HK1008548A - Transgenic plants expressing dna constructs containing a plurality of genes to impart virus resistance - Google Patents

Description

Transgenic plants expressing DNA constructs comprising multiple genes with antiviral resistance

Technical Field

The present invention relates to plant genetic engineering and means and methods for conferring multiple traits (including resistance to viruses) to plants using vectors encoding multiple genes, such as coat protein genes, protease genes or replicase genes.

Background

Most important agricultural crops are susceptible to attack by plant viruses, which can cause severe damage to the crop, decrease growers' economic income, and increase consumer costs. Efforts have been made to control or prevent plant viral infestation of crops, but viral pathogens remain a significant problem in agriculture.

Scientists have recently developed methods for conferring antiviral resistance to plants using genetic engineering techniques. This development is very advantageous because the genetic material which confers protection to the plant is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it has the ability to inhibit or retard viral proliferation or the ability to prevent the onset of pathogenic symptoms. "resistant" is an "sensitive" antisense word and can be classified as (1) highly resistant, (2) moderately resistant, or (3) poorly resistant, depending on its effect. Importantly, resistant plants show reduced or no symptoms and have a reduced or negligible proliferation of virus in vivo. Several different host resistances against viruses are recognized. The host may be resistant to (1) the occurrence of an infestation, (2) the propagation of a virus, or (3) the action of a virus.

Potyviruses are a group of plant viruses that cause disease in a variety of crops and have been shown to cause cross-infection between plant members of different families. Potyviruses include watermelon mosaic virus-2 (WMV-2), papaya ringspot of papaya ringspot virus and watermelon mosaic I virus (PRV-p and PRV-w) strains, Zucchini Yellow Mosaic Virus (ZYMV), potato virus Y, tobacco etch virus, and many others; among them, PRV-p and PRV-w are two members of the plant potyvirus group that are very closely related, and they were once classified into different virus types, but recently, they were classified into different strains of the same virus. See, for example, table I in published european patent application No.578,627.

These viruses consist of jagged filamentous particles with a diameter of about 780X 12 nm. The viral particles contain a single-stranded RNA genome containing about 10,000 positive (+, coding sense) nucleotides. Translation of the potyvirus RNA genome indicates that the RNA encodes a large polyprotein of about 330 kD. The multimeric protein comprises several proteins, one of which is a 49kD protease that specifically cleaves the multimeric protein into at least 6 other peptides. These proteins are found in infected plant cells and are essential components of viral replication, and one of the proteins contained within the polyprotein is a 35kD capsid or coat protein, which coats and protects viral RNA from degradation. Another protein is the nuclear inclusion body protein, also known as replicase, which is believed to play a role in the replication of viral RNA. During potyvirus infection, replicase (60kD, also known as intranuclear inclusion body B protein) and protease (50kD, also known as intranuclear inclusion body I or intranuclear inclusion body a protein) are transported post-translationally, cross the nuclear membrane into the plant nucleus in the later stages of virus infection, and accumulate to higher levels.

The coat protein gene is usually located at the 3' end of the RNA, just before a stretch of terminal adenylate residues (200-300 bases). In these viruses, the position of the 49kD protease gene appears to be conserved. In tobacco etch virus, the protease cleavage site has been identified as the dipeptide Gln-Ser, Gln-Gly or Gln-Ala. The conservation of these dipeptides as cleavage sites in these viral polyproteins is evident from the sequence of the potyviruses listed above.

Expression of coat protein genes from tobacco mosaic virus, papaya mosaic virus, cucumber mosaic virus, potato virus X and other viruses in transgenic plants confers resistance to infection by the respective viruses. Some evidence of heterologous protection has been reported. For example, it has been reported by Namba et al Phytophology, 82, 940(1992) that expression of the coat protein gene from watermelon mosaic virus-2 or zucchini yellow mosaic virus in transgenic tobacco plants confers protection against the other 6 potyviruses (bean yellow mosaic virus, potato virus Y, Venezetia virens, clover yellow vein virus, pepper mottle virus and tobacco etch virus) on the plants. According to Stark et al Biotechnology, 1, 1257(1989), it was reported that expression of potyvirus soybean mosaic virus in transgenic plants confers protection against two serologically unrelated potyviruses (tobacco etch Virus and Potato Virus Y).

However, expression of the preselected coat protein gene does not reliably confer heterologous protection to the plant. For example, transgenic squash plants that contain the CMV-C coat protein gene and that have been shown to be resistant to CMV-C are not resistant to infection by several virulent strains of CMV (including CMV-V-27 and CARNA-5). Therefore, there is a need for an improved method for obtaining resistance of plants against potyvirus.

Summary of the invention

The present invention provides a recombinant chimeric DNA molecule comprising a plurality of DNA sequences, each DNA sequence comprising a promoter operably linked to a DNA sequence encoding a virus-associated protein, such as a Coat Protein (CP), a protease or a replicase, wherein said DNA sequences are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule, thereby conferring to the plant resistance to infection by each of said viruses. The DNA sequences are preferably joined in tandem, i.e.head to tail of one sequence. Furthermore, it is preferred that substantially equal amounts of resistance to infection by each of said viruses is present in plant cells transformed with said plurality of DNA sequences.

Each DNA sequence is also preferably linked to a 3 ' untranslated DNA sequence, which 3 ' untranslated DNA sequence plays a role in plant cells, causing termination of transcription and addition of polyadenylic acid to the 3 ' end of the transcribed mRNA sequence. The virus is preferably a plant-associated virus such as potyvirus.

Thus, the DNA molecules of the present invention may be used as chimeric recombinant "expression constructs" or "expression cassettes" (expression cassettes) for the preparation of transgenic plants having increased resistance to at least two plant viruses, such as potyviruses. The expression cassette also preferably contains at least one selectable marker gene or reporter gene which, together with the viral gene, is stably integrated into the genome of the transformed plant cell. The selectable marker and/or reporter gene facilitates identification of transformed plant cells and plants. Preferably, the viral gene is flanked by two or more selectable marker genes, reporter genes, or a combination thereof. Another aspect of the present invention is to provide a method of making a virus-resistant plant, such as a dicot, comprising:

(a) transforming plant cells with a chimeric recombinant DNA molecule comprising a plurality of DNA sequences, each DNA sequence comprising a promoter functional in said plant cells, said promoter being linked to a DNA sequence encoding a protein associated with a virus capable of infecting said plant;

(b) regenerating said plant cells to obtain differentiated plants; and

(c) transformed plants expressing the DNA sequence to confer upon the plant resistance to infection by said virus are identified, preferably the plants are substantially equally resistant to infection by each virus.

It is another object of the present invention to provide a method of conferring resistance to viral infection to a virus-sensitive cucurbitaceae plant, the method comprising:

(a) transforming cucurbit plant cells with a DNA molecule encoding a plurality of proteins of a virus capable of infecting said cucurbit plant;

(b) regenerating said plant cells to obtain differentiated plants;

(c) selecting a transformed cucurbitaceae plant that expresses viral proteins at a level sufficient to render the plant resistant to infection by said virus.

It is still another object of the present invention to provide a transformed plant having multiple virus resistance, said transformed plant comprising stably integrated DNA sequences encoding viral proteins.

It is a further object of the present invention to provide virus-resistant transformed plant cells containing multiple (i.e., 2-7 or more) viral genes expressed as viral proteins from the same viral strain or as viral proteins from different viral strains, as are different members of a viral population (e.g., potyvirus population).

The present invention was primarily described by inserting multiple viral coat protein expression cassettes into binary plasmids and then characterizing the resulting plasmids. The combination of CMV, ZYMV, WMV-2, SQMV and PRV coat protein expression cassettes were placed in the binary plasmid pPRBN. Subsequently, binary plasmids containing multiple coat protein expression cassettes were transferred into Agrobacterium (Agrobacterium) for use in plant transformation procedures. Two or more viral coat protein transformation susceptible genes are transferred into plants, such as plants of the Cucurbitaceae family, together with associated selectable markers and/or reporter genes using binary plasmids containing multiple expression cassettes.

