MXPA00003537A - Binary viral expression system in plants - Google Patents

Abstract

This invention relates to a regulated binary plant viral expression system. It is comprised of two chromosomally-integrated components. One component is a proreplicon, which contains i(cis)-acting viral sequences required for replication and a contains a target gene. The other component is a chimeric i(trans)-acting replication gene comprising a regulated promoter operably-linked to the coding region for a viral replication protein. The proreplicon lacks the replication gene essential for replicon replication, and thus cannot undergo autonomous episomal replication. However, regulated expression of the i(trans)-acting replication protein in plant cells also containing the proreplicon will trigger the release of free replicon from the integrated proreplicon, result in its episomal replication i(in trans), and result in the expression of the target gene, if present, through gene amplification. The expression system is useful for both production of foreign proteins as well as silencing endogenous genes and transgenes in plant tissue. Tissue-specific expression is controlled by the choice of promoter controlling the transcription of the i(trans)-acting replication gene.

Description

SYSTEM OF BIRTH VIRAL EXPRESSION IN PLANTS FIELD OF THE INVENTION The present invention relates to the field of molecular biology and to the genetic transformation of plants with foreign or different gene fragments. More particularly, this invention relates to a heritable system of viral expression in plants, used for the expression of transgenes in plants.

BACKGROUND OF THE INVENTION The transgenic work of plants is surrounded by low and inconsistent levels of transgenic expression. Episomal vectors are expected to overcome these problems. In microbes, episomal vectors (plasmids) are possible, because these vectors can be maintained by selection. Although plant viruses have been used as episomal expression vectors, their use has been restricted to expression REF: 32891, due to lack of selection and / or cellular toxicity (U.S. Patent No. 4,855,237, WO 9534668).

Plant Virus Viruses are infectious agents with a relatively simple organization and unique modes of replication. A virus of a given plant can contain either RNA or DNA, which can be either single-stranded or double-stranded. Rice dwarf virus (RDV) and lesion-causing tumor virus (WTV) "are examples of double-stranded RNA plant viruses, and single-stranded RNA plant viruses include tobacco mosaic virus. (TMV), turnip yellow mosaic virus (TYMV), rice necrosis virus (RNV), and brome mosaic virus (BMV) RNA in single-stranded RNA virus can be either , a positive (+) or negative thread (-) Although many plant viruses have RNA genomes, the organization of genetic information differs between groups. The genome of the majority of RNA viruses of monopartite plants is a molecule of a single strand of sense (+). There are at least 11 major groups of viruses with this type of genome. An example of this type of virus is TMV. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two different RNA molecules, of a single strand of sense (+) encapsulated in separate particles. Both RNAs are required for their infectious activity. The cowpea mosaic virus (or chickpea species) (CPMW) is an example of a bipartite plant virus. A third major group, containing at least six major types of plant viruses, is tripartite, with three RNA molecules of a single strand of sense (+). Each strand is encapsulated separately, and all three are required for their infectious activity. An example of a tripartite plant viurus is the alfalfa mosaic virus (AMV). Many plant viruses also have smaller subgenomic ARNAs, which are synthesized to amplify a specific gene product. Plant viruses with double-stranded DNA genomes include Cauliflower Mosaic Virus (CaMV).

Geminivirus Plant viruses with single-stranded DNA genomes are represented by geminivirus and include the African Yucca Mosaic Virus (ACMV), the Tomato Golden Mosaic Virus (TGMV), and the Striped Maize Virus (MVS). ). Geminiviruses are subdivided on the basis of whether they infect monocots or dicots, and whether their vector insect is a hopper or a whitefly. He Subgroup I of geminiviruses are transmitted by leaflets that infect monocotyledonous plants (for example, the wheat dwarf virus), the subgroup II of geminivirus are transmitted by leaflets that infect monocotyledonous plants (for example, the Virus on the Top of the Beet Enchinamiento), and the subgroup III of geminivirus is transmitted by the whitefly, which infects dicotyledonous plants (for example, the Tomato Golden Mosaic Virus, TGMV, and the African Yucca Mosaic Virus, ACMV). He Subgroup I and II of geminivirus have a single genome (monopartite). Subgroup III of geminivirus has a bipartite genome. For example, TGMV and ACMV that consist of two DNA genomes of a single strand, circular, A and B of approximately, 2.8 kB each in size. The DNA of the A genome and the DNA of the B genome of a given Subgroup III virus have little sequence similarity, except for an almost identical common region of approximately 200 bp. While both DNA genome A and DNA genome B are required for infection, only the DNA of the A genome is necessary and sufficient for replication, and the DNA of the B genome encodes the functions required for the movement of the virus through the plant. infected In both TGMV and ACMV, DNA A contains four open reading structures (ORFs) that are expressed in a bidirectional manner and arranged in a similar manner. The ORFs are named according to their orientation relative to the common region, that is, complementary (C) against viral (V) in MCA and to the left (L) or to the right (R) in TGMV. Thus, the ORFs AL1, AL2, AL3 and AR1 of TGMV are homologous to AC1, AC2, AC3 and AVI, respectively, of ACMV. Three major transcripts in the ACMV DNA A have been identified and these mimic the AVI and AC1 ORFs separately and the AC2 / AC3 ORFs together. There is experimental evidence for the function of these ORFs. Thus, in ACMV AC1 encodes a replication protein that is' essential and sufficient for replication, AC2 is required by transactivation of the coat protein gene, AC3 encodes a protein that is not essential for replication, but increases the accumulation of Viral DNA, and AVI is the coat protein gene. Except for the essential viral replication protein (coding for AC1 and AL1 in ACMV and TGMV, respectively), the replication of geminivirus is transmitted in the host's replication and transcription machinery. Although gemniviruses are DNA viruses from single-stranded plants, they are replicated via the double-stranded DNA intermediate by "rolling circle replication".

