Genetics Computer Group, Inc., University Research Park, Madison, WI) and DNASTAR biocomputing software (DNASTAR Inc., Madison, WI).

Protein purification and microsequencing of the N-terminus of purified cp. Purified virions (3 mg) were dialyzed overnight in 0.1 M KH2PO4 (pH 7.0) and dissociated by heating for 2.5 hours at 55°C. Denatured protein was centrifuged at 5,000 x g for 10 minutes, washed twice with distilled water, and resuspended in 0.125 M Tris buffer (pH 7.0), 0.5% sodium dodecyl sulfate ("SDS"), and 10% glycerol. Undigested and Staphylococcus aureus V8 protease digested protein fractions were separated on a 12% denaturing SDS-polyacrylamide gel electrophoresis ("SDS-PAGE") and transferred to an Immobilon Milipore membrane (Millipore Corp., Bedford, MA) using a Bio-Rad miniblotter apparatus (Bio-Rad Laboratories, Richmond, CA). An Applied Biosystems model 470A protein sequencer with a model 120A PTH analyzer (Applied Biosystems, Inc., Foster City, CA) was used to determine the amino terminal sequence of the cp by Edman degradation. The Pico-tag method was used for total amino acid analysis with a detection limit of 1 pmol. Amino terminal protein sequencing and total amino acid analysis of the cp were conducted at the Analytical/Synthesis Facility of the Biotechnology Center, Cornell University, Ithaca, NY.

In vitro transcription and translation. In vitro transcripts were synthesized using T7 RNA polymerase from NotI-linearized recombinant pGMM plasmids containing the TomRSV 1.7-kbp RT-PCR cDNA product. The pGMM

plasmid is a pBluescript derivative engineered containing multiple cloning sites and the leader sequence of the cp gene of cucumber mosaic virus strain white leaf ("CMV-WL"). Transcripts were analyzed by electrophoresis on formaldehyde- containing agarose gels (Pinck, L., et al.,"A Satellite RNA in Grapevine Fanleaf Virus Strain F13,"J. Gen. Virol., 69: 233-239 (1988), which is hereby incorporated by reference). Transcripts (l llg/pl) were translated at 30°C in a nuclease-treated rabbit reticulocyte lysate system (Promega Corp., Madison, WI) containing 35S methionine.

Translation reactions were stopped after 1 hour by the addition of denaturing buffer (10% SDS and 25% (3-mercaptoethanol), and 35S-labeled translation products were analyzed by SDS-PAGE.

Engineering of the cp gene constructs into plant transformation vectors. The 1.7-kbp RT-PCR cDNA product corresponding to the cp gene was digested with XbaI and ligated in both sense and antisense orientation into the XbaI site of the plant expression vector pNYS. This vector is a pUCl 8 derivative made by custom PCR engineering (Slightom, J. L.,"Custom Polymerase-Chain-Reaction Engineering of a Plant Expression Vector,"Gene, 100: 251-255 (1991), which is hereby incorporated by reference), and contains the cauliflower mosaic virus ("CaMV") 35S promoter sequence, the CMV-WL leader sequence, a multiple cloning site, and the nopaline synthase ("NOS") terminator sequences. Identification of sense and antisense constructs was done by BamHI or KpnI digestion. The TomRSV cp expression cassette was excised by partial HindlII digestion from pNYS and ligated into the Hindlll site of the binary vectors pBI121 (Clontech Labs Inc., Palo Alto, CA) or pGA482GG, a derivative of pGA482 modified by the insertion of the P-glucuronidase ("GUS") gene in the BgII site, and a gentamycin resistance gene in the SalI site (Quemada, H. D., et al.,"Expression of Coat Protein Gene from Cucumber Mosaic Virus Strain C in Tobacco: Protection Against Infection by CMV Strains Transmitted Mechanically or by Aphids,"Phytopathology, 81: 794-802 (1991), which is hereby incorporated by reference) (Fig. 1). Both binary vectors contain the GUS gene and the neomycin phosphotransferase (NPT II) gene that confers resistance to kanamycin.

Transformation of N. benthamiana and N. tabacum. The binary vectors pGA482GG or pBI121 containing the TomRSV cp gene constructs in sense or

antisense orientation were electroplated into the disarmed A. tumefaciens strains LBA4404 (Clontech Labs Inc., Palto Alto, CA), C58Z707/C58sZ707 (Hepburn, A. G., et al.,"The Use of pNJ5000 as an Intermediate Vector for the Genetic Manipulation of Agrobacterium Ti Plasmids,"J. Gen. Microbiol., 131: 2961-2969 (1985), which is hereby incorporated by reference), and EHA101/EHA105 (Hood, E. E., et al.,"The Hypervirulence of Agrobacterium tumefaciens A281 is Encoded in a Region of pTIBo542 Outside the T-DNA,"J. Bacteriol., 168: 1291-1301 (1986), which is hereby incorporated by reference). Leaf disc-transformation by Agrobacterium was done following the procedure described by Horsch et al.,"Leaf Disc Transformation," Plant Molecular BiologY Manual, Kluwer Academic Publishers, Dordrecht, Netherlands, pp. A5: 1-9 (1993), which is hereby incorporated by reference). Leaves for transformation experiments were collected from seedlings germinated in vitro or in the greenhouse. Selection of transformants was done using kanamycin at 300 mg/liter. Some plants were transformed with binary vectors that did not harbor any cp gene constructs, and were used as controls for resistance evaluation.

Characterization of transgenic N. tabacum and N. benthamiana Ro plants. Expression of GUS in transformed regenerants was assayed using the histological and fluorimetric assays (Jefferson, R. A.,"Assaying Chimeric Genes in Plants: The GUS Gene Fusion System,"Plant Mol. Biol. Rep., 5: 387-405 (1987), which is hereby incorporated by reference). Expression of the NPT II gene was assayed by enzyme-linked immunosorbent assays ("ELISA") using commercial y-globulins (5'Prime 3'Prime, Inc., Boulder, CO). Coat protein expression in transgenic plants containing sense cp constructs was determined by direct double- antibody sandwich ELISA with y-globulins against TomRSV produced as described in Bitterlin, M. W., et al.,"Serological Grouping of Tomato Ringspot Virus Isolates: Implications for Diagnosis and Cross-Protection,"Phytopathology, 78: 278-285 (1988), which is hereby incorporated by reference. Extraction buffer, healthy Nicotiana tissue, TomRSV-infected tissue, and/or purified virus were included as controls in each ELISA plate (Immulon 2, Dynatech Labs Inc., Chantilly, VA).

Replicated wells were loaded for each plant sample and optical density was read at 450 nm.

Detection of the TomRSV cp gene in selected transgenic plants was corroborated by PCR and Southern blot analysis after isolation of plant genomic DNA (Murray, M. G., et al.,"Rapid Isolation of High Molecular Weight Plant DNA," Nucleic Acids Res., 8: 4321-4325 (1980), which is hereby incorporated by reference).

PCRs using oligonucleotide primers specific for the TomRSV cp, NPT II, and GUS genes were run using the reagents and instructions of the Perkin-Elmer PCR kit (Perkin-Elmer Corp.). Total plant RNA was extracted from actively growing leaves prior to inoculation (Pinck, L., et al.,"A Satellite RNA in Grapevine Fanleaf Virus Strain F13,"J. Gen. Virol., 69: 233-239 (1988), which is hereby incorporated by reference), and used for Northern blot analysis. Southern and Northern blot analyses were conducted using a 32P-labeled cloned probe for the cp gene that also contained the first 43 nt of the untranslated 3'end common region of RNA 1 and RNA 2.

