Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
  • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
  • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
  • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
  • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
  • C12N15/8283—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for virus resistance
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/34011—Potyviridae
  • C12N2770/34022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

• the present invention relates to the isolation and purification of nucleic acid sequences encoding for papaya ringspot virus coat proteins, a method of conferring resistance to papaya ringspot virus by transforming plants with a construct containing one or more isolated viral coat protein nucleic acid sequences, and transgenic plants and seeds transformed with such multiple virus nucleic acid constructs.
• Papaya (Carica papaya L.) is an important fruit crop grown widely in tropical and subtropical lowland regions (Manshardt, "Papaya in Biotechnology of Perennial Fruit Crops," ed. Hammerschlag. 21:489-511, CAB Int., Wallingford, UK (1992)). Worldwide, Brazil, India, and Mexico are the largest producers of papaya. Hawaii, the largest producer of papaya in the United States, exports 66% of the total fresh production, primarily to the U.S. mainland and to Japan (Martin, “Papaya Production Statistics,” Proc. Annu. Hawaii Papaya Ind. Assoc. Conf, 39th. Kihei, pp. 31-36, Sept.
• Papaya ringspot virus is a member of the potyvirus group of plant viruses, which are pathogenic to several crop plants, and which exhibit cross-infectivity between members of different plant families.
• a potyvirus is a single-stranded (+) R ⁇ A plant virus.
• the viral genome is approximately 10,000 bases in length.
• the expression strategy of potyviruses includes translation of a complete polyprotein from the positive sense viral genomic R A. PRSV is by far the most widespread and damaging virus that infects papaya, occurring worldwide wherever papaya is grown (P ⁇ rcifull, "Papaya Ringspot Virus,” CMI/AAB Descr. Plant Viruses. No. 292 (No. 84 Revis., July 1984) 8 pp.
• PRSV infections have resulted in the devastation of the papaya industry in Brazil, Taiwan, and Hawaii in recent years (Gonsalves, D., "Control of Papaya Ringspot Virus in Papaya: A Case Study," Annu. Rev. Phytopathol. 36:415-37 (1998)).
• Various attempts have been made to control or prevent infection of crops by PRSV, but these have been largely unsuccessful.
• Parasite-derived resistance is a phenomenon whereby transgenic plants containing genes or sequences of a parasite are protected against detrimental effects of the same or related pathogens.
• viral genes can be effective in the translatable and non-translatable sense forms, and, less frequently, antisense forms (Baulcombe, D.C., "Mechanisms of Pathogen-Derived Resistance to Viruses in Transgenic Plants," Plant Cell 8:1833-44 (1996); Dougherty et al., “Transgenes and Gene Suppression: Telling us Something New?” Current Opinion in Cell Biology 7:399-05 (1995); Lomonossoff, G.P., "Pathogen-Derived Resistance to Plant Viruses," Ann. Rev. Phytopathol. 33:323-43 (1995)).
• the present invention relates to isolated nucleic acid molecules encoding a viral coat protein of papaya ringspot virus and the protein encoded by those nucleic acid molecules.
• Another aspect of the present invention pertains to nucleic acid constructs containing the isolated nucleic acid molecules of the present invention operably linked to 5' and 3' regulatory regions.
• the present invention also relates to nucleic acid constructs containing a plurality of trait DNA molecules, wherein at least some of the plurality of trait DNA molecules have a length that is insufficient to independently impart that trait to plants transformed with that trait DNA molecule.
• the plurality of trait DNA molecules are capable of collectively imparting their traits to plants transformed with the DNA construct and thereby effecting the silencing of the DNA construct.
• the trait associated with the DNA molecules of this construct is disease resistance, and the trait DNA molecules are derived from a gene encoding a papaya ringspot virus coat protein in a papaya ringspot virus strain selected from the group consisting of Thailand (“TH”), Keaau (“KE”), Kapoho (“KA”), Mexico (“ME”), Taiwan (“YK”), Brazil (“BR”), Jamaica (“JA”), Oahu (“OA”), and Panaewa (“PA”).
• the present invention also relates to a DNA construct containing a fusion gene which includes a trait DNA molecule which has a length insufficient to independently impart a desired trait to plants transformed with the trait molecule, operatively coupled to a silencer molecule effective to achieve post- transcriptional gene silencing.
• the trait DNA molecule and the silencer molecule collectively impart the trait to plants transformed with the construct.