Viruses as Expression Vectors The construction of plant viruses to introduce and express foreign non-viral genes in plants has been demonstrated (US Patent No. 4,855,237, WO 9534668). When the virus is a DNA virus, the constructions can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid for easy construction of the desired viral vector with the foreign DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into the plasmid. The plasmid is then used to make all constructions. The RNA virus is then produced by transcription of the viral sequence of the plasmid and translation of the viral genes to produce the protein or coat proteins, which encapsulate the viral RNA. Alternatively, the cDNA can be cloned behind a heterologous plant promoter, introduced into a plant cell, and used to transcribe the viral RNA that can be replicated autonomously [Sablowski et al. (1995) Proc. 'Na you. Acad. Sci. USA vol 92, pp 6901-6905]. Geminiviruses have many advantages as potential vectors of expression in plants. These include 1) replication for high numbers of copies asymptomatically, 2) few well-characterized genomes, 3) assembly into nucleosomes, and 4) nuclear transcription. The DNA A component of these viruses is capable of autonomous replication in the cells of plants, in the absence of B DNA. Vectors have been developed in which the ORF of the coat protein has been replaced by a heterologous coding sequence, and the heterologous coding sequence expressed from the coat protein promoter [Hayes et al. (1989) Nucl ei c Aci ds Res. Vol. 17, pp. 2391-403; Hayes et al. (1988) Na t ure (London) vol. 334, pp. 179-82]. The larger full-length copies of the native type of the TGMV genomes A and B were transformed into petunia [Rogers et al. (1986) Cell (Cambridge, Mass) vol. 45, pp. 593-600]. Replication was reported in the primary transformants and in some of the progenies themselves consistent with their Mendelian inheritance, indicating that the chromosomal non-integrated master copy, to the replicon, is hereditary. This suggests that the gametophyte and / or the development of seed tissues lacks the capacity to support replication. The report did not show whether the virus replicated in the seed tissue did not germinate. The prior art shows that geminiviruses are not transmitted in the seed in nature [Goodman, (1981) J. Gen. I saw role. Vol. 54, pp. 9-21]. Thus, there is no evidence that they can replicate in the gametophytic tissue or develop in the seed. The DNA A of the Tomato Golden Mosaic Virus (TGMV), modified by replacing its sequence that encodes the protein of the envelope with that of the NPT II or GUS reporter genes or with that of the 35S: NPT II gene and a full-length copy greater than that of modified viruses, was transformed into tobacco [Hayes et al. (1989) Nucl ei c Aci ds Res. Vol. 17, pp. 2391-403; Hayes et al. (1986) Na t ure (London) vol. 334, pp. 179-82]. The leaves of the transgenic plants showed that the high levels of the reporter enzymes were copies dependent on the number. However, the replication of vector expression and the reporter gene was not reported in the seeds and the genetic stability of the vector in the transgenic plants in subsequent generations was not reported. The use of the African Yucca Mosaic Virus (ACMV) in a similar way has not been reported and it is unknown that ACMV DNA or protein or replication proteins can be stably maintained in the progeny of plants if they can replicate in the tissues of the seed. In one report, the chimeric gene in which the constitutive promoter of the 35S plant, fused to the TGMV sequence containing ORFs AL1, AL2, and AL3, was transformed into Ni coti ana ben thami ana. The different transgenic lines did not show significant uniformity in the expression levels of the 35S: ALl-3 gene as well as their capacity to complement viral replication [Hanley-Bowdoin et al. (1989) Pl an t Cell vol. 1, pp. 1057-67]. In another report, the chimeric genes in which the constitutive promoter of the plant, 35S, was fused to the coding sequence of the AL1 protein of TGMV replication, were transformed into tobacco. The expression of the TGMV replication protein in the primary transformants, supported the replication of a mutant A genome, lacking the replication protein [Hanley-Bowdoin et al. (1990) Proc. Na ti. Acad. Sci. USES. vol. 87, pp. 1446-50], however, in none of both reports, the genetic stability of the chimeric replication protein gene was reported through subsequent generations or its ability to support viral replication in the seed tissue. In another report, the chimeric genes in which the constitutive promoter of the plant, 3S5, separately fused to the coding sequences of the replication proteins AL1, AL2, and AL3 of TGMV, was transformed into tobacco [Hayes et al. (1989) Nucl ei c Aci ds Res. Vol. 17, pp. 10213-22]. The TGMV replication protein was shown to have been expressed in the progeny, but the genetic stability of the chimeric replication protein gene was not reported through the subsequent generations. Furthermore, it was not reported whether the transgenic plants would support replication in the tissue of the cells. In another description, Roger et al. (EP 221044) demonstrates the expression of foreign proteins in the plant tissue using a modified "A" genome of the TGMV geminivirus. The foreign gene was inserted in place of the gene encoding the viral coat protein and the resulting plasmid was transformed into the tissue of the plant. Roger et al. did not report the specific expression of the foreign protein tissue and are silent for the genetic stability of the transforming plasmid. All viral vectors reported have a greater disadvantage. Any of them did not appear to be stably maintained in the transgenic plants and / or are not practically employed. A) Yes, despite intensive efforts to develop plant vectors and viruses, recombinant vectors based on commercially used plant viruses, which are heritable and capable of episomal expression and replication in desired tissue or tissues of the transgenic host plant, have not been developed. without the need of infection to the whole generation. In effect, the replication of plant viruses is expected to be detrimental to the growth and development of plant cells. For example, when a full-length copy larger than the TGMV A genome is introduced into a one-tenth plant cell like many transgenic plants obtained when the B genome is used or when the control transformations are made [Roger et al. . (1986) Cell (Cambridge, Mass.) Vol. 45, pp-600]. The authors suggest that this may be due to the expression of a gene in TGMV DNA A. In addition, the crude extract of the plants that express copies one after the other of genomes whether TGMV A and TGMV B are unable to infect the plants of Ni coti ana benthami ana. This is consistent for having a low virus titrator. Thus, transgenic plants that regenerate, could be selected by low level of expression of a toxic viral gene product and low level of viral replication. This is also consistent with the authors' findings that relatively few cells initiate virus release, a conclusion based on their observation that most tissues remain viable and non-symptomatic. Similarly, the sparse replication in transgenic plants containing 35S: replication protein in other reporters suggests that the plants are either selected for the poor expression of the replication protein, presumably because of their toxicity, or that the profiles tissue-specific expression of the replication gene are different from those of viral replication. Recently, it has been reported that 6 of 11 transgenic tobacco plants stably transformed with a nomopartite geminivirus (Yellow Tobacco Dwarf Virus) with a functional replication gene, showed episomal replication [Needham et al. (1998) Pl ant Cell -Rep. Vol. 17, pp. 631-639]. Genes and inactive endogenous transgenes are an important technology [see Senior et al. (1998) Bi otechnol. Genetl Eng. Rev. Vol. 15, pp. 79-119; Thomas et al. (1998) Pl an t Growth Regul. Vol. 25, p. 205]. Inactivity of endogenous genes or transgenes by viral infection or by viruses stably incorporated in transgenic plants, has been reported by RNA virus [Baulcombe et al. PCT Int. Appl. (1998), Ruiz et al. (1998) Pl an t Cel l vol. 10, pp. 937-946], Geminivirus [Kjemtrup e t al. (1998) Pl an t. J. vol. 14, pp. 91-100, Atkison et al. Pl an t J. vol. 15, pp. 593-604], and Cauliflower Mosaic Virus [Al-Kaff et al.