Labeling was done using the oligolabeling method (Feinberg, A. P., et al.,"A Technique for Radiolabeling DNA Restriction Endonuclease Fragments to High Specific Activity,"Anal. Biochem., 137: 266-267 (1984), which is hereby incorporated by reference).

Challenge inoculation of transgenic Nicotiana plants. The TomRSV peach inoculum was prepared by harvesting symptomatic N. benthamiana leaves 5 days after inoculation and grinding them in phosphate buffer (0.01 M K2HP04, pH 7.0). Several inoculum dilutions (wt/vol) were used to screen the transgenic plants, e. g., 1: 25 (1 g of tissue/25 ml of buffer), 1: 50, or 1: 100 for N. tabacum, a local lesion host for TomRSV. Lower inoculum doses were used on the systemic host N. benthamiana because of the severity of the virus isolate, e. g., 1: 50,1: 100; 1: 250, 1: 500,1: 1,000,1: 1,500, and 1: 2,000 (wt/vol). Plants were inoculated when they were between 8 to 12 cm in height. The inoculum was applied by gently rubbing the upper surface of leaves predusted with Corundum (Universal Photonics, Inc., Hicksville, NY). For N. benthamiana, only the youngest, fully expanded apical leaves (three to four leaves/plant) were inoculated, while all the leaves were inoculated for N. tabacum. Seedlings of Chenopodium quinoa, a local lesion host for TomRSV, were inoculated at each inoculum dose to monitor inoculum strength from experiment to experiment. Plants were observed daily for symptom development. Number of lesions per leaf were counted for N. tabacum, and days required for necrosis to occur

for N. benthamiana. Resistant Ro and Rl transgenic lines were carried on to Rs and R2 generations, respectively, for further evaluation and segregation studies. RI and R2 seeds were germinated in vitro on kanamycin 300 mg/liter before establishing the plants in the greenhouse.

Example 1-RNA sequence analysis.

Three overlapping RT-PCR products (2.2,1.7, and 1.5 kbp) were sequenced to determine the nucleotide sequence of the cp gene and the 3'end untranslated region of a peach isolate of TomRSV (Fig. 2). The peach isolate cp gene was 1,689 nt in length with the following nucleotide composition: % A = 24.63, % G = 23.92, % U = 29.96, % C = 21.49 [% A + U = 54.59, and % C + G = 45.41].

Comparison of the nucleotide sequence for the cp of the peach isolate with the raspberry isolate of TomRSV sequenced by Rott et al.,"Nucleotide Sequence of Tomato Ringspot Virus RNA-2,"J. Gen. Virol., 72: 1505-1514 (1991) indicated 96.9% identity at the nucleic acid level (51 nt differences) and 96.6% identify at the protein level (19 amino acid differences). From the deduced amino acid sequence, the molecular mass of the cp of the peach isolate was 62.0 kDa with 562 amino acids, 6.65 isoelectric point, and-2.18 charge at pH 7.0. The TomRSV raspberry isolate had the same number of amino acid residues and molecular mass, but a different isoelectric point (7.81) and charge (+3.80 at pH 7.0), because of 19 amino acid substitutions scattered throughout the cp (Fig. 2). Ten of the amino acid differences observed between the cp of the peach and raspberry isolates of TomRSV corresponded to changes in amino acid groups and charge (hydrophobic to acidic or polar/basic to polar or polar to hydrophobic) and accounted for the difference in isoelectric point and charge. The calculated size for the cp of the peach isolate was similar to that determined by SDS-PAGE (described below). Comparison of the nucleotide sequence for the 3'end noncoding region of the peach (1,522 nt) and the raspberry (1,547 nt) isolates of TomRSV indicated only 90.3% identity (151 nt differences with 49 nt representing purine-pyrimidine changes, and 4 nt deletions plus 9 nt insertions accounting for the 5 nt difference in length). The nucleotide composition for the 3'end untranslatable region of the peach isolate was as follows:

% A = 24.61, % G = 24.23, % U = 30.48, and % C = 20.68 [% A +U = 55.09, % C + G = 44.91].

Example 2-Coat protein analysis.

SDS-PAGE of purified cp indicated that the TomRSV peach isolate had a cp with molecular mass of approximately 60 kDa. The amino acid composition obtained from acid hydrolysis of purified cp was in close agreement with the values expected from the RNA sequence (data not shown). The N-terminal region of the cp was microsequenced in order to identify the protease cleavage site and the amino acids at the cp N-terminus. The sequence determined for the first 22 amino acids at the N-terminal region (SEQ. ID. NO. 10) was as follows: Gly Gly Ser Trp Gln Glu Gly Thr Glu Ala Ala Phe Leu Gly Lys Val 1 5 10 15 Thr Cys Ala Lys Asp Ala 20 The amino acid cysteine in the deduced sequence could not be determined by the direct sequencing method used.

The cp cleavage site Q-G (Gln-Gly) was determined by comparing the cp N-terminal amino acid sequence with the residues deduced from the nucleotide sequence (Fig. 2). The predicted size for a protein released at the Q-G cleavage site was in agreement with the expected molecular mass of the cp. In addition, the amino acid sequencing data obtained for the N-terminal region of a V8 protease-generated polypeptide of molecular mass of 47 kDa matched perfectly the amino acid sequence for cp residues 133 to 155 (sequence underlined in Fig. 2). This confirmed that the open reading frame analyzed was indeed encoding the cp.

Example 3-Characterization of the engineered cp constructs.

In vitro translated 32S protein products were observed for the sense TomRSV cp construct, while antisense constructs gave no translated protein products, as expected. The in vitro translation product comigrated with purified cp, supporting

the proposed identity of the cp cleavage site and demonstrating the functionality of the cp sense constructs.

Example 4-Characterization of transgenic N. tabacum and N. benthamiana Ro plants.

Transgenic N. benthamiana and N. tabacum plants containing sense and antisense cp constructs were obtained using A. tumefaciens. A total of 313 'putative'transgenic cp lines were recovered, from which 173 sense and 140 antisense transgenic lines were transferred to the greenhouse for resistance evaluation.

Once transferred to the greenhouse, leaf samples were collected from each plant and tested by NPT II and GUS assays. Most tested plants (86%) were determined to be NPT II positive by ELISA prior to inoculation. GUS assays (X-Gluc and MUG) gave less consistent results for the same population of transgenic plants (73 and 82% positive, respectively). Coat protein gene expression was not detectable by ELISA in any of the transgenic N. benthamiana and N. tabacum sense lines tested. However, PCR analysis of several NPT II positive transgenic sense and antisense lines confirmed the presence of the transferred TomRSV cp gene (Fig. 3A). Similarly, Southern blot analysis demonstrated the presence of the TomRSV cp gene in several transgenic lines analyzed. Interestingly, the cp transcripts were detectable in Northern blot analysis only at low levels in some, but not all, of the transgenic sense and antisense lines analyzed (Fig. 3B). Two hundred sixty-five independent transgenic cp plants that were NPT II ELISA positive were further analyzed for resistance in the greenhouse.

Example 5-Evaluation of transgenic N. benthamiana for resistance.

The systemic host N. benthamiana was highly susceptible to the peach isolate of TomRSV. Control (nontransformed and vector-transformed) plants developed local chlorotic lesions on the inoculated leaves 3 days after inoculation.

Necrosis of the apical leaves was initiated 4 days postinoculation. Necrosis was observed on the apical tip after 4 to 5 days at 1: 50 dilutions, and after 6 to 7 days at 1: 250 to 1: 2,000 dilutions. Severe necrosis of the apex led to plant death 7 to 8 days after inoculation regardless of the inoculum dose.