Thus, the present invention provides a method of genetic engineering by which multiple traits can be manipulated and tracked as individual gene inserts (i.e., constructs that behave as if a single gene segregates as a single Mendelian locus). Although the present invention is exemplified by the use of virus resistance genes, virtually any combination of genes may be linked. Thus, by simply tracking the linked selectable marker or reporter gene that has been inserted into the transformed vector, one can track a cluster of genes that exhibit traits such as disease resistance, increased herbicide resistance, increased shelf life, and the like.

It has also been found that when multiple genes in tandem are inserted, their potencies are preferably substantially identical, more preferably their potencies are substantially equal; wherein the term "substantially" when referring to viral resistance, is as defined in the examples below. For example, if one examines multiple transgenic lines containing intact ZYMV and WMV-2 coat protein inserts, one would find that if one line is immune to infection by ZYMV, it will also be immune to infection by WMV-2. Similarly, if a line shows a delay in the development of ZYMV, it shows a delay in the development of WMV 2. Finally, if a line is susceptible to ZYMV, it is also susceptible to WMV-2. This phenomenon was not expected. This initiative as a plant breeding tool may be difficult to apply if there is no correlation between the efficacy of each gene in these multi-gene constructs. Even for single gene constructs, one must test multiple transgenic plant lines to find one that exhibits the appropriate level of efficacy. Depending on the species used, the probability of finding lines with useful expression levels is in the range of 10-50%.

If the efficacy of each gene on a Ti plasmid containing multiple genes is not mutually relevant, the probability of finding transgenic lines against each target virus will decrease dramatically. For example, if a species uses a single gene insert, a resistant line is identified with a probability of 10%, the species is transformed with the three-gene construct CZW and the levels of potency of the genes are not mutually correlated, then the probability of finding a line resistant to CMV, ZYMV and WMV-2 would be 0.1 × 0.1 × 0.1 ═ 0.001 or 0.1%. However, despite the fact that the efficacy of multivalent genes is not mutually coherent with each other, the probability of finding lines resistant to CMV, ZYMV and WMV-2 is still 10% but 0.1%. This advantage becomes more pronounced when the construct used contains 4 or more genes.

Brief description of the drawings

FIG. 1 shows the structure of binary vector pPRBoriGN.

FIG. 2 shows the structure of binary vector pPRBN.

FIG. 3 shows the structure of pPRCPW.

FIG. 4 shows the structure of binary plasmid pEPG 321.

FIG. 5 shows the structure of binary plasmid pEPG 106.

FIG. 6 shows the structure of binary plasmid pEPG 111.

FIG. 7 shows the structure of binary plasmid pEPG 109.

FIG. 8 shows the structure of binary plasmid pEPG 115.

FIG. 9 shows the structure of binary plasmid pEPG 212.

FIG. 10 shows the structure of binary plasmid pEPG 113.

FIG. 11 shows the structure of binary plasmid pGA482 GG.

FIG. 12 shows the structure of binary plasmid pEPG 382.

Detailed description of the invention

The plants of the invention are rendered resistant to viruses by expressing in the plant an isolated DNA sequence comprising nucleotides encoding a plurality (i.e., 2-7) of viral proteins such as coat proteins, proteases and/or replicases.

Representative viruses from which these DNA sequences can be isolated include, but are not limited to, potato virus x (pvx), potyviruses such as potato virus Y (pvy), cucumber virus (CMV), tobacco vein mottle virus, Watermelon Mosaic Virus (WMV), Zucchini Yellow Mosaic Virus (ZYMV), common bean mosaic virus, bean yellow mosaic virus, soybean mosaic virus, peanut mottle virus, beet mosaic virus, wheat streak mosaic virus, maize dwarf mosaic virus, sorghum mosaic virus, sugarcane mosaic virus, sorghum mosaic virus, prunella poxvirus, tobacco etch virus, white potato feathers, yam mosaic virus and Papaya Ringspot Virus (PRV), cucumber viruses including CMA and comovirus.

In summary, potyviruses are single-stranded RNA viruses that are enclosed within a monomer of repetitive proteins, called coat protein (cp). The encapsulated virus has a curved rod-like morphology. The vast majority of potyviruses are transmitted by aphids in a non-sustained manner. The range of crops that can be infected by potyvirus is wide, and from this it can be seen that the range of hosts includes plants from multiple families, but is not limited to solanaceae, chenopodiaceae, gramineae, asteraceae, leguminosae, dioscoreaceae, cucurbitaceae, and papaycaceae.

As used herein, the term "isolated" in reference to a DNA sequence or gene means that the sequence is chemically extracted from the genome of the virus and purified and or modified to the extent that it can be inserted into a vector of the invention in the appropriate orientation (i.e., sense or antisense). The term "chimeric" as used herein means that two or more DNA sequences from different sources, strains or species (i.e., from bacteria or plants) are linked together, or that the manner in which two or more DNA sequences from the same species are linked has not been found in a native genome. Thus, the DNA sequences useful in the present invention may be naturally occurring, semi-synthetic or fully synthetic DNA. The DNA sequence may be linear or circular, i.e.may be located in a complete or linear plasmid such as the binary plasmid described below. The term "heterologous" as used herein means non-identical, e.g., differing in nucleotide and/or amino acid sequence, phenotype or independent isolate. The term "expression" as used herein refers to transcription or transcription prior to translation of a particular DNA molecule.

Most of the recombinant DNA methods used in the practice of the present invention are standard and well known to those skilled in the art and are described in detail in, for example, European patent application publication No.223,452, published on 29.11.1986, which is incorporated herein by reference. Enzymes are commercially available and used according to the Vendor's recommendations or other modifications known in the art. Documents containing these standard techniques generally include the following: wu editor (1979) Methods in Enzymology (Methods in Enzymology), Vol.68; miller (1972) Experiments in Molecular Genetics (Molecular Genetics Experiments); sambrook et al (1989) Molecular Cloning: a Laboratory Manual, second edition; glover editor (1985) DNA Cloning Vol.II (DNA clone Vol.II); h.g.polites and k.r.marotti (1987) "a step-wise protocol for cdnsynthesis," Biotechniques (biotechnology), 4, 514-; gelvin and R.A.Schilperoot editions, Introduction, Expression, and analysis of GeneProducts in Plants (Introduction, Expression and analysis of gene products in Plants); all of these documents are incorporated herein by reference.

To practice the invention, the viral gene must be isolated from the viral genome and inserted into a vector containing the genetic control sequences necessary for expression of the inserted gene. When the expression vector/insert construct is assembled, it is used to transform plant cells, which are then used to regenerate plants. These transgenic plants carry the viral genes present in the expression vector/insert construct. The gene is expressed in plants and thus provides plants with increased resistance to viral infection.

There are several different methods for isolating viral genes. To this end, one of ordinary skill in the art can use information on the composition of potyviruses, cucumber viruses, or comoviruses genomes to locate and isolate coat protein genes or nuclear inclusion body genes. The coat protein gene within the potyvirus is located at the 3' end of the RNA, just before a stretch of about 200-300 adenylate residues. The nuclear inclusion body B (NIb) gene is located at the 5 'end of the coat protein gene, while the nuclear inclusion body A (NIa) gene is adjacent to the 5' end of the NIb gene. In addition, information on the proteolytic cleavage site was used to determine the N-terminus of the potyvirus coat protein gene and the N-terminus and C-terminus of the non-coat protein gene. The recognition site for proteases is conserved in the potyvirus group and has been determined to be a dipeptide Gln-Ser, Gln-Gly or Gln-Ala. The nucleotide sequences encoding these dipeptides can be determined.

A quantity of virus is cultured and harvested using methods well known in the art. Viral RNA is then isolated and viral genes isolated using a number of known procedures. Using the viral RNA, a cDNA library was prepared by methods known in the art. The viral RNA is incubated with primers that hybridize to the viral RNA and reverse transcriptase to produce complementary DNA molecules. Generating a complementary DNA strand of the complementary DNA molecule; and the sequence is a DNA copy (cDNA) of the original viral RNA molecule. The complementary strand of DNA may be produced in such a way that a double-stranded cDNA is produced, or the DNA encoding the cDNA may be amplified by polymerase chain reaction using oligonucleotide primers specific for the viral gene. In addition to virus-specific sequences, these primers also include novel restriction sites for subsequent cloning steps. Thus, a double stranded DNA molecule containing the sequence information of the viral RNA is produced. After addition of the restriction enzyme linker molecule by ligase, these DNA molecules can be cloned into plasmid vectors of E.coli. Various fragments are inserted into a cloning vector such as a plasmid whose characteristics are well known, and then Escherichia coli is transformed with the plasmid to prepare a cDNA library.