Sci ence (Washington, D.C.) 279: 2113-2115 (1998)].

However, it has not been reported that the regulated virus induces inactivity in transgenic plants, which are expected to provide inactivity of the regulated gene. To date, there are no known recombinant virus-based vectors in plants, which are heritable and capable of episomal replication and expression of foreign proteins in tissue or tissues of a transgenic host plant, without the need for an infection in all of them. generation.

BRIEF DESCRIPTION OF THE INVENTION In one embodiment, the present invention provides a binary transgenic viral expression system for the replication and increased expression of a target gene comprising a) a heritable proreplicon lacking a functional replication gene for autonomous episomal replication, and comprising: ) Vi-cis-acting elements required for viral replication; ii) an objective gene comprising at least one suitable regulatory sequence; and iii) flanking sequences that allow excision or cleavage of the elements of (i) and (ii), and, b) an inheritable chimeric trans-acting replication gene, comprising a regulated plant promoter, operably linked to a Viral protein replication code sequence. In another embodiment, the present invention provides the binary transgenic viral expression system, described above but without a target gene, wherein the expression of the trans-acting replication gene in cells containing the proreplicon, results in the replication of the replicon without an objective gene. The expression system of the present invention is employed for the controlled replication of viral vectors in transgenic plants. Both system components are chromosomally integrated. One component is a chimeric trans-acting replication gene, in which the code sequence of the protein or replication proteins of the geminivirus is placed under the control of an induced and / or specific developed tissue or promoter. The second component is a proreplicon, which is unable to replicate itself, but does so in the presence of protein or viral replication proteins. The two components can be introduced together in a transgenic plant or brought together by cross-transgenic plants, which carry separate components. Also provided are methods for making the expression cassettes and methods of using them, to produce cells from transformed plants that have an altered genotype and / or phenotype. The main aspect of the invention is illustrated in Figure 1. Figure 1 illustrates a scheme for transactivating replication of in trans proreplicon. The regulated expression of a chromosomally integrated chimeric replication gene will result in the release and replication of the replicon from a chromosomally integrated master copy of the proreplicon. The different components of the invention are independently heritable and can be introduced together into a transgenic plant or taken together by crossing transgenic plants carrying the separated components, such as by the method to produce TopCross® high oil corn seed [Patent North American No. 5,704,160]. Methods for the production of expression cassettes and methods of using them to produce cells of transformed plants having an altered genotype and / or phenotype are also provided.

BRIEF DESCRIPTION OF THE FIGURES AND DESCRIPTIONS OF THE SEQUENCES Figure 1 illustrates the excision or cutting and activation of a proreplicon via the expression of a chimeric trans-acting replication gene.

The invention can be more fully understood from the following detailed description and the accompanying sequence listing. The sequence listings appended thereto comply with the governing rules of the amino acid and / or nucleotide sequence described in the patent applications as set forth in 37 C.F.R. §1.821-1.825. SEQ ID NOs: 1-14 refer to primers used in the Examples.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a regulated binary expression system that uses several genetic elements of a plant virus. The expression system is used for the replication and expression of the regulated replicon of target genes in plants, either to produce foreign proteins or to inactivate the genes of endogenous plants and particularly to achieve stable expression in terminally differentiated cells. The Applicant has resolved that the problem stated in providing a chromosomally integrated two-component viral expression system comprises a replicon and a trans-acting replication gene. The proreplicon contains the cis-acting viral sequences required for replication and a target gene under the control of suitable regulatory sequences. Proreplicon is incapable of self replication in plant cells, because it lacks a chimeric trans-activation replication gene. The second component of the system, a chimeric trans-acting replication gene, consists of a regulated promoter, operably linked to the code region for a viral replication protein. The expression of the replication protein results in the release and replication of the replicon from the proreplicon. Plant cells containing the proreplicon replicate the replicon only in the presence of the replication protein. Thus, the regulated expression of the chimeric replication gene in such cells results in a replication of the regulated replicon and amplification of the target gene.

Using the current system, the Applicant has demonstrated that (i) the tissues of corn and soybean seeds will support the replication of the geminivirus; Y (ii) that the expression system will effect the expression of target genes in transgenic plants. The present invention advances in the art, by providing viral vectors of plants (a) which are stably maintained in the chromosome of transgenic plants; (b) whose replication is controlled by the regulated expression of the replication proteins; and (c) they contain nucleic acid sequences that are either homologous to the genes or endogenous transgenes of the plants, or that encode the foreign proteins that can be produced in the transgenic plant. The following definitions are used to understand the meaning of the terms used in this description. The term "gene" refers to a fragment of nucleic acid that expresses mRNA, functional RNA, or a specific protein, which includes regulatory sequences. The term "native gene" refers to a gene as found naturally. The term "chimeric gene" refers to any gene that contains 1) regulatory and coding sequences that are not found together in nature, or 2) sequences that encode parts of proteins not naturally added, or 3) parts of promoters that they are not added naturally. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or that comprise regulatory sequences and coding sequences derived from the same source, but arranged in a different manner from those found in nature. A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. The transgenes can include, for example, genes that are either homologous or heterologous to the genes of a particular plant to be transformed. Additionally, transgenes can comprise native genes inserted in a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural site in the genome of an organism. The term "wild type" refers to a gene, virus, or normal organism, found in nature without any known mutation. The term "genome" refers to the complete genetic material of an organism. The term "coding sequence" refers to a DNA or RNA sequence that codes for a specific amino acid sequence and that excludes the non-coding sequences. The terms "open reading structure" and "ORF" refer to the amino acid sequence encoded between the initiation of translation and the stop codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to a unit of three adjacent nucleotides (? Codon ') in a sequence coding for the specific initiation and chain termination, respectively, of protein synthesis (mRNA translation). ). A "functional RNA" refers to an antisense RNA, ribozyme, or other RNA that is not translated. It can be derived from any part of a gene, including its open reading structure, the 5 'non-coding sequence, or the 3' non-coding sequence. The terms "regulatory sequences" or "suitable regulatory sequences" refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which includes the RNA transcription, processing or stability, or translation of the associated coding sequence.