One hundred sixty-six Ro transgenic N. benthamiana cp lines (representing 73 sense and 93 antisense lines) were inoculated with TomRSV at inoculum doses from 1: 50 to 1: 2,000. Most of the inoculated transgenic N. benthamiana Ro plants (60 to 87%) showed no delay or a short delay in symptom development (2 to 5 days), but later on became necrotic and died. Approximately 10% of the 166 transgenic Ro lines screened were resistant (16 lines-7 sense and 9 antisense). These resistant lines never developed any visible symptoms or systemic necrosis, and the virus could not be detected by ELISA in the youngest, noninoculated leaves. All 16 Ro resistant transgenic plants survived when reinoculated at 1: 25 inoculum dose to ensure that they were not escapes, and were kept for seed production.

Rl seedlings from 8 of the 16 Ro resistant lines (four sense lines designated S1, S5, S9, and S12 ; and four antisense lines designated AS3, AS4, ASH, and AS 13) were inoculated at 1: 50 dilution (20 seedlings per line) to evaluate whether the resistance was inherited. Out of the eight Rs lines tested, three lines (S5, S 12, and AS4) were completely resistant with none of the tested plants developing symptoms (Fig. 4), three lines (S9, AS11, and AS13) were highly resistant with 80% of the plants remaining symptomless, and two lines (S1 and AS3) were intermediately resistant with 60% of asymptomatic plants (Fig. 4).

R2 progeny of these Rl resistant plants were inoculated to study the inheritance of their resistance. R2 seedlings (20 per line) were inoculated at 1: 25 or 1: 50 dilution. Out of the eight R2 lines screened, six lines (Sl, S5, S12, AS4, ASH, and AS 13) were completely resistant even at high inoculum doses with 100% of the plants remaining symptomless. One sense line (S9) was highly resistant with 75% asymptomatic plants, and one antisense line (AS3) was intermediately resistant with 30 and 60% asymptomatic plants following inoculation at 1: 25 and 1: 50, respectively.

In addition, the other eight Ro resistant transgenic lines were screened only at the R2 seedling stage. Three of the lines (one sense and two antisense) were highly resistant with 75% of the plants remaining symptomless, and five lines (two sense and three antisense) were intermediately resistant with 40 to 60% asymptomatic plants.

Example 6-Evaluation of transgenic N. tabacum for resistance.

N. tabacum developed only local lesions and no systemic necrosis when inoculated with the TomRSV peach isolate. Chlorotic lesions appeared in control (nontransformed and vector-transformed) plants 3 days postinoculation, and because necrotic with typical rings around the central necrotic spot. The virions could not be detected by ELISA in the new, noninoculated leaves. Sixteen transgenic Ro lines containing sense and antisense cp constructs were screened: 12 sense and four antisense Ro lines. From the 16 Ro lines screened at a 1: 50 inoculum dose, only two lines (one sense [L7] and one antisense [L9]) displayed complete resistance with no local lesions developing. The remaining 14 Ro lines screened developed local lesions ranging from numbers similar to the control to significantly reduced number of lesions. No local lesions developed in the inoculated Rs progeny from the two resistant Ro lines L7 and L9.

In a separate experiment, 12 Ro transgenic lines (seven sense and five antisense) were carried on to seeds without prior screening. The Ri seedlings (average five plants per line) were inoculated at a 1: 50 inoculum dose. Two sense transgenic lines (L3 and L38) and one antisense line (L42) showed no local lesions, while most other lines tested showed a significant reduction in the number of local lesions, as well as delay in symptom appearance (1 to 2 days), compared with the nontransformed controls. The number of local lesions for R, transgenic N. tabacum sense and antisense lines varied in range from numbers similar to the control (65 to 70 local lesions per leaf for the control versus 37 to 48 local lesions per leaf for the transgenic lines L33 and L11), to intermediate numbers (25 local lesions per leaf for line L8), to lines with few local lesions (5 to 13 for lines LI and L41), to no local lesions (L38) (Fig. 5). For N. tabacum, 12% of the Ro lines screened (2/16) and 25% of the Rl lines (3/12) were completely resistant to TomRSV, because they did not develop any local lesions.

The 3,247 nt at the 3'end of RNA 2 of a peach isolate of TomRSV were cloned and sequenced, the precise location of the polyprotein cleavage site was determined, the cp gene was cloned, and direct functional evidence of the engineered cp gene by in vitro transcription and translation was determined. There is a high percent nucleotide identity (97%) between the nucleotide and amino acid sequences

of the raspberry and the peach isolates for the cp gene. Interestingly, the percent identity for the 3'end untranslated region (1.5 kbp) was significantly lower (90%).

Comparison of the 3'end untranslated and cp nucleotide and amino acid sequences among the different nepoviruses sequenced so far (Bacher, J., et al.,"Sequence Analysis of the 3'Termini of RNA 1 and RNA 2 of Blueberry Leaf Mottle Virus,"J.

Gen. Virol., 75: 2133-2138 (1994); Bertioli, D. J., et al.,"Arabis Mosaic Nepovirus Coat Protein in Transgenic Tobacco Lessens Disease Severity and Virus Replication," Ann. Appl. Biol., 120: 47-54 (1992); Blok, V. C., et al.,"The Nucleotide Sequence of RNA 2 of Raspberry Ringspot Nepovirus,"J. Gen. Virol., 73: 2189-2194 (1992); Blok, V. C., et al.,"Sequence Analysis of the 3'Termini of RNA 1 and RNA 2 Blueberry Mottle Virus,"Virus Res., 33: 145-156 (1994); Brault, V., et al., "Nucleotide Sequence and Genetic Organization of Hungarian Grapevine Chrome Mosaic Nepovirus RNA 2,"Nucleic Acids Res., 17: 7809-7819 (1989); Buckley, B., "Nucleotide Sequence and In Vitro Expression of the Capsid Protein Gene of Tobacco Ringspot Virus,"Virus Res., 30: 335-349 (1993); Demangeat, G., et al., "Analysis of the In Vitro Cleavage Products of the Tomato Black Ring Virus RNA 1 Encoded 250 K Polyprotein,"J. Gen. Virol., 72: 247-252 (1990); Everett, K. R., et al., "Nucleotide Sequence of the Coat Protein Genes of Strawberry Latent Ringspot Virus: Lack of Homology to the Nepoviruses and Comoviruses,"J. Gen. Virol., 75: 1821-1825 (1994); Kreiah, S., et al.,"Sequence Analysis and Location of Capsid Proteins Within RNA 2 of Strawberry Latent Ringspot Virus,"J. Gen. Virol., 75: 2527-2532 (1994); Meyer, M., et al.,"The Nucleotide Sequence of Tomato Black Ring Virus RNA-2,"J. Gen. Virol., 67: 1257-1271 (1986); Rott, M. E., et al., "Nucleotide Sequence of Tomato Ringspot Virus RNA-2,"J. Gen. Virol., 72: 1505- 1514 (1991); Scott, N. W., et al.,"The Identification, Cloning, and Sequence Analysis of the Coat Protein Region of a Birch Isolate (I2) of Cherry Leaf Roll Nepovirus," Arch. Virol., 131: 309-315 (1993); Scott, N. W., et al.,"A 1.5 kb Sequence Homology in 3'-Terminal Regions of RNA-1 and RNA-2 of a Birch Isolate of Cherry Leaf Roll Nepovirus is also Present, in Part, in a Rhubarb Isolate,"J. Gen. Virol., 73: 481-485 (1992); Serghini, M. A.,"RNA 2 of Grapevine Fanleaf Virus: Sequence Analysis and Coat Protein Cistron Location,"J. Gen. Viro., 71: 1433-1441 (1990), which are hereby incorporated by reference) indicated higher similarity among members of the same

nepovirus subgroups, with the exception of strawberry latent ringspot virus ("SLRV"). Even though SLRV is transmitted by nematodes, its classification in the nepovirus group is debated because of the low similarity and the presence of two rather than one cp species (43 and 29 kDa) (Everett, K. R., et al.,"Nucleotide Sequence of the Coat Protein Genes of Strawberry Latent Ringspot Virus: Lack of Homology to the Nepoviruses and Comoviruses,"J. Gen. Virol., 75: 1821-1825 (1994); Kreiah, S., et al.,"Sequence Analysis and Location of Capsid Proteins Within RNA 2 of Strawberry Latent Ringspot Virus,"J. Gen. Virol., 75: 2527-2532 (1994), which are hereby incorporated by reference).

