CUCUMBER MOSAIC VIRUS COAT PROTEIN GENE FIELD OF INVENTION This invention relates to a coat protein gene of cucumber mosaic virus strain c (CMV-C) . More specifically the invention relates to a process for preparing said gene as well as its incorporation into a transfer vector and to its use to produce transformed plant cells and transformed plants which are resistant to CMV viral infections.

BACKGROUND OF THE INVENTION

Cucumber mosaic virus (CMV) is a single-stranded (+) RNA plant virus which has a functionally divided genome. The virus genome contains four RNA species designated RNAsl-4; 3389 nucleotides (nt),

3035 nt, 2193 nt and 1027 nt, respectively (Peden and Symons, 1973;

Gould and Symons, 1982; Rezaian et al., 1984; Rezaian et al., 1985).

Only RNAεl-3 are required for infectivity (Peden and Symons, 1973) because the coat protein, which is encoded by RNA 4, is also encoded by RNA 3. Translations of CMV RNAS yield a 95KDal polypeptide from

RNA 1, a 94kDal polypeptide from RNA 2, (Gordon et al., 1983) and two polypeptides from RNA 3: its 5' end encodes a 35KDal polypeptide, and its 3" end encodes a 24.5kDal polypeptide (Gould and Symons, 1982). The 24.5kDal polypeptide is identical to that encoded by RNA

4 and is the coat protein.

The CMV coat protein gene does not contain the signals necessary for its expression once transferred and integrated into a plant genome. It must be engineered to contain a constitutive promoter 5' top its translation initiation codon (ATG) and a poly(A) addition signal (AATAAA) 3' to its translation termination codon. Several promoters which function in plants are available, but we believe that the best promoters are the constitutive promoters from CaMV, the Ti genes nopaline synthase (Bevan et al.,1983) and octopine synthase (Depicker et al., 1982), and the bean storage protein gene phaseolin. The poly (A) addition signals from these genes are suitable for our purposes as well.

INFORMATION DISCLOSURE Plants that are resistant to virus diseases and methods for producing them, are described in EP 223,452.

An, G., et al. (1985) "New Cloning Vehicles for Transformation Of Higher Plants". EMBO J. 4:277-284; Dodds , J.A., et al. (1985) Cross protection between strains of cucumber mosaic virus: effect of

host and type of inoculum on accumulation of virions and double- stranded RNA of the challenge strain. Virology 144:301-309. Pietrzak, M. , et al. (1986) Expression in plants of two bacterial antibiotic resistant genes after protoplast transformation with a new plant expression vector. Nuc. Acids Res 14:5857-5868.

SUMMARY OF THE INVENTION This invention provides: The coat protein gene from the C strain of cucumber mosaic virus (CMV-C) .

A plant transformation vector comprising the coat protein gene from CMV-C, the promoter of the 35S gene of cauliflower mosaic virus and the polyadenylation signal of either the cauliflower mosaic virus 35S gene or the phaseolus vulgaris seed storage protein gene.

A bacterial cell containing a plant transformation vector comprising the coat protein gene from CMV-C, the 35S promoter of cauliflower mosaic virus and the polyadenylation signal of either the cauliflower mosaic virus 35S gene or the Phaseolus vulgaris seed storage protein gene.

A transformed plant cell containing the coat protein gene from CMV-C, the cauliflower mosaic virus 35S promoter and the polyadenyl- ation signal of either the cauliflower mosaic virus gene or the Phaseolus vulgaris seed storage protein gene (phaseolin) .

A plant comprising transformed cells containing the coat protein gene of CKV-C, the cauliflower mosaic virus 35S promoter and the polyadenylation signal of either the cauliflower mosaic virus gene or the Phaseolus vulparis seed storage protein gene (phaseolin) . Transformed plants of this invention include beets, citrus fruit, corn, cucumber, peppers, potatoes, soybean, squash and tomatoes. Especially preferred are members of the cucurbitaceae (squash, cucumber, i.e.,) and solanaceae (peppers, tomatoes, i.e.) family. A process for producing virus-resistant plants comprising propagating a plant expressing the coat protein gene from the C strain of cucumber virus. Especially preferred is the process for producing members of the cucurbitacaea and solanacaed families. DESCRIPTION OF THE PREFERRED EMBODIMENT Charts 1 to 5 are set forth to illustrate the constructions of this invention. Certain conventions are used to illustrate plasmids and DNA fragments as follows:

(1) The single line figures represent both circular and linear

double-stranded DNA.

(2) Asterisks (*) indicate that the molecule represented is circular. Lack of an asterisk indicates the molecule is linear.