• the DNA molecules of this DNA construct are derived from a gene encoding a papaya ringspot viral coat protein from a papaya ringspot virus strain selected from the group consisting of TH, KE, KA, ME, YK, BR, JA, OA, and VE.
• the present invention also relates to host cells, plant cells, transgenic plants, and transgenic plant seeds containing the nucleic acid constructs of the present invention.
• the present invention also relates to a method of imparting resistance against papaya ringspot virus to papaya plants. This involves transforming a papaya plant with the constructs of the present invention.
• Figures 1 A-B show the cloning vectors used for the DNA constructs of the present invention.
• Figure 1 A shows the expression cassette, pEPJ-YKT, containing the PRSV-CP variable regions of the YK, KE, and TH strains ligated into the pEPJ vector.
• Figure IB shows the transformation vector pGA482G.
• Figures 2A-B show the expression vectors used for cloning and subcloning the silencer-PRSV-CP construct.
• Figure 2A shows the pNP-YKT vector, containing the silencer DNA molecule (M1/2NP) and the PRSV-CP variable regions of PRSV strains YK, KE, and TH.
• Figure 2B shows the pGFP- YKT vector, containing the silencer molecule GFP ligated to the PRSV-CP variable regions of PRSV strains YK, KE, and TH PRSV strains.
• Figures 3 A-G show various PRSV-CP DNA molecules ligated to the silencer molecule (M 1/2 NP) in an expression vector.
• Figure 3 A shows clone pNP-K
• Figure 3B shows clone pNP-KK
• Figure 3C shows clone pNP-EE
• Figure 3D shows clone pNP-KKTC
• Figure 3E shows clone pNP-KKTV
• Figure 3F shows clone pNP-EETC
• Figure 3G shows clone pNP-EETV.
• Figure 4A shows the a full-length (1 Kb) KE-CP DNA molecule encoding a translatable RNA for PRSV-CP ligated into the expression vector pEPJ.
• Figure 4B shows a full-length (1 Kb) KE-CP DNA molecule encoding a non-translatable RNA for PRSV-CP ligated into the expression vector pEPJ.
• Figure 5 shows a 855 bp NcoVBamHI Mexico PRSV-CP DNA molecule ligated into the expression vector pEP J.
• the present invention relates to nucleic acids which encode for a viral coat protein ("CP") of papaya ringspot virus (“PRSV”).
• CP viral coat protein
• PRSV papaya ringspot virus
• nucleic acid of the present invention is the CP gene isolated from the PRSV strain Kapoho ("KA"), which has a nucleic acid sequence corresponding to SEQ ID NO: 1 as follows:
• the present invention also relates to an isolated nucleic acid molecule encoding a CP gene isolated from the Thailand ("TH") strain of PRSV, which has a nucleic acid sequence corresponding to SEQ ID NO: 3 as follows:
• nucleic acid which encodes a CP gene isolated from the Keaau (“KE") strain of PRSV.
• PRSV-KE contains two "cut-sites", i.e., two potential cleavage sites for a mature coat protein.
• SEQ ID NOS: 5 and 6 contain, respectively, the N terminus and C terminus cleavage sites for PRSV-KE coat protein. Both cleavage sites result in proteins that appear to be functional in viral replication in the plant.
• SEQ ID NO: 5 encodes the first coat protein cleavage site product, CP 1 , of the KE strain of
• KE-CP 1 has an amino acid sequence corresponding to SEQ ID NO: 7, as follows:
• SEQ ID NO: 6 encodes the second coat protein cleavage site product, CP2, ofthe KE strain ofPRSV.