Microsequencing data of the N-terminus of the cp of the peach isolate confirmed that the proteolytic cleavage site between the movement protein and the cp was Q-G at position 1,320 to 1,321 of the polyprotein, as recently reported (Hans, F., et al.,"Tomato Ringspot Nepovirus Protease: Characterization and Cleavage Site Specificity,"J. Gen. Virol., 76: 917-927 (1995), which is hereby incorporated by reference). This cleavage site differed from the R-A, R-G, K-A, and C-A sites proposed for other nepoviruses in subgroups I and II (Bertioli, D. J., et al.,"Transgenic Plants and Insect and Cells Expressing the Coat Protein of Arabis Mosaic Virus Produce Empty Virus-Like Particles,"J. Gen. Virol., 72: 1801-1809 (1991); Blok, V. C., et al.,"The Nucleotide Sequence of RNA 2 of Raspberry Ringspot Nepovirus," J. Gen. Virol., 73: 2189-2194 (1992); Brault, V., et al.,"Nucleotide Sequence and Genetic Organization of Hungarian Grapevine Chrome Mosaic Nepovirus RNA 2," Nucleic Acids Res., 17: 7809-7819 (1989); Buckley, B.,"Nucleotide Sequence and In Vitro Expression of the Capsid Protein Gene of Tobacco Ringspot Virus,"Virus Res., 30: 335-349 (1993); Demangeat, G., et al.,"Analysis of the In Vitro Cleavage Products of the Tomato Black Ring Virus RNA 1 Encoded 250 K Polyprotein,"J.

Gen. Virol., 72: 247-252 (1990); Serghini, M. A.,"RNA 2 of Grapevine Fanleaf Virus: Sequence Analysis and Coat Protein Cistron Location,"J. Gen. Viro., 71: 1433-1441 (1990), which are hereby incorporated by reference), but was one of the proteolytic cleavage sites (Q-M, Q-S, Q-G, E-S, and E-G) described previously for como, poty-, and picornaviruses (Hellen, C. U. T., et al.,"Proteolytic Processing of Polyproteins in the Replication of RNA Viruses,"Biochemistry, 28: 9881-9890 (1989), which is hereby incorporated by reference). Cherry leaf roll virus ("CLRV"), which like

TomRSV is in subgroup III, has also a picornavirus proteolytic site Q-S (Scott, N. W., et al.,"The Identification, Cloning, and Sequence Analysis of the Coat Protein Region of a Birch Isolate (I2) of Cherry Leaf Roll Nepovirus,"Arch. Virol., 131: 309-215 (1993), which is hereby incorporated by reference). Nepoviruses in general share similarities in genomic structure and translational strategies with the plant como-and potyviruses, as well as, the animal picornaviruses (Goldbach, R.,"Genome Similarities Between Plant and Animal RNA Viruses,"Microbiol. Sci., 4: 197-202 (1987), which is hereby incorporated by reference).

Previous attempts to engineer resistance to nepoviruses into Nicotiana spp. focused on transgenic lines expressing high cp levels (Bardonnet, N., et al., "Protection Against Virus Infection in Tobacco Plants Expressing the Coat Protein of Grapevine Fanleaf Nepovirus,"Plant Cell Rep., 13: 357-360 (1994); Bertioli, D. J., et al.,"Arabis Mosaic Nepovirus Coat Protein in Transgenic Tobacco Lessens Discase Severity and Virus Replication,"Ann. Appl. Biol., 120: 47-54 (1992) ("Bertioli 1992"); Brault, V., et al.,"Genetically Engineered Resistance Against Grapevine Chrome Mosaic Nepovirus,"Plant Mol. Biol., 21: 89-97 (1993) ("Brault 1993"), which are hereby incorporated by reference) using members of subgroups I and II. Bertioli 1992 inoculated N. tabacum cv. Xanthi plants cloned from two Ro transgenic lines expressing the cp of ArMV, and observed that lesions developed only on the inoculated leaves, but no systemic virus infection was detectable on the newly formed leaves when challenged by virions or purified RNA and the resistance was durable with time. Interestingly, transgenic plants expressing the ArMV cp produced empty virus particles (Bertioli, D. J., et al.,"Transgenic Plants and Insect and Cells Expressing the Coat Protein of Arabis Mosaic Virus Produce Empty Virus-Like Particles,"J. Gen. Virol., 72: 1801-1809 (1991), which is hereby incorporated by reference).

Evaluation of the resistance in transgenic Nicotiana lines expressing the cp genes of grapevine fanleaf ("GFLV") and grapevine chrome mosaic ("GCMV") was based on studying the inhibition of viral replication as assessed by dot blot (Brault 1993, which is hereby incorporated by reference) or Northern blot analysis (Bardonnet, N., et al.,"Protection Against Virus Infection in Tobacco Plants Expressing the Coat Protein of Grapevine Fanleaf Nepovirus,"Plant Cell Rep.,

13: 357-360 (1994) ("Bardonnet 1994"), which is hereby incorporated by reference), because GFLV and GCMV do not produce symptoms on Nicotiana spp. The use of dot and Northern blots greatly restricted the number of transgenic lines that were analyzed. Brault 1993, produced transgenic N. tabacum cv. Xanthi plants with a construct containing the cp gene and the complete 3'end untranslated region of GCMV. Two out of nine transgenic lines characterized showed reduction in viral replication (monitored by counting the number of dot blot positive plants) at a low inoculum dose (1 g/ml), but the difference vanished at a higher inoculum (10 pg/ml), although the overall accumulation of viral RNAs seemed to be reduced.

Data were presented only for 10 days postinoculation, so evaluation of durability of the reduction in replication overtime could not be made. The authors suggested that reduced viral replication in those two transgenic lines was correlated with high cp expression levels. However, all the lines analyzed had high cp levels, and no lines with low or nondetectable cp levels were tested.

Bardonnet 1994 transformed N. benthamiana with the GFLV cp gene.

Virus replication was assayed by Northern blot analysis or by immunodetection of the 5'end genome-linked protein VPg. Only cp ELISA positive lines (11/40 transgenic lines obtained) were analyzed, from which one transgenic line that expressed the highest cp level by ELISA was selfed and its R2 progeny characterized. Although significant delay in systemic infection occurred in this line, with nearly 65% of the plants protected 24 days after inoculation, the protection was finally overcome after 60 days when all the plants became infected.

The present invention relates to cp-engineered protection against a nepovirus of subgroup III. All cp transgenic plants were analyzed for resistance to TomRSV, regardless of their lack of detectable cp expression by ELISA.