Since potyvirus genes are generally conserved, oligonucleotide based similar genes or similar gene fragments from previous isolates can be used as hybridization probes to screen cDNA libraries to determine whether any transformed bacteria contain DNA fragments with the appropriate viral sequences. The cDNA inserts hybridized to these probes in any bacterial colony can be sequenced. The viral gene is present in its entirety in colonies, 5 'of which extends to the sequence encoding the N-terminal protease cleavage site of the gene of interest, and 3' of which extends to the sequence encoding the C-terminal proteolytic cleavage site.

Alternatively, the cDNA fragment may be inserted into an expression vector in the sense orientation. Antibodies against the viral protein can be used to screen a cDNA expression library and the gene can be isolated from colonies expressing the protein.

The nucleotide sequences encoding the coat protein gene and the nuclear inclusion body gene of many viruses have been determined and inserted into expression vectors. Expression vectors contain the genetic control sequences necessary for expression of the inserted gene. The coat protein gene is inserted in such a way that the regulatory sequences are functional and that the gene is expressed when transferred into the genome of the plant. Several references that have been selected to isolate, clone and express viral genes are listed in table I below.

TABLE I

Genes cloned from RNA viruses viral genes reference the literature pawpaw ringspot cp m.m. fitch et al, Bio/Technology, 10,

1466(1992) Potato Virus X K. Ling et al, Bio/Technology 9, 752cp (1991)

Hoekema et al, Bio/Technology, 7, 273

(1989) Watermelon mosaic virus h.ouemada et al, j.gen.virol. (virogenetics), IIcp 71, 1451

(1990)

Namba et al, Phytopathology 82, 940

(1992) Cucurbita pepo yellow leaf flower s.namba et al, Phytopathology 82, 940 virus cp (1992) tobacco mosaic virus cp r.s.nelson et al, Bio/Technology 6, 403

(1988)；

P. powell Abel et al, Science 232, 738

(1986) Alfalfa mosaic virus cp Loesch-Fries et al, EMBO j., 6, 1845

(1987)

N.E.Turner et al.，EMBO J.，6，1181

(1987) Soybean mosaic virus cp d.m. stark et al, Biotechnology, 7, 1257

(1989) Cucumber mosaic virus C h.q.ouemada et al, molecular plant pathol.p. (molecular plant disease lines cp physiology), 81, 794(1991) cucumber mosaic virus WL UpJohn Co (PCT WO90/02185) line cp tobacco etch virus cp Allison et al, Virology, 147, 309(1985) tobacco etch virus core j.c.carrington et al, j.virol., 61, 2540 internal inclusion body protein (1987) pepper mottle virus cp w.g.dougherty et al, Virology, 146, 282, 1987

(1985) Potato virus Y cp d.d. shukla et al, Virology, 152, 118

(1986) Inclusion of the protein Potato Virus X cp C.Lawson et al, Biotechnology (Biotechnology), 8, 127 in the Potato Virus Y Nuclear European Patent Application 578,627

(1990) Tobacco streak Virus C.M.Van Dun et al virology, 164, 383(TSV) cp (1988)

In order for viral genes to be expressed, the necessary genetic control sequences must be employed. Since the proteins encoded by the potyvirus genome are produced by post-translational processing of polyproteins, the viral genes isolated from the viral RNA do not contain the transcriptional and translational signals necessary for their expression once they have been transferred and integrated into the plant genome. Therefore, it must be genetically engineered to contain a plant expressible promoter, a translation initiation codon (ATG) and a plant functional Poly (A) addition signal (AATAAA) 3' of its translation stop codon. In the present invention, the viral gene is inserted into a vector containing a cloning site inserted 3 'to the initiation codon and 5' to the Poly (A) signal. The 5' end of the start codon is a promoter, so that when the structural gene is inserted at the cloning site, a functional unit is formed in which the inserted gene is expressed under the control of various genetic control sequences.

The DNA segment called the promoter is responsible for the regulation of the transcription of DNA into mRNA. Many promoters that function in plant cells are known in the art and can be used in the practice of the present invention. These promoters may be obtained from a variety of sources such as plants or plant viruses, including, but not limited to, promoters isolated from the caulimovirus population such as the cauliflower mosaic virus 35S promoter (CaMV35S), the cauliflower mosaic virus 35S strong promoter (enhCaMV35S), the figwort mosaic virus full length transcription promoter (FMV35S), and promoters isolated from chlorophyll a/b binding proteins. Other useful promoters include those that are capable of expressing potyvirus proteins in certain types of cells in which infection is known to occur, either in an inducible manner or in a tissue-specific manner. For example, inducible promoters from phenylalanine ammonia lyase, phenylketene synthetase, hydroxyproline-rich glycoproteins, extensins, pathogenesis-related proteins (e.g., PR-1a), and potato-derived injury-inducing protease inhibitors may be used.

Preferred promoters for use in the viral gene expression cassettes of the invention include the nopaline synthase genes from the CaMV, Ti genes (Bevan et al, Nucleic Acids Res. II (Nucleic Acids Res. Vol. II), 369-. Poly (A) addition signals derived from these genes are also suitable for use in the expression cassettes of the invention. The particular promoter selected is preferably one that is capable of providing sufficient expression of the DNA coding sequence to which it is linked to produce a large amount of protein or RNA to provide effective antiviral resistance, but not so much as to cause damage to the cell in which the protein or RNA is expressed. The promoter selected should be capable of functioning in tissues including, but not limited to, epidermal tissues, vascular tissues, and mesophyll tissues. In fact, the choice of promoter is not critical, as long as it has sufficient transcriptional activity to achieve expression of the preselected protein or antisense RNA and subsequently confer resistance to viruses on the plant.

The untranslated leader sequence may be derived from any suitable source and may be specifically modified so that it facilitates translation of the mRNA. The 5' untranslated region may be obtained from a promoter selected to express the gene, from an unrelated promoter, from the natural leader or coding region of the gene to be expressed, from viral RNA, from a suitable eukaryotic gene, or from a synthetic gene sequence. The invention is not limited to the constructs presented in the examples below.

The termination region or 3 'untranslated region used is a sequence that causes termination of transcription and addition of polyadenylic acid (Poly (A)) to the 3' end of the transcribed mRNA sequence. The termination region may be naturally occurring with the promoter region or structural gene or from another source, and preferably includes a terminator and a sequence encoding polyadenylation. Suitable 3' untranslated regions of chimeric plant genes include (but are not limited to): (1) a 3' transcribed untranslated region comprising a polyadenylation signal from a Ti plasmid gene that induces Agrobacterium tumefaciens formation, such as the nopaline synthase (NOS) gene; and (2) plant genes like the soybean 7S storage protein gene.

Selectable marker genes can be incorporated into the expression cassettes of the invention and used to select for those cells and plants that have been transformed. The marker gene used may be expressed as resistance to antibiotics such as kanamycin, gentamicin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol and the like. Other markers such as, for example, genes encoding herbicide tolerance, such as genes resistant to glyphosate, sulfonylurea, phosphinothricin or bromoxynil, may also be used in combination or alone. Additional selection modalities include methotrexate resistance, heavy metal resistance, complementation to prototroph an auxotrophic host, and the like. See, for example, PCT WO/91/10725 in Table 1, cited above. The present invention also contemplates replacing all virus-associated genes with a string of selectable marker genes.

The particular marker used will be one that enables selection of transformed cells, as the marker for transformed cells is the opposite of the marker for those cells that have not been transformed. Depending on the number of different host species, one or more markers may be used, wherein different hosts are selected using different selection conditions, which are known to the person skilled in the art. The selectable marker may be replaced by, or used in conjunction with, a selectable marker or reporter gene, such as the β -glucuronidase gene or luciferase gene. Cells transformed with this gene were identified by the formation of blue colored products by treatment with 5-bromo-4-chloro-3-indol- β -D-glucuronide (X-Gluc).

