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EP1086237A2 - Methodes et materiels ameliores de transformation - Google Patents

Methodes et materiels ameliores de transformation

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Publication number
EP1086237A2
EP1086237A2 EP99953356A EP99953356A EP1086237A2 EP 1086237 A2 EP1086237 A2 EP 1086237A2 EP 99953356 A EP99953356 A EP 99953356A EP 99953356 A EP99953356 A EP 99953356A EP 1086237 A2 EP1086237 A2 EP 1086237A2
Authority
EP
European Patent Office
Prior art keywords
dna
rna
launching platform
plant
trans
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP99953356A
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German (de)
English (en)
Inventor
Lada Rasochova
Thomas L. German
Paul G. Ahlquist
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Wisconsin Alumni Research Foundation
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Wisconsin Alumni Research Foundation
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Filing date
Publication date
Application filed by Wisconsin Alumni Research Foundation filed Critical Wisconsin Alumni Research Foundation
Publication of EP1086237A2 publication Critical patent/EP1086237A2/fr
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    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8203—Virus mediated transformation
    • 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)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells

Definitions

  • RNA viruses have been found to be valuable tools in the phenotypic and genotypic transformation of targeted cells and tissues. See, e.g., U.S. Patent No. 5,500,360, which teaches novel viral RNA expression vectors. It has been shown that the RNA of the genome of an RNA virus can be modified to include an exogenous RNA segment and that the modified RNA can be introduced into a host cell, replicated therein, and thereby express the exogenous RNA segment.
  • RNA virus-based vectors can spread to non-target plants by mechanical means and/or by insects. Such spread can be prevented by using vectors that can replicate and/or move only in target plants expressing the appropriate trans-acting factors.
  • the subject invention pertains to improved materials and methods for transforming host cells which involve transfecting said cells with a DNA-launching platform.
  • One aspect of the subject invention pertains to a DNA-launching platform which encodes a modified viral RNA molecule downstream of DNA-dependent RNA polymerase (pol) promoter, whereby the DNA- launching platform is capable of being introduced into a host cell and effectively "launching" said modified viral RNA molecule into the host cell such that it is replicated and expressed therein.
  • poly DNA-dependent RNA polymerase
  • modified viral RNA molecule refers to a viral RNA which has been changed from its natural state. Examples of changes of viral RNA include, but are not limited to, removal of a part of viral RNA genome, insertion or substitution of an exogenous RNA, etc.
  • the exogenous RNA segment can be located in a region of the viral RNA molecule such that it does not disrupt the RNA replication. Techniques for such manipulations have been well known to those of ordinary skill in the art for many years.
  • the modified viral RNA molecule further comprises a ribozyme which is located in the proximity of the 3' end of the modified viral RNA molecule.
  • the viral segment may have the ability to be replicated with or, alternatively, without the presence of trans-acting viral replicating elements.
  • Another aspect of the subject invention pertains to a method of genotypically or phenotypically modifying a host cell, comprising introducing a DNA-launching platform which encodes a viral RNA molecule and an exogenous RNA segment in a location which does not disrupt the replication of said viral RNA segment or said exogenous RNA segment, whereby the exogenous RNA segment confers a detectable trait in the host cell.
  • the subject invention applies to a wide array of plant cells.
  • Still a further aspect of the subject invention pertains to cells in which the DNA- launching platform of the subject invention has been introduced. Yet another aspect of the subject invention pertains to a plant comprising cells transfected with the DNA-launching platform.
  • the novel methods and materials of the subject invention provide a greater inoculation efficiency of RNA viruses because use of DNA-launching platforms of the subject invention are more resistant to degradation than RNA inocula, and because each DNA platform produces multiple RNA transcripts over an extended period of time.
  • the DNA-launching platform provides a genetically stable inplanta archive copy of a desired vector construct, the continuing transcription of said DNA platform will repeatedly reinoculate the host cell with the desired construct. This serves to counteract genetic instability problems that have inhibited the expression of some genes from vectors based on plant and animal RNA viruses.
  • the inoculation methods of the subject invention provide a much simpler means of producing inocula in bulk for large scale use, which is cheaper and more efficient than inoculating with in vitro RNA transcripts.
  • Figure 1 represents the schematic for producing the la and 2a proteins in the host cell.
  • Figure 2 illustrates an example of an Agrobacterium transformation vector containing an expression cassette capable of expressing la and/or 2a BMV proteins.
  • Figure 3 illustrates several Agrobacterium vectors that were produced to transform host plant cells (black rectangles indicate T-DNA borders).
  • Figure 4 represents the general mechanism of BMV RNA3 launching, and replication.
  • Figure 5 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • the BMV and CCMV designations denote cis-acting elements.
  • Figure 6 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • Figure 7 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • Figure 8 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • Figure 9 depicts Agrobacterium vector for delivery of DNA-launching platforms to plant cells (open triangles represent T-DNA borders).
  • Figure 10 depicts DNA-launching platforms which can be used in accord with the teachings contained herein.
  • Figure 11 shows that BMV replication factors support efficient RNA3 replication in protoplasts.
  • Figure 12 shows the efficient replication of launched BMV RNA3 in protoplasts.
  • Figure 13 shows transgenic expression of BMV 1 a and 2a mRNAs in N. tabacum and N. benthamiana.
  • Figure 14 shows the efficient replication of launched BMV RNA3 in (la + 2a)- transgenic plants.
  • Figure 15 shows the successful GUS expression from the launched BMV RNA3 in (la + 2a)- transgenic plants.
  • Figure 16 shows the successful GUS expression from the launched BMV RNA3 in protoplasts.
  • Figure 17 shows the successful GFP expression from the launched BMV RNA3 in (la
  • Figure 18 shows the successful GFP expression from the launched BMV RNA3 in protoplasts.
  • Figure 19 shows the efficient replication of the launched BMV RNA3 in (la + 2a)- transgenic N. benthamiana using Agrobacterium inoculation.
  • Figure 20 shows the successful GUS expression from the launched BMV RNA3 having the SHMV coat protein in (la + 2a)-transgenic plants.
  • Figure 21 shows that launched BMV replicates, moves cell-to-cell, and spreads long distances in (la+2a)-transgenic plants.
  • Figure 22 shows transfection of progeny from (la+2a)-transgenic N. benthamiana with
  • SEQ ID NO. 1 pBlLR2 — partial nucleotide sequence includes BMV la expression cassette.
  • SEQ ID NO. 2 pBlLR3 — partial nucleotide sequence includes BMV la expression cassette.
  • SEQ ID NO. 3 pB2LR4 — partial nucleotide sequence includes BMV 2a expression cassette.
  • SEQ ID NO. 4: pB2LR5 partial nucleotide sequence includes BMV 2a expression cassette.
  • SEQ ID NO. 5 pB12LR6 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • SEQ DD NO. 6 pB12LR7 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • SEQ DD NO. 7 pB12LR8 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • SEQ DD NO. 8 pB12LR9 — partial nucleotide sequence includes BMV la and 2a expression cassettes.
  • RNA virus as used herein means a virus whose genome is RNA in a double-stranded or single-stranded form, the single strand being a (+) strand or (-) strand.
  • transfection means an introduction of a foreign DNA or RNA into a cell by mechanical inoculation, electroporation, agroinfection, particle bombardment, microinjection, or by other known methods.
  • transformation means a stable incorporation of a foreign DNA or RNA into the cell which results in a permanent, heritable alteration in the cell. Accordingly, the skilled artisan would understand that transfection of a cell may result in the transformation of that cell.
  • launched refers to a polynucleotide that has been transcribed from a DNA-launching platform, as described herein and, preferably, replicated.