Nevertheless, the level of resistance obtained appeared to be much higher than that obtained so far for other nepoviruses. Direct evaluation of symptoms allowed a large number of transgenic lines (over 250 independent sense and antisense transgenic lines) to be analyzed. From the N. tabacum and N. benthamiana transgenic sense and antisense lines analyzed, several lines were obtained that showed delay in symptom appearance, reduction of symptom severity, or appeared completely resistant. A high proportion of the resistant lines (40 to 56%) were antisense. Similar results have been

reported for tobacco etch virus ("TEV"), for which antisense and truncated cp genes were in some way dysfunctional and more effective at disrupting the normal virus- host relationship than full cp genes (Lindbo, J. A., et al.,"Pathogen-Derived Resistance to a Potyvirus: Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,"Mol.

Plant-Microbe Interact., 5: 144-153 (1992), which is hereby incorporated by reference).

Although the TomRSV cp gene was detected by PCR in DNA extracted from transgenic plants, Northern blot analysis showed very low levels of cp transcript and cp expression was not detectable by ELISA. The reasons for the lack of cp detection in the transgenic plants need further investigation. However, since the expression of the engineered cp gene was detected in vitro, possible explanations are that the pNYS expression cassette gives very low transcription rates or the cp gene is cosuppressed as has been reported for TEV (Lindbo, J. A., et al.,"Pathogen-Derived Resistance to a Potyvirus: Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,"Mol.

Plant-Microbe Interact., 5: 144-153 (1992), which is hereby incorporated by reference). Thus, the cp is produced at levels below the threshold level of detection by ELISA. Another possibility is that the cp is being degraded in the transgenic plants. Despite the lack of cp detection by ELISA, several of the transgenic lines of the present invention were highly resistant to TomRSV.

There are other reports in the literature of transgenic plant lines that are highly resistant to virus inoculation, yet the expected protein product is not observed.

In those instances, the resistant phenotype may be mediated through a defective RNA species and not the expected translation product (Dougherty, W. G., et al.,"RNA- Mediated Virus Resistance in Transgenic Plants: Exploitation of a Cellular Pathway Possibly Involved in RNA Degradation,"Mol. Plant-Microbe Interact., 7: 544-552 (1994); Lindbo, J. A., et al.,"Pathogen-Derived Resistance to a Potyvirus: Immune and Resistant Phenotypes in Transgenic Tobacco Expressing Altered Forms of a Potyvirus Coat Protein Nucleotide Sequence,"Mol. Plant-Microbe Interact., 5: 144- 153 (1992); Lindbo, J. A., et al.,"Induction of a Highly Specific Antiviral State in Transgenic Plants: Implication for Regulation of Gene Expression and Virus

Resistance,"Plant Cell, 5: 1749-1759 (1993); Smith, H. A., et al.,"Transgenic Plant Virus Resistance Mediated by Untranslatable Sense RNAs : Expression, Regulation, and Fate of Nonessential RNAs,"Plant Cell, 6: 1441-1453 (1994), which are hereby incorporated by reference). These studies hypothesized that RNA transcripts derived from antisense and cp genes produced in transgenic plants may interfere with viral replication by annealing to the viral RNA, thereby interfering with transcription or translation, or interfering with binding of the viral replicase or viral assembly. They also suggested that transgenic plants expressing defective RNAs or proteins will be among the most effective polyvirus control strategies.

The results of the method of imparting resistance to a plant to TomRSV achieved by the present invention are important for a number of reasons.

As discussed above, the development of resistance to nepoviruses in group I and II has previously been reported. Bertioli 1992 reported that Ro tobacco plants expressing high levels of cp of Arabis mosaic nepovirus developed symptoms on inoculated leaves but no systemic symptoms. However, these researchers did not bring the plants to R, stage to determine if the lines would be resistant. In a report for resistance to Chrome mosaic nepovirus, Brault 1993 showed that two out of nine transgenic lines of tobacco showed reduction in virus replication that was overcome when inoculated with higher inoculum doses. Furthermore, these lines had high levels of coat protein expression. With grapevine fanleaf, Bardonnet 1994 showed that R2 plants of a single line of N. benthamiana expressing high levels of cp expression, but had only delay of virus infection. For example, 65% of the plants showed delay of infection until 24 days but all plants had become infected by 60 days after inoculation.

The present invention differs from the these reports regarding nepoviruses (and from reports for other types of viruses) in several very fundamental ways. Firstly, the above-mentioned cases are for nepoviruses in group I and II. The present invention is the first for the development of virus resistant transgenic plants for a nepovirus in group III. Secondly, the previously reported references dealt with plants showing high levels of cp expression and can be regarded as the classical coat protein mediated protection. This work, therefore, did not teach that plants with low or zero levels of cp expression would give resistance to nepoviruses. Thus, within the

realm of the nepoviruses, the present invention is the first to show that plants that do not have detectable levels of cp expression do show high resistance and that plants that cannot produce cp (transgenic plants with antisense gene constructs) also show extremely high resistance. Lastly, the cases relating to nepoviruses show that resistance was merely a delay of infection or local infection. In contrast, the present invention shows that some of the transgenic lines are virtually immune to infection.

Based on the results from the previous references, such results would not have expected.

With respect to virus resistance to transgenic plants in general (Lomonossoff GP,"Pathogen-derived Resistance to Plant Viruses"Ann. Rev.

Photopathol., 33: 323-343 (1995), which is hereby incorporated by reference), others have amply shown that high expression of cp will give resistance that can be overcome by increasing the strength of the inoculum dose, but that RNA-mediated resistance will give strong resistance that is not overcome by increase in inoculum strength. It also generally known that antisense resistance is not as effective as nontranslatable sense RNA-mediated resistance where the potential coding sense RNA is changed (by frame shift, for example). Recently, it has also been shown that the sense RNA mediated resistant plants with potyviruses will have low but detectable levels of RNA. The underlying resistance is due to post transcriptional gene silencing (Baulcombe DC,"Mechanisms of Pathogen-derived Resistance to Viruses in Transgenic Plants,"Plant Cell 8: 1833-1844 (1996), which is hereby incorporated by reference). However, even though there are low levels of RNA production, it is the rule that these transgene RNA transcripts can be detected by Northern Blot analysis.

Thus, the present invention gives completely unexpected results from published results of transgenic resistance. First, cp expression in the transgenic plants was not detected, which shows that very little, if any, messenger RNA were being produced in the cytoplasm. Then, Northern blot analysis showed very low levels of RNA in some lines and no RNA detection in other lines, even though they were resistant to infection. Thus, RNA levels were not detectable or very, very low. Also, there was no correlation of resistance to RNA expression. For example, line S 1 had over 40% of the progenies infected (Fig. 5) and very low levels of RNA transcripts were detected in transgenic plants (lane 5 of Figure 3), but line S5 was completely

resistant (Fig. 5) and yet RNA transcripts were not detected. These results were unexpected.

Although the present invention does not describe the mechanism of the form of resistance that was observed, the results do show, however, that the results do not fit the normal trend of results that one skilled in the art would expect when transforming plants with cp transgenes of plant viruses. In particular, one would not have expected that the transgenic plants have mainly no detectable levels of transcript RNA, but yet a percentage of the plants show excellent resistance that is inherited.

Lastly, there are concerns regarding the risks of transgenic plants to the environment. One of the big risk is via heteroencapsidation and recombination between the transgene RNA and viral RNA. The present invention eliminates the risk of heteroencapsidation, because there is no detectable levels of cp produced to encapsidate attacking viruses, and, thus, virtually eliminates the risk of recombination. There is no detectable transcript RNA to recombine with the attacking virus genomic RNA. The present invention allows the practice of controlling TomRSV without any risk of heteroencapsidation and recombination.

Heretofore, no one has taught this for TomRSV, nor for other viruses.