In forming the expression constructs of the present invention, the components of the expression construct, such as the DNA sequences, linkers or fragments thereof, are routinely inserted into conventional cloning vectors, such as plasmids or phages, which are capable of replication in bacterial hosts, such as E.coli. There are a large number of cloning vectors which are described in the literature. In order to meet the specific requirements of the vector, after each cloning, the cloning vector may be isolated and subjected to further procedures such as restriction, insertion of new fragments, ligation, deletion, excision, insertion, in vitro mutagenesis, addition of polylinker fragments, and the like.

For Agrobacterium-mediated transformation, the expression cassette will be included within a vector flanked by fragments of the Agrobacterium Ti or Ri plasmid, representing the right and/or left border of the Agrobacterium Ti or Ri plasmid transfer DNA (T-DNA). This facilitates the integration of the chimeric DNA sequences of the invention into the genome of the host plant cell. The vector also contains sequences that facilitate replication of the plasmid in Agrobacterium cells as well as in E.coli cells.

All DNA manipulations are usually performed in e.coli cells, and the final plasmid with the potyvirus expression cassette is transferred into agrobacterium cells by direct DNA transformation, conjugation, etc. These Agrobacterium cells contain a second plasmid also derived from the Ti or Ri plasmid. This second plasmid carries all the viral genes required for the transfer of foreign DNA into plant cells.

Suitable plant transformation cloning vectors include those derived from the Ti plasmid of Agrobacterium tumefaciens, which are generally disclosed in U.S. Pat. No.5,258,300 to Glassman et al. In addition to those disclosed, can be found in, for example, Herrera-Estralla, Nature (Nature), 303, 209(1983), Biotechnica (published PCT application PCTWO/91/10725), and U.S. Pat. No.4,940,838 to Schilperoort et al.

Various techniques may be utilized to introduce genetic material into or transform a plant cell host. However, the particular manner in which the plant vector is introduced into the host is not critical to the practice of the present invention, and any method which allows for efficient transformation may be employed. In addition to transformation with plant transformation vectors derived from the root cancer-inducing (Ti) or hairy root-inducing (Ri) plasmids of Agrobacterium, additional methods can be employed to introduce the DNA constructs of the invention into plant cells. Such methods may include, for example, the use of liposomes, transformation with viruses or pollen, the use of chemicals that increase direct uptake of DNA (Paszkowski et al, EMBOJ. (EMBO J., 3, 2717(1984)), microinjection (Crossway et al, mol. Gen. Genet. (Gen. Genet., 202, 179(1985)), electroporation (Klomm et al, Proc. Natl. Acad. Sci. USA (Advance of national academy of sciences), 82, 824(1985)) or high-speed microprojectile bombardment (Klein et al, Nature (Nature), 327, 70 (1987)).

The source of the plant tissue used for transformation or the choice of plant cells in culture depends on the nature of the host plant and the transformation protocol. Useful tissue sources include callus, suspension culture cells, protoplasts, leaf fragments, stem fragments, male inflorescences, pollen, embryos, hypocotyls, tuber fragments, meristematic regions, and the like. The tissue source is renewable because it retains the ability to regenerate into a whole fertile plant after transformation.

Transformation is carried out under conditions suitable for the plant tissue of choice. The plant cells or tissues are contacted with the DNA carrying the multiple gene expression cassette of the invention for an effective period of time. Ranging from an electric pulse of less than 1 second to electroporation to co-culture in the presence of plasmid-containing Agrobacterium cells for 2-3 days. The buffers and media used will also vary depending on the source of the plant tissue and the transformation protocol. Many transformation protocols employ a feeder layer of suspension cultured cells (e.g., from tobaco or Black Mexican Sweet Corn.) on a solid medium plate, separated from the plant cells or tissues to be transformed by a sterile piece of filter paper.

After treatment with DNA, the plant cells or tissues may be cultured for various times prior to selection, or the cells or tissues may be immediately contacted with a selection agent such as those described above. Protocols involving contact with Agrobacterium will also include agents that inhibit the growth of Agrobacterium cells. Commonly used compounds are antibiotics such as cefotaxime and carbenicillin. The medium used in the selection may be formulated so as to maintain the transformed callus or suspension culture cells in an undifferentiated state, or to allow the callus, the fragment of leaf or stem, the tuber piece, etc. to grow roots.

Cells or calli grown in the presence of conventional inhibitory concentrations of the selection agent are considered to have been transformed and subculture can be continued on the same medium for several generations to remove non-resistant parts. The cells or callus are then assayed for the presence of viral gene (expression) cassettes or subjected to known plant regeneration procedures. In the protocol involving direct shoot growth, those shoots that grow on selective media are considered to have been transformed and can be excised and rooted on selective media suitable for root growth, or by simply immersing the excised shoot in a rooting-inducing compound and planting it directly in vermiculite.

In order to produce transgenic plants with multiple virus resistance, the viral genes must be taken up by the plant cells and stably integrated into the genome of the plant. The plant cells and tissues selected for resistance to the inhibitor are considered to have acquired the selectable marker gene encoding for that resistance during the transformation process. Since marker genes are usually linked to viral genes, it can be assumed that pathogen genes are also obtained as well. Southern blot hybridization analysis using probes specific for the viral genes can then be performed to confirm that the foreign gene has been taken up by the plant cell and integrated into the genome of the plant cell. This technique will also give the copy number of the gene that has been incorporated. The same assay can be used for the successful transcription of the foreign gene into mRNA using Northern blot hybridization analysis of total cellular RNA and/or cellular RNA enriched in the polyadenylation region. mRNA molecules comprised within the scope of the present invention are those which contain virus-specific sequences derived from viral genes present in the transformed vector which are of the same polarity as the viral genomic RNA, so that they can base pair with virus-specific RNA of polarity opposite to that of the viral genome under the conditions described in chapter 7 of Sambrook et al (1989). mRNA molecules also included within the scope of the invention are those which contain virus-specific sequences derived from viral genes present in the transformed vector which are of opposite polarity to the viral genomic RNA, so that they can base pair with the viral genomic RNA under the conditions described in chapter 7 of Sambrook et al (1989).

The presence of viral genes can also be detected by immunological tests such as the double antibody sandwich assay described by Namba et al (Gene, 107, 181(1991)), modified by Clark et al (J.Gen.Virol. (J.Virol., 34, 475 (1979)). See also Namba et al, Phytopathology 82, 940 (1992).

Resistance to viruses can be determined by the infectivity study test disclosed by Namba et al in ibid, where plants are rated according to symptoms when any inoculated leaf shows clear veins, mosaic or necrotic symptoms.

It is understood that the present invention is possible when sense or antisense virus-specific RNA is transcribed from an expression cassette as described above. That is, there is no particular molecular mechanism responsible for the phenotype and/or genotype of interest exhibited by the transgenic plant. Thus, protection against viral attack may be achieved by one or several mechanisms.

It is also understood that resistance to viruses may be achieved by expression of any virus-encoded gene. Thus, transgenic plants expressing a coat protein gene or a non-coat protein gene may be resistant to infection by homologous or heterologous viruses. For example, Transgenic Plants carrying the PRV NIa protease gene were found to be resistant to attack by PRV (see Table 7, example III), Transgenic Plants carrying the coat protein gene of the WMV-2 FL line were found to be resistant to attack by the Heterologous line of virus WMV-2 NY (tables 1-8, examples I-IV), and Transgenic Plants carrying the CMV-C coat protein gene were found to be somewhat resistant to attack by ZYMV (for further information on ZYMV, see the applicants' co-assigned pending patent application Ser. No. one entitled "Transgenic Plants Exhibiting Heterologous Viral protein protocol", filed day 12, 30, 1994, which is incorporated herein by reference).

Seeds from tissue culture regenerated plants are planted in soil and pollinated in white flowers to produce true-breeding plants. Progeny from these plants become inseparable lines that are used to assess resistance to viruses in the field under a range of environmental conditions. The commercial value of antiviral plants is extremely great if many different hybrid combinations with resistance are available for sale. Typically depending on differences in maturity, disease and pest resistance, color, or other agronomic traits, a grower typically plants more than one hybrid. In addition, because of differences in traits such as maturity, disease and pest tolerance, or public demand for a particular variety in a given geographic location, hybrids that are adapted to one region of a country are not adapted to another region of the country. For this purpose, resistance to viruses is bred into a large number of parental lines so that many hybrid combinations can be produced.