(3) Junctions between natural boundaries of functional com- ponents are indicated by vertical lines along the horizontal lines.

(4) Genes or functional components are indicated.

(5) Distances between genes and restriction sites are not to scale. The figures show the relative positions only unless indicated otherwise. Most of the recombinant DNA methods employed in practicing the present invention are standard procedures, well known to those skilled in the art, and described in detail, for example, EP-223452 which is incorporated herein by reference. Enzymes are obtained from commercial sources and are used according to the vendor's recommen- dations or other variations known to the art. Reagents, buffers, and culture conditions are also known to those in the art. General references containing such standard techniques include the following: R.Wu, ed. (1979) Methods in Enxymolo v Vol.68; J.H. Miller (1972) Experiments in Molecular Genetics: T. Maniatis et al.(1982) Molecular Cloning: & Laboratory Manual: and D.M. Glover, ed. (1985) DNA Cloning Vol II, all of which are incorporated by reference. Example 1 Isolation of CMV RNAs

Cucumber mosaic virus strain C (CMV-C) was propagated in tobacco plants and RNA was isolated by the method of Lot et al. (Annals of Phytopathology 4:25, 1972). Example 2 Cloning of CMV-C (a) Synthesis of double-stranded cDNA3

Total CMV-C RNA was polyadenylated in order to provide a site for the annealing of an oligo dT primer. The reaction buffer was as follows: 5 μl, 1 M Tris pH 7.9; 1 μl, 1 M MgCl ₂ ; 2.5 μl, 0.1 M MnCL ₂ ; 5 μl, 5 μ NaCl; 0.5 μl, 100 mM ATP; 18 μl, 2.8 mg/ml bovine serum albumin. 3.2 μl of this buffer were mixed with 2 μg of CMV-C total RNA. 3.8μl H2O and 1 μl of poly-A polymerase were added, and the reaction mixtures were incubated at 37*C. for 10 minutes. The resulting polyadenylated RNA was used in the cDNA synthesis protocol of Polites and Marotti (Biotechniques 4:514, 1986), except that 0.75 mM KC1 was used instead of 50 mM NaCl, and 130 uCi/100 μl ³² P-dCTP was used instead of 10-50 uCi/100 μl ^*

After dε-cDNA was synthesized, it was purified by G-100 column chroma ography, precipitated with ethanol, and suspended in 20. μl of IX Eco Rl methylase buffer (100. nM NaCl,; 100. mM:Tris-HCL pH .8.0, 1 mM EDTA.,80 μM S-adenosyl methionine, 100 μg/ml bovine serum albumin). After removal of a 2 μl aliquot for subsequent gel analysis, an additional 1 μl of 32 mM S-adenosyl methione was added to the reaction mixture mix, and 1 μl (20 units) of Eco RI methylase. The reaction was incubated at 37 ^β C. for 30 minutes and stopped by incubation at 70°C. for 10 minutes. Two μl were removed from the above reaction, and 1 μl (5 units) of £j. coli DNA polymerase I klenow fragment was added. The reaction was incubated at 37°C. for 10 minutes, then extracted with phenol/- chlorofor before precipitating with ethanol. The pellet was washed in 70% ethanol, then in 70% ethanol/0.3 M sodium acetate. The pellet was dried and resuspended in 8 μl 0.5 μg/μl phos- phorylated Eco RI linkers (available from Collaborative Research, Inc, 128 Spring Street, Lexington, MA 02173). One μl 10X ligase buffer (800 mM Triε-HCl pH 8.0, 200 mM MgCl ₂ , 150 mM DTT, 10 mM ATP) and 1 μl of T4 DNA ligase (4 units/μl) were added, and the reaction was incubated overnight at 15"C.

The ligation reaction was then stopped by incubation at 65°C. for 10 minutes. Sixty μl of water, 10 μl of 10X Eco RI salts(900 mM Tris pH 8.0, 100 mM MgC12, 100 mM NaCl), and 10 μl of EcoRI (10 units/μl) were added, and the reaction was incubated at 37°C. for 1 hr (a 5 μl aliquot was removed at the beginning for subsequent gel analysis) . The reaction was stopped by phenol/chloroform and chloroform extraction. A 5 μl aliquot was removed for gel analysis, and half of the remainder was frozen for future use. The other half was purified by G-100 column chromatograph . The G-100 fractions containing the cDNA were concentrated by butanol extraction, precipi¬ tated with ethanol, and resuspended in 10 μl of H2O. After removing 3 μl for subsequent analysis, 1 μl lambda gtll arms (available from Stratagene Co., 3770 Tandy St, San Diego, Ca. 92121), 1 μl of 10X ligase buffer, and 1 μl T4 DNA ligase were added, and the reaction was incubated at 15°C. overnight.

