WO1994028147A1

WO1994028147A1 - Dna sequence comprising at least two coat protein genes

Info

Publication number: WO1994028147A1
Application number: PCT/EP1994/001786
Authority: WO
Inventors: Kirsten Bojsen; Janne Brunstedt
Original assignee: Sandoz Ltd.; Sandoz-Patent-Gmbh; Sandoz-Erfindungen Verwaltungsgesellschaft M.B.H.
Priority date: 1993-06-02
Filing date: 1994-06-01
Publication date: 1994-12-08
Also published as: AU7122694A; ZA943863B; GB9311332D0; IL109847A0

Abstract

The present invention provides a recombinant DNA sequence comprising nucleotide sequences encoding the coat proteins of at least one luteovirus and at least one closterovirus, or a recombinant sequence comprising nucleotide sequences encoding at least a part of both proteins. The recombinant sequence may additionally comprise a nucleotide sequence encoding a coat protein, or a part thereof, of a furo-virus, such as beet necrotic yellow vein virus (BNYVV).

Description

DNA sequence comprisine at least two coat protein genes

The present invention relates to transformed plants, particularly sugar beet.

According to the present invention there is provided a recombinant DNA sequence comprising nucleotide sequences encoding the coat proteins of at least one luteovirus and at least one closterovirus, or a recombinant sequence comprising nucleotide sequences encoding at least a part of both proteins. The recombinant sequence may additionally comprise a nucleotide sequence encoding a coat protein, or a part thereof, of a furo-virus, such as beet necrotic yellow vein virus (BNYVV).

A taxonomy of plant viruses is given by T. Wilson (1989) in Bioessays (Vol 10, No. 6, pp 179-185).

By "part" is meant a peptide encoded by the sequence depicted in SEQ ID Nos 1, 2 or 3, and having at least 10 amino acids. More preferably the peptide has at least 15 amino acids, and still more preferably the peptide has at least 20 amino acids.

The invention also includes a recombinant DNA sequence according to the invention, which is modified in that codons which are preferred by the organism into which the recombinant DNA is to be inserted are used so that expression of the thus modified DNA in the said organism yields substantially similar proteins to those obtained by expression of the unmodified recombinant DNA in the organism in which the protein-encoding components of the recombinant DNA are found endogenously.

In a preferred embodiment of the invention, the closterovirus is beet yellows virus (BYV), and the luteovirus is beet western yellows virus (BWYV).

The nucleotide sequence of the cDNA encoding the coat protein of BYV has been published in "Virus Genes" (Brunstedt et al. (1991) Vol 5:3, pp 267-272). The nucleotide sequence of

BWYV RNA has been published in Nucleic Acids Research (Veidt et al. (1988), Vol 16, pp

9927 - 9932); and the nucleotide sequence of BYNVV RNA-2 has been published in J. Gen Virol. (Bouzoubba et_al. (1986) Vol 67, pp 1689-1700).

Transcription of all the coat protein genes may be under control of a single promoter. It is preferred, however, that transcription of each coat protein gene, or part thereof, is under control of a seperate promoter.

Such promoters are known and may be constitutive and or inducible. Suitable promoters include the 35 S promoter, the hsp80 promoter (US Patent Application No 791929), the hsr203 promoter (European Patent Application No. 93102887.2), and promoters regulating expression of octipine genes, rubisco gene(s), and the promoters of genes encoding plant wound-inducible proteins, such as the WUN promoter (European Patent Application No. 89912967.0), and promoters of pathogene sis-related inducible proteins.

It is particularly preferred that the transcription of at least the BYV coat protein gene and the BWYV coat protein gene is under control of an enhanced 35 S promoter or the hsr203 promoter or the hsp80 promoter.

The recombinant DNA sequence may further comprise other gene regulator sequences, typically down stream of the promoters. Such sequences are known and are preferably positioned between the promoter and the structural gene regulated thereby, and include the omega sequence from tobacco mosaic virus which may function as part of a leader sequence in front of the coat protein genes.