  • cis-acting element denotes that portion of the RNA genome of an RNA virus which must be present in cis, that is, present as a part of each viral strand as a necessary condition for replication of that strand.
  • Virus replication may depend upon the existence of one or more trans (diffusible) elements which interact with the cis-acting element to carry out RNA replication. If trans-acting elements are necessary for replication, they need not be present or coded for on the modified viral RNA provided, but may be made available within the infected cell by some other means.
  • the trans-acting replication functions may be provided by other, unmodified or modified, components of the viral genome transfected into the cells simultaneously with the modified RNA.
  • the target cell may also be premodified, for example, cells may have been previously transformed to provide constitutive expression of the trans-acting functions from a chromosome.
  • the cis-acting element is composed of one or more segments of viral RNA which must be present on any RNA molecule that is to be replicated within a host cell by RNA replication. The segment will most likely be the 5 ' and 3 ' terminal portions of the viral RNA molecule, and may include other portions and/or virus open reading frames as well.
  • the cis-acting element is accordingly defined in functional terms: any modification which destroys the ability of the RNA to replicate in a cell known to contain the requisite trans-acting elements, is deemed to be a modification in the cis- acting element. Conversely, any modification, such as deletion or insertion in a sequence region which is able to tolerate such deletion or insertion without disrupting replication, is a modification outside the cis-acting element. As is demonstrated herein, using the example of BMV which is known and accepted by those skilled in the art to be a functional example from which substantial portions of an RNA virus molecule may be modified, by deletion, insertion, or by a combination of deletion and insertion, without disrupting replication.
  • Exogenous RNA is a term used to describe a segment or component of RNA to be inserted into the virus RNA to be modified, the source of the exogenous RNA segment being different from the RNA virus itself.
  • the source may be another virus, an organism such as a plant, animal, bacteria, virus, or fungus.
  • the exogenous RNA may be a chemically synthesized RNA, derived from a native RNA, or it may be a combination of the foregoing.
  • the exogenous RNA may provide any function which is appropriate and known to be provided by an RNA segment.
  • Such functions include, but are not limited to, a coding function in which the RNA acts as a messenger RNA encoding a sequence which, when translated by the host cell, results in synthesis of a peptide or protein having useful or desired properties; the RNA segment may also be structural, as for example in ribosomal RNA; it may be regulatory, as for example with small nuclear RNAs or anti-sense RNA; or it may be catalytic.
  • the exogenous RNA may encode, for example, a protein which is a key enzyme in a biochemical pathway, which upon expression effects a desirable phenotypic characteristic, such as altering cell metabolism.
  • exogenous RNA may encode a protein involved in transcriptional regulation, such as zinc finger, winged-helix, and leucine-zipper proteins.
  • anti-sense RNA sometimes termed (-) strand RNA, which is in fact a sequence complementary to another RNA sequence present in the target cell which can, through complementary base pairing, bind to and inhibit the function of the RNA in the target cell.
  • non- viral is used herein in a special sense to include any RNA segment which is not normally contained within the virus whose modification is exploited for replication and expression, and is therefore used synonymously with "exogenous".
  • a gene derived from a different virus species than that which is modified is included within the meaning of the terms "non-viral” and "exogenous” for the purposes of describing the invention.
  • a non-viral gene as the term is used herein could include a gene derived from a bacterial virus, an animal virus, or a plant virus of a type distinguishable from the virus modified to effect transformation.
  • a non-viral gene may be a structural gene derived from any prokaryotic or eukaryotic organism.
  • the subject invention concerns a novel method of transfecting a host cell which uses a DNA-launching platform to introduce viral RNA into the cell.
  • the subject invention is directed towards a method of transfection employing a DNA-launching platform which encodes a modified viral RNA molecule comprising an RNA viral component attached to an exogenous RNA component and a DNA-dependent RNA pol promoter.
  • the DNA-dependent RNA pol promoter is preferably but not necessarily fused within up to 10 nucleotides of the 5 ' transcriptional start site of the modified viral RNA molecule, and more preferably within up to 5 nucleotides of the 5' transcriptional start site.
  • the DNA-launching platform produces transcripts of the modified viral RNA molecule that are then capable of RNA replication in the presence of replication factors, which can be present in the modified viral RNA and/or may be supplied in trans by other means including expression from chromosome or supplied on different launching plasmids.
  • the exogenous RNA can be replicated as well. Further, the exogenous RNA can be expressed in the cell, thereby providing a predetermined phenotypic characteristic.
  • the DNA launching platform further comprises a nucleotide sequence encoding a self-cleavable ribozyme situated proximate to the 3 ' end of said RNA molecule.
  • the ribozyme cleaves the modified RNA viral molecule at the 3' region.
  • the 3' region can consist of up to 30 nucleotides upstream or downstream of the 3' end; and preferably consists of up to 10 nucleotides upstream or downstream of the 3' end.
  • the ribozyme cleaves the modified RNA viral molecule precisely at the 3' end.
  • Other known regulatory sequences e.g., promoters and/or termination sequences, may also be substituted for and/or included on the DNA-launching platform.
  • a suitable restriction site can be introduced proximate to the 3 ' end of the modified viral RNA molecule sequence and the DNA molecule can be cleaved by an appropriate restriction enzyme prior to transfection.
  • DNA-launching platform as used herein is intended to mean a DNA molecule, circular or linear, which has a coding region comprising a segment encoding a modified viral RNA segment, and further, which is capable of being delivered into a cell and subsequently transcribed.
  • Possible regulatory sequences can include, but are not limited to, any promoter already shown to be constitutive for expression, such as those of viral origin (CaMV 19S and 35S) or so-called “housekeeping" genes (ubiquitin, actin, tubulin) with their corresponding termination/polyA + sequences.
  • seed-and/or developmentally-specific promoters such as those from plant fatty acid/lipid biosynthesis genes (ACPs, acyltransferases, desaturases, lipid transfer protein genes) or from storage protein genes (zein, napin, cruciferin, conglycinin, phaseolin, or lectin genes, for example), with their corresponding termination/polyA + sequences can be used for targeted expression.
  • the gene can be placed under the regulation of inducible promoters and their termination sequences so that gene expression is induced by light (rbcS-3A, cab- 1), heat (hsp gene promoters) or wounding (mannopine, HGPGs). It is clear to one skilled in the art that a promoter may be used either in native or truncated form, and may be paired with its own or a heterologous termination/polyA + sequence.
  • the subject invention is directed toward a method of genotypically or phenotypically modifying a cell comprising the following steps: a) forming a cDNA molecule of a virus RNA, or of at least one RNA component if the RNA virus is multipartite, the viral RNA having been modified to contain a DNA segment encoding a non- viral RNA component situated in a region able to tolerate such insertion without disrupting replication of the RNA product encoded thereby; b) cloning modified cDNA into a DNA- launching platform; and c) transfecting a suitable host cell with said DNA-launching platform.
  • the method further comprises pretransforming a plant with trans-acting viral replication factors and/or other trans-acting factors.
  • trans-acting factors may include viral movement proteins(s), coat protein(s), viral protease(s), and other structural and nonstructural genes.
  • trans-acting factors may be introduced on separate expression plasmids or may be expressed from RNA transcripts. In a preferred embodiment such trans-acting factors do not replicate.
  • Suitable host cells may include protoplasts, cells in suspension, or cells in tissues or whole organisms.
  • the RNA viral segment can be derived from brome mosaic virus (BMV), whereby the DNA- launching platform comprises DNA encoding the RNA3 segment of the virus.
  • Brome mosaic virus (BMV) is a member of the ⁇ virus-like super family of positive-strand RNA viruses of animals and plants, and has a genome divided among three RNAs.