Regulatory sequences include, enhancers, promoters, leader translation sequences, introns and polyadenylation signal sequences. They include natural and synthetic sequences as well as sequences which can be a combination of synthetic and natural sequences. The "non-coding sequence 5 '" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. It is presented in the fully processed mRNA, upstream of the initiation codon and can affect the processing of the primary transcript to mRNA, stability or translation efficiency of the mRNA. (Turner et al. (1995) Mol ecul ar Bi o technol ogy vol.3, p.225). The "non-coding sequence 3 '" refers to nucleotide sequences located 3' (downstream) to a coding sequence and includes the polyadenylation signal sequences and other sequences encoding the regulatory signals capable of affecting the processing of the mRNA or the expression of the gene. The polyadenylation signal is usually characterized by the affectation of the addition of polyadenylic acid tracts to the 3 'end of the mRNA precursor. The use of different 3 'non-coding sequences is carried out by Ingelbrecht et al. (1989) Pl an t Cell vol. 1, pp. 671-680. The "promoter" refers to a nucleotide sequence, usually upstream (50) to its coding sequence, which controls the expression of the coding sequence by providing recognition for the RNA polymerase and other factors required for proper transcription. "Promoter" includes a minimal promoter that is a short DNA sequence comprising a TATA box and other sequences that serve to specify the transcription initiation site, to which the regulatory elements are added for the control of expression. "Promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that are capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal elements upstream, the latter elements often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence which can stimulate the activity of the promoter and can be an innate element of the promoter or a heterologous element inserted to increase the level or specificity of the tissue of a promoter. It is capable of operating in both orientations (normal or loose), and is capable of operating even when moving either upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to specific sequence DNA binding proteins that mediate their effects. The promoters can be derived in either whole from a native gene, or can be composed of different elements derived from different promoters found in nature, or even, comprise of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors, which control the effectiveness of transcription initiation in response to physiological or experimental conditions. "Constitutive expression" refers to the expression that uses a constitutive promoter "Regulated expression" or "conditional" refers to the expression that uses a regulated promoter, respectively. "Constitutive promoter" refers to promoters that direct the expression of the promoter. gene in all tissues and at all times Examples of constitutive promoters include the CaMV 35S promoter, and the nopaline synthase promoter. "Regulated promoter" refers to promoters that direct expression of the gene not constitutively but spatially and / or temporarily regulated and includes either inducible and tissue-specific promoters.Synthetic and natural sequences are also included as sequences which may be a combination of natural and synthetic sequences.Different promoters may direct the expression of a gene in cell types or different tissues, or to different stages of development, or in response to different environmental conditions The new promoters of various types used in plant cells are constantly being discovered; Numerous examples can be found in the compilation by Okamuro et al. (1989) Bi ochemi stry of Plan t s vol. 15, pp. 1-82. Since in many cases, the boundaries of the regulatory sequences have not been fully defined, DNA fragments of different lengths may have identical promoter activity.

"Specific tissue promoter" refers to regulated promoters that are not expressed in all plant cells, but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as an embryo or cotyledon) , or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporarily regulated (such as in early or late embryogenesis), during the maturation of the fruit in the development of seeds or fruits, in the fully differentiated leaf, or in the beginning of aging. "Inducible promoter" refers to those regulated promoters, which can be changed into one or more cell types by an external stimulus (such as a chemical, light, hormone, stress or pathogen). The term "operably linked" refers to the association of the nucleic acid sequences in a single nucleic acid fragment, such that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence or a functional RNA, when it is capable of affecting the expression of such a coding sequence or functional RNA (i.e., the coding sequence or functional RNA is under the transcriptional control of the promoter). The coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. The term "expression" refers to the stable accumulation and transcription of functional or sense RNA (mRNA). The expression can also refer to the production of the protein. The "altered levels" refer to the level of expression in transgenic organisms, which differ from that of normal or non-transformed organisms. "Overexpression" refers to the level of expression in transgenic organisms that exceeds the levels of expression in normal or untransformed organisms. "Antisense inhibition" refers to the production of antisense RNA transcripts, capable of suppressing the expression of .protein from an endogenous gene or transgene. The terms "co-suppression" and "transchange" refers to the production of sense RNA transcripts, capable of suppressing the expression of endogenous genes or identical or substantially similar transgenes (US Patent No. 5,231,020). The term "inactivated gene" refers to the inhibition or down-regulation of an endogenous gene or a transgene that is substantially similar to the target gene. The term "homologue a" refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of the two protein molecules. Estimation of such homology is provided by either DNA-DNA or DNA-RNA hybridization under conditions of stringency or rigidity as is well understood by those skilled in the art [as described in Hames and Higgins (eds.). Nucleic Acid Hybridization, IRL Press, Oxford, U.K.]; or by comparing the sequence similarity between two nucleic acids or proteins. The homologous genes will be "substantially similar" to each other. "Substantially similar" refers to fragments of nucleic acids where changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate the alteration of gene expression by antisense or co-suppression technology. "Substantially similar" also refers to modifications of the nucleic acid fragments of the present invention, such as the deletion or insertion of one or more nucleotide bases that do not substantially affect the functional properties of the resulting transcript. For example, substantially similar sequences can be defined by either their ability to inhibit, under stringent conditions (0.1X SSC, 0.1% SDS, 65 ° C), with specifically identified sequences. The preferred substantially similar nucleic acid fragments of the present invention, are those fragments of nucleic acid, whose DNA sequences are at least 80% identical to the sequences specifically identified, either over the entire length of the sequence or over a portion of the sequence. The most preferred nucleic acid fragments are at least 90% identical to the specifically identified sequences, either over the entire length of the sequence, or over a portion of the sequence. The most preferred nucleic acid fragments are at least 95% identical to the specifically identified sequences, either over the entire length of the sequence or over a portion of the sequence. The term "binary transgenic viral expression system" describes the expression system comprised of the proreplicon and the chimeric trans-acting replication gene, which function together to effect the expression of a target gene in a plant. Both elements of the system will be chromosomally integrated and hereditary. The stimulation of the regulated promoter that drives the trans-acting gene will cause the expression of the viral replication proteins, which will be in turn, cut or excised to the replicon from the proreplicon and initiate the replication of the replicon. The "binary transgenic viral replication system" refers to a replication system comprised of two chromosomally integrated elements. The first element is a proreplicon which lacks an objective gene that encodes a foreign protein. The second element is comprised of a regulated promoter operably linked to a chimeric trans-acting replication gene. The proreplicon and a chimeric trans-acting gene that work together, will replicate the proreplicon in a plant in a regulated manner.