The recombinant sequence may further comprise known marker genes and/or at least one gene encoding a known anti-microbial protein. An anti-microbial protein includes a protein (alone or in combination with another material) which is toxic or growth inhibitory under any circumstances to any micro-organism, including bacteria, viruses and particularly fungi. Such anti-microbial proteins include those that exhibit anti-microbial activity upon contact with a micro-organism and those that are anti-microbial as a consequence of assimilation or respiration thereof.

Examples of suitable anti-microbial proteins are given in British Patent Application No. 9303725.7, filed February 24, 1993, corresponding to European Patent Application No. 94810103.5.

Infection of plants with fungal or viral pathogens may induce a systemic synthesis of about 10 families of homologous pathogenesis-related proteins (PR proteins) in vegetative tissues. Such PR-proteins have been classified into 5 groups. The PR-2, PR-3 and PR-5 proteins are beta- 1,3 glucanase, chitinases and thaumatin-like proteins respectively. Specific functions have not been assigned to the PR-1 and PR-4 groups of proteins. The PR-4 proteins are similar to C-terminal domains of prohevein and the putative wound-induced WIN proteins of potato, thus lacking the N-terminal hevein domain. "Basic counter-part of the acidic pathogenesis-related 4 group of proteins" thus includes the basic counter part of proteins similar to the C-terminal domains of prohevein and the putative wound-induced WIN proteins of potato.

The recombinant DNA sequence according to the present invention may still further comprise at least one gene encoding a protein which is the basic counter-part of the acidic pathogenesis- related 4 group of proteins. The preferred such protein is a chitin-binding WIN protein, preferably similar if not identical to that extractable from barley grain or stressed barley leaves.

It is particularly preferred that the recombinant DNA sequence is modified, wherein - at least in the coat protein genes - known plant preferred codons are substituted for viral codons so that transcription of the thus modified DNA in plants and translation of the resulting RNA yields coat proteins having an amino acid sequence identical or substantially similar to the native virus coat proteins. Further preferred modifications to the DNA sequence include removal or deactivation of open reading frames within the viral coat protein genes, particularly removal or deactivation of the internal open reading frame at nucleotides 120-122 in SEQ ID No. 2 within the gene encoding the BWYV coat protein, and alteration of the coat protein gene stop codons.

The preferred such alterations include replacement of leaky stop codons (for example TAG) by non-leaky such codons (for example TAA).

The invention further includes nucleic acid sequences which are similar to the recombinant DNA sequence according to the invention. By similar is meant a test sequence which is capable of hybridizing to the inventive sequence. When the test and inventive sequences are double stranded the nucleic acid constituting the test sequence preferably has a TM within 20°C of that of the inventive sequence. In the case that the test and inventive sequences are mixed together and denatured simultaneously, the TM values of the sequences are preferably within 10°C of each other. More preferably the hybridization is performed under stringent conditions, with either the test or inventive DNA preferably being supported. Thus either a denatured test or inventive sequence is preferably first bound to a support and hybridization is effected for a specified period of time at a temperature of between 50 and 65°C in double strength citrate buffered saline containing 0.1%SDS followed by rinsing of the support at the same temperature but with a buffer having a reduced SC concentration. Depending upon the degree of stringency required, and thus the degree of similarity of the sequences, such reduced concentration buffers are typically single strength SC containing 0.1%SDS, half strength SC containing 0.1%SDS and one tenth strength SC containing 0.1%SDS. Sequences having the greatest degree of similarity are those the hybridization of which is least affected by washing in buffers of reduced concentration. It is most preferred that the test and inventive sequences are so similar that the hybridization between them is substantially unaffected by washing or incubation in one tenth strength sodium citrate buffer containing 0.1%SDS.

The present invention also includes a vector containing any of the above-mentioned DNA sequences, as well as a biological system which includes any of said DNA sequences and which allows expression of the DNA .

The biological system may be a micro-organism, such as an Agrobacterium strain, for example, or a plant.