  • RNA1 and RNA2 encode the la and 2a proteins, respectively, which are necessary for a genomic RNA replication and subgenomic mRNA synthesis (see, e.g., U.S. Patent No. 5,500,360, which to the extent not inconsistent herewith, is incorporated herein by reference).
  • la and 2a interact with each other and with cell factors to form a membrane bound viral RNA replication complex associated with the endoplasmic reticulums of infected cells.
  • BMV RNA3, a 2.1-kb RNA encodes the 3a protein (32kDa) and coat protein (20kDa), which are involved in the spread of BMV infection in its natural plant hosts but are dispensable for RNA replication. See U.S. Patent No. 5,500,360.
  • the 3a or coat protein gene of the RNA3 viral segment can be replaced with exogenous RNA, whereby it does not interfere with the replication element. Further, the exogenous RNA segment can be inserted downstream of an additional subgenomic promoter.
  • cells or tissues can be pretransformed to express la, 2a, 3a, and coat protein, or any combination thereof, wherein DNA-launching platforms containing a foreign gene(s) with the necessary cis-acting components is transfected, such that the foreign gene is replicated and/or expressed.
  • the host cell is pretransformed with BMV 1 or BMV2 such that it is transgenically engineered to express la and 2a proteins.
  • BMV1 and BMV2 are removed such that they are incapable of replication, but can express la and 2a to form a viral RNA replication complex associated with the endoplasmic reticulum of the host cell.
  • RNA-launching platform comprising the RNA3 viral replication segment, as well as the exogenous RNA of interest
  • RNA3 viral replication segment as well as the exogenous RNA of interest
  • DNA-launching platforms could be also derived from either RNA1 or RNA2.
  • sequence encoding the la protein could be replaced with an exogenous RNA; replication would require the expression of la (e.g., separate expression plasmid).
  • the DNA- launching platform also comprises a ribozyme situated proximate to the 3' end of the modified RNA3, wherein said ribozyme cleaves the RNA3 at the 3' end.
  • viral segments from other known viruses, and/or subviral agents can be used to formulate DNA-launching platforms of the subject invention.
  • BMV is merely one representative example of the many viruses suitable for practicing the subject invention. It is widely accepted that principles on which the subject invention is based are broadly applicable to a myriad of viruses.
  • viruses examples include, but are not limited to, alfalfa mosaic virus (AMV), barley stripe mosaic virus, cowpea mosaic virus, cucumber mosaic virus, reoviruses, polio virus, Sindbis virus, vesicular stomatitis virus, influenza virus, retroviruses, and cowpea chlorotic mottle virus (CCMV) and any other viruses that replicate through RNA intermediates and from which a cDNA copy can be obtained.
  • AMV alfalfa mosaic virus
  • barley stripe mosaic virus cowpea mosaic virus
  • cowpea mosaic virus cucumber mosaic virus
  • reoviruses polio virus
  • Sindbis virus vesicular stomatitis virus
  • influenza virus retroviruses
  • cowpea chlorotic mottle virus CCMV
  • the inserted gene need not be a naturally occurring gene, but may be modified, a composite of more than one coding segment, or it may encode more than one protein.
  • the RNA may also be modified by combining insertions and deletions in order to control the total length or other properties of the modified RNA molecule.
  • the inserted non- viral gene may be either prokaryotic or eukaryotic in origin.
  • the inserted gene may contain its own translation start signals, for example, a ribosomal binding site and start (AUG) codon, or it may be inserted in a manner which takes advantage of one or more of these components preexisting in the viral RNA to be modified.
  • AUG ribosomal binding site and start
  • Certain structural constraints must be observed to preserve correct translation of the inserted sequence, according to principles well understood in the art. For example, if it is intended that the exogenous coding segment is to be combined with an endogenous coding segment, the coding sequence to be inserted must be inserted in reading frame phase therewith and in the same translational direction.
  • RNA segments or genes which fail to confer a readily observable phenotypic trait on recipient cells or plants is irrelevant to the invention, and in any case will be readily recognizable by those of ordinary skill in the art without undue experimentation.
  • An exogenous RNA segment may be inserted at any convenient insertion site in any of the cDNA sequences corresponding to a viral RNA, or component RNA of a multipartite RNA virus, provided the insertion does not disrupt a sequence essential for replication of the RNA within the host cell.
  • an exogenous RNA segment may be inserted within or substituted for the region which normally codes for coat protein.
  • regions which contribute to undesirable host cell responses may be deleted or inactivated, provided such changes do not adversely affect the ability of the RNA to be replicated in the host cell.
  • RNA replication For many single component and multipartite RNA viruses, a reduction in the rate of normal RNA replication is tolerable and will in some instances be preferred, since the amount of RNA produced in a normal infection is more than enough to saturate the ribosomes of the transformed cell.
  • Plant cells which are inoculated in culture will normally remain transfected as the cells grow and divide since the RNA components expressed from the DNA-launching platform are able to replicate and thus become distributed to descendant cells upon cell division. Plants regenerated from phenotypically modified cells, tissues, or protoplasts remain phenotypically modified. Similarly, plants transfected as seedlings remain transfected during growth. Optimal timing of application of the transfecting components will be governed by the result which is intended and by variations in susceptibility to the transfecting components during various stages of plant growth.
  • RNA viruses are seed transmitted from one generation to the next. This property can be exploited to effect genotypic transformation of a plant. That is to say, the modified RNA remains transmissible from one generation to the next, just as seed-borne virus infections are transmitted from one generation to the next.
  • Binary vectors for expressing the BMV la and 2a proteins in plants were constructed. Starting with the pBI101.2 construct (Clontech, Palo Alto, CA), the GUS gene was removed by first cutting the construct with EcoRI and SnaBI. The overhanging restriction fragment ends were filled in by treatment with Klenow fragments and dNTPs. The restriction fragment ends were religated forming the pB 101.2LR1.
  • the 2a expression cassette was inserted into pBI 101.2 LR1.
  • First the pBI 101.2LR1 was cut with Hind III and dephosphorylated.
  • pB2PA17 (Dinant et al, 1993) was cut with Hind III and the 2a insert was purified using a low melting agarose gel. The restriction fragment ends were ligated forming the pB2LR4 and pB2LR5 ( Figures 3c and 3d).
  • the 1 a expression cassette was inserted into pBI 101.2LR1 by first cutting pBI 101.2LR1 with SnaBI and dephosphorylated.
  • pBlPA17 (Dinant et al, 1993) was cut with Pstl and the extra nucleotides were removed with T4 DNA polymerase.
  • the la insert was purified using a low melting agarose gel. The restriction fragment ends were ligated forming the pBlLR2 and pBlLR3 vectors ( Figures 3a and 3b).
  • the 1 a expression cassette was inserted into pB2LR4 and pB2LR5 by cutting pB2LR4 or pB2LR5 with SnaBI and dephosphorylated.
  • PBI PA 17 (Dinant etal, 1993) was cut with Pstl, and the extra nucleotides were removed with T4 DNA polymerase.
  • the la insert was purified using low melting agarose gel and ligated with the cut pB2LR4 or pB2LR5 vectors to form pB12LR6, pB12LR7, pB12LR8, and pB12LR9 vectors ( Figures 3e-3h).
  • Example 2 Construction of DNA-launching Platform for wtRNA3 of BMV and for RNA Derivatives Containing Foreign Sequences
  • Vector pRTlOl (Topfer et al, 1987) was cut with PpuMI and the restriction fragment ends were filled in with Klenow fragment and dNTPs, and cut with BamHI and dephosphorylated.
  • Vector pB3RQ39 (Ishikawa et al, 1997) was cut with SnaBI and BamHI; the B3 fragment was isolated from a low melting agarose gel. This fragment was ligated to the cut pRTlOl thereby forming pB3LR10 ( Figure 4).