Such a system is used where the replication of the virus is desired in a regulated manner, but where an expression of the foreign gene is not sought. For example, regulated expression of the virus can be used to confer plant resistance or viral infection. The term "target gene" refers to a gene in the replicon that expresses the desired target sequence, which is either a functional RNA or an mRNA that encodes a protein. The target gene is not essential for replicon replication; additionally, the target genes may comprise active non-viral genes within a non-native organism, or chimeric genes and will be under the control of the appropriate regulatory sequences. Thus, the regulatory sequences in the target gene can come from any source, including the virus. The target genes may include coding sequences that are either heterologous or homologous to the genes of a particular plant to be transformed. However, the target genes do not include native viral genes. The proteins encoded by objective genes are known as "foreign or foreign proteins." The terms "in ci s" ev in trans "refer to the presence of DNA elements such as the viral origin of the replication and the gene of the protein or replication proteins, in the same DNA molecule or different DNA molecules, respectively. - The terms "cis-acting sequence" and "cis-acting element" refer to a DNA or RNA sequence, whose function is required to be in the same molecule. An example of a sequence ci s - ~ acting in the replicon is the origin of viral replication. The terms "trans-acting sequence" and "trans-acting element" refer to a DNA or RNA sequence, whose function is not required to be in the same molecule. Examples of trans-acting sequences are gene replication (ACI or AL1 in ACMV or TGMV geminivirus, respectively), which can function in replication without being in the replicon. The term "viral sequences" is the viral sequences that are necessary for viral replication (such as the origin of replication) and that are in the cis orientation. The terms "episomal" and "replicon" refer to a DNA or RNA vector that undergoes episomal replication in plant cells. It contains cis-acting viral sequences, such as the origin of replication, necessary for replication. They may or may not contain the trans-acting viral sequences necessary for replication, such as the viral replication genes (e.g., the AC1 and AL1 genes in the geminivirus ACMV and TGM, respectively). They may or may not contain a target gene for expression in the host plant. The term "replication-defective replicon" refers to a replicon containing viral cis-acting sequences, such as the origin of replication, necessary for replication, but defective in an essential replication gene. Consequently, a replication-defective replicon can replicate episomally only when provided with the essential in trans replication protein. The term "episomal replication" and "replicon replication" refers to the replication of DNA or RNA viruses or replicons derived from viruses that are not chromosomally integrated. It is required that the presence of protein or proteins of viral replication be independent of chromosomal replication, and result in the production of multiple copies of viruses or replicons per copy of the host genome. The term "autonomous episomal replication" refers to the replication of a replicon containing all the ci s and trans-acting sequences, such as the replication gene, required for replication. Episomal replication does not require the presence of a replication gene provided in trans. The term "origin of replication" refers to a cis-acting replication sequence essential for viral or episomal replication. The term "proreplicon" refers to a replication-defective replicon that is integrated into a bacterial plasmid or a host plant chromosome. It comprises viral sequences that are required for replication, and flanking sequences that allow the release of the replicon from there. In addition, the proreplicon may contain a target gene. In the case of RNA viruses, flanking sequences include regulatory sequences that allow the generation of RNA transcripts that can replicate in the presence of a replication protein. These regulatory sequences may be for constitutive or regulated expression. Proreplicon lacks a gene that encodes a replication protein essential for replication. Therefore, it allows subjecting it to autonomous episomal replication but it can subject it to episomal replication in the presence of the replication protein provided in trans. Thus, this replication requires both releases from the integration and presence of the essential trans replication gene. The release from the integration can be triggered in different ways. For example, in the case of a geminivirus, the proreplicon may be present as a partial or complete duplication one after another, such that a full length replication sequence is flanked by the virus sequences and such that the sequence viral duplication includes the origin of viral replication. Thus, in this case, the proreplicon serves as a master copy from which the replicons can be excised or cut by replication in the presence of protein or replication proteins [Bisaro, David. Recombination in geminivirus: Mechanisms to maintain the size of the genome and the generation of genomic diversity. Homologous Recomb. Gene Sil encing Plants (1994), 219-70. Editor (s): Paszkowski, Jerzy, Publisher: Kluwer, Dordrecht, Germany]. In the case of proreplicons of the RNA virus (for example, Brome Mosaic Virus), the amplicon sequences flank the inactive replicon, which include regulatory sequences, allowing the generation of the replicon as RNA transcripts that can replicate in trans in the presence of the replication protein. These regulatory sequences may be for constitutive or regulated expression. The terms "viral replication protein" and "" replicase "refer to the viral replication protein, essential for viral replication. It can be provided in trans to the replicon to support its replication. Examples include, viral replication proteins, encoded by the AC1 and AL1 genes in the geminivirus ACMV and TGMV, respectively. Some viruses have only one replication protein, others may have more than one. Viral replication proteins can also include single-stranded RNA virus replication proteins, such as RNA-dependent RNA polymerases, when they can support i n trans viral replication, for example, the Brome Mosaic Virus (BMV). The term "replication gene" refers to a gene that encodes a viral replication protein. In addition to the ORF of the replication protein, the replication gene may also contain other ORF (s) of overlap or non-overlap, such as found in viral sequences in nature. These additional ORFs (while not essential for replication) can increase the replication and / or accumulation of viral DNA. Examples of such additional ORFs are AC3 and AL3 in the geminivirus ACMV and TGMV, respectively. The term "chimeric trans-acting replication gene" refers to a replication gene in which the sequence encoding a replication protein is under the control of a regulated plant promoter, rather than the native viral replication gene. . The term "chromosomally integrated" refers to the integration of a foreign gene or a DNA construct into the host DNA by covalent linkages. The term "transformation" refers to the transfer of a foreign gene into the genome of a host organism. Examples of plant transformation methods include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol, Vol 43, p 277) and accelerated particle or "gene trip" transformation technology (Klein. et al. (1987) Nature (London) vol 327, pp. 70-73; US Patent No. 4,945,050). The terms "transformed", "transformants" and "transgenic" refer to plants or all those that have had through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not been through the transformation process. The term "temporarily transformed" refers to cells in which transgenes and foreign DNA have been introduced (by such methods as agrobact-mediated transformation or biolistic bombardment), but not by being selected for stable maintenance. The term "stably transformed" refers to cells that have been selected and regenerated in a selection medium after transformation. The terms "genetically stable" and "hereditary" refer to chromosomally integrated genetic elements that are stably maintained in the plant and stably inherited by the progeny through successive generations. The terms "primary transformant" and "TO generation" refers to transgenic plants that are of the same genetic generation as the tissue from which they were initially transformed (ie, they do not pass through eosis and fertilization due to transformation). The terms "secondary transformants" and the generations "Tl, T2, T3, etc." they refer to transgenic plants derived from primary transformants through one or more fertilization and meiotic cycles. Secondary transformants can be derived by self-fertilization of primary and secondary transformants or crossings of primary or secondary transformants with other transformed or untransformed plants. The term "derived from" refers to the obtaining of genetic material from an identified species or source. The invention provides a two component viral expression system, for the production of transgenic plants. Both components are chromosomally integrated, and stably maintained by themselves. One component is the proreplicon that is unable to replicate by it. The second component is a chimeric trans-acting replication gene in which the sequence encoding a viral replication is operably linked to a regulated promoter. Expression of the viral replication protein under appropriate stimulation will result in the release of the replicon from the proreplicon and its episomal replication in an autonomous cellular fashion. Thus, replicon replication can be targeted for specific plant cells by controlling the expression of protein or protein replication to such cells. The plants will be more sensitive to cellular toxicity and / or to the detrimental effect of viral replication and / or protein or replication proteins in early stages of plant growth and differentiation, which involve cell division and differentiation. Thus, the control of the expression of the replication protein and the replicon replication completely or extensively so as not to terminally divide the differentiated cells, will reduce the deleterious effect of replicon replication on the growth and development of the plant. Examples of such terminally differentiated cells include, but are not limited to, the storage cells of seed and root tissue, and of mature leaf cells. In addition, the chromosomally integrated proreplicon serves as a master copy for replicons, not only in different generations, but also in the same gene if the cell divisions occur after the onset of episomal replication. This strategy will also solve the problem of episomal instability through cell divisions, since episomes are unstable in the absence of selection. In addition, replicon replication is expected to reach the high expression level of target genes through the amplification of gene that is hereditary and autonomic cells. The expression of the target gene may involve either the production of foreign proteins or the inactivation of the endogenous nuclear gene as well as the transgenes by inhibition or antisense co-suppression. In accordance with the subject invention, new recombinant virus constructs (including vectors and transfer methods for making them and their uses) are described. When used to transform a plant cell, the vectors provide a transgenic plant capable of amplifying the gene through the high level of regulated expression. This regulated expression could be in response to a particular stimulus, such as the stage of development, healing of the plant (for example, by insect or pathogen attack), environmental stress (such as heat or high salinity), or chemicals , that induce specific promoters. Subjects in which the particular tissues and / or parts of the plants have a new or altered phenotype can be produced by the subject method. The constructs include vectors, expression cassettes and binary plasmids that depend on the proposed use of a particular construct. Two basic DNA constructs are required, which can be combined in a variety of ways for the transformation of a plant cell, and the obtaining of a transgenic plant. For the agrobac teri um mediated transformation, the proreplicon and chimeric replication gene can be combined in a binary plasmid or both can be introduced into a cell in binary plasmids separated by either co-transformation or sequential transformations. Alternatively, the two constructs can be combined by crossing two transgenic lines containing one or the other construct. The termination region used in the target gene in the replicon, also in the chimeric replication protein gene, will be selected mainly for convenience, since the termination regions appear to be relatively interchangeable. The termination region which is used may be native to the transcriptional initiation region, may be native to the DNA sequence of interest, or may be derived from another source. The termination region may occur naturally, or completely or partially synthetic. Suitable termination regions are available from the Ti plasmid of A. t umefa ci ens, such as the octopine synthase and nopaline synthase termination regions or genes for β-phaseolin, the latent chemically inducible gene, pIN (Hershey et al. (1991) Pl an t Mol. Bi ol. Vol 17 (4), pp. 679-90; U.S., 5,364,780).