• KE-CP2 has an amino acid sequence corresponding to SEQ ID NO: 8, as follows:
• nucleic acid suitable in the present invention is the CP gene isolated from the Taiwan (“YK”) strain of PRSV, corresponding to SEQ ID NO: 9, as follows:
• SEQ ID NO: 9 encodes the CP of the YK strain of PRSV which has an amino acid sequence corresponding to SEQ ID NO: 10, as follows:
• nucleic acid suitable in the present invention is the CP gene isolated from the Mexico (“ME”) strain of PRSV, corresponding to SEQ ID NO: 11, as follows:
• SEQ ID NO: 11 encodes the CP of the ME strain of PRSV which has an amino acid sequence corresponding to SEQ ID NO: 12, as follows:
• nucleic acid suitable in the present invention is the CP gene isolated from the Brazil (“BR”) strain of PRSV, corresponding to SEQ ID NO: 13, as follows:
• SEQ ID NO: 13 encodes the CP of the BR strain of PRSV which has an amino acid sequence corresponding to SEQ ID NO: 14, as follows:
• nucleic acid suitable in the present invention is a CP gene isolated from the Jamaica (“JA") strain of PRSV, corresponding to SEQ ID NO: 15, as follows:
• nucleic acid suitable in the present invention is a CP gene isolated from the Oahu ("OA") strain of PRSV, corresponding to SEQ ID NO: 17, as follows:
• SEQ ID NO: 17 encodes the CP of the OA strain of PRSV which has an amino acid sequence corresponding to SEQ ID NO: 18, as follows: Ser Lys Asn Glu Ala Val Asp Ala Gly Leu Asn Glu Lys Phe Lys Glu 1 5 10 15
• nucleic acid suitable in the present invention is the CP gene isolated from the Venezuela (“VE") strain of PRSV, corresponding to SEQ ID NO: 19, as follows:
• SEQ ID NO: 19 encodes the CP of the VE strain of PRSV which has an amino acid sequence corresponding to SEQ ID NO: 20, as follows:
• nucleic acid molecules shown above are variants of the nucleic acid molecules shown above.
• An example of a suitable nucleic acid is a nucleic acid molecule which has a nucleotide sequence that is at least 85% similar to the nucleotide sequence of the SEQ ID NOS: 1, 3, 5, 6, 9, 11, 13, 15, 17, and 19 by basic BLAST using default parameters analysis, or which hybridizes to the nucleotide sequence of SEQ ID NOS: 1, 3, 5, 6, 9, 11, 13, 15, 17, and 19 under stringent conditions characterized by a hybridization buffer comprising 5X SSC buffer at a temperature of about 42°-65°C, preferbly 45°C.
• Fragments of genes encoding PRSV-CP are particularly useful in the present invention. Fragments capable of use in the present invention can be produced by several means. In one method, subclones of the gene encoding the CP of choice are produced by conventional molecular genetic manipulation by subcloning gene fragments. In another approach, based on knowledge of the primary structure of the protein, fragments of a PRSV-CP encoding gene may be synthesized by using the PCR technique together with specific sets of primers chosen to represent particular portions of the protein. These, then, would be cloned into an appropriate vector in either the sense or antisense orientation.
• fragments of the nucleic acids of the present invention are fragments of the genes which have been identified as conserved ("con") regions of the CP proteins, or alternatively, those portions of PRSV-CP nucleotide sequences that have been identified as variable (“var”) regions.
• Sequences identified using DNAStar Mega alignment program as either variable or conserved in a PRSV-CP gene can be amplified using standard PCR methods using forward and reverse primers designed to amplify the region of choice and which include a restriction enzyme sequence to allow ligation of the PCR product into a vector of choice. Combinations of amplified conserved and variable region sequences can be ligated into a single vector to create a "cassette" which contains a plurality of DNA molecules in one vector.
• the present invention also relates to a DNA construct that contains a DNA molecule encoding for a PRSV-CP isolated from any of a variety of PRSV strains, most preferably the TH, KA, KE, YK, ME, BR, JA, OA, and VE strains.
• this involves inserting the nucleic acid molecule into an expression system to which the nucleic acid molecule is heterologous (i.e., not normally present).
• the heterologous nucleic acid molecule is inserted into the expression system which includes the necessary elements for the transcription and translation of the inserted protein coding sequences.
• the nucleic acid molecules of the present invention may be inserted into any of the many available expression vectors and cell systems using reagents that are well known in the art.
• Suitable vectors include, but are not limited to, the following viral vectors such as lambda vector system gtl 1, gt WES.tB, Charon 4, and plasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9, pUC18, pUC19, pLG339, pR290, pKC37, pKClOl, SV 40, pBluescript II SK +/- or KS +/- (see "Stratagene Cloning Systems"
• Recombinant molecules can be introduced into cells via transformation, particularly transduction, conjugation, mobilization, or electroporation.
• DNA sequences are cloned into the vector using standard cloning procedures in the art, as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Press, NY (1989), and Ausubel, F. M. et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety.
• the various DNA sequences may normally be inserted or substituted into a bacterial plasmid.
• Any convenient plasmid may be employed, which will be characterized by having a bacterial replication system, a marker which allows for selection in a bacterium, and generally one or more unique, conveniently located restriction sites.
• Numerous plasmids referred to as transformation vectors, are available for plant transformation. The selection of a vector will depend on the preferred transformation technique and target species for transformation.