When the genetic control of antiviral resistance is understood, the addition of antiviral resistance to agronomically elite lines can be accomplished extremely efficiently. This requires crossing resistant and sensitive plants and studying the genetic type of segregating generations to determine whether the trait is expressed as a dominant or negative trait, the number of genes involved, and if more than one gene is required for expression, any interaction that may occur between the genes. With respect to transgenic plants of the types disclosed herein, the transferred gene exhibits dominant single-gene mendelian genetic behavior. This genetic analysis may be part of an initial effort to change an agronomically elite but sensitive line to a resistant line. The transformation process (backcrossing) is performed by crossing the original resistant line with the sensitive elite line and crossing the progeny with the sensitive parent. Progeny from this cross are segregating, resulting in some plants carrying the resistance gene and some without the band. Plants carrying the resistance gene are re-crossed with the sensitive parent and the progeny produced are re-segregating for resistance and sensitivity. This process is repeated until the original sensitive parent has been transformed into a resistant line that also possesses all of the other important traits originally found in the sensitive parent. Independent backcrossing procedures were performed for each susceptible elite line to be converted into a virus-resistant line.

After backcrossing, the appropriate combination of new resistant lines and lines that produce commercially valuable hybrids is evaluated for antiviral resistance and a set of agronomic traits. Non-segregating resistant lines and hybrids are produced, which are representative of the original sensitive lines and hybrids. This needs to be assessed under a range of environmental conditions under which the lines and hybrids are grown commercially. The number of satisfactory hybrid parent lines is increased and hybrid production is carried out using standard hybrid production methods.

The present invention will be further illustrated with reference to the following detailed examples.

Example I pumpkin variety A. binary plasmid vector with Multi-Virus resistance

The DNA transferred into the plant genome is contained in binary plasmids (m. bevan, Nucleic Acids Res. (Nucleic Acids research), 11, 369 (1983)). The parent binary plasmid was PGA482, constructed from g.an (g.an Plant physics, (Plant physiology), 81, 86 (1986)). The vector contains the T-DNA border sequence from pTiT37, the selectable marker gene Nos-NPTII containing a plant expressible nopaline gene promoter fused to the bacterial NPTII gene from Tn5, the multiple cloning region and the sticky end of phage lambda.

The procedure for deriving plasmid pPRBoriGN (FIG. 1) from plasmid PGA482 was as follows: a bacterial selection marker, gentamicin resistance (r. almansberger et al, mol. gen. gene. (molecular genetics of genes), 198, 514(1985)) was inserted near the right Border (BR), but outside the T-DNA region. The Nos-NPTII gene was then excised, and placed in B_RA Multiple Cloning Site (MCS) was regenerated in the T-DNA region. Next, a plant expressible β -Glucuronidase (GUS) gene cassette (R.A. Jefferson et al, EMBO J., 6, 3901(1987)) was inserted into the T-DNA region near the origin of replication of pBR 322. Finally, the plant expressible NPTII gene was inserted in the left border (B)_L) In the nearby T-DNA region. The NPTII gene was generated by inserting the NPTII coding region into the expression cassette of the E.coli plasmid pDH51 (R.Kay et al, nucleic acids. Res. (nucleic acids research), 15, 2778 (1987)). This provides a polyadenylation signal for the 35S promoter of cauliflower mosaic virus (CaMV).

Plasmid pPRBN was derived from pPRBoriGN (FIG. 2) as follows: from the beginning of the GUS coding sequence to B on pPRBoriGN_LIs missing. Thus, the GUS gene and the 35S/NPTII cassette were removed as a unit. This region was then replaced by a fragment consisting of only the 35S/NPTII cassette. The result of these steps was the removal of a short stretch of the GUS gene and pBR322 homologous sequences, leaving the plant expressible in B_LNearby NPTII genes. B. Donor gene

1. Watermelon mosaic virus 2

By usingSpecific oligonucleotide primers generate a fragment consisting of the coding region for the WMV2 coat protein from the WMV-2 FL line and flanking AatII (5 ') and BglII (3') restriction enzyme sites, thereby constructing a plant-expressible WMV2 gene. This fragment was ligated with AatII/BglII digested pUC19B₂Ligation, pUC19B₂Is a modified pUC19 plasmid containing a BglII restriction enzyme site in its multiple cloning region. To generate a plant-expressible coat protein expression cassette, the resulting plasmid (designated pUCXM 2P)₂₅) Further modifications were made, i.e.the CaMV35S promoter and polyadenylation signals from pUC1813/CP19 were increased (J.LSLightom, Gene, 100, 251 (1991)). The protein produced by the gene expression should be a fusion protein of WMV2 coat protein fused to the amino-terminal portion of CMV coat protein. The expression Cassette (CPW) was then excised by BamHI digestion and ligated to the BglII site of pPRBoriGN to generate the binary plasmid designated pPRCPW (FIG. 3).

2. Summer squash yellow leaf mosaic virus

The cloning and characterization of the ZYMV coat protein gene of the ZYMV FL line used herein is described in h.quemada et al, j.gen.virol (journal of viral genetics), 71, 1451 (1990). Strategies for constructing plants expressing ZYMV coat protein genes are described in J.L Slightom (1991) and S.Namba et al Phytopathology 82, 945(1992) cited above.

3. Cucumber mosaic virus

The cloning, characterization and genetic engineering of the CMV coat protein gene used in our experiments is described in h.quemada et al, j.gen.virol (journal of viral genetics), 70, 1065(1989) and the paper of 1991 cited above.

4. Mosaic virus of pumpkin

SQMV is a comovirus transmitted by seeds and transmitted by the striped or spotted cucumber beetles (Acalymma species and Diabrotica species). Within 5 minutes the insects become infected with the virus and the virus is retained until day 20. The range of the host is limited to cucurbitaceae plants. The virus consists of fully symmetric particles 30nm in diameter, containing single-stranded RNA, which is divided into two functional segments called M-RNA and B-RNA (Provvidenti, plant viruses of the Horticulture crop in Tropics and Subtropics, Taiwan (1986), pp.20-36).

Isolation, DNA sequences, modifications and expression of these genes in plant cells are described by Hu et al in Arch.Virol., 130, 17 (1993). Briefly, after isolation and sequencing, the genes were genetically engineered into the plant expression cassette pUC18cpexpress as described by Slightom, Gene, 100, 251 (1991). The methodology and use of the expression cassette resulted in a SQMV coat protein clone linked to the cucumber mosaic virus 5' untranslated leader sequence. The fusion was driven by the 35S promoter and a 35S terminator was used (FIG. 4). After HindIII digestion, the modified gene was isolated and introduced in a single step into the Upjohn binary plasmid pGA482GG (FIG. 11). This plasmid is a derivative of PGA482(An, Methods in Enzymol. (Methods in enzymology), 153, 292 (1987)). The two coat protein expression cassettes are oriented in the same direction as the NPTII gene. These genes exist as single copy genes.

5. Papaya ringspot virus

Plant-expressible PRV genes were isolated by polymerase chain reaction using specific oligonucleotide primers. For further information, see the applicants 'assignee's co-pending patent application entitled "Papaya Ringspot Virus Coat Protein Gene", filed 1994, 12, 30, which is incorporated herein by reference. This gene was inserted into the plant expression cassette pUC18cpexprers by genetic engineering after isolation and sequencing according to the method of Slightom, ibid (1991).

6. Constructs of multiple coat proteins

To obtain binary plasmids capable of transferring more than one plant expressible gene into the genome of a plant, the expression cassettes of the individual coat proteins are linked together in various combinations, respectively.

(a)ZYMV72/WMBN-22

ZYMV72/WMBN22 is derived from the binary plasmid pPRBN into which expression cassettes for ZYMV and WMV2 coat proteins have been inserted. The expression cassettes were inserted sequentially into the unique BglII restriction site of pPRBN. To accomplish this insertion, a BamHI site was introduced at the 5 'end of the 35S promoter and a BglII site was introduced at the 3' end of the poly (A) addition sequence of the WMV-2 and ZYMV expression cassettes. BamHI and BglII sites were introduced by using appropriate oligonucleotide primers during PCR amplification of the expression cassette. The PCR product was digested with BamHI and BglII to give appropriate ends. The WMV-2 expression cassette with BamHI/BglII termini was inserted into the unique BamHI/BglII terminal site described above to generate ZYMV72/WMBN22 (FIG. 5). The binary expression cassette was designated ZW.