The Constructo Proreplicon A basic construct is proreplicon. In the case of geminivirus, the proreplicon is preferably present as a partial or complete dimer one after another, in T-DNA, such as a single replicon is flanked by the viral cis-acting sequences necessary for viral replication, which include the origin of replication (Figure 1). These dimers can serve as master copies from which replicons can be excised or excised by replicative release (Bisaro, David.) Geminivirus recombination: Mechanism to maintain the size of the genome and the generation of genomic diversity. Homologous Recomb. Gene Silencing Plants (1994), 219-70. Editor (s): Paszkowski, Jerzy. Publisher: Kluwer, Dordrecht, Germany) in the presence of the replication protein. The preferred source of proreplicon sequences is ACMV and TGMV, in which the essential replication gene (e.g., AC1) is proposed no. functional by mutation (ie, addition, rearrangement, or partial or complete deletion of nucleotide sequences). The mutation can be in the non-coding sequence, such as the promoter, or it can be in the coding sequence of the replication protein, such that it results in either one or more amino acids altered in the replication protein or in a structure exchange. Preferably, the total replication gene is deleted from the proreplicon so that there is no homology between the transacting replication gene and the replicon, to reduce the inactivation of the transacting replication gene based on the virus-induced homology, during the replication of the replicon In addition, the proreplicon preferentially has the majority or all of the coat protein genes which can be deleted and replaced by a restriction site by a cloning target gene. Proreplicons may also contain objective genes in the replicon sequence. The coding sequence in these target genes is operably linked to the regulatory sequences that are of viral and / or plant origin. One or more introns may also be present in the cassette. Other sequences, including those encoding temporal peptides, leader secretory sequences, or introns, may also be present in the proreplicon and replicon as desired. How to obtain and use these sequences is well known to those skilled in the art. The target gene can encode a peptide of interest (e.g., an enzyme), or a functional RNA, whose sequence results in antisense inhibition or co-suppression. The nucleotide sequences of this invention can be synthetic, naturally derived or combinations thereof. Depending on the nature of the nucleotide sequence of interest, it may be desirable to synthesize the codon sequence of preferred plants. It is contemplated that the modified proreplicons may cause them to have only the minimum origin of sequence (ori) of replication. This will allow a maximum site for the cloning of target genes as well as to eliminate all or almost all homology between the proreplicon and the replicase gene to reduce the inactivation of the chimeric replication gene gene by the chromosomal proreplicon or the episomally replicating replicon. The source of the minimal sequence of bipartite geminiviruses can be either DNA from the A genome or the DNA from the B genome. The target genes can encode functional RNAs to genes or transgenes endogenous silent homologs or can encode foreign proteins. The foreign proteins will typically be non-viral encoded proteins and proteins that may be foreign to the host plant. Such foreign proteins will include, for example, enzymes for primary or secondary metabolism in plants, proteins that confer diseases or resistance to herbicides, enzymes without commercially used plants, and proteins with desired properties used in animal feed or for human food. Additionally, foreign proteins encoded by the target genes will include seed storage proteins with improved nutritional properties, such as high-sulfur 10 kD corn seed protein, or zein sulfur proteins.