• a variety of vectors are available for stable transformation using Agrobacterium tumefaciens, a soilborne bacterium that causes crown gall. Crown gall are characterized by tumors or galls that develop on the lower stem and main roots of the infected plant. These tumors are due to the transfer and incorporation of part of the bacterium plasmid DNA into the plant chromosomal DNA.
• T-DNA This transfer DNA
• the plasmid DNA, pTi, or Ti-DNA, for "tumor inducing plasmid” contains the vir genes necessary for movement of the T-DNA into the plant.
• the T-DNA carries genes that encode proteins involved in the biosynthesis of plant regulatory factors, and bacterial nutrients (opines).
• the T-DNA is delimited by two 25 bp imperfect direct repeat sequences called the "border sequences.”
• a constitutive promoter is a promoter that directs expression of a gene throughout the development and life of an organism.
• constitutive promoters that are widely used for inducing expression of transgenes include the nopoline synthase ("NOS”) gene promoter, from Agrobacterium tumefaciens, (U.S. Patent 5034322 to Rogers et al., which is hereby incorporated by reference in its entirety), the cauliflower mosaic virus (“CaMV”) 35S and 19S promoters (U.S. Patent No. 5,352,605 to Fraley et al., which is hereby incorporated by reference in its entirety), the enhanced CaMV35S promoter ("enh CaMN35S”), the figwort mosaic virus full-length transcript promoter
• NOS nopoline synthase
• FMN35S those derived from any of the several actin genes, which are known to be expressed in most cells types (U.S. Patent No. 6,002,068 to Privalle et al., which is hereby incorporated by reference in its entirety), and the ubiquitin promoter ("ubi”), which is a gene product known to accumulate in many cell types.
• ubi ubiquitin promoter
• An inducible promoter is a promoter that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, the DNA sequences or genes will not be transcribed.
• the inducer can be a chemical agent, such as a metabolite, growth regulator, herbicide or phenolic compound, or a physiological stress directly imposed upon the plant such as cold, heat, salt, toxins, the action of a pathogen or disease agent such as a virus or fungus.
• a plant cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating, or by exposure to the operative pathogen.
• an appropriate inducible promoter for use in the present invention is a glucocorticoid-inducible promoter ("GIP") (Schena et al., " A Steroid-Inducible Gene Expression System for Plant Cells," Proc. Natl. Acad. Sci. 88:10421-5 (1991), which is hereby incorporated by reference in its entirety).
• GIP glucocorticoid-inducible promoter
• Other useful promoters include promoters capable of expressing potyvirus proteins in an inducible manner or in a tissue-specific manner in certain cell types where infection is known to occur.
• tissue specific promoters include seed, flower, or root specific promoters as are well known in the field (U.S. Patent No. 5,750,385 to Shewmaker et al., which is hereby incorporated by reference in its entirety).
• Roberts and Lauer Methods in Enzymology 68:473 (1 79), which is hereby incorporated by reference in its entirety.
• the particular promoter selected is preferably capable of causing sufficient expression of the DNA coding sequences to which it is operably linked, to result in the production of amounts of the proteins effective to provide viral resistance, but not so much as to be detrimental to the cell in which they are expressed.
• the actual choice of the promoter is not critical, as long as it has sufficient transcriptional activity to accomplish the expression of the preselected proteins, where expression is desired, and subsequent conferral of viral resistance to the plants.
• the promoters selected should be capable of functioning in tissues including, but not limited to, epidermal, vascular, and mesophyll tissues.
• the nucleic acid construct of the present invention also includes an operable 3' regulatory region, which provides a functional poly(A) addition signal (AATAAA) 3 ' of its translation termination codon. This is selected from among those which are capable of providing correct transcription termination and polyadenylation of mRNA for expression in the host cell of choice, operably linked to a DNA molecule which encodes for a protein of choice.
• a number of 3' regulatory regions are known to be operable in plants. Exemplary 3' regulatory regions include, without limitation, the nopaline synthase 3' regulatory region (Fraley, et al., "Expression of Bacterial Genes in Plant Cells," Proc. Nat'l Acad. Sci.
• a vector of choice, suitable promoter, and an appropriate 3' regulatory region can be ligated together to produce the expression systems which contain the nucleic acids of the present invention, or suitable fragments thereof, using well known molecular cloning techniques as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition Cold Spring Harbor Press, NY (1989), and Ausubel et al. (1989) Current Protocols in Molecular Biology, John Wiley & Sons, New York, N.Y., which are hereby incorporated by reference in their entirety.