Although the invention has been described in detail for the purpose of illustration, it is understood that such detail is solely for that purpose, and variations can be made therein by those skilled in the art without departing from the spirit and scope of the invention which is defined by the following claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Cornell Research Foundation, Inc.

(ii) TITLE OF INVENTION: DNA MOLECULE ENCODING TOMATO RINGSPOT VIRUS PROTEINS AND USES THEREOF (iii) NUMBER OF SEQUENCES: 10 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Nixon, Hargrave, Devans & Doyle LLP (B) STREET: Clinton Square, P. O. Box 1051 (C) CITY: Rochester (D) STATE: New York (E) COUNTRY: U. S. A.

(F) ZIP: 14603 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1. 0, Version #1. 30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: US 60/042,658 (B) FILING DATE: 04-APR-1997 (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Goldman, Michael L.

(B) REGISTRATION NUMBER: 30,727 (C) REFERENCE/DOCKET NUMBER: 19603/952 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (716) 263-1304 (B) TELEFAX: (716) 263-1600 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 3247 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GTTCAGGGCG GGTCCTGGCA AGAAGGTACT GAAGCCGCTT TTCTAGGCAA AGTTACCTGT 60 GCGAAGGACG CCAAGGGTGG AACTTTATTG CACACTTTGG ATATTATAAA AGAGTGCAAA 120 TCCCAAAATT TATTAAGGTA TAAAGAATGG CAACGTCAAG GCTTTCTTCA TGGAAAGCTT 180 AGATTGCGCT GCTTCATACC CACTAACATT TTTTGTGGGC ATTCCATGAT GTGTTCTTTG 240 GACGCGTTTG GTCGTTATGA TTCGAACGTG CTAGGTGCTA GTTTTCCAGT GAAGTTGGCA 300 AGTTTATTGC CAACGGAGGT GATTAGTCTA GCTGATGGAC CCGTGGTCAC GTGGACGTTT 360 GATATTGGAC GTCTGTGTGG CCATGGTCTC TATTATTCCG AGGGCGCTTA TGCGAGGCCC 420 AAAATTTATT TTTTAATTCT TTCTGATAAT GATGTTCCTG CAGAAGCAGA TTGGCAATTT 480 ACCTATCAGC TTTTGTTTGA GGATCATACG TTTTCGAATT CCTTTGGGGC GGTTCCTTTT 540 ATTACCTTAC CCCATATTTT TAATAGATTA GATATAGGTT ATTGGCGCGG GCCAACAGAG 600 ATAGATTTAA CATCAACTCC CGCACCAAAC GCCTATCGTT TACTTTTCGG CTTGTCCACT 660 GCTATTAGTG GTAACATGTC GACTTTGAAT GCCAATCAAG CCCTATTGCG TTTTTTTCAG 720 GGCTCGAATG GCACTTTACA TGGGCGCATT AAAAAGATAG GGACAGCACT TACAACTTGT 780 TCCCTTTTAT TATCGTTGCG CCACAAAGAT GCGAGTCTCA CATTGGAGAC CGCATATCAA 840 AGGCCCCATT ACATTTTGGC TGATGGACAA GGGGCTTTTT CACTACCAAT TTCTACCCCC 900 CATGAAGCAA CCTCCTTTGT GGAGGACATG TTGCGCCTGG AGATTTTTGC TATTGCTGGG 960 CCTTTTAGTC CCAAAGATAA TAAAGCAACA TACCAATTCA TGTGTTATTT CGATCACATA 1020 GAATCGGTTG AGGGGGTACC TAGAACTATA GCAGGCGAGC AGCAGTTCAA CTGGTGTAGT 1080 TTAACAAATT CCACAATCGA TGACTGGAGG TTTGAGTGGC CGGCTCGCCT ACCAGATATA 1140 CTTGATGATA AGTCAGAAGT GCTTTTAAGG CAACATCCTT TATCTCTGCT TATCTCATCT 1200 ACCGGTTTTT TTACGGGTAG AGCCATTTTT GTTTTCCAGT GGGGTGTGAA TACTACTGCT 1260 GGGAATATGA AAGGCTCATT TTCTGCGCGC CTGGCCTTTG GCAAGGGCGT GGAGGAAATT 1320 GACCAGACGT CAACAGTGCA ACCACTTGTT GGCGCTTGTG AAGCCCGCAT ACCCGTGGAG 1380 TTTAAGACTT ACACGGGTTA TACTACTTCG GGTCCTCCTG GATCCATGGA ACCATACATT 1440 TACGTGAGGC TTACGCAACC TACGCTTGTG GATAGGCTTT CTGTGAATGT TATTTTACAG 1500 GAGGGATTTT CTTTCTATGG ACCTAGCGTC AAACATTTTA AGAAAGAAGT CGGCACGCCT 1560 AGTGCCACCC TAGAGACAAA TAACCCCGTT GGGCGCCCAC CTGAGAATAT CGATACAGGG 1620 GGTCCCGGCG GCCAGTATGC AGCTGCCTTA CAAGCAGCTC AGCAAGCTGG GAGAAATCCT 1680 TTTGGGCGTG GCTAAGTTGG CTTCCTGAAA GGCGAGTAGC TGCCGTTAGC AGCTTCCAAA 1740 AGGTGGCCTC TTAATTAGCT TTTAATAGGG GTTATCCAGC CTTAAGCAAG CTGGCACCGG 1800 TCCTGATGGA CTACCAGGAA AGCACCTGGT TTGGAAGAAT TCGAGTAAAA TTCTTAAATC 1860 TTGTTTACTC GTGACTTATA GTACATTCAA GAGGAATGAC TCATGTTTTG TCCATTTACA 1920 TGATGGCATA AAGAGTTAAC GGCTCATATG GTGCTCATTA CGTTCAAGTG TTGAAGGATC 1980 CAATAGCCTT GAACTGTGGT GCCATGTGAG GAGATCCACG TTATCTCTGA TTGTCAAAAT 2040 AGACTAGTCT AGGAGACGAT AAATCCTATG TGGGTGAGTC CCATTCTGGC GAGACACGCA 2100 ATGCCTTTTA TTTGTTTGAG GTTATCAAAC ATCATATCTT GAGTCTGCAT TTAAATTCCA 2160 ATAATGTAGT TGTCATAGCC TACCGATGAG CCTGCGAGAA AGGTTCCATG AGGACTAGGG 2220 TTGGCTAACC CTCACTTAAT CTCTCTATTG GTCATTCGAC AGTGCGTCGA GAATTCATGG 2280 GTTTCATCAC CCACATTGAA GCGAGTGTCT CGTAAGAAAC CCACTCGGAT TGATGTACTT 2340 ACCATGCATC CTTTCGAGTA AAGCATCGAT TCCGTCGTTG TGGTTCTTCA ACTGTGGTTT 2400 TAGATGAGCG ATGAGTTGCG CTGCCCGCGT ATGAAGCGTG GAAAAGTAGT CTGAAACGAA 2460 CTTAGTACCA GAGGTAGGAC GCCATTGTTC CAGGCGTTTT TTATGGACAT AAGCTGTAAA 2520 CTTGGTTTCG CAAGCCATGC AGCACCTCCC TTTATTCGTG TACTATCCAG GGGCTCCCGG 2580 TTCTTTCTTA CCGGTACAAT ACCTGGTGAA GCGAATACTT GCGTCGAGGG