The invention also includes plants, particularly sugar beet, transformed with the above disclosed recombinant DNA, and plants transformed with a recombinant DNA sequence including a portion encoding the coat proteins of both the beet yellows virus and the beet western virus, and optionally the coat protein of the beet necrotic yellow vein virus, or similar proteins functionally equivalent thereto in which one or more amino acids have been added, substituted or removed without substantially reducing the capacity of the protein(s) to function as a viral coat protein and/or to elicit a plant mediated^λanti-viral response. It is preferred that such functionally equivalent proteins have an amino acid sequence which is at least 70% similar to the sequence of at least one of the viral coat proteins encoded by the recombinant DNA sequence. It is more preferred that the degree of similarity is at least 80%, still more preferred that the degree of similarity is at least 85% and still more preferred that the degree of similarity is at least 90%.

In the context of the present invention, two amino acid sequences with at least 70% similarity to each have at least 70% identical or conservatively replaced amino acid residues in a like position when aligned optimally allowing for up to 5 gaps with the proviso that in respect of the gaps a total not more than 10 amino acid residues are affected.

For the purpose of the present invention conservative replacements may be made between amino acids within the following groups:

(i) Alanine, Serine and Threonine;

(ii) Glutamic acid and Aspartic acid;

(iii) Arginine and Lysine;

(iv) Asparagine and Glutamine;

(v) Isoleucine, Leucine, Valine and Methionine;

(vi) Phenylalanine, Tyrosine and Tryptophan

Such plants are made by known methods and include regeneration of plant cells or protoplasts transformed with the recombinant DNA of the invention according to a variety of known methods (Agrobacteriwn Ti and Ri plasmids, electroporation, micro-injection, micro-projectile gun etc). The transformed cells may in suitable cases be regenerated into whole plants in which the nuclear material is stably incorporated into the genome. Both monocot and dicot plants may be obtained in this way.

Examples of genetically modified plants according to the present invention include: fruits, including tomatoes, mangoes, peaches, apples, pears, strawberries, bananas, and melons; field crops such as canola, sunflower, tobacco, sugar beet, small grain cereals such as wheat, barley and rice, maize and cotton, and vegetables such as potato, carrot, lettuce, cabbage and onion. The preferred plant is sugar beet.

The invention also includes the progeny of plants according to the present invention, which progeny expresses the said recombinant DNA sequences, and the seeds of the plants or the progeny thereof.

The invention still further includes protein derived from expression of the recombinant DNA according to the present invention, and anti-viral protein produced by expression of the recombinant DNA within plants.

The invention still further includes a process for combatting viruses in sugar beet which comprises transforming sugar beet with a recombinant DNA sequence according to the present invention and obtaining transcription of the said DNA. It is particularly preferred that the RNA resulting from the transcription of said recombinant DNA is translated into protein.

The invention may be further understood by reference to the following text, including Examples, Sequence Identifications, and the accompanying Drawings.

SEQ ID No. 1 shows the nucleotide sequence of the BYV coat protein gene.

SEQ ID No. 2 shows the nucleotide sequence of the BWYV coat protein gene, in which a start codon of an internal open reading frame in the native viral gene has been removed and a leaky stop codon replaced.

SEQ ID No. 3 shows the nucleotide sequence of the BNYVV coat protein in which PCR has been used to replace a leaky stop codon (TAG) at bp. 654-656 with the codon TAA.

Figure 1 shows the vector pUC19ES35S-35St into which the sequence depicted in SEQ ID No.

1 is cloned as a Kpnl-BamHl fragment using a Pstl-Kpnl linker.

Figure 2 shows the vector pPS48 into which the sequence depicted in SEQ ID No 2 is cloned as a Hindlll-EcoRI blunt ended fragment in the Smal site, and into which the BNYVV coat protein sequence of SEQ ID No. 3 is cloned as a Hindlll blunt ended fragment.