• nucleic acid molecules encoding the various papaya ringspot virus coat proteins or polypeptides, as described above, have been cloned into an expression system, they are ready to be incorporated into a host cell. Such incorporation can be carried out by the various forms of transformation noted above, depending upon the vector/host cell system.
• Suitable host cells include, but are not limited to, bacteria, virus, yeast, mammalian cells, insect, plant, and the like. Accordingly, another aspect of the present invention relates to a recombinant plant cell containing one or more of the PRSV-CP nucleic acids of the present invention.
• this method is carried out by transforming a plant cell with a nucleic acid construct of the present invention under conditions effective to yield transcription of the DNA molecule in response to the promoter.
• Methods of transformation may result in transient or stable expression of the DNA under control of the promoter.
• the nucleic acid construct of the present invention is stably inserted into the genome of the recombinant plant cell as a result of the transformation, although transient expression can serve an important purpose, particularly when the plant under investigation is slow- growing.
• Plant tissue suitable for transformation include without limitation, leaf tissue, root tissue, meristems, zygotic and somatic embryos, callus, protoplasts, tassels, pollen, embryos, anthers, and the like.
• the means of transformation chosen is that most suited to the tissue to be transformed.
• Transient expression in plant tissue is often achieved by particle bombardment (Klein et al., "High- Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells," Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety).
• particle bombardment Klein et al., "High- Velocity Microprojectiles for Delivering Nucleic Acids Into Living Cells," Nature 327:70-73 (1987), which is hereby incorporated by reference in its entirety.
• tungsten or gold microparticles (1 to 2 ⁇ m in diameter) are coated with the DNA of interest and then bombarded at the tissue using high pressure gas. In this way, it is possible to deliver foreign DNA into the nucleus and obtain a temporal expression of the gene under the current conditions of the tissue.
• Biologically active particles e.g., dried bacterial cells containing the vector and heterologous DNA
• can also be propelled into plant cells U.S. Patent Nos. 4,945,050, 5,
• An appropriate method of stably introducing the nucleic acid construct into plant cells is to infect a plant cell with Agrobacterium tumefaciens or Agrobacterium rhizogenes previously transformed with the nucleic acid construct.
• the Ti (or RI) plasmid of Agrobacterium enables the highly successful transfer of a foreign DNA into plant cells.
• Yet another method of introduction is fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies (Fraley, et al., Proc. Natl. Acad. Sci. USA 79:1859-63 (1982), which is hereby incorporated by reference in its entirety).
• the DNA molecule may also be introduced into the plant cells by electroporation (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824 (1985), which is hereby incorporated by reference in its entirety).
• plant protoplasts are electroporated in the presence of plasmids containing the expression cassette. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and regenerate.
• the precise method of transformation is not critical to the practice of the present invention. Any method that results in efficient transformation of the host cell of choice is appropriate for practicing the present invention.
• transformed cells are first identified using a selection marker simultaneously introduced into the host cells along with the nucleic acid construct of the present invention.
• selection markers include, without limitation, markers encoding for antibiotic resistance, such as the nptll gene which confers kanamycin resistance (Fraley, et al., Proc. Natl. Acad. Sci. USA 80:4803- 4807 (1983), which is hereby incorporated by reference in its entirety), and the genes which confer resistance to gentamycin, G418, hygromycin, streptomycin, spectinomycin, tetracycline, chloramphenicol, and the like.
• Cells or tissues are grown on a selection medium containing the appropriate antibiotic, whereby generally only those transformants expressing the antibiotic resistance marker continue to grow.
• Other types of markers are also suitable for inclusion in the expression cassette of the present invention.
• a gene encoding for herbicide tolerance such as tolerance to sulfonylurea is useful, or the dhfr gene, which confers resistance to methotrexate (Bourouis et al., EMBO J. 2:1099-1104 (1983), which is hereby incorporated by reference in its entirety).
• reporter genes which encode for enzymes providing for production of an identifiable compound are suitable.
• uidA a gene from Escherichia coli that encodes the ⁇ -glucuronidase protein, also known as GUS (Jefferson et al., "GUS Fusions: ⁇ Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plants," EMBO J. 6:3901-3907 (1987), which is hereby incorporated by reference in its entirety).
• GUS ⁇ -glucuronidase protein
• enzymes providing for production of a compound identifiable by luminescence, such as luciferase, are useful.