The Construction of the Chimeric Trans-acting Replication Gene.

The other basic construct is a chimeric trans-acting replication gene, consisting of a regulated plant promoter, operably linked to the coding sequence of a replication protein. For the ACMV and TGMV geminivirus, the replication proteins are encoded by the ORFs AC1 and AL1, respectively. Also included are single-stranded RNA virus replication proteins, such as RNA-dependent RNA polymerase, encoded by the first ORF of potato X virus (PVX) [Angeli et al. (1997) The EMBO Journal, vol. 16, pp. 3675-3684]. Regulated expression of the protein or proteins of viral replication is possible, by placing the coding sequence of the replication protein under the control of the promoters that are tissue-specific, developmentally specific, or inducible. Several genes regulated by specific tissues and / or promoters have been reported in plants. These include genes that encode seed storage proteins (such as napkin, cruciferin, beta-conglycinin, phaseolin), proteins from zein bodies or oils (such as oleosin), or genes involved in fatty acid biosynthesis, including acyl carrier proteins, desaturase stearoyl ACP, and fatty acid desaturases (fad 2-1), and other genes expressed during embryonic development, such as Bce 4, in which the EA9 gene is expressed at high levels in the cells of the Brassica seed coat [see, for example, EP 255378 and Kridl et al. (1991) Seed Sci en Research vol. 1, pp. 209-219]. Particularly used for the specific expression of the seed is the alveja vicilina promoter [Czako et al. (1992) Mol. Gen Genet Vol. 235 (1), pp. 33-40] that has been shown to be specific to the seed by the use of diphtheria toxin. Other promoters used for expression in mature leaves are those that are changed at the beginning of sensing or aging, such as the SAG promoter from Arabidopsis [Gan et al. (1995) Science (Washington, D.C.) vol. 270 (5244), pp. 1986-8]. A class of fruit-specific promoters, expressed at or during anthesis or flowering until fruit development, at least until the beginning of ripening, is discussed in US 4,943,674, a description of which is incorporated herein by reference. The cDNA clones that are preferentially expressed in cotton fibers have been isolated [John et al. (1992) Proc. Na ti. Acad. Sci. USES. vol. 89 (13), pp. 5769-73]. The tomato cDNA clones, which present differential expression during the development of the fruit, have been isolated and characterized [Manson et al. (1985) Mol. Gen Gene t. Vol. 200 / pp. 356-361; Slater et al. (1985) Pl an t Mol. Bi ol. Vol. 5, pp. 137-147]. The promoter for the polygalacturonase gene is active in the ripening of the fruit. The polygalacturonase gene is described in U.S. Pat. No. 4,535,060 (published August 13, 1985), U.S. Pat. No. 4,769,061 (published September 6, 1988), U.S. Pat. No. 4,801,590 (published January 31, 1989), and U.S. Pat. No. 5,107,065 (published April 21, 1992), descriptions of which are incorporated herein for reference. The mature plastid mRNA for psbA (one of the components of photosystem II), reaches its highest level in the development of the fruit, in contrast to the plastido ARNASm for other components of photosystem I and II, which decline to levels not detectable in the chromoplasts, after the beginning of maturation [Piechulla et al. (1986) Plan t Mol. Bi ol. Vol 7, pp. 367-376].

Recently, cDNA clones representing genes apparently involved in pollen interactions have been isolated and characterized [McCormick et al., Toma to Bi or technolgy (1987) Alan R. Liss, Inc., New York) and tomato pistil. (Gasser et al., (1989) Plant Cell vol. 1, pp. 15-24.] Other examples of specific tissue promoters include those that direct expression in leaf cells after damage to the leaf (eg from insects). chewable); in tubers (for example, the promoter of the patatin gene); and in fibrous cells (an example of a cell-regulated fibrous protein is E6 [John et al. (1992)) Expression of the gene in cotton fiber (Gossypi um hirsutum L.): cloning of RNAs Proc. Na ti. Acad Sci. USA vol 89 (13), pp. 5769-73]). The E6 gene is more active in fiber, despite the low levels of transcripts found in the leaf, ovule and flower. The tissue specificity of some "tissue-specific" promoters may not be absolute and may be assayed by one skilled in the art using the diphtheria toxin sequence. One can also achieve specific tissue expression with "weak" expression by a combination of different specific tissue promoters (Beals, et al (1997) Pl an t Cell vol.9, pp. 1527-1545). Other specific tissue promoters can be isolated by one skilled in the art (see U.S. 5,589,379). Several inducible promoters ("changing genes") have been reported. Many are described in the review by Gatz (1996) Current Opinion in Biotechnology ogy vol. 48, pp. 89-108]. These include the tetracycline repressor system, the Lac repressor system, inducible copper systems, inducible salicylate systems, such as the PRla system, [Aoyama et al. (1997) N-H Plant Journal vol. 11, pp. 605-612] and inducible systems ecdysomes and glucocorticoids. Also included are benzenesulfonamides (U.S. 5,364,780) and inducible alcohol systems (WO 97/06269 and WO 97/06268) and glutathione S-transferase promoters. Other studies have focused on inducibly regulated genes in response to stress or environmental stimuli, such as increased salinity, drought, pathogens and wounds. For example, the genes that code for serine proteinase inhibitors, which are expressed in response to tomato lesions, CGraham et al. (1985) J. Bi ol. Ch em. Vol. 260, pp. 6555-6560; Graham et al. (1985) J. Bi ol. Ch em. Vol. 260, pp. 6561-6554) and in ARNSm correlated with the synthesis of ethylene in the maturation of fruits and leaves, after the lesions (Smith et al. (1986) Plan ta vol 168, pp. 94-10). The accumulation of a metallocarboxypeptidase inhibitory protein has been reported in several leaves of injured potato plants [Graham et al. (1981) Bi och em Bi ophys Res. Comm. Vol. 101, pp. 1164-1170]. Other plant genes have been reported to be induced by methylj asmonate, promoters, heat impact, anaerobic stress, or harmless to herbicides. A variety of techniques for introducing contrusts into a plant cell host are available and are known to those skilled in the art. These techniques include transformation with DNA, using A. tumefaci ens o A. rhi zogenes as the transformation agent, electroporation, particle acceleration, etc. [see for example the patents EP 295959 and EP 138341]. Particularly preferred is the use of the binary type vectors of Tplasmidos Ti and Ri of Agroba cterium spp. The vectors derived from Ti transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, turnip, tobacco and corn [Pacciotti et al. (1985) Bio / Technology vol. 3, pp. 241; Byrne et al. (1987) Plant Cell, Tissue and Organ Culture vol. 8, p.3; Sukhapinda et al. (1987) Plant Mol. Biol. Vol. 8, pp. 209-216]; Lorz et al. (1985) Mol. Gen. Genet.