Figure 3 shows the BNYVV coat protein sequence of SEQ ID No. 3 cloned according to

Figure 2 and inserted into the plasmid pGNBNYVV. Figure 4 shows the plasmid pGNBWYVBYV comprising the EcoRl fragment resulting from digestion of the vector of Figure 1 and the Hindlll fragment comprising the BWYV coat protein gene resulting from digestion of the vector of Figure 2. In Figures 3 and/or 4 the following abbreviations are used:

OCSt: Octipine synthetase terminator sequence

NPTII: Neomycin phosphotransf erase encoding gene

35S: Cauliflower Mosaic Virus 35S promoter

35St: Cauliflower Mosaic Virus 35S terminator sequence

E35S: Enhanced Cauliflower Mosaic Virus 35S Promoter

Omega: Omega leader sequence from Tobacco Mosaic Virus

NOSt: Noplaine synthetase terminator sequence

BMYV: Beet Mild Yellows Virus coat protein

BYV: Beet Yellows Virus coat protein

BNYVV: Beet Necrotic Yellow Vein Virus coat protein

GUS: Beta-glucuronidase encoding protein

RB: Right Border sequence

LB: Left Border sequence

Figure 5 shows a PCR analysis of sugar beets transgenic for both the BWYV and the BYV coat proteins according to the nucleotide sequences depicted in SEQ ID Nos 1 and 2.

The present invention provides, inter alia, a recombinant DNA sequence comprising nucleotide sequences encoding the coat proteins of at least one luteovirus and at least one closterovirus, or a recombinant sequence comprising nucleotide sequences encoding at least a part of both proteins. The recombinant sequence may additionally comprise a nucleotide sequence encoding a coat protein, or a part thereof, of a furo-virus, such as beet necrotic yellow vein virus (BNYVV). The preferred luteovirus is beet western yellows virus (BWYV), and the preferred closterovirus is beet yellows virus (BYV).

A vector comprising at least the coat protein genes for the BYV and the BWYV is produced and inserted into a suitable Agrobacteriwn strain according to known methods. The thus transformed Agrobacteriwn is then used to transform plant cells, preferably sugar beet. The transformed cells are then regenerated into plants transgenic for both coat proteins.

SEQ ID No. 1 shows the nucleotide sequence of the BYV coat protein gene. The omega sequence from Tobacco Mosaic Virus is inserted as a leader sequence (bp. 13-78) in front of the coat protein gene (between the promoter and the coat protein gene). The start codon is given by bp. 89-91 and the stop codon by bp. 701-703.

The BYV coat protein sequence depicted in SEQ ID No. 1 is cloned as a KpnI-BamHI fragment in Pstl-BamHI in pUC19ES35S-35St using a Pstl-Kpnl linker. The EcoRI fragment comprising the BYV coat protein gene resulting from digestion of this vector is inserted into pGNBWYVBYV together with the Hindlll fragment comprising the BWYV coat protein resulting from digestion of the vector depicted in Figure 2 (see Figure 4).

SEQ ID No. 2 shows the nucleotide sequence of a modified BWYV coat protein gene. The leaky stop codon (TAG) in the native viral gene sequence is replaced by the codon TAA at bp. 695-697 as can be seen from the sequence given in SEQ ID No. 2, and the start codon of an internal Open Reading Frame (ORF) of the coat protein gene (bp. 120-122. in SEQ ID No. 2) is changed from ATG to ACG in order to prevent translation of this ORF. Both alterations are performed using PCR. SEQ ID No. 2 also shows that the omega sequence from tobacco mosaic virus is inserted as a leader sequence (bp. 7-72) in front of the coat protein gene. The start codon is bp. 89-91.

The modified BWYV coat protein gene of SEQ ID No. 2 is cloned as a Hindlll-EcoRI blunt ended fragment in the Smal site of pPS48 (Fig. 2). The Hindlll fragment comprising the BWYV coat protein gene resulting from digestion of the vector of Figure 2 is inserted into the plasmid pGNBWYVBYV together with the EcoRI fragment comprising the BYV coat protein gene resulting from digestion of the vector of Figure 1 (see Figure 4).