ATGAGAGTAG 2640 CATGTTCCTA CTCATTGAAG GAATATGTCG TGTTTTCCAC ACGTTAGTGT TAAATGCAGT 2700 ACCCAGCGCC ATAGTGCAAG AATGGTTCCC AGCCACTTTT TCTGGGATTC TAATCGTACG 2760 ACACAATTGC ATGTGTATCG TTGACGGAGG AGTAGCGATC CTCTACCACG CGAGCCTGGA 2820 AGTAATTGCC GGGGCCGAAG AAGGCCAGCA TGCGGTACGA TTAACTTTAG CTGTAATGTA 2880 GTGGTATGTT AAGTTGAGAC TAACTTACCG TACGAGTCAA ACTCCTTGGG TGGATGTGTG 2940 TTCTGCCACC TTGGAGGAAG TAGATGTGAT TTTACCAGTC TGAGACGAGC CATTAATTTG 3000 GTGCTTTTAT TCATTGATGA TAATACTCGT GCAGTTGCAG CTGCACGAGT ATGTTGGTAC 3060 GCACAGTCTA CTCGGATACG GCCGAGTTGC CCTCACAACA GGGATTATCT CTCAATCTTA 3120 ACTACTGCCA GGACGTTGTT TTCGCAGGGT TTTGTTGGTC CGTTTGTGTT TCAAAACGCT 3180 GCTTTGCAAT TTTCTATTTT GTTTTATTGC TTTCGGAGTG TCGAACTTTG TCCAAGTTCA 3240 TAAAAGC 3247 (2) INFORMATION FOR SEQ ID NO : 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1689 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: GGCGGGTCCT GGCAAGAAGG TACTGAAGCC GCTTTTCTAG GCAAAGTTAC CTGTGCGAAG 60 GACGCCAAGG GTGGAACTTT ATTGCACACT TTGGATATTA TAAAAGAGTG CAAATCCCAA 120 AATTTATTAA GGTATAAAGA ATGGCAACGT CAAGGCTTTC TTCATGGAAA GCTTAGATTG 180 CGCTGCTTCA TACCCACTAA CATTTTTTGT GGGCATTCCA TGATGTGTTC TTTGGACGCG 240 TTTGGTCGTT ATGATTCGAA CGTGCTAGGT GCTAGTTTTC CAGTGAAGTT GGCAAGTTTA 300 TTGCCAACGG AGGTGATTAG TCTAGCTGAT GGACCCGTGG TCACGTGGAC GTTTGATATT 360 GGACGTCTGT GTGGCCATGG TCTCTATTAT TCCGAGGGCG CTTATGCGAG GCCCAAAATT 420 TATTTTTTAA TTCTTTCTGA TAATGATGTT CCTGCAGAAG CAGATTGGCA ATTTACCTAT 480 CAGCTTTTGT TTGAGGATCA TACGTTTTCG AATTCCTTTG GGGCGGTTCC TTTTATTACC 540 TTACCCCATA TTTTTAATAG ATTAGATATA GGTTATTGGC GCGGGCCAAC AGAGATAGAT 600 TTAACATCAA CTCCCGCACC AAACGCCTAT CGTTTACTTT TCGGCTTGTC CACTGCTATT 660 AGTGGTAACA TGTCGACTTT GAATGCCAAT CAAGCCCTAT TGCGTTTTTT TCAGGGCTCG 720 AATGGCACTT TACATGGGCG CATTAAAAAG ATAGGGACAG CACTTACAAC TTGTTCCCTT 780 TTATTATCGT TGCGCCACAA AGATGCGAGT CTCACATTGG AGACCGCATA TCAAAGGCCC 840 CATTACATTT TGGCTGATGG ACAAGGGGCT TTTTCACTAC CAATTTCTAC CCCCCATGAA 900 GCAACCTCCT TTGTGGAGGA CATGTTGCGC CTGGAGATTT TTGCTATTGC TGGGCCTTTT 960 AGTCCCAAAG ATAATAAAGC AACATACCAA TTCATGTGTT ATTTCGATCA CATAGAATCG 1020 GTTGAGGGGG TACCTAGAAC TATAGCAGGC GAGCAGCAGT TCAACTGGTG TAGTTTAACA 1080 AATTCCACAA TCGATGACTG GAGGTTTGAG TGGCCGGCTC GCCTACCAGA TATACTTGAT 1140 GATAAGTCAG AAGTGCTTTT AAGGCAACAT CCTTTATCTC TGCTTATCTC ATCTACCGGT 1200 TTTTTTACGG GTAGAGCCAT TTTTGTTTTC CAGTGGGGTG TGAATACTAC TGCTGGGAAT 1260 ATGAAAGGCT CATTTTCTGC GCGCCTGGCC TTTGGCAAGG GCGTGGAGGA AATTGACCAG 1320 ACGTCAACAG TGCAACCACT TGTTGGCGCT TGTGAAGCCC GCATACCCGT GGAGTTTAAG 1380 ACTTACACGG GTTATACTAC TTCGGGTCCT CCTGGATCCA TGGAACCATA CATTTACGTG 1440 AGGCTTACGC AACCTACGCT TGTGGATAGG CTTTCTGTGA ATGTTATTTT ACAGGAGGGA 1500 TTTTCTTTCT ATGGACCTAG CGTCAAACAT TTTAAGAAAG AAGTCGGCAC GCCTAGTGCC 1560 ACCCTAGAGA CAAATAACCC CGTTGGGCGC CCACCTGAGA ATATCGATAC AGGGGGTCCC 1620 GGCGGCCAGT ATGCAGCTGC CTTACAAGCA GCTCAGCAAG CTGGGAGAAA TCCTTTTGGG 1680 CGTGGCTAA 1689 (2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 562 amino acids (B) TYPE: amino acid (C) STRANDEDNESS : single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: Gly Gly Ser Trp Gln Glu Gly Thr Glu Ala Ala Phe Leu Gly Lys Val 1 5 10 15 Thr Cys Ala Lys Asp Ala Lys Gly Gly Thr Leu Leu His Thr Leu Asp 20 25 30 Ile Ile Lys Glu Cys Lys Ser Gln Asn Leu Leu Arg Tyr Lys Glu Trp 35 40 45 Gln Arg Gln Gly Phe Leu His Gly Lys Leu Arg Leu Arg Cys Phe Ile 50 55 60 Pro Thr Asn Ile Phe Cys Gly His Ser Met Met Cys Ser Leu Asp Ala 65 70 75 80 Phe Gly Arg Tyr Asp Ser Asn Val Leu Gly Ala Ser Phe Pro Val Lys 85 90 95 Leu Ala Ser Leu Leu Pro Thr Glu Val Ile Ser Leu Ala Asp Gly Pro 100 105 110 Val Val Thr Trp Thr Phe Asp Ile Gly Arg Leu Cys Gly His Gly Leu 115 120 125 Tyr Tyr Ser Glu Gly Ala Tyr Ala Arg Pro Lys Ile Tyr Phe Leu Ile 130 135 140 Leu Ser Asp Asn Asp Val Pro Ala Glu Ala Asp Trp Gln Phe Thr Tyr 145 150 155 160 Gln Leu Leu Phe Glu Asp His Thr Phe Ser Asn Ser Phe Gly Ala Val 165 170 175 Pro Phe Ile Thr Leu Pro His Ile Phe Asn Arg Leu Asp Ile Gly Tyr 180 185 190 Trp Arg Gly Pro Thr Glu Ile Asp Leu Thr Ser Thr Pro Ala Pro Asn 195 200 205 Ala Tyr Arg Leu Leu Phe Gly Leu Ser Thr Ala Ile Ser Gly Asn Met 210 215 220 Ser Thr Leu Asn Ala Asn Gln Ala Leu Leu Arg Phe Phe Gln Gly Ser 225 230 235 240 Asn Gly Thr Leu His Gly Arg Ile Lys Lys Ile Gly Thr Ala Leu Thr 245 250 255 Thr Cys Ser Leu Leu Leu Ser Leu Arg His Lys Asp Ala Ser Leu Thr 260 265 270 Leu Glu Thr Ala Tyr Gln Arg