  • the pB3LR15 ( Figure 4) that is a pB3LR10 derivative has the Clal-Kpnl fragment replaced with the corresponding fragment from pB3TP8 (Janda et al, 1987).
  • PCR was performed on pRTlOl to amplify an EcoRV and EcoRI fragment.
  • a one nucleotide deletion was performed during the PCR process.
  • the resulting PCR product was cut with EcoRV and EcoRI and inserted into dephosphorylated pRTlOl cutwith EcoRV and EcoRI to form pRTlOlLRl l.
  • the pRTlOlLRl l was cut with Stul and BamHI and dephosphorylated.
  • PB3RQ39 was cut with SnaBI and BamHI and a B3 fragment was isolated using a low melting agarose gel.
  • a DNA-launching platform wherein the BMV RNA3 coat protein was replaced with GUS was also constructed.
  • the pB3MI22 (Ishikawa et al, 1997) was cut with Clal and Stul and a B3GUS insert was isolated.
  • the pB3LR10 or pB3LR14 DNA-launching constructs were cut with Clal and Stul and dephosphorylated.
  • the B3GUS fragment was then ligated to the cut pB3LR10 or pB3LR14 thereby forming the pB3GUSLR17 and pB3GUSLR18 DNA-launching constructs ( Figure 5).
  • a DNA-launching platform having a BMV RNA3 with a GUS gene insertion wherein the GUS is downstream of an additional BMV subgenomic promoter was constructed.
  • the pB3LRl 5 construct was cut with Aval and the restriction fragment ends were filled in with Klenow fragment and dNTPs. Construct was then cut with Clal and dephosphorylated.
  • the pB3MI22 was cut with Clal and Stul and a B3GUS fragment was isolated. The isolated B3GUS fragment was then ligated to the cut pB3LR15 construct to form a new construct of pB3GUSCPLRl 9 ( Figure 5).
  • a BMV RNA3 based DNA-launching platform with a CP gene inserted downstream of an additional cowpea chlorotic mottle virus (CCMV) subgenomic promoter was constructed.
  • the pB3GUSLRl 7 construct was cut with Stul and Kpnl and dephosphorylated.
  • the pBC3 AJ14 (Pacha and Ahlquist, 1991) was cut with Ndel, the ends were blunted by known methods in the art, and then cut with Kpnl.
  • a coat protein fragment was then isolated. The coat protein fragment was then ligated to the cut pB3GUSLRl 7 to form a new construct of pB3GUSCPLR22 ( Figure 5).
  • a DNA-launching platform was constructed having a subgenomic RNA4.
  • the pB4MK2 The pB4MK2
  • RNA4 fragment was then isolated.
  • the pRTlOlLRl 1 construct was cut with Stul and BamHI and dephosphorylated.
  • a DNA-launching platform wherein the BMV coat protein was replaced with GFP was constructed.
  • pEGFP (Clontech, CA) was cut with Notl, filled in with Klenow fragment and dNTPs, cut with Sail, and GFP insert was isolated using low-melting agarose gel.
  • the pB3LRl 5 was cut with Sail and Stul and dephosphorylated.
  • the GFP fragment was then ligated to the cut pB3LR15 thereby forming the pB3GFPLR48 ( Figure 6e).
  • a DNA-launching platform having a BMV RNA3 with a GFP gene insertion wherein the CP is downstream of an additional CCMV subgenomic promoter was constructed.
  • the pBC3AJ14 (Pacha and Ahlquist, 1991) was cut with Ndel and EcoRI and the ends were blunted by known methods in the art.
  • the coat protein fragment was then isolated and ligated into dephosphorylated and blunted pEGFP cut with Notl and Stul forming pEGFPCPLR49.
  • pEGFPCPLR49 was cut with Kpnl and the EGFPCP fragment was isolated using low-melting agarose gel.
  • PB3GFPLR48 was cut with Kpnl and dephosphorylated.
  • the EGFPCP fragment was then ligated to the cut pB3GFPLR48 thereby forming the pB3GFPCPLR50 ( Figure 6a).
  • RNA transcription vector wherein the GFP gene is expressed as a translational fusion with BMV 3a was constructed.
  • the pB3TP10 (Pacha and Ahlquist, 1991) was cut with BamHI and dephosphorylated.
  • the GFP fragment was amplified from pEGFP (Clontech, CA) using PCR and the following primers:
  • the amplified product was cut with BamHI and purified using low-melting agarose gel.
  • the GFP fragment was ligated to the cut pB3TP10 forming pB3GFPLR47 ( Figure 6d).
  • the pB3GFPLR47 was cut with EcoRI and transcribed using T7 RNA polymerase.
  • An Agrobacterium vector containing BMV RNA3 DNA-launching platform was constructed.
  • the pBI101.2LRl was cut with Smal and dephosphorylated.
  • the pB3LR15 was cut with Pvull and the B3 fragment was purified using a low-melting agarose gel.
  • the B3 fragment was then ligated to the cut pBI 101.2LR1 thereby forming pB3LR42 ( Figure 9).
  • a DNA-launching platform wherein the BMV RNA3 coat protein was replaced with the SHMV (Sunn hemp mosaic virus) coat protein and the GUS gene was inserted downstream of an additional BMV subgenomic promoter was constructed.
  • the pB3RS4 (Sacher et al, 1988) was cut with Aval, blunted with Klenow fragment and dNTPs, and cut with Kpnl.
  • the SHMV coat protein fragment was isolated using a low-melting agarose gel.
  • the pB3GUSLRl 7 was cut with Stul and Kpnl and dephosphorylated.
  • the SHMV coat protein fragment was ligated to the cut pB3GUSLRl 7 thereby forming pB3GUSCPLR24 ( Figure 7).
  • NTl Medium (1 liter) was made with Gibco-BRL (MS salt, catalog #1 1118-031), 3ml of 6% KH2P04, and 0.2 ⁇ g/ml 2,4D (final concentration). The pH was adjusted to 5.5-5.7 using KOH, and the resulting mixture was autoclaved.
  • NTl Plating Medium (1 liter) was made with NTl medium and 72.86 g mannitol, the pH was adjusted to 5.5-5.7, and the resulting mixture was autoclaved.
  • Electroporation Buffer was made with 0.8% NaCl, 0.02% KC1, 0.02% KH2P04, 0.1 1%
  • Enzyme Solution was made with 0.4M mannitol, and 20mM MES. The pH was adjusted to 5.5, and the resulting mixture was autoclaved. Growth conditions: Cells (Nicotiana tabacum) were grown at room temperature in NTl media with constant shaking (about 200 rpm).
  • the cells were centrifuged at 800 rpm for 5 min. The supernatant was discarded. About 40 ml of wash solution was added, cells were resuspended and were centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were then resuspended in three volumes of enzyme digestion solution, and incubated for 60 min. at room temperature.
  • the cells were transferred into 50 ml plastic tube and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 40 ml of wash solution and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in 40ml of electroporation buffer and centrifuged at 800 rpm for 5 min. The supernatant was discarded. The cells were resuspended in four volumes of electroporation buffer.
  • Electroporation One ml of cells containing the RNA or DNA inocula was transferred into electroporation cuvettes and placed on ice for 10 min. The cells were then mixed and electroporated at 500 microF, 250V. The cuvettes were placed on ice for 10 min. The cells were transferred into 10 ml of NTl plating media.
  • RNA Analysis RNA extraction, denaturing 1 % agarose gel electrophoresis and Northern blot hybridization were performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 ⁇ g) of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 10 6 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment.
  • RNA3 Replication of RNA3 was confirmed by detection of sgRNA4, thus showing that BMV RNA replication factors la and 2a expressed from expression plasmid(s) support efficient replication of RNA3 supplied as in vitro transcript ( Figure 11) as well as launched from DNA-launching platform ( Figure 12).