SEQ LD No. 3 shows the nucleotide sequence of a modified BNYVV coat protein gene, in which PCR is used to replace a leaky stop codon (TAG) in the viral nucleotide sequence at bp. positions 654-656 with TAA. The start codon of the gene depicted in SEQ ID No. 3 is bp. 90-92. It is preferred that the nucleotide sequence of the BNYVV coat protein is further modified, preferably by insertion of the omega sequence from Tobacco Mosaic Virus as a leader sequence (bp- 7-72) in front of the coat protein gene, between it and the promoter. The BNYVV virus coat protein sequence is cloned as a Hindlll blunt-ended fragment in pS48 (Figure 2) and inserted into pGNBNYVV (see Figure 3).

Production of transformed plants.

A disarmed Agrobacteriwn twnefaciens strain, for example, is transformed with the vector of Figure 4 or a like vector further comprising the BNYVV coat protein gene. Plant cells are then treated with such transformed disarmed Agrobacteriwn, and the thus transformed plant cells are regenerated into whole plants (optionally via callus), in which the new nuclear material is stably incorporated into the genome. Morphologically normal plants which do not contain Agrobαctewm-originating oncogenes result from such transformation. It will be appreciated, however, that the recombinant DNA of the invention may be introduced into plant cells by other known methods, including use of a micro-projectile gun, electroporation, electro- transformation, incubation with plant protoplasts in the presence of polyethylene glycol, and micro-injection etc, and that regeneration of transformed plant cells or protoplasts is carried out according to methods known to the skilled man, including treatment of the cells or protoplasts with cytokines and/or low carbohydrate content media where this is necessaiy or desirable in order to improve the regeneration frequency.

Plants transformed to comprise the DNA sequences of the present invention express the introduced coat protein genes (Figure 5) and exhibit an improved response to challenge with the respective viruses. A particularly beneficial improvement is seen when the transformed plants are challenged with both viruses, either simultaneously by way of a mixed infection, or concomitantly as could occur in nature. Plants subjected to a concomitant infection exhibit a surprisingly improved resistance to the second viral infection in comparison with non- transformed control plants.

It will be appreciated that the invention is not limited to the above examples only, many variations thereto being possible. For example the DNA sequence may comprise, or further comprise the nucleotide sequence which is complementary to the nucleotide sequence encoding at least one of the virus coat proteins. Furthermore, the DNA sequence may comprise a complementary nucleotide sequence as a substitute for at least one of the genes encoding at least one of the coat proteins of luteo- clostero- or furo- viruses.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Sandoz Ltd

(B) STREET: Lic tstrasse 35

(C) CITY: Basel

(D) STATE: BS

(E) COUNTRY: Switzerland

(F) POSTAL CODE (ZIP) : CH-4002

(G) TELEPHONE: 061-324-1111 (H) TELEFAX: 061-322-7532 (I) TELEX: 965-05055

(A) NAME: Sandoz Erfindungen Verwaltungsgesellschaft

MBH

(B) STREET: Brunnerstrasse 59

(C) CITY: Vienna

(E) COUNTRY: Austria

(F) POSTAL CODE (ZIP) : A-1230

(A) NAME: Sandoz Patent GMBH

(B) STREET: Humboltstrasse 3

(C) CITY: Loerrach

(E) COUNTRY: Germany

(F) POSTAL CODE (ZIP) : D-7850

(ii) TITLE OF INVENTION: DNA sequence comprising at least two coat protein genes

(iii) NUMBER OF SEQUENCES: 3

(iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25 (EPO)

(2) INFORMATION FOR SEQ ID NO: 1:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 783 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beet Yellows Virus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

GGTACCCAGC TTATTTTTAC AACAATTACC AACAACAACA AACAACAAAC AACATTACAA 60

TTACTATTTA CAATTACAGT CGACTGACAT GGGATCAGCC GAACCTATAA GTGCAATCGC 120

GACTTTTGAA AACGTGAGTC TCGCAGACCA AACGTGTTTG CACGGTGAAG ACTGCGACAA 180 ACTACGGAAG AATTTCGAAG AGTGTTTGAA ATTGAAAGGG GTTCCGGAAG ACAAACTCGG 240