• the selection marker employed will depend on the target species; for certain target species, different antibiotics, herbicide, or biosynthesis selection markers are preferred.
• Plant cells and tissues selected by means of an inhibitory agent or other selection marker are then tested for the acquisition of the viral gene by Southern blot hybridization analysis, using a probe specific to the viral genes contained in the given cassette used for transformation (Sambrook et al., "Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor, New York: Cold Spring Harbor Press (1989), which is hereby incorporated by reference in its entirety).
• the presence of a viral coat protein gene can also be detected by immunological assays, such as the double-antibody sandwich assays described by Namba et al., "Expression of the Gene Encoding the Coat Protein of Cucumber Mosaic Virus (CMV) Strain WL appears to Provide Protection to Tobacco Plants against Infection by Several Different CMV Strains," Gene 107:181-188 (1991), which is hereby incorporated by reference in its entirety, as modified by Clark et al., " Characteristics Of the Microplate Method for Enzyme-Linked Immunosorbent Assay For the Detection of plant Viruses," J. Gen. Virol. 34, 475- 83 (1977), which is hereby incorporated by reference in its entirety.
• Potyvirus resistance can also be assayed via infectivity studies as generally described by Namba et al., "Protection of Transgenic Plants Expressing the Coat Protein Gene of Watermelon Virus ii or Zucchini Yellow Mosaic Virus against Potyviruses," Phytopath. 82:940946 (1992), which is hereby incorporated by reference in its entirety, wherein plants are scored as symptomatic when any inoculated leaf shows veinclearing, mosaic, or necrotic symptoms. After the expression cassette is stably incorporated in transgenic plants, it can be transferred to other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
• transgenic plants of this type are produced, the plants themselves can be cultivated in accordance with conventional procedure so that the nucleic acid construct is present in the resulting plants.
• transgenic seeds or propagules are recovered from the transgenic plants. These seeds can then be planted in the soil and cultivated using conventional procedures to produce transgenic plants.
• the present invention also relates to DNA constructs which contain a plurality of DNA molecules which are derived from one or more genes which encode a papaya ringspot viral coat protein.
• the PRSV-CP DNA molecules may be derived from one or more strains, including, but not limited to, TH, KE, KA, ME, YK, BR, JA, OA, and VE.
• Some of the PRSV-CP DNA molecules may be a fragment of the nucleic acid sequence of the CP(s) of choice which by itself is too short, i.e., does not contain sufficient nucleotide sequence, to impart its respective trait when placed in an vector and used to transform plant cells as described above. Collectively, however, this plurality of DNA molecules impart their trait to the transformed plant.
• Suitable nucleic acids for this construct include fragments of a PRSV CP-encoding DNA molecule, of any strain, including but not limited to, TH, KE, KA, ME, YK, BR, JA, OA, and VE.
• the DNA molecules are inserted in the construct as less than full-length DNA, preferably in the range of about 200 bp of the full-length PRSV- CP DNA molecule.
• the 200 bp fragments are preferably chosen from the conserved and variable regions of CP-encoding DNA. There is no need to include separate promoters for each of the fragments; only a single promoter is required.
• viral gene fragments can preferably be incorporated in a single expression system to produce transgenic plants with a single transformation event.
• the present invention also relates to a DNA construct containing a fusion gene which includes a trait DNA molecule which has a length insufficient to independently impart a desired trait to plants transformed with the trait molecule, operatively coupled to a silencer molecule effective to achieve post- transcriptional gene silencing.
• the trait DNA molecule and the silencer molecule collectively impart the trait to plants transformed with the construct.
• the trait DNA molecules of this DNA construct are derived from a gene encoding a papaya ringspot viral coat protein from a papaya ringspot virus strains which include, but are not limited to TH, KE, KA, ME, YK, BR, JA, OA, and VE.
• the fragments of trait DNA molecules are subcloned into the fusion gene cassette. Suitable DNA fragments are those of about 200 bp which derive from the variable and conserved regions of the CP-encoding molecules of choice.
• the silencer molecule of the construct of the present invention can be selected from virtually any nucleic acid which effects gene silencing. This involves the cellular mechanism to degrade mRNA homologous to the transgene mRNA.
• the silencer DNA molecule can be heterologous to the plant, need not interact with the trait DNA molecule in the plant, and can be positioned 3' to the trait DNA molecule.
• the silencer DNA molecule can be a viral cDNA molecule, including, without limitation, a gene encoding a replicase, a movement protein, or a nucleocapsid protein; a green fluorescence protein encoding DNA molecule, a plant DNA molecule, or combinations thereof.