  • Agrobacterium tumefaciens by the freeze-thaw method.
  • Vectors pBlLR2, pB2LR4, pB12LR6, and pB12LR7 were all individually used.
  • An Agrobacterium strain LBA 4404 containing an appropriate helper Ti plasmid was grown in 5 ml of YEP medium overnight at 28 °C. Two ml of the overnight culture were added to 50 ml YEP medium in a 250-ml flask and shaken vigorously (250 rpm) at 28 °C until the culture grew to an OD 500 of 0.5 to 1.0. The culture was chilled on ice.
  • the cell suspension was centrifuged at 3000 g for 5 min. at 4°C. The supernatant solution was discarded. The cells were resuspended in 1 ml of ice-cold 20 mM CaCl 2 solution. 0.1 -ml aliquots were dispensed into prechilled eppendorf tubes. About 1 ⁇ g of plasmid DNA was added to the cells. The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37 °C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28 °C for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes.
  • the tubes were centrifuged for 30 s and the supernatant solution was discarded.
  • the cells were resuspended in 0.1 ml YEP medium, plated on a YEP agar plate containing selection antibiotic(s), and incubated at 28°C. Transformed colonies appeared in 2-3 days.
  • Agrobacterium tumefaciens strain LBA 4404 containing the preselected binary vector was used for plant transformation. Explants were placed in - 10 ml of overnight grown Agrobacterium culture for 30 min. Leaf explants were then blotted on filter paper and placed on TB2 (plus 1.0 mg/1 6-benzyl-aminopurine and 0.1 mg/1 -naphthalene acetic acid) media for 4 days, abaxial side down. Explants are then rinsed three times in sterile water, blotted on filter paper, and placed on TB2 media for regeneration with 100 mg/1 kanamycin and 400 mg/1 carbenicillin at 25 °C, 16 hour photo period, abaxial side down.
  • Explants were transferred to fresh TB2 media with 100 mg/1 kanamycin and 400 mg/1 carbenicillin every 10 to 14 days until plantlets developed. Plantlets typically developed at 10-14 days. Plantlets were cut from the callus and placed on MST media containing 100 mg/1 kanamycin and 400 mg/1 carbenicillin to induce rooting. Rooted plants were transferred to soil.
  • TB1 (1 liter) included 4.30 g MS salts, 100 mg myo-inositol, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose, 2 mg BAP, 0.10 mg of NAA, and 8g Noble agar. The media was adjusted to a pH 5.7 and autoclaved.
  • TB2 (1 liter) included 4.30 g MS salts, 100 mg myo-inositol, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose, 1.0 mg BAP, 0.10 mg NAA, and 8 g Noble agar. The media was adjusted to pH 5.7 and autoclaved.
  • MST (1 liter) included 4.30 g MS salts, 1.0 ml Nitsch and Nitsch vitamins, 30 g sucrose,
  • YEP 100 ml included 1.0g Bacto-peptone, 1.0 g Bacto-yeast extract, and 0.5 g NaCl. The media was autoclaved.
  • RNA Analysis Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 ⁇ g) of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 10 6 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment. Figure 13a shows the successful expression of BMV la and 2a mRNA in transgenic N. tabacum.
  • Coating Microcarriers with DNA The following was sequentially and quickly added: 5 ⁇ l DNA (l ⁇ g/ ⁇ l), 50 ⁇ l of 2.5M CaCl 2 , and 20 ⁇ l of 0.1 M Spermidine.
  • RNA Analysis Total RNA extraction, denaturing 1% agarose gel electrophoresis and Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 ⁇ g) of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 10 6 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment.
  • Figure 14a shows that the launched BMV RNA3 replicates efficiently in transgenic plants expressing BMV replication factors 1 a and 2a and that the launched RNA3 is unable to replicate in the absence of BMV la and/or 2a.
  • Agrobacterium tumefaciens Vectors pBlLR2, pB2LR4, pB12LR6, and pB12LR7 were all individually used.
  • An Agrobacterium strain LBA 4404 containing an appropriate helper Ti plasmid was grown in 5 ml of YEP medium overnight at 28°C. Two ml of the overnight culture were added to 50 ml YEP medium in a 250-ml flask and shaken vigorously (250 rpm) at 28 °C until the culture grew to an OD 500 of 0.5 to 1.0. The culture was chilled on ice. The cell suspension was centrifuged at 3000 g for 5 min.
  • the cells were resuspended in 1 ml of ice-cold 20 mM CaCl 2 solution. 0.1 -ml aliquots were dispensed into prechilled eppendorf tubes. About 1 ⁇ g of plasmid DNA was added to the cells. The cells were frozen in liquid nitrogen. The cells were thawed by incubating the test tube in a 37°C water bath for 5 min. 1 ml of YEP medium was added to the tube and incubated at 28°C for 2-4 h with gentle shaking to allow the bacteria to express the antibiotic resistance genes. The tubes were centrifuged for 30 s and the supernatant solution was discarded. The cells were resuspended in 0.1 ml YEP medium. The cells were plated on a YEP agar plate containing selection antibiotic(s) and incubated at 28 °C. Transformed colonies appeared in 2-3 days.
  • Agrobacterium tumefaciens strain LBA 4404 containing the preselected binary vector was used. Explants were placed in ⁇ 10ml of overnight grown Agrobacterium culture for 30 min. Leaf explants were then blotted on filter paper and placed abaxial side down on MS 104 media for 4 days. Explants were then rinsed three times in sterile water, blotted on filter paper, and placed on MS 104 media for regeneration with 300 mg/L kanamycin and 400 mg/L carbenicillin. Explants were transferred to fresh MS 104 media with 300 mg/L kanamycin and 400 mg/L carbenicillin every 10-14 days until plantlets developed. Plantlets typically developed at 31-50 days. Plantlets were cut from the callus and placed on MST media plus 300 mg/L kanamycin and 400 mg/L carbenicillin to induce rooting. Rooted plants were transferred to soil.
  • MS104 included 4.3 g MS salt mixture, 1.0 ml B5 vitamin solution, 30 g sucrose, 1.0 mg BA, 0.1 mg NAA , and 8.0 g Phytagar. The media was adjusted to pH 5.8 and autoclaved. 100 ml of YEP included 1.0 g Bacto-peptone, 1.0 g Bacto-yeast extract, 0.5 g NaCl. The media was autoclaved.
  • RNA Analysis Total RNA extraction, denaturing 1% agarose gel electrophoresis and
  • Northern blot hybridization was performed by known methods, such as that performed in Rasochova and Miller (1996). Each lane was loaded with equal amounts (approx. 5 ⁇ g) of total RNA as determined by spectrophotometry and confirmed by ethidium bromide staining of ribosomal RNA before Northern blot hybridization. 1 X 10 6 cpm/ml of radioactive probe in hybridization buffer was used per hybridization experiment.
  • Figure 13b shows the successful expression of BMV la and 2a mRNA in transgenic N. benthamiana.
  • Microcarrier Sterilization of Microcarriers 80 mg of gold microcarriers were resuspended in 1 ml of 70% ethanol, soaked for 15 min., and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Particles were resuspended in 1 ml of sterile distilled, deionized water and centrifuged at 13,000 x g for 5 min. The supernatant was carefully removed and discarded. Water washing of particles was repeated 2 more times. After final rinse, particles were resuspended in 1 ml of sterile 50% glycerol.
  • Coating Microcarriers with DNA To the 50 ⁇ l of particles the following was sequentially and quickly added: 5 ⁇ l DNA (l ⁇ g/ ⁇ l), 50 ⁇ l of 2.5M CaCl 2 , and 20 ⁇ l of 0.1 M Spermidine.