The resulting ligated lambda gtll/cDNA molecules were packaged according to the procedure recommended by the manufacturer of the packaging extract (Gigapack plus, also frorc Stratagene). This

yielded recombinant lambda phage, which were plated according to methods known to those skilled in the art.

Lambda clones containing the coat protein gene were identified by hybridization with radioactively labelled single-stranded cDNA from purified RNA 4 of the whiteleaf strain of CMV (obtained from Dr. D. Gonsalveε, Cornell University, Geneva, NY). This RNA 4 single- stranded cDNA was synthesized as follows: RNA 4 molecules were polyadenylated as described above for total CMV-C RNA, except that 5.8 μg RNA 4 was used. First strand synthesis was as described by Polites and Marotti (Biotechniques 4:514, 1986) except that non- radioactive dCTP was not included. Instead, 260 uCi/100 μl of radioactive dCTP was used.

The labelled single-stranded cDNA was purified by P6 column chromatography and used to probe replicate filters lifted from the lambda phage plates mentioned above. The single-stranded cDNA hybridized with DNA from several phage clones, indicating that they contained at least a part of the CMV-C coat protein gene. Several of these lambda clones were grown, and DNA from them was isolated according to methods known to those skilled in the art. Example 2 Construction of a pUC19 Clone containing the CMV-C coat protein gene Several of the cloned cDNA's mentioned in the above example were transferred to the plasmid vector, pUC19 (available from Bethesda Research, P.O. Box 6009, Gaithersburg, Md 20877), using standard methods. The cloned fragments in pUC19 were then sequenced by the technique described by Maxam and Gilbert (Methods in Enzvπtologv 65:499, 1980). Based on this information, one clone was identified as containing the entire coat protein gene. The sequence of this clone (designated pCMV9.9) is shown in Chart 1. Example 4. Construction of a micro T-DNA plasmid containing a plant- expressible CMV-C coat protein gene with the CaMV 35S polyadenylation signal In order to attach the CaMV 35S promoter and polyadenylation signal, a fragment extending from the Ace I site (at position 311) to the Eco RI site (at position 1421) was removed from pCMV .9 and ligated into the multiple cloning site of the vector pDH51 (Pietrzak et al., 1986) (available from Thomas Hohn, Friedrich Miescher Insti¬ tute, P.O. Box 2543, CH-4002, Basel, Switzerland). This was accom-

plished by complete Eco RI and partial Ace I digestion of pCMV9.9, creating a blunt-ended molecule out of the appropriate Ace I-Eco RI fragment (using the Klenow fragment of E. Coli DNA polymerase I), and ligating it into the Sma I site of pDH51 Chart 2). This clone., designated pDH51/CP19, was sequenced by the Maxam-Gilbert technique to confirm its suitability for expression in plants.

The plant expressible coat protein gene was then moved into a vector suitable for Agrobacterium-mediated gene transfer. An Eco RI- Eco RI fragment was removed from pDH51/CP19 and placed into the Eco RI site of the plasmid, pUC1813 (available from Robert Kay, Dept. of Chemistry, Washington State University, Pullman, Washington) , creating the plasmid pUC1813/CP19. A 1.8 k.b. fragment containing the plant expressible gene was removed by partial Hind 3 digestion and ligated into the Hind 3 site of the vector, pGA482 (An et al. , 1985) (available from Gynehung An, Institute of Biological Chemistry. Washington State University) .

The resulting plasmid was designated pGA482/CP19H (Chart 3). The plant expressible gene for glucuronidase (available from Clontech Laboratories, Inc., 4055 Fabian Way, Palo Alto, CA) was then added to pGA482/CP19H by removing a partial Bam HI-Bam HI fragment from a pUC1813 clone containing the gene and inserting it into the Bgl II site of pGA482/CP19. This final construction was designated pGA4- 82/CP19/GUS (Chart 3) and was confirmed by Maxam-Gilbert sequencing. This plasmid, or its derivatives, can be transferred into Agrobacteritjm strains A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRi- 278b) and others. Strains A208, C58, LBA4404, and A4RS are available from ATCC, 12301 Parklawn Drive, Rockville, MD. A4RS(pRi278b) is available from Dr. F. Casse-Delbart, C.N.R.A., Routede Saint Cyr, F78000, Versailles, France. C58Z707 is available from Dr. A.G. Hepburn, University of Illinois, Urbana, IL.