Pro His Tyr Ile Leu Ala Asp Gly Gln 275 280 285 Gly Ala Phe Ser Leu Pro Ile Ser Thr Pro His Glu Ala Thr Ser Phe 290 295 300 Val Glu Asp Met Leu Arg Leu Glu Ile Phe Ala Ile Ala Gly Pro Phe 305 310 315 320 Ser Pro Lys Asp Asn Lys Ala Thr Tyr Gln Phe Met Cys Tyr Phe Asp 325 330 335 His Ile Glu Ser Val Glu Gly Val Pro Arg Thr Ile Ala Gly Glu Gln 340 345 350 Gln Phe Asn Trp Cys Ser Leu Thr Asn Ser Thr Ile Asp Asp Trp Arg 355 360 365 Phe Glu Trp Pro Ala Arg Leu Pro Asp Ile Leu Asp Asp Lys Ser Glu 370 375 380 Val Leu Leu Arg Gln His Pro Leu Ser Leu Leu Ile Ser Ser Thr Gly 385 390 395 400 Phe Phe Thr Gly Arg Ala Ile Phe Val Phe Gln Trp Gly Val Asn Thr 405 410 415 Thr Ala Gly Asn Met Lys Gly Ser Phe Ser Ala Arg Leu Ala Phe Gly 420 425 430 Lys Gly Val Glu Glu Ile Asp Gln Thr Ser Thr Val Gln Pro Leu Val 435 440 445 Gly Ala Cys Glu Ala Arg Ile Pro Val Glu Phe Lys Thr Tyr Thr Gly 450 455 460 Tyr Thr Thr Ser Gly Pro Pro Gly Ser Met Glu Pro Tyr Ile Tyr Val 465 470 475 480 Arg Leu Thr Gln Pro Thr Leu Val Asp Arg Leu Ser Val Asn Val Ile 485 490 495 Leu Gln Glu Gly Phe Ser Phe Tyr Gly Pro Ser Val Lys His Phe Lys 500 505 510 Lys Glu Val Gly Thr Pro Ser Ala Thr Leu Glu Thr Asn Asn Pro Val 515 520 525 Gly Arg Pro Pro Glu Asn Ile Asp Thr Gly Gly Pro Gly Gly Gln Tyr 530 535 540 Ala Ala Ala Leu Gln Ala Ala Gln Gln Ala Gly Arg Asn Pro Phe Gly 545 550 555 560 Arg Gly (2) INFORMATION FOR SEQ ID NO : 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1552 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4: GTTGGCTTCC TGAAAGGCGA GTAGCTGCCG TTAGCAGCTT CCAAAAGGTG GCCTCTTAAT 60 TAGCTTTTAA TAGGGGTTAT CCAGCCTTAA GCAAGCTGGC ACCGGTCCTG ATGGACTACC 120 AGGAAAGCAC CTGGTTTGGA AGAATTCGAG TAAAATTCTT AAATCTTGTT TACTCGTGAC 180 TTATAGTACA TTCAAGAGGA ATGACTCATG TTTTGTCCAT TTACATGATG GCATAAAGAG 240 TTAACGGCTC ATATGGTGCT CATTACGTTC AAGTGTTGAA GGATCCAATA GCCTTGAACT 300 GTGGTGCCAT GTGAGGAGAT CCACGTTATC TCTGATTGTC AAAATAGACT AGTCTAGGAG 360 ACGATAAATC CTATGTGGGT GAGTCCCATT CTGGCGAGAC ACGCAATGCC TTTTATTTGT 420 TTGAGGTTAT CAAACATCAT ATCTTGAGTC TGCATTTAAA TTCCAATAAT GTAGTTGTCA 480 TAGCCTACCG ATGAGCCTGC GAGAAAGGTT CCATGAGGAC TAGGGTTGGC TAACCCTCAC 540 TTAATCTCTC TATTGGTCAT TCGACAGTGC GTCGAGAATT CATGGGTTTC ATCACCCACA 600 TTGAAGCGAG TGTCTCGTAA GAAACCCACT CGGATTGATG TACTTACCAT GCATCCTTTC 660 GAGTAAAGCA TCGATTCCGT CGTTGTGGTT CTTCAACTGT GGTTTTAGAT GAGCGATGAG 720 TTGCGCTGCC CGCGTATGAA GCGTGGAAAA GTAGTCTGAA ACGAACTTAG TACCAGAGGT 780 AGGACGCCAT TGTTCCAGGC GTTTTTTATG GACATAAGCT GTAAACTTGG TTTCGCAAGC 840 CATGCAGCAC CTCCCTTTAT TCGTGTACTA TCCAGGGGCT CCCGGTTCTT TCTTACCGGT 900 ACAATACCTG GTGAAGCGAA TACTTGCGTC GAGGGATGAG AGTAGCATGT TCCTACTCAT 960 TGAAGGAATA TGTCGTGTTT TCCACACGTT AGTGTTAAAT GCAGTACCCA GCGCCATAGT 1020 GCAAGAATGG TTCCCAGCCA CTTTTTCTGG GATTCTAATC GTACGACACA ATTGCATGTG 1080 TATCGTTGAC GGAGGAGTAG CGATCCTCTA CCACGCGAGC CTGGAAGTAA TTGCCGGGGC 1140 CGAAGAAGGC CAGCATGCGG TACGATTAAC TTTAGCTGTA ATGTAGTGGT ATGTTAAGTT 1200 GAGACTAACT TACCGTACGA GTCAAACTCC TTGGGTGGAT GTGTGTTCTG CCACCTTGGA 1260 GGAAGTAGAT GTGATTTTAC CAGTCTGAGA CGAGCCATTA ATTTGGTGCT TTTATTCATT 1320 GATGATAATA CTCGTGCAGT TGCAGCTGCA CGAGTATGTT GGTACGCACA GTCTACTCGG 1380 ATACGGCCGA GTTGCCCTCA CAACAGGGAT TATCTCTCAA TCTTAACTAC TGCCAGGACG 1440 TTGTTTTCGC AGGGTTTTGT TGGTCCGTTT GTGTTTCAAA ACGCTGCTTT GCAATTTTCT 1500 ATTTTGTTTT ATTGCTTTCG GAGTGTCGAA CTTTGTCCAA GTTCATAAAA GC 1552 (2) INFORMATION FOR SEQ ID NO : 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5: AGCACCATGG TCTGTCGAAA ACAAAACTTG C 31 (2) INFORMATION FOR SEQ ID NO : 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 35 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6: AGCTGACCAT GGCTTGGACA AAGTTCGACA CTACG 35 (2) INFORMATION FOR SEQ ID NO : 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7: AGCTGACCAT GGAAGCTTCC ATTAGAGCTT ATC 33 (2) INFORMATION FOR SEQ ID NO : 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 39 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 8: AGCTAGTCTA GACCATGGTT CAGGGCGGGT CCTGGCAAG 39 (2) INFORMATION FOR SEQ ID NO : 9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 41 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 9: GCATGATCTA GACCATGGTA AAAGCTAATT AAGAGGCCAC C 41 (2) INFORMATION FOR SEQ ID NO : 10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 10: Gly Gly Ser Trp Gln Glu Gly Thr Glu Ala Ala Phe Leu Gly Lys Val 1 5 10 15 Thr Cys Ala Lys Asp Ala 20