  • the mixture was incubated for 10 min. on a vortex shaker at room temperature. Particles were pelleted by centrifugation at 13,000 x g for 5 sec. Supernatant was carefully removed and discarded. Particles were resuspended in 140 ⁇ l of 70% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discarded. Particles were resuspended in 140 ⁇ l of 100% ethanol and centrifuged at 13,000 x g for 5 sec. Supernatant was removed and discarded. Particles were resuspended in 50 ⁇ l of 100% ethanol.
  • RNA Analysis Total RNA extraction, denaturing 1% agarose gel electrophoresis and
  • Transgenic N. tabacum and N. benthaniana plants were produced according to the procedures discussed above.
  • the plants were transfected with a DNA-launching platform containing a GUS gene (Figure 5a) by particle bombardment as described in Examples 5 and 7.
  • the plants were incubated for 3-5 days and then assayed for ⁇ -glucuronidase (GUS) activity using 1 mg/ml X-Gluc (5-bromo-4-chloro-3-indolyl glucucuronide) as substrate in 0.1M potassium phosphate buffer, pH 7.0, 50 ⁇ M potassium ferrocyanide, and 2% Triton® X-100.
  • GUS ⁇ -glucuronidase
  • RNA3 derivatives Following an overnight incubation at 37°C, cells replicating launched RNA3 derivatives and expressing the GUS reporter gene from a subgenomic RNA4 gave rise to blue spots ( Figure 15).
  • the launched RNA3 derivative did not replicate and express GUS reporter gene in the absence of BMV RNA replication factors la and 2a (e.g., in wt N. benthamiana and in wt N. tabacum).
  • Example 9 Transfection of Transgenic Plants Expressing BMV la. 2a. 3 a. and CP
  • a plant is transformed with BMV la, 2a, 3a, and CP genes whereby those genes are stably expressed in said plant. This can be done with the procedures outlined above. Any modifications that would be needed would be readily apparent to those skilled in the art in light of the teachings contained herein.
  • a DNA-launching platform encoding an RNA replicon which contains a foreign gene and necessary BMV or CCMV cis-acting replication signals to replicate said replicon is constructed ( Figure 10b).
  • Foreign genes to be included in said replicon could include, for example, a Bacillus thuringiensis polynucleotide that codes for a B.t. protein. Other sequences would include, e.g., sequences that encode herbicide resistance, or any other known sequence that encodes peptides or proteins having desired qualities in plants.
  • plants can be transformed to express BMV la, 2a, 3a, and a TMV coat protein in place of the BMV coat protein.
  • a DNA-launching platform is then made containing one or more foreign genes and the necessary cis-acting replication signals, either BMV or CCMV, and a TMV origin of assembly ( Figures 8a, 8b, and 10a).
  • This launching platform provides a distinct advantage as TMV is a rod-shaped virus which has no strict limit on the size of RNA that can be encapsidated.
  • TMV movement protein can be used in place of BMV3a ( Figure 7c). Hybrids between tobamo and bromoviruses were shown to be viable (Sacher et al, 1988; De Jong and Ahlquist, 1992).
  • CCMV subgenomic promoter can be substituted for BMV sequences in a desired DNA-launching platform. Because the sequence of CCMV subgenomic promoter differs from the sequence of BMV subgenomic promoter, the probability of recombination that would result in loss of a foreign gene is much lower in a construct having a combination of these two different promoters.
  • trans-acting components may include, but are not limited to, replication factors, components responsible for cell to cell movement, or components such as the coat protein which may be required for long distance spread, viral proteases responsible for post translational processing, or other known trans-acting functions.
  • BMV RNA3 derivatives contained the GUS gene in place of the coat protein ORF ( Figure 5a) (these were inoculated with or without coat protein expression plasmid, Figure 5b), or had the BMVCP gene translated from an additional subgenomic RNA driven from BMV or CCMV subgenomic promoter ( Figures 5c and 5d), or had the SHMV coat protein translated from an additional BMV subgenomic RNA ( Figure 7b).
  • Protoplasts were collected by centrifugation (800 rpm, 5 min.) 24 hours post inoculation.
  • the chemiluminescent GUS assay was performed using GUS-LightTM (Tropix, MA) according to manufacturer's instructions. Protein concentrations were determined using the Bio-Rad protein kit (Bio-Rad Laboratories, Hercules, CA). The GUS values, determined by luminometer, were adjusted to the same total protein concentration.
  • Figures 16a and 16b show successful GUS expression in protoplasts in the presence of trans-acting BMV replication factors la and 2a.
  • N. tabacum protoplasts isolated by using the above described methods were transfected by electroporation with expression plasmids for trans-acting BMV replication factors 1 a and 2a and with DNA-launching platforms for RNA3 derivatives having the GFP gene in place of BMV coat protein ORF (Figure 6e), the CP gene translated from an additional subgenomic RNA ( Figure 6a) or with an RNA transcript having the GFP expressed as a fusion protein with BMV 3a ORF ( Figure 6d).
  • Protoplasts were incubated for 24 hrs and examined for GFP expression using a fluorescent microscope.
  • Figure 18 shows the successful expression of GFP in protoplasts.
  • Example 12 Transfection of (la + 2a)-Transgenic Plants with BMV RNA3-Based DNA- Launching Platform Containing GFP
  • N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 having the GFP gene in place of BMV coat protein (Figure 6e).
  • the GFP expression was determined 24 hrs post inoculation using a fluorescent microscope.
  • Figure 17 shows the successful expression of GFP in (la + 2a)- transgenic N. benthamiana.
  • Example 13 Transfection of (la + 2aVTransgenic N. benthamiana with BMV RNA3 DNA- Launching Platform Using Agrobacterium
  • N. benthamiana plants were inoculated with BMV RNA3 DNA-launching platform using Agrobacterium tumefaciens. Once the desired construct (pB3LR42) was obtained in E. coli it was transferred to A. tumefaciens strain LBA4404 using a thaw-freeze method as described above. The Agrobacterium was grown overnight in 28 °C under constant shaking. A single lower leaf of N. benthamiana were punctured with a needle multiple times and submerged in Agrobacterium culture. The plants were grown at 23 °C with a 16 hr photoperiod. The inoculated leaves were harvested 14 days post-inoculation. The total RNA extraction and northern blot hybridization were performed as described above. Figure 19 shows replication of launched BMV RNA3 in inoculated (la + 2a)-transgenic N. benthamiana.
  • Example 14 Transfection of (la + 2aVTransgenic Plants with BMV RNA3-Based DNA- Launching Platform Containing GUS and SHMV Coat Protein
  • N. benthamiana plants were transfected using a particle bombardment as described above with a DNA-launching platform for BMV RNA3 wherein the BMV coat protein was replaced with the SHMV coat protein (Sunn-hemp mosaic virus) and the GUS gene was inserted downstream of an additional BMV subgenomic promoter (Figure 7b).
  • the GUS expression was determined by histochemical GUS assay described above.
  • Figure 20 shows the successful expression of GUS in (la + 2a)-transgenic plants.
  • Example 15 Movement of Launched BMV RNA 3
  • FI progeny plants from self-fertilized (la+2a)-transgenic N. benthamiana BP14 were inoculated with BMV RNA3 DNA launching platform using Agrobacterium tumefaciens. Seedlings were germinated on Smurf media containing Kanamycin. Plants were grown at 23 °C with a 16 hr photoperiod. Once the desired construct (pB3LR42) was obtained in E. coli it was transferred to A. tumefaciens strain LBA4404 using a thaw-freeze method as described above. The Agrobacterium was grown overnight at 28 °C under constant shaking. A single lower leaf of N.