Example 5. Construction of a micro T-DNA plasmid containing a plant- expressible CMV coat protein Eene with the phaseolin polyadenylation signal

The 35S polyadenylation signal was replaced by that of phaseolin by removing an Ayr II-Xba fragment from ρDH51/CP19. This digestion removes a DNA region which includes the 3' terminus of the coat protein clone (however, all of the CMV coat protein coding region remains intact), an artificially introduced stretch of A-residues,

and part of the pDH51 polylinker. An Xba I-Xba I fragment from a pUC1813 clone containing the phaseolin polyadenylation signal was used to replace the Ayr II-Xba I fragment. This construction was then removed by digestion with Eco-RI. and cloned into the Eco RI site of pUC1813. This pUC1813 clone was then digested with Xba I. and the fragment consisting of the 35S promoter, the coat protein coding region, and the phaseolin polyadenylation signal was ligated into Xba I site of pGA482. The glucuronidase gene was then added as described above to produce the plasmid pGA482/CP19A-/411GUS (Chart 4) . The structure of the plasmid was confirmed by Maxam-Gilbert sequencing.

An alternative plasmid (designed to test the effect that the stretch of A-residues have on expression) was constructed by placing the phaseolin polyadenylation signal from pUC1813 (see above) into the Xba I site of pDH51/CP19. A fragment containing the 35S pro¬ moter, coat protein coding sequence, and the phaseolin and 35S polyadenylation signals were then removed from pDH51/CP19 by Eco RI digestion, and ligated into pUC1813. A fragment containing the 35S promoter, coat protein coding sequence (including the artificially synthesized stretch of A-residues) , and the phaseolin polyadenylation signal was then removed from that plasmid by partial Xba I digestion and ligated to the Xba-I site of pGA482. The glucuronidase gene was subsequently inserted as described above. The resulting plasmid was designated pGA482/CP19/411/GUS (Chart 5), and the structure was confirmed by Maxam-Gilbert sequencing.

These plasmid, or their derivatives, are transferred into Agrobacterium strains A208, C58, LBA4404, C58Z707, A4RS, A4RS(pRi- 278b) and others which in turn are utilized to insert the CMV-C coat protein genes into plant cells by methods described in Example 4 and Chart 3 of U.S. Patent application Serial No. 135,655 filed December 21, 1987, entitled "Agrobacterium Mediated Transformation of Germi¬ nating Plant Seeds". The Example and Chart are appended hereto as Exhibit A.

EXHIBIT A

The purpose of this exanple is to incorporate * modified seed storage protein which encodes * higher percentage of sulfur-contain¬ ing aaino acids; such a gene is referred to as High Sulfur Storage Protein (HSSP)-gene. This gene is constructed so that it is develop- mentally expressed in the seeds of dicotyledonous plants; this has been accomplished by using the phaseolin promoter. The modified gene must encode a substantial number of sulfur-containing amino acids. Naturally occurring HSSP-genes can also be used. The two best naturally occurring HSSP-genes are the beta zein gene (15 kD) (Peder- sen et al (1986) J.. Btol. Chen. 201:6279) and the Brazil nut protein (Altenbach et al. (1987) plant Mo . Bio. 8:239). However, any other natural or synthetic gene derivative of an HSSP-gene can be used for the improvement of the nutritional value of seeds.

EXHIBIT A

Chart 4

11

BR Plant Mutant Plant BL PGA 82 Nps-Nptll I Promoter ALS or EPSPS gene Poly(A) | I or other herbicide resistant or detoxgene

Chart 5

BR Plant Bacillus or Plant BR pGA482 j I Nos-Nptll | Promoter other toxgene polyfA'- 1 j for insect pest

SUBSTITUTE SHEET

Qiart 1

Cmvccoat.Seq Length: 1423 December 1, 1987 12.49 Cheek: 215.