TCTCGCGTTA GAACTTTGTT TGTATTCCTG TGCGACGATA GGTACTTCTA ATAAAGTTAG 300

TGTCCAACCG ACGTCTACTT TCATCAAAGC TTCGTTCGGT GGTGGGAAGG AATTGTTCCT 360

CACTCACGGT GAACTGAGGT CTTTTCTGGA CTCTCAGAAA CTTTTGGAAG GAAAGCCTAA 420

CAAGTTGCGT TGTTTCTGCC GCACTTTTCA GAAGGACTAC ATATCCTTCG CGAAGGAATA 480

CCGAGGAAGA CTGCCTCCGA TTGCTAGAGC CAACCGTCAC GGTCTACCTG CTGAAGATCA 5 0

CTACTTAGCT GCTGATTTCA TATCGACATC AACAGAACTT ACTGACCTAC AACAAGGTCG 600

TCTGCTGTTG GCGCGCGAAA ACGCCACTCA CACAGAGTTC TCGTCTGAAT CACCAGTAAC 660

TAGTTTGAAA CAGCTGGGTC GTGGTCTAGC CACCGGAAGA TGATTGGCTC TGTCGAACTA 720

GCTCAGACGA GACCCTTTTT TAGAGTATTA CTGTTAAAGG GTTTCGTTTA TTATATTGGA 780

TCC 783 (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 718 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beet Western Yellows Virus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

AAGCTTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 60

TTTACAATTA CAGTCGACGA AATCGTTAAT GAATACGGTC GTGGGTAGGA GAACAATCAA 120

CGGAAGAAGA CGACCACGGA GGCAAACACG ACGCGCTCAG CGCTCTCAGC CAGTGGTTGT 180

GGTCCAAACC TCTCGGGCAA CACAACGCCG ACCTAGACGA CGACGAAGAG GTAACAACCG 240

GACAAGAGGA ACTGTTCCTA CCAGAGGAGC AGGCTCAAGC GAGACATTTG TTTTCTCGAA 300

AGACAATCTC GCGGGATCCT CCAGCGGACG AATCACGTTC GGGCCGAGTC TATCAGACTG 360

CCCAGCATTC TCTAATGGAA TACTCAAGGC CTACCATGAG TATAAAATCT CGATGGTCAT 420

TTTGGAGTTC GTCTCCGAAG CCTCTTCCCA AAACTCCGGT TCCATCGCTT ACGAGCTGGA 480

CCCACACTGT AAACTCAACT CCCTTTCCTC AACTATCAAC AAGTTCGGGA TCACAAAGCC 540

CGGGAAAGCG GCGTTTACAG CGTCTTACAT CAATGGAAAG GAATGGCACG ACGTTGCCGA 600

GGACCAATTC AGGATCCTCT ACAAAGGCAA TGGTTCTTCA TCGATAGCTG GTTCTTTTAG 660

AATCACCATC AAGTGCCAAT TCCACAATCC CAAATAAGTA GACGAGGAAC CCGAATTC 718 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 771 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(iii) ANTI-SENSE: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Beet Necrotic Yellow Vein Virus