  • benthamiana was punctured with a needle multiple times and submerged in Agrobacterium culture. The inoculated, middle, and upper leaves were harvested 14 days post- inoculation. Total RNA extraction and northern blot hybridization were performed as described above. RNA3 replication was detected in all leaves tested (Fig. 21). It shows that BMV RNA3 is able to replicate, move cell-to-cell and spread long distance in (la+2a)-transgenic plants.
  • Example 16 Transfection of Progeny From ( 1 a+2a -Transgenic N. benthamiana With BMV R ⁇ A3 DNA-Launching Platform
  • Progeny plants from self-fertilized (la+2a)-transgenic N. benthamiana (designated BP14) were inoculated with BMV RNA3 DNA-launching platform using Agrobacterium as described in Example 13.
  • Control plants (non-transgenic N. benthamiana) were inoculated with the sap from BMV infected barley using inoculation buffer composed of 50mM NaP0 4 , pH7.0, and 1% celite. Root samples were harvested 6 weeks post inoculation. RNA extraction and northern blot hybridization were performed as described above.
  • Figure 22 shows that BMV RNA3 replicated to very high levels in roots.
  • BSMV Barley stripe mosaic virus
  • RNA alpha, beta, and gamma tripartite genome
  • These genomic RNAs have an m7Gppp cap at the 5' end and a t-RNA like structure at the 3' end (Jackson and Hunter, 1989).
  • a DNA-launching plasmid for BSMV RNA alpha, RNA beta, and RNA gamma containing BSMV RNA cDNA is constructed by precisely fusing at its 5' end to a DNA- dependent RNA polymerase promoter and to a self-cleaving ribozyme at its 3' end.
  • a polyadenylation signal may be also included.
  • a convenient restriction site may be engineered at the 3' end of viral cDNAs.
  • Foreign genes or sequences may be expressed in several ways. For example, DNA-launching plasmids based on BSMV RNA beta may contain a foreign gene or sequence expressed in place of ORF beta a.
  • Transgenic plants having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator are obtained.
  • Such trans-acting factors may include parts of the viral RNA replicase (ORFs alpha a and/or gamma a) or other trans-acting factors.
  • the trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • Cis-acting sequences necessary for BSMV RNA replication are removed from transgenes.
  • the full-length RNA alpha is expressed from the chromosome.
  • ORF gamma a including the 5' untranslated region and ORF gamma b from a seed transmitted strain, such as ND18, are also expressed (Edwards, 1995).
  • a DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5' end of the BSMV RNA beta, cis-acting elements important for BSMV RNA beta life cycle, such as the 5' and 3' ends, the intercistronic region between the beta a and beta b ORFs (Zhou and Jackson, 1996) and a foreign gene or sequence in place of ORF beta a (coat protein) which is dispensable for BSMV replication and movement (Petty and Jackson, 1990).
  • DNA-launching plasmids may lack the internal poly(A) region as this region is dispensable for replication and contain a ribozyme or a convenient restriction site at the 3' end of the modified viral RNA.
  • a DNA-launching plasmid is constructed from RNA gamma in which ORFs gamma a and/or gamma b are replaced with foreign genes or sequences which may also include the triple gene block genes (ORFs beta b, beta c, and beta d) or a heterologous movement protein (TMV 30K, RCNMV 35K).
  • TMV Tobacco mosaic virus
  • TMV has a single-stranded positive sense RNA genome.
  • the 5' end has an m7Gppp cap and the 3 ' end contains a t-RNA like structure.
  • a DNA-launching plasmid is constructed based on TMV RNA containing TMV cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and at its 3' end to a self-cleaving ribozyme.
  • a polyadenylation signal may be also included.
  • a convenient restriction site may be engineered at the 3 ' end.
  • Foreign gene may be expressed from an additional subgenomic RNA by including an additional subgenomic RNA promoter on the (-) strand.
  • Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
  • Such factors may include the viral replicase (126K/183K), movement protein (30K), or coat protein (17.6K).
  • At least one cis- acting sequence necessary for TMV RNA replication is removed from transgenes.
  • the transacting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • a DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5 ' end of the TMV cDNA, cis-acting elements important for the TMV life cycle, such as the 5 ' and 3 ' ends, origin of assembly, etc., at least one foreign gene or sequence in place of the trans-acting factor that is expressed from the chromosome, and a ribozyme or a convenient restriction site at the 3' end.
  • the foreign gene sequence can be expressed from an additional subgenomic RNA promoter and the sequence coding for the trans-acting factor that is expressed from the transgene can be deleted from the DNA-launching plasmid.
  • the DNA-launching plasmid will have a deletion of nucleotides 3420-4902, which appears to be a region that inhibits replication in trans. (Lewandowski et al, 1998).
  • PVX Potato virus X
  • the 5 ' end has an m7Gppp cap and the 3' end is polyadenylated.
  • a full-length cDNA clone of PVX has been constructed and infectious RNA transcripts obtained (Hemenway et al, 1990).
  • a DNA-launching plasmid is constructed based on PVX RNA containing PVX cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and having a polyadenylation site at its 3 ' end.
  • a convenient restriction site may also be included at the 3 ' end.
  • a foreign gene may be expressed from an additional subgenomic RNA.
  • Transgenic plants are obtained having one or more trans-acting factors fused to the
  • DNA-dependent RNA polymerase promoter and terminator may include the viral
  • RNA polymerase gene ORF1-147K
  • coat protein ORF5-21K
  • triple gene block ORF2- 25K, ORF3-12K, ORF4-8K
  • the triple gene block genes can be expressed individually. Alternatively, they can be expressed as negative sense transcripts from which plus sense subgenomic RNA for ORFs 2, 3, and 4 can be transcribed by the viral replicase.
  • Such transgene will have a DNA-dependent RNA polymerase promoter fused to sequence of ORFs 2, 3, and 4 in the minus sense orientation and the transcribed sequence will include a subgenomic RNA promoter.
  • At least one cis-acting sequence necessary for PVX RNA replication is removed from transgenes.
  • the trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • a DNA-launching plasmid is constructed containing the DNA-dependent RNA polymerase promoter precisely fused to the 5 ' end of the PVX genome, cis-acting elements important for PVX life cycle, such as the 5' and 3 ' ends, origin of assembly, etc., at least one foreign gene or sequence in place of the trans-acting factor that is expressed from the chromosome and a polyadenylation signal.
  • the foreign gene sequence can be expressed from an additional subgenomic RNA promoter and the sequence coding for the trans- acting factor that is expressed transgenically can be deleted from the DNA-launching plasmid.
  • a DNA-launching plasmid is constructed having a DNA-dependent RNA polymerase promoter, polyadenylation site, and the PVX cDNA sequence in which the ORF2
  • DNA- launching plasmid is inoculated to transgenic plants expressing movement protein from heterologous virus, such as tobacco mosaic virus (TMV 30K), tomato mosaic virus (ToMV 30K), or red clover necrotic mosaic virus (RCNMV 35K).
  • TMV 30K tobacco mosaic virus
  • ToMV 30K tomato mosaic virus
  • RPNMV 35K red clover necrotic mosaic virus
  • FHV Flock House Virus Flock house virus
  • RNA1 encodes protein A, involved in RNA replication, and protein B that is translated from sg RNA3 and is dispensable for RNA replication.
  • RNA2 encodes virion capsid precursor protein alpha.
  • FHV is infectious to insect, plant, mammalian, and yeast cells (Selling et al, 1990; Price et al, 1996).
  • a DNA-launching plasmid is constructed for FHV RNA1 and RNA2 containing FHV
  • RNA cDNA precisely fused at its 5 ' end to a DNA-dependent RNA polymerase promoter and at its 3' end to a self-cleaving ribozyme.
  • a polyadenylation signal may be also included.
  • a convenient restriction site may be engineered at the 3' end.