Vol. 199, p. 178; Potrykus (1985) Mol. Gen. Genet.

Vol. 199, p. 183; Park et al. (1995) J Plant Biol.

Vol. 38 (4), pp. 365-71; Hiei et al. (1994) Plant J. Vol. 6, pp. 2271-282]. The use of T-DNA for the transformation of plant cells has received an intense study and is widely described in EP 120516, Hoekma, In: The Binary Plant Vector System Offset-drukkerij Kanters BV, Albasserdam, 1985, Chapter V , Knauf et al., Genetic Analysis of Hos Range Expression by Agrobacterium, In: Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed., Springer-Verlag, New York, 1983, p. 245, and An et al. (1985) EMBO J. Vol. 4, pp. 2-284. For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples. A variety of techniques are available and are known to those skilled in the art for the introduction of constructs into a plant cell host. These techniques include transformation with DNA using A. turne fasciens or A. rhizogenes as the transformation agent, electroporation, particle acceleration, etc. [see for example, Patents EP 295959 and EP 138341]. Particularly preferred is the use of the binary-type vectors of the Ti and Ri plasmids of Agrobacterium spp. The vectors derived from Ti transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, * such as soy, cotton, turnip, tobacco and corn [Pacciotti et al. (1985) Bio / Technology vol. 3, pp. 241; Byrne et al. (1987) Plant Cell, Tissue and Organ Culture vol. 8, p.3; Sukhapinda et al. (1987) Plant Mol. Biol. Vol. 8, pp. 209-216]; Lorz et al. (1985) Mol. Gen. Genet. Vol. 199, p. 178; Potrykus (1985) Mol. Gen. Genet. Vol. 199, p. 183; Park et al. (1995) J Plant Biol. Vol. 38 (4), pp. 365-71; Hiei et al. (1994) Plant J.

Vol. 6, pp. 2271-282]. The use of T-DNA for the transformation of plant cells has received an intense study and is widely described [Patent EP 120516, Hoekma, In: The Binary Plant Vector System Offset-drukkerij Kanters BV, Albasserdam, 1985, Chapter V, Knauf et al., Gene ti c Analyzes of Range Express by Agrobacterium, In: Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed., Springer-Verlag, New York, 1983, p. 245, and An et al. (1985) EMBO J. Vol. 4, pp. 2-284. For the introduction in plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples. - The cells of transgenic plants are then placed in an appropriate selective medium for the selection of transgenic cells, which are then grown up to the callus. From the projections of calluses, they are grown and plants of the projection are generated by growing in a root medium. The various constructs will normally be attached to a selection marker in the plant cells. Conveniently, the label can be resistant to a biocide, particularly an antibiotic (such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide or the like). The particular marker used will allow the selection of transformed cells compared to cells lacking DNA which have been introduced.

The components of the DNA constructs including transcription cassettes of this invention can be prepared from. the sequences which are native (endogenous) or foreign (exogenous) to the host. By "strange" it is suggested that the sequence is not found in the wild type or native host within which the construct is introduced. The heterologous constructs will contain at least one region which is not native to the gene from which the transcription initiation region is derived. To confirm the presence of transgenes in transgenic cells and plants, a Southern spotting assay can be performed, using methods known to those skilled in the art. Replicons can be detected and quantified by Southern staining, since they can be readily distinguished from the proreplicon sequences by the use of appropriate restriction enzymes. The expression products of the transgenes can be detected in any variety of ways, which depend on the nature of the product, and include the enzyme assay and Western staining. A particularly useful way to quantify protein expression and detect replication in different plant tissues is with the use of a reporter gene, such as GUS. Once the transgenic plants have been obtained, they can be grown to produce tissues or parts of plants that have the desired phenotype. The tissue or parts of the plant can be harvested, and / or the seed collected. The seed can serve as a source for the further growth of plants with tissues or parts that have the desired characteristics. The present viral expression system has been used to demonstrate that (i) the corn and soybean tissue will support the replication of the geminivirus; and (ii) that the expression system will effect the expression of foreign genes in tobacco. More specifically, the Applicant has used a temporary trial using biolistic bombardment, to show that the development of corn and soybean seeds can support the replication of the vector derived from ACMV-A in which, the gene for the coat protein was suppressed The Applicant has tested the episomal replication of the mutant ACMV-A ADNAs in mature tobacco leaves by developing the soybean embryo (50-150 mg of weight), developing maize seed, and cell cultures of corn suspensions. For this, a full-length copy larger than that of the mutant ACMV-A DNA is introduced into the tissues by logistic bombardment and the temporarily transformed tissues are analyzed by Souther stain for replication. In a mutant, a mutant named CP, a deletion of 784 bp is made in the coat protein gene from a Bam Hl site at position 142 to the Pml I site at position 926 (with reference to SspI in the structure of one of the ori being in position 1). In another mutant, called CP + REP mutant, there is a deletion in the AC1 replication gene that is elaborated by the deletion of the 651 bp Bgl II fragment that covers the ORFs of ACl, C2, and AC3 in addition to the suppression of the protein of the cover, mentioned above. While the CP mutant can replicate episomally by itself, the CP + REP mutant can not. However, the CP + REP mutant can replicate episomally in all tissues when co-bombarded with either the CP mutant or a chimeric replication gene under the control of the constitutively expressed 35S promoter or the seed-specific phaseolin promoter. Since all these tissues except the suspension culture, are from the last state of cell