• the DNA molecule conferring disease resistance can be positioned within the DNA construct in the sense (5'— >3') orientation. Alternatively, it can have an antisense (3'-»5') orientation.
• Antisense RNA technology involves the production of an RNA molecule that is complementary to the messenger RNA molecule of a target gene. The antisense RNA can potentially block all expression of the targeted gene.
• plants are made to express an antisense RNA molecule corresponding to a viral RNA (that is, the antisense RNA is an RNA molecule which is complementary to a "plus" (+) sense RNA species encoded by an infecting virus). Such plants may show a slightly decreased susceptibility to infection by that virus.
• antisense RNA is termed antisense RNA.
• the DNA construct of the present invention is configured so that the trait and silencer DNA molecules encode RNA molecules which are translatable. As a result, that RNA molecule will be translated at the ribosomes to produce the protein encoded by the DNA construct. Production of proteins in this manner can be increased by joining the cloned gene encoding the DNA construct of interest with synthetic double-stranded oligonucleotides which represent a viral regulatory sequence (i.e., a 5' untranslated sequence) (U.S. Patent No. 4,820,639 to Gehrke, and U.S. Patent No. 5,849,527 to Wilson, which are hereby incorporated by reference in their entirety).
• a viral regulatory sequence i.e., a 5' untranslated sequence
• the DNA construct of the present invention can be configured so that the trait and silencer DNA molecules encode mRNA which is not translatable. This is achieved by introducing into the DNA molecule one or more premature stop codons, adding one or more bases (except multiples of 3 bases) to displace the reading frame, removing the translation initiation codon, etc. See U.S. Patent No. 5,583,021 to Dougherty et al., which is hereby incorporated by reference in its entirety.
• the subject DNA construct can be incorporated in cells using conventional recombinant DNA technology, such as described in detail above.
• Another aspect of the present invention is a method to confer resistance to PRSV to plants.
• the expression system of the present invention can be used to transform virtually any plant tissue under suitable conditions. Transformed cells can be regenerated into whole plants such that the PRSV-transgene imparts resistance to PRSV in the intact transgenic plants. In either case, the plant cells transformed with the recombinant DNA expression system of the present invention are grown and caused to express the DNA molecule or molecules in the constructs of the present invention, and, thus, to impart papaya ringspot resistance.
• the silencer DNA molecule is believed to boost the level of heterologous RNA within the cell above a threshold level. This activates the degradation mechanism by which viral resistance is achieved.
• Transgenic plants which show post-transcription gene silencing- derived resistance establish the highly resistant state and prevent virus replication.
• a chimeric transgene consisting of a silencer DNA (e.g., GFP) fused with various small nontranslatable fragment viral genome would be prefened for viral resistance.
• the silencer DNA can increase the induced gene silencing.
• the chimeric nature of the gene would provide multiple virus resistance.
• nontranslatable construction produces no protein, thus reducing the possible complementation of naturally occurring mutants and transencapsidation of other viruses.
• the small fragment also reduces the possibility of recombination with other viral genomes.
• transgenic line exhibits a delay in symptom development to one virus, it will also exhibit a delay in symptom development to the second virus. Finally, if a transgenic line is susceptible to one of the viruses it will be susceptible to the other. This phenomenon is unexpected. If there were not a correlation between the efficacy of each gene in these multiple gene constructs, this approach as a tool in plant breeding would probably be prohibitively difficult to use. The probability of finding a line with useful levels of expression can range from 10-50%, depending on the species involved (U.S. Patent No. 6,002,072 to McMaster et al., which is hereby incorporated by reference in its entirety).
• Example 1- Amplification and Cloning of CP Variable Region DNAs Total RNA was extracted from PRS V-infected papaya plants.
• Restriction enzyme sequence is shown in small letters; the stop codon is shown in caps, without underline; viral sequences are underlined.
• the amplified fragments were digested with the appropriate restriction enzymes.
• a restriction enzyme Xbal-Xhol digested YK fragment (209 bp) was first ligated into the pEPJ vector.
• AXhol-Smal digested KE fragment (209 bp) was ligated behind (i.e., at the 3' end of) the YK fragment and then a Smal-BamHI digested TH fragment (206 bp) was ligated behind the KE.
• the resultant clone, pEPJ- YKT, shown in Figure 1A contains the variable region of CP from YK-KE-TH in the 5'-> 3' direction.