  • Foreign genes or sequences may be expressed in several ways. For example, DNA-launching plasmids based on FHV RNA1 may contain a foreign gene or sequence expressed from subgenomic RNA3 as ORF B replacement or as a translational fusion with ORF B. Alternatively, a foreign gene may be expressed from an additional sg RNA.
  • DNA-launching plasmids based on FHV RNA2 may contain a foreign gene(s) or sequence(s) expressed as a part of polyprotein alpha. Foreign gene(s) in such construct may include sequences necessary for polyprotein clevage.
  • DNA- launching plasmids will preferably also express a movement protein of a heterologous plant virus, such as 30K of TMV or 35K of RCNMV. Alternatively, DNA-launching plasmids will be inoculated onto transgenic plants expressing such movement protein.
  • Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator.
  • Such factors may include protein A or capsid protein precursor alpha, and preferably will also include a movement protein from a plant virus, such as 30K of TMV or 35K of RCNMV.
  • Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • Transgenically expressed trans-acting factors preferably lack at least one cis-acting factor which is necessary for their replication, such as the 5 ' and/or 3 ' end.
  • a DNA-launching plasmid is constructed based on FHV RNAl or FHV RNA2 containing a DNA-dependent RNA polymerase promoter precisely fused to the 5' end of RNAl (or RNA2), cis-acting elements important for FHV RNAl (or RNA2) replication, such as the 5 ' and 3' ends, at least one foreign gene or sequence and a self-cleaving ribozyme at the 3' end. Polyadenylation signal may also be included. Alternatively, a convenient restriction site may be engineered at the 3' end of the modified viral RNA sequence of the DNA-launching plasmid.
  • DNA-launching plasmids based on FHV RNAl may contain a foreign gene or sequence in place of ORF A.
  • the ORF A may be deleted and the foreign gene may be expressed from subgenomic RNA3, for example as an ORF B replacement or as a translational fusion with ORF B.
  • DNA-launching plasmid may contain two exogenous RNA sequences, one in the place of ORF A and the other expressed from the subgenomic RNA3.
  • DNA- launching plasmids based on FHV RNA2 may contain a foreign gene(s) or sequence(s) in place of ORF alpha or expressed as a part of polyprotein alpha. Foreign gene(s) in such a construct may include sequences necessary for polyprotein clevage.
  • Tomato spotted wild virus is a tripartite (RNA L, M, S), negative sense and ambisense, single stranded RNA virus.
  • Transgenic plants are obtained having one or more trans-acting factors fused to the DNA-dependent RNA polymerase promoter and terminator. Such factors include the putative TSWV polymerase gene (ORF L), ORF N, and possibly other trans-acting factors (NSm or NSs). At least one cis-acting sequence, such as 5' and/or 3' ends, which are necessary for TSWV RNA replication are removed from the transgene.
  • Trans-acting factors are stably expressed in the plant cell or their expression may be induced if an inducible promoter is used.
  • a DNA-launching plasmid is constructed based on TSWV RNA M in which the Gl and G2 coding sequences are replaced with at least one foreign gene or sequence.
  • Such DNA- launching plasmid contains a DNA-dependent RNA polymerase promoter and TSWV RNA M cDNA fused to the self-cleaving ribozymes at the 5' and 3' ends.
  • a DNA- launching plasmid is constructed based on TSWV RNA S in which the N coding region is replaced with a foreign gene or sequence.
  • Example 22 Barley Mild Mosaic Virus Genome of barley mild mosaic virus (BaMMV) consists of two positive sense, single- stranded, 3'-polyadenylated RNAs.
  • the RNAl encodes proteins related to the potyviral P3, 6K1, Cl, 6K2, NIa-VPg, NIa-Pro, Nib and capsid protein (Kashiwazaki etal, 1990).
  • the RNA2 encodes PI and P2 protein (Kashiwazaki et al, 1991).
  • the PI protein is related to the potyviral HC-Pro and the P2 protein is important for fungal transmission.
  • An isolate was obtained containing a deletion in the P2 protein (Timpe and Kuhne, 1995) thus indicating that P2 is dispensable for viral RNA replication.
  • a DNA-launching plasmid is constructed for BaMMV RNAl and RNA2 containing BaMMV RNA cDNA precisely fused at its 5' end to a DNA-dependent RNA polymerase promoter and a polyadenylation site at its 3' end.
  • Foreign genes or sequences may be expressed in several ways.
  • DNA-launching plasmids based on BaMMV RNA2 may contain a foreign gene or sequence expressed as a part of polyprotein which can be cleaved and a foreign protein can be released.
  • Transgenic plants are obtained having the BaMMV RNAl cDNA lacking the 5' and 3' ends fused to the DNA-dependent RNA polymerase promoter and terminator.
  • a DNA-launching plasmid is constructed based on BaMMV (isolate M) RNA2.
  • Such plasmid contains a DNA-dependent RNA polymerase promoter precisely fused to the 5' end of RNA2, RNA2 cis-acting replication signals located in the 5' and 3' ends, PI ORF and a foreign gene in place of P2 ORF or expressed as a part of P1 P2 polyprotein which can be cleaved and a foreign protein can be released.

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Abstract

L'invention concerne de nouvelles méthodes et de nouveaux matériels destinés à la transformation d'une cellule hôte et à l'expression dans celle-ci d'ARN exogène. Spécifiquement, l'invention concerne des plates-formes de lancement d'ADN utilisées pour introduire un segment viral réplicant fixé à un polynucléotide exogène dans une cellule, de manière que le polynucléotide exogène soit exprimé dans ladite cellule et confère un trait détectable.
EP99953356A 1998-05-22 1999-05-21 Methodes et materiels ameliores de transformation Ceased EP1086237A2 (fr)

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US8652698P 1998-05-22 1998-05-22
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GB0007231D0 (en) * 2000-03-24 2000-05-17 Chiron Spa Modified rna for gene delivery
US6800748B2 (en) 2001-01-25 2004-10-05 Large Scale Biology Corporation Cytoplasmic inhibition of gene expression and expression of a foreign protein in a monocot plant by a plant viral vector
AU2004209660A1 (en) 2003-02-03 2004-08-19 Fraunhofer Usa, Inc. System for expression of genes in plants
WO2005033306A1 (fr) 2003-10-01 2005-04-14 Japan Science And Technology Agency Cellule transformee, procede de production de proteine faisant intervenir cette cellule, kit de production de proteine, fragment adn permettant d'exprimer un vecteur viral, utilisation de celui-ci, procede de production de transformant pour la production de proteine, transformant pour la production de proteine obtenu par le

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CA1288073C (fr) * 1985-03-07 1991-08-27 Paul G. Ahlquist Vecteur de transformation de l'arn
WO1990012107A1 (fr) * 1989-03-31 1990-10-18 The Salk Institute Biotechnology/Industrial Associates, Inc. Systeme d'expression par recombinaison base sur le virus de la mosaique du tabac satellitaire
JPH04121200A (ja) * 1990-09-07 1992-04-22 Nippon Nohyaku Co Ltd 植物細胞におけるポリペプチドの製造法
CA2158937C (fr) * 1993-09-15 2006-01-03 Thomas W. Dubensky, Jr. Vecteurs d'alphavirus recombinant
GB9703146D0 (en) * 1997-02-14 1997-04-02 Innes John Centre Innov Ltd Methods and means for gene silencing in transgenic plants

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WO1999061597A3 (fr) 2000-03-30
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JP2002516089A (ja) 2002-06-04
CA2329509A1 (fr) 1999-12-02
BR9911065A (pt) 2001-02-06
AR020324A1 (es) 2002-05-08
WO1999061597A9 (fr) 2000-02-24
WO1999061597A2 (fr) 1999-12-02
AU4310199A (en) 1999-12-13

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