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

AAGCTTATTT TTACAACAAT TACCAACAAC AACAAACAAC AAACAACATT ACAATTACTA 60

TTTACAATTA CAGTCGAGTA CCCACCAACA TGTCGAGTGA AGGTAGATAT ATGACATGGA 120

AGGATATGTC ACATAATAAG TTTATGACCG ATCGATGGGC TCGTGTTTCG GACGTCGTGA 180

GTGTTATTAA ACAATCGCAT GCTATGGACT TGTCCAAGGC TGCGAATCTA TCTATAATTA 240

AAACTGCTTT GGCAGGATTA GGCTCGGGTT GGTCTGACAG TAATCCTTTT GTGTCTCCGA 300

TGACCCGTTT TCCACAGACA CTAACTATGT ATGGTGCACT TGTGTTATAT GTTAATCTGT 360

CTGACCCAGA ATTTGCGTTG ATAATGACTA AGGTAAATAC TTTAACTGAT TCAGGGTTAG 420

CAGATAATGC ATCTGCTAAT GTACGTAGAG ATGTGGTGTC TGGAAATAAA GCTGAATCAT 480

CCGGTAAAAC TGCTGGTACT AATGAGAACT CTGCTTATAC GCTTACTGTC AGTCTTGCTG 540

GTTTAGCTCA AGCTCTTAGG CTTGAAGAAT TAATGTGGAC CCGGGATAAA TTTGAGGACC 600

GGTTCAAATT ACCATGGACA CCTGTTCAAG GTAGAACCAG TCCACCCGGA CAATAACAAT 660

TAGCTGCTGC TCGGGTGACG GCACACATTC GAGCGGCGAA GCGGGCACTA TTATATCCTG 720

GTGATAGTCC CGAGTGGGTT GGTTGGAAAC GACCTGCAGG CATGCAAGCT T 771

Claims

1. A recombinant DNA sequence comprising nucleotide sequences encoding the coat proteins of at least one luteovirus and at least one closterovirus, or a recombinant sequence comprising nucleotide sequences encoding at least a part of both proteins.

2. DNA sequence according to claim 1, further comprising a nucleotide sequence encoding the coat protein of a furo-virus or a sequence encoding at least a part of the protein.

3. DNA sequence according to claim 1 wherein the closterovirus is beet yellows virus (BYV) and the luteovirus is beet western yellows virus (BWYV), or according to claim 2, wherein the furo-virus is beet necrotic yellow vein virus (BNYW).

4. DNA sequence according to any one of claims 1 - 3, wherein transcription of each coat protein gene is under control of a seperate promoter, preferably selected from the group consisting of constitutive and inducible promoters, including the 35S promoter, the enhanced 35 S promoter; the hsp80 promoter, the hsr203 promoter, and promoters regulating expression of: octipine genes, rubisco gene(s), and the genes encoding wound and pathogenesis-related inducible proteins.

5. DNA sequence according to any one of claims 1-4, further comprising at least one translation enhancing sequence down stream of at least one of the promoters controlling expression of at least one of the viral coat protein genes.

6. DNA sequence according to any preceding claim, further comprising at least one of the following: known marker genes; known genes encoding anti-bacterial or anti-fungal proteins.

7. DNA sequence according to any preceding claim, wherein - at least in the coat protein genes - known plant preferred codons are substituted for viral codons so that transcription of the thus modified DNA in plants and translation of the resulting RNA yields coat proteins having an amino acid sequence identical or substantially similar to the native virus coat proteins.

8. DNA sequence according to any preceding claim, wherein at least one viral coat protein gene stop codon has been modified, and/or at least one open-reading frame has been deactivated or removed from at least one of the viral coat protein genes.

9. A vector containing a DNA sequence as claimed in any one of claims 1-8.

10. A biological system which includes a DNA sequence as claimed in any one of claims 1 - 9.

11. A biological system according to the preceding claim, selected from the group consisting of plants and micro-organisms.

12. Plants transformed with recombinant DNA as claimed in any one of claims 1 to 9.

13. Plants according to the preceding claim, wherein the plants are sugar beet.

14. Plants transformed with a recombinant DNA sequence including a portion encoding the coat proteins of both the beet yellows virus and the beet western yellows virus, and optionally the coat protein of beet yellow necrotic vein virus, or proteins functionally equivalent thereto, in which one or more amino acids have been added, substituted or removed without substantially reducing the capacity of the protein to function as a viral coat protein and/or to elicit a plant mediated anti-viral response.

15. The progeny of plants according to any one of claims 12-14, which progeny expresses the said recombinant DNA sequences, and the seeds of such plants and of such progeny.

16. Protein derived from expression of the DNA as claimed in any one of claims 1 - 8.

17. A process for combatting viruses in sugar beet which comprises transforming sugar beet with a recombinant DNA sequence according to any one of claims 1 - 8, and obtaining transcription of said DNA.