(b)CMV73/ZYMV72/WMBN22

The CMV-c coat protein expression cassette was inserted into the unique HindIII site of ZYMV72/WMBN22 to give CMV73/ZYMV72/WMBN22 (FIG. 5). The ternary expression cassette is designated as CZW.

(c)CMV-WL41/ZYMV72/WMBN22(C-WLZW)

The expression cassette for the CMV-white leaf line, ZYMV and WMV2 coat protein genes were inserted into the binary plasmid pPRBN to obtain CMV-WL41/ZYMV72/WMV2 (C-WLZW). To place this combination of viral coat protein expression cassettes within pPRBN, the expression cassette for the coat protein gene of the CMV-white leaf line was inserted into ZYMV72/WMVN22 (see above for construction of ZYMV72/WMBN 22). To construct the CMV-WL cp expression cassette, Namba et al (Namba et al, Gene, 107, 181(1991) inserted the coat protein coding region of the CMV-WL with a cpexpress. the HindIII fragment containing the CMV-WL expression cassette was inserted into the HindIII site of ZYMV72/WMBN22 to give CMV-WL41/ZYMV72/WMBN22 (FIG. 7). the binary plasmid was designated CwLZW.

(d)WM310/ZYMV47/482G

The HindIII fragment with the ZYMV cp expression cassette (as described above) was inserted into the unique HindIII site of pGA482G to give ZYMV 47/482G. Next, the BamHI fragment with WMV2 expression cassette described above was inserted into the unique BglII site of ZYMV47/482G to obtain WMV 310/482G. This construct is designated as WZ.

(e)PRVcpwm16S/WLWL41/ZY72/WMBN22

The expression cassettes for the PRV-FL line, CMV-white leaf line, ZYMV and WMV-2 coat protein genes were inserted into binary plasmid pPRBN to obtain PRVcpwm16S/C_WL41/ZY72/WNBN22 (FIG. 9). For further information on the expression cassette of the PRV-FL line, see the co-pending patent application entitled "PapayaRingspot Virus Coat Protein Gene", filed 1994, 12, 30, assigned to the assignee of the present application and incorporated herein by reference. PRVcpwm16S/C_WL41/ZY72/WNBN22 this construct is designated PCZW.

(f)PNIa22/C_WL41/ZY72/WMBN22

Inserting the expression cassette of the PNIa gene of the PRV-P strain and the expression cassettes of the coat protein genes of the CMV-WL strains ZYMV and WMV-2 into a binary plasmid pPRBN to obtain PNIa22/C_WL41/ZY72/WMBN22 (FIG. 10). This construct is designated PNIa CZW. For further information on the PRV-P line PNIa Gene expression cassette, see the applicants co-pending patent application entitled "Papaya Ringspot Virus Protease Gene", 1994, 12, 30, which is assigned to the assignee hereof and is incorporated herein by reference.

(g)SQ21/SQ42/WMBN22/ZY72/PRVcpwm16s/C_WL41

Expression cassettes were prepared for the coat protein genes of WMV-2, ZYMV, PRV-FL and CMV-WL lines and for the two coat protein genes from SqMV, which were inserted into the binary plasmid pGA482GG (FIG. 12). This construct is designated SWZPC. C. Transformation of pumpkin

After peeling the seeds, the seeds were surface-sterilized in 20% sodium hypochlorite solution containing Tween 20 (200. mu.l/1000 ml) for 20-25 minutes. Then rinsed 3 times with 100ml of distilled water. Seeds were germinated in 150X 25mm culture tubes containing 20ml of 1/4 strength Murashige and Skoog minimal organics (MS) medium solidified with 0.8% Difco Bacto agar. After 5-7 days, cotyledons were removed from the seedlings, shoot tips were cut off and transferred into a GA7 incubator (Magenta Corp.) containing 75ml of MS medium (solidified with 1.5% Difco Bacto agar). All cultures were incubated in the culture chamber at 25 ℃ for 16 hours, unless otherwise indicated. Light was provided by cold fluorescent lamps (Phillips F40CW) and plant growth lamps (General Electric F40-PF).

Leaf fragments of the plants were harvested in vitro and soaked in a broth culture of Agrobacterium tumefaciens (OD 600, 0.1-0.2) and transferred to a 100X 20mm petri dish containing 40ml of MS medium (MS-I) supplemented with 1.2 mg/l 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-T) and 0.4 mg/l Benzylamino Acid (BAP) and 200. mu.M AS. The plates were incubated at 23 ℃. After 2-3 days, the leaf fragments were transferred to MS-I medium (MS-IA) containing carbenicillin (500 mg/liter), cefotaxime (200 mg/liter) and kanamycin sulfate (150 mg/liter). After 10 days, the leaves were transferred to fresh MS-IA cultures. Thereafter, the tissues were transferred to fresh MIS-IA medium every 3 weeks. After about 16-24 weeks, kanamycin-resistant embryonic callus was obtained and transferred to a rotary culture tube supplemented with carbenicillin (500 mg/liter), kanamycin sulfate (150 mg/liter) and CaCl₂·2H₂O (1.03 mg/l) in liquid MS minimal organic medium. Developing embryos were harvested and transferred to a medium containing 20mg AgNO₃In the MS basic organic medium. The germinated embryos were subcultured in fresh medium until seedlings with roots were formed. Transferring the embryo seedling into soil for R₁And (4) producing seeds. D. Plant analysis

The expression of NPTII gene by kanamycin-resistant transformants was analyzed by ELISA using a commercially available ELISA kit (5-Prime 3-Prime, Boulder, CO). Using appropriate primers, the polymerase chain reaction was performed to amplify the NPTII gene (near the right border) and the coat protein gene closest to the left border. Some lines were further characterized by Southern blot analysis. Expression of the viral coat protein gene in presumably transformed plants was probed by ELISA using an antibody conjugated to alkaline phosphatase following the procedure of m.f. clark et al, j.gen.virol. (journal of viral genetics), 34, 475 (1977). Antiserum against CMV-C, WMV-2-NY and ZYMV-FL was supplied by D.Gonsalves (Comelluniversity, Geneva, New York).

T-DNA in R₁And the presence or absence thereof in the progeny thereof is determined by an ELISA assay measuring the selectable NPTII marker gene. The inheritance in line ZW20 (the lack of the NPTII gene in advanced generations of ZW 20) was followed by PCR or Southern analysis. D. Inoculation step

The isolated R1 or R2 progeny were germinated in the greenhouse together with appropriate control lines. Before inoculation with virus, cotyledons were harvested for NPTII ELISA assays. The CMV C line, ZYMV FL line and WMV-2 NY line, which were propagated in Cucumis Sativus, Cucurbita Pepo and Phaseolus Vulgaris, respectively, at 1X 10^-1Dilutions (weight/volume) and cotyledons sprinkled with carborundum were mechanically inoculated on 6-day-old seedlings. Plants were inoculated in the greenhouse. Approximately 7-10 days after inoculation, plants were transplanted into the field. To monitor some transmission of the virus by aphids, several trials included non-inoculated control plants. Data on symptom development was collected prior to examining the results of NPTII ELISA, so scoring was performed without knowledge of the transgene status of each isolate to be evaluated.

Plants were scored for a disease severity score of 0-9 based on symptoms on leaf (0 ═ no symptoms, 3 ═ symptoms on inoculated leaves and/or very mild symptoms on newly emerged leaves, 5 ═ moderate systemic spread, 7 ═ severe systemic spread, 9 ═ severe systemic spread and developmental disorders). The fruits were also scored according to the severity of symptoms (0 ═ no symptoms, 3 ═ fruits with slight green spots, 5 ═ moderate discoloration, 7 ═ severe discoloration, 9 ═ fruits discolored and deformed). The disease scores for the fruits and leaves of each plant were then assessed, giving the disease grade for each line on average. E. Design of field test area

The field trials were conducted under the approval of the animal and plant health quarantine Agency (APHIS) of the United States Department of Agriculture (USDA). The design used was that each row consisting of transgenic lines was paired with a row containing the non-transgenic counterpart as its control. Each row consisted of 15 plants (two plants spaced 2 feet apart) with 5 feet spacing between rows. In each trial, 2-3 replicates were set for each transgenic line. The test area was surrounded by a minimum of 30 feet of non-transgenic pumpkin plant isolation belts to reduce transgenic pollen flying out of the test site and to monitor the spread of the virus in the field. Transgenic material used in this experiment included R from a self-pollinated or backcrossed R0 yellow tortilla (crooknegk) inbred line₁And R₂The progeny. In some cases, transgenic inbred lines are crossed with non-transgenic inbred lines in order to produce transgenic versions of the commercial pumpkin hybrids Pavor or Dixie. F. Results

AW constructs

Line ZW20 was derived from R transformed with ZYMV72/WMBN22₀And (5) plant growing. For R during field trials 1 and 2₁Plant observations revealed that plants containing the NPTII insert maintained resistance to ZYMV and WMV-2 throughout the experiment.