1 GAATTCCCGC CGCAATCGGG AGTTCTTCCG CGTCCCGCTC CGAAGCCTTC

51 AGACCGCAGG TGGTTAACGG TCTTTAGCAC TTTGGTGCGT ATTAGTATAT

101 AAGTATTTGT GAGTCTGTAC ATAATACTAT ATCTATAGAG TCCTGTGTGA

151 GTTGATACAG TAGACATCTG TGACGCGATG CCGTGTTGAG AAGGGATCAC

201 ATCTGGTTTT AGTAAGCCTA CATCATAGTT TTGAGGTTCA ATTCCTCTTA

251 CTCCCTGTTG AGTACCTTAC TTTCTCATGG ATGCTTCTCC GCGAGATTGC

301 QTTATTGTCT ACTGACTATA TAGAGAGTGT GTGTGCTGTG TTTTCTCTTT

351 TGTGTCGTAG AATTGAGTCG AGTCATGGAC AAATCTGAAT CAACCAGTGC

401 TGGTCGTAAC CATCGACGTC GTCCGCGTCG TGGTTCCCGC TCCGCCCCCT

451 CCTCCGCGGA TGCTAACTTT AGAGTCTTGT CGCAGCAGCT TTCGCGACTT

E01 AATAAGACGT TAGCAGCTGG TCGTCCAACT ATTAACCACC CAACCTTTGT

551 AGGGAGTGAA CGCTGTAGAC CTGGGTACAC GTTCACATCT ATTACCCTAA

601 AGCCACCAAA AATAGACCGT GAGTCTTATT ACGGTAAAAG GTTGTTACTA

651 CCTGATTCAG TCACGGAATA TGATAAGAAG CTTGTTTCGC GCATTCAAAT

701 TCGAGTTAAT CCTTTGCCGA AATTTGATTC TACCGTGTGG GTGACAGTCC

751 GTAAAGTTCC TGCCTCCTCG GACTTATCCG TTGCCGCCAT CTCTGCTATG

801 TTCGCGGACG GAGCCTCACC GGTACTGGTT TATCAGTATG CCGCATCTGG

851 AGTCCAAGCC AACAACAAAC TGTTGTTTGA TCTTTCGGCG ATGCGCGCTG

901 ATATAGGTGA CATGAGAAAG TACGCCGTCC TCGTGTATTC AAAAGACGAT

951 GCGCTCGAGA CGGACGAGCT AGTACTTCAT GTTGACATCG AGCACCAACG

1001 CATTCCCACA TCTGGAGTGC TCCCAGTCTG ATTCCGTGTT CCCAGAACCC

1051 TCCCTCCGAT CTCTGTGGCG GGAGCTGAGT TGGCAGTTCT ACTACAAACT

1101 GTCTGGAGTC ACTAAACGTT TTACGGTGAA CGGGTTGTCC ATCCAGCTTA

1151 CGGCTAAAAT GGTCAGTCGT GGAGAAATCC ACGCCAGCAG ATTTACAAAT

1201 CTCTGAGGCG CCTTTGAAAC CATCTCCTAG GTTTCTTCGG AAGGGCTTCG

1251 GTCCGTGTAC CTCTAGCGCA ACGTGCTAGT TTCAGGGTAC GGGTGCCCCC

1301 CCACTTTCGT GGGGGCCTCC AAAAGGAGAC CAAAAAAAAA AAAAAAAAAA

1351 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA

1401 AAAAAAAAAA AAAAAAAGAA TTC

SUBSTITUTE SHEET

Chart 2

pCMV9 .9 ^ — i coat protein gene t

A residues

CaMV 35S poly-A signal pDH51/CP19

CaMV I coat protein gene 1 — t -l

35S A residues promoter

* ^{» •,} τ ^* *"r3"-- - ^~~ - ^~~ c i-&'z?__"

Chart

Construction of pGA482/CP19H/GUS

CaMV 35S

NOA- poly-A signal NPT11 I pGA482/CP19H

CaMV coat protein t B

35S gene A residues promoter

* **-__ :. - ___ i

- 13- Chart 4

Construction of pGA482/CP19A-/411/GUS

CaMV 35S poly-A signal pDH51/CP19

CaMV coat protein gene * 35S A residues promoter

pDH51/CP19A-/411

CaMV coat protein gene phaseolin

35S poly-A promoter signal (411)

] p6A482/CP19A-/411

NOS* B NPTII CaMV coat protein gene phaseolin B R 35S poly-A L promoter signal (411)

pGa482/CP19A-/411/6US

NOS

B NPTII CaMV coat pro- phaseolin gus -O R 35S tein gene poly-A gene promoter signal(411)

_ ^" .___. _* -_ .

Chart 5 Construction of pGA482/CP19/411/GUS

CaMV 35S poly-A signal pDH51/CP19"[ |-

CaMV coat protein gene t

35S A residues promoter

pDH51/CP19/411 CaMV 35S poly-A signal

1

CaMV coat protein t phaseolin 35S gene A poly-A signal (411) promoter residues

PGA482/CP19/411

NOS-

-L -H f-LZJ

B NPTII CaMV coat protein t phaseolin R 35S poly-A promoter A signal (411) residues

PGA482/CP19/411/GUS

NOS*

B H I-

NPTII CaMV coat pro- t phaseolin gus HZ

35S A poly-A gene promoter resi- signal dues (411)