• Fragments XbaVBamHl from pEPJ-YKT were ligated into other expression vectors pNP, shown in Figure 2A, and pGFP, shown in Figure 2B, creating pNP-YKT and pGFP-YKT, respectively.
• "Ml/2 NP” shown in Figure 2 A refers to a fragment consisting of approximately one half (387-453 bp) of the gene encoding the nucleocapsid protein ("N" or "NP" gene) of the viral genome of the tomato spotted wilt virus (“TSWV”), a tospovirus that causes crop damage worldwide.
• the N gene of TSWV is an example of a gene derived from the viral genome that is useful as a silencer molecule in the nucleic acid constructs of the present invention.
• Restriction enzyme HindlH/Kpnl digested fragments from these two expression vectors were then ligated into the Hindl ⁇ UKpnl cloning site of the transformation vector pGA482G, resulting in clones pTi-NP-YKT and pTi-GFP- YKT.
• Cesium chloride purified pTi-NP-YKT and pTi-GFP-YKT were then used for host cell transformation by particle gun bombardment.
• Restriction enzyme sequence is shown in small letters; the stop codon is shown in caps, without underline; viral sequences are underlined.
• the pNP clones were Hindlll IKpnl digested from the expression vectors, and ligated into the Hind ⁇ WKpn ⁇ cloning site of the transformation vector pGA482G, resulting in clones pTi-NP-K, pTi-NP-KK, pTi-NP-EE, pTi-NP- KKTC, pTi-NP-KKTV, pTi-NP-EETC and pTi-NP-EETV. Cesium chloride purified pTi-NP-clones were then used for host cell transformation by particle gun bombardment.
• Two full-length KE-CP constructs start from the first CP cut site which is 60 nt upstream from the second CP cut site.
• the primers used for amplification and construction of pEP J-TL KE and pEP J-NTL KE are shown in Table 3.
• Restriction enzyme sequence is shown in small letters; the stop codon is shown in caps, without underline; viral sequences are underlined.
• the Ncol/Bamt ⁇ l digested PCR KECP fragments were ligated into pEPJ vector, as shown in Figure 4.
• the expression cassette was then subcloned into the transformation vector ⁇ GA482G.
• Restriction enzyme sequence is shown in small letters; the stop codon is shown in caps, without underline; viral sequences are underlined.
• Papaya embryos were bombarded with DNA constructs prepared as described above and shown in Figures 2-5.
• the transformation procedure was followed as described in Cai et al., "A Protocol for Efficient Transformation and Regeneration of Carica papaya L. In Vitro " Cell Devel. Biol-Plant 35: 61-69 (1999), which is hereby incorporated by reference in its entirety.
• Plasmid DNA was purified by ethidium bromide CsCl gradient (Ausubel et al., "CsCl/Ethidium Bromide Preparations of Plasmid DNA," Current Protocols in Molec Biol. unit 2.9.1-2.9.20 (1995), which is hereby incorporated by reference in its entirety), ethanol precipitated and suspended in water.
• Immature zygotic embryos were extracted from seeds of immature green 'Sunrise' or 'Kapoho' papaya and placed on induction medium and kept in the dark. Zygotic embryos with their somatic embryo clusters were placed on Whatman #2 filter paper and spread. The somatic embryos were allowed to proliferate, and following this, the embryos were spread firmly onto fresh filter paper and bombarded with tungsten-coated plasmid DNA. Seven days after bombardment, materials were transferred to induction medium containing kanamycin at 75 mg/L. After four weeks, the kanamycin level was raised to 150 mg/L. After a few weeks in kanamycin medium, actively growing embryo clusters were transferred to kanamycin-free medium.
• Transgenic lines from the germination medium were analyzed by PCR to confirm that the virus gene was in the plantlets.
• Northern blots were carried out to detect the level of RNA expressed in transgenic lines, and the copy number of the transgene in the transgenic plants was determined by Southern blot analysis.
• transgenic plants were challenged with the KE strain of PRSV. Plants were thereafter monitored for viral symptoms. If no disease symptoms appeared after approximately 4 weeks post- inoculation, those plants were challenged with a different PRSV strain to test for cross-resistance.
• transgenic lines containing the various PRSV DNA constructs of the present invention, as described above, were transferred to the greenhouse. Inoculation with KE virus was carried out on 90 plant lines transformed with at least one KE-containing DNA construct. Of those 90 lines challenged with PRSV- KE, 26 lines showed resistance and 64 lines were susceptible.

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