Line ZW19 was also derived from R transformed with ZYMV72/WMVN22₀And (5) plant growing. ZW-19 showed reduced symptom development when inoculated with ZYMV or WMV-2, compared to ZW-20 (Table 2).

TABLE 1 in our experiments conducted in 1991 and 1992, transgenic yellow pumpkin tortilla (YC) line showed symptoms (# symptomatic plants (%)) 40-47 days after inoculation of ZYMV-FL line or WMV-2 line at a dilution of 1/10 (weight/volume)
								Line of	NPTII	1991WMV-2 test		1992WMV-2 test		1992ZYMV test
ZW-19	+-	----	----	14/1416/16	(100)(100)	11/11*19/19	(100)(100)
								ZW-20	+-	0/145/18	(0)(28)	0/510/25	(0)(40)	----	----
Light and slight

CW constructs

The transgenic pumpkin plants with expression vectors containing CMV-C and WMV-2 CP genes are inoculated by CMV V27, V33 or V34 lines, and the CMVV27, V33 or V34 lines can infect the transgenic plants expressing CMV-C coat protein. For further information on CMV V27, V33, or V34 strains, see co-pending patent application entitled "plantations to V27, V33, or V34 strains of Cucumber Mobile Virus", filed 30, 1994, which is hereby incorporated by reference. Most transgenic plants are resistant to attack by these heterologous CMV lines, unlike transgenic lines with CMV-C CP alone. The symptoms of the remaining infected plants are greatly reduced relative to transgenic plants carrying CMV-C CP alone.

CZW constructs

The CZW-3 line was transformed from R with CMV73/ZYMV72/WMBN22₀The plant develops. This line had been asymptomatic for infection by CMV-C, ZYMV-FL or WMV-2 in field trials, either individually or as a mixture containing all three viral agents (Table 2).

CZW-40 strain Slave C_WL41/ZWMV-72/WMBN22 converted R₀The plant develops. When infected with CMV-C, ZYMV-FL or WMV-2 NY, it did not have any protection against infection (Table 2).

TABLE 2 symptoms exhibited by pumpkin plants after inoculation with CMV-C, ZYMV-FL or WMV-2-NY at a dilution of 1/10 (weight/volume)
					Line of	NPTII	Attack of	The proportion of the existing symptoms%	Disease grade leaf fruit
CZW-3	+-	CMV-C	1/13 0.76/6 100	0.4 0.08.5 --
					CZW-40*	+-	CMV-C	4/4 10011/11 100	5.0 --9.0 --
CZW-3	+-	ZYMV-FL	0/14 04/4 100	0.0 0.08.0 7.0
					CZW-40	+-	ZYMV-FL	9/9 1005/5 100	9.0 --7.0 --
CZW-3	+-	WMV-2-NY	0/40 015/15 100	0.0 0.07.0 7.0
					CZW-40	+-	WMV-2-NY	11/11 1003/3 100	7.0 --7.0 --
*Greenhouse screening

The disease grade of leaves and fruits was 0. In contrast, 100% of cp-isolates showed severe symptoms of viral infection on their leaves and fruits. The disease grade of CP-segregants is in the range of 7.0-9.0. This experiment shows that by combining the coat protein genomes, one can obtain simultaneous resistance to infection by different viruses in inbred and hybrid lines.

TABLE 3 pumpkin plants show symptoms 55 days after simultaneous inoculation of a mixture of CMV-C, ZYMV-FL and WMV-2-NY at a dilution of 1/10(w/v)
						Line of	CP	The proportion of the existing symptoms%		Disease grade leaf fruit
Dixie CZW-3	+-	0/2730/30	0100	0.08.4	0.07.0
						Pavo CZW-3	+-	0/2733/33	0100	0.07.0	0.0--
YS20CZW-3	+-	0/4015/15	0100	0.07.0	0.07.0
						Dixie	+-	--14/14	--100	--8.7	--7.0
Pavo	+-	--24/24	--100	--8.9	--7.0

Positive R by CP in season 3₁Selfing of CZW-3 segregants to yield R for CZW-3₂And (4) generation. This transgenic inbred line was used as the parent and two transgenic hybrid lines were also generated, corresponding to Asgrow's commercial hybrids Pavo and Dixie. Progeny from the inbred line showed the expected segregation ratio of 3: 1 (based on NPTII ELISA) for the inserted gene, while both hybrid lines showed the expected ratio of 1: 1. The transgenic selfed and hybrid progeny were inoculated with an inoculation mixture containing all three viruses CMV-C, WMV-2-NY and ZYMV-FL at a dilution of 1/10 (w/v). Table 3 shows that the isolates were completely resistant to infection by all 3 viruses.

The results herein demonstrate that when the coat protein genes are inserted in combination, they confer resistance to multiple viruses on plants. For example, the transgenic line CZW-3 was asymptomatic in all 3 virus trials (CMV, ZYMV and WMV-2) in which both single and simultaneous inoculation of each virus and all three were asymptomatic. Because infection by more than one virus is often found during the growing season under commercial field conditions, the ability to obtain strains resistant to multiple virus infections is critical to the development of commercially valuable pumpkin cultivars.

In these experiments, it was also observed that when multiple coat protein genes were inserted into the same construct, all genes exhibited approximately equal levels of potency in the construct. The CZW-3 line, which has a high level of resistance to CMV-C, also has a high level of resistance to ZYMV-FL and WMV-2-NY. In contrast, transgenic lines such as ZW-19 showed only moderate resistance (with less marked symptoms of resistance) to WMV-2, and the line also showed only moderate resistance to ZYMV. Furthermore, greenhouse screening of transgenic line CZW-40 demonstrated that this line, which was not resistant to CMV-C, was also not resistant to ZYMV-FL and WMV-2-NY. This level of interpordination of effects between genes on a multigene construct may be a reflection of the effect of the location of gene insertion into the plant genome. In any event, this phenomenon provides a means to greatly increase the probability of finding transgenic lines with high levels of resistance to multiple viruses.

Example II introduction of a Multi-CP Gene expression cassette into the cucumis melo

1. Transformation of cucumis melo

The polygenic constructs listed above were used to transform cucurbita pepo inbred lines using the method of Fang and Grumet (Fang and Grumet, molecular biology of plant microbial interaction, 6, 358 (1993)). Transplanting rooted transformed plants into a greenhouse and allowing them to produce R₁。

Plant analysis/inoculation procedure

Transgenic plants were analyzed and inoculated as described in example I above.

2. Results

The ZW construct. Line CA76-ZW-102-29 is resistant to infection by either ZYMV-FL or WMV-2-NY. In contrast, all other lines were not resistant to infection by ZYMV or WMV-2 (Table 4).

TABLE 4 symptoms exhibited by Cucumis sativus plants after inoculation with ZYMV-FL or WMV-2-NY at a dilution of 1/10 (weight/volume)
					Line of	NPTII	Attack of	The proportion of the existing symptoms%	Grade of disease
CA-ZW-102-29	+-	ZYMV-FL	0/30 0	0.0
						+-	WMV-2-NY	0/9 02/2 100	0.05.0
CA-ZW-115-38	+-	ZYMV-FL	0/30 0	0.0
						+-	WMV-2-NY	9/17 06/8 75	0.04.0

PCZW constructs. Line CA95 PXZW-1 was resistant to infection by CMV-C, ZYMV-FL and WMV-2. This line has conventional resistance to PRV, so that the efficacy of the PRV insert can be determined. In contrast, most PCZW transgenic lines do not have resistance to CMV-C or ZYMV-FL. By WMV-2Inoculation of these lines by NY is still ongoing (Table 5).

TABLE 5 symptoms exhibited by Cucumis sativus plants after inoculation with ZYMV-FL, WMV-2-NY or CMV-C at a dilution of 1/10 (weight/volume)
						Line of	NPTII	Attack of	The proportion of the existing symptoms%		Grade of disease
CA95-PCZW-93351-1	+-	CMV-C	1/123/3	(08)(100)	0.66.3
							+-	WMV-2-NY	1/92/2	(11)(100)	0.55.0
	+-	ZYMV-FL	6/86/6	(75)(100)	4.29.0
						CZ95-PCZW-93356-1	+-	CMV-C	9/94/4	(100)(100)	7.07.0
	+-	ZYMV	10/105/5	(100)(100)	7.07.0
						CA95-PCZW-93356-6	+-	CMV-C	9/91/1	(100)(100)	7.09.0
	+-	ZYMV	10/101/1	(100)(100)	7.07.0

SWZPC constructs. Although resistance has not been assessed for these lines, PCR analysis has confirmed that 27/36 (75%) of the c.cucumis strain produced by this construct contains all 6 CP genes, plus an NPTII selectable marker gene. This demonstrates that Agrobacterium-mediated transformation can be used to transfer at least 7 (and possibly more) linked genes from a binary plasmid into plant cells, and subsequently obtain complete plants containing all 7 linked gene inserts.

Example III introduction of multiple CP Gene expression cassettes into cucumber

1. Transformation of cucumber

Cucumber inbred lines were transformed with the various coat protein gene constructs listed above using the method of modified Sarmento et al (Sarmento et al, Plant Cell Tissue and organ Culture, 31, 185 (1992)). Transfer rooted plants to greenhouse and produce R₁And (4) seeds. Transgenic plants were analyzed and inoculated as described in example I above.

2. Results

The CZW construct. Lines GA715 CZW7, 95, 33, 99 were resistant to both ZYMV-FL and WMV-2-NY (these lines were traditionally bred against CMV-C and therefore the potency of the CMV coat protein insert could not be determined) (Table 6)

TABLE 6 cucumber plants showing symptoms after inoculation with ZYMV-FL, ZYMV-CA or WMV-2-NY at a dilution of 1/10 (weight/volume)
						Line of	NPTII	Attack of	The proportion of the existing symptoms%		Grade of disease
GA715 CZW-7	+-	ZYMV-FL	0/87/7	0100	0.06.4
							+-	WMV-2-NY	0/58/9	089	0.04.4
CA715-CZW-33	+-	ZYMV-FL	0/69/9	0100	0.06.9
							+-	WMV-2-NY	0/68/8	0100	0.04.3
GA715-CZW-95	+-	ZYMV-FL	0/112/2	0100	0.05.0
							+-	WMV-2-NY	0/102/2	0100	0.05.0
GA715-CZW-99	+-	ZYMV-F	0/86/6	0100	0.06.7
							+-	WMV-2-NY	0/75/5	0100	0.03.0

The PNIa CZW construct. Line GA715 PNIa CZW-21 was resistant to CMV-C, ZYMV-FL and PRV-P-HA, whereas line GA715 PNIa CZW-15 was sensitive to ZYMV-FL and WMV-2-NY (Table 7)

TABLE 7 symptoms exhibited by cucumber plants after inoculation with ZYMV-FL, WMV-2-NY or PRV-P-HA at a dilution of 1/10 (weight/volume)
					Line of	NPTII	Attack of	The proportion of the existing symptoms%	Grade of disease
GA715 PNIaCZW-21	+-	ZYMV-FL	0/7 02/2 100	0.07.0
						+-	CMV-Carna-5	0/4 04/11 44	0.02.2
	+-	WMV-2-NY	NT NTNT NT	NTNT
						+-	PRV-P-HA	0/3 06/6 100	0.03.0
GA715 PNIa CZW-15	+-	ZYMV-FL	2/2 10012/12 100	3.05.0
						+-	WMV-2-NY	4/4 10010/10 100	3.03.0
	+-	PRV-P-HA	NT NTNT NT	NTMT
						+-	CMV-CCarna-5	NT NTNT NT	NTNT

Example iV introduction of a Multi-CP Gene expression cassette into watermelon

1. Transformation of watermelon

The watermelon inbred lines were transformed with the multiple coat protein gene expression cassettes WZ listed above using the method described by modified Choi et al (Plant Cell Reons, 344 (1994)).

2. Plant analysis/inoculation procedure

Transgenic plants were analyzed and inoculated as described in example I above.

3. Results

The WZ construct. Line WA₃WZ-20-14 was resistant to ZYMV-FL and WMV-2-NY (Table 8).

TABLE 8 symptoms exhibited by transgenic watermelon lines after inoculation of ZYMV-FL or WMV-2-NY at a dilution of 1/10 (weight/volume)
					Line of	NPTII	Attack of	The proportion of the existing symptoms%	Disease grade
WA3WZ-20-14	+-	ZYMV-FL	0/14 011/11 100	0.09.0
						+-	WMV-2-NY	0/13 01/10 100	0.09.0

All publications, patents, and patent documents are incorporated by reference herein, as if each individual publication, patent, or patent document were individually incorporated. The invention has been described with reference to specific and preferred embodiments and techniques. It will be understood, however, that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims (20)

1. A chimeric recombinant DNA molecule comprising:

a plurality of DNA sequences, each sequence comprising a promoter linked to a DNA sequence encoding a viral protein, wherein said plurality of DNA sequences are expressed in virus-susceptible plant cells transformed with said recombinant DNA molecule to confer resistance to infection by said respective virus to said cells.

2. The chimeric DNA molecule according to claim 1, characterized in that said DNA sequences are concatenated in tandem.

3. The chimeric DNA molecule according to claim 1, characterized in that the expression of said DNA sequence confers a substantially equivalent resistance to infection.

4. The chimeric DNA molecule according to claim 1, characterized in that the expression of at least one of said plurality of DNA sequences confers resistance against a plurality of viruses.

5. The DNA molecule of claim 1, characterized in that said DNA sequence further comprises a selectable marker gene, a reporter gene or a combination thereof, said marker gene, reporter gene or combination thereof being capable of being identified in a plant cell transformed with said DNA molecule.

6. The DNA molecule of claim 5, characterized in that said plurality of DNA sequences are flanked by two selectable marker genes, two reporter genes, or a combination thereof.

7. The DNA molecule of claim 1, characterized in that said viral proteins comprise at least two of the coat proteins of watermelon mosaic virus II, cucumber mosaic virus or zucchini yellow mosaic virus.

8. The DNA molecule of claim 1, characterized in that said plant cell is from the Cucurbitaceae family.

9. A method of conferring resistance to a plurality of viruses to a virus-sensitive plant, the method comprising:

(a) transforming cells of said susceptible plant with a chimeric recombinant DNA molecule comprising a plurality of DNA sequences, each DNA sequence comprising a promoter functional in cells of said plant and linked to a DNA sequence encoding a viral protein, said virus being capable of infecting said plant;

(b) regenerating said plant cells to obtain differentiated plants; and

(c) identifying a transformed plant which expresses said DNA coding sequence to confer to the plant resistance to infection by said virus.

10. The method of claim 9 wherein expression of the DNA coding sequences confers substantially equal amounts of resistance to infection by each virus.

11. The method of claim 9 wherein expression of at least one of said DNA coding sequences confers resistance to a plurality of said viruses.

12. The method of claim 9, characterized in that said plant is a dicotyledonous plant.

13. The method of claim 9, wherein said DNA molecule is part of a binary Ti plasmid and said plant cell is transformed by agrobacterium tumefaciens mediated transformation.

14. The method of claim 9, wherein said DNA sequence further comprises a selectable marker gene or reporter gene that identifies said transformed plant.

15. Method according to claim 9, characterized in that the DNA sequence further comprises at least two of the coat protein genes of watermelon mosaic virus II, cucumber mosaic virus or zucchini yellow mosaic virus.

16. Method according to claim 9, characterized in that the sensitive plant is a plant of the cucurbitaceae family.

17. A transformed plant prepared by the method of claim 9.

18. A transformed plant cell prepared by the method of claim 9.

19. A transformed seed of the transformed plant of claim 17.

20. A hybrid plant prepared from the transformed plant of claim 17, which plant is resistant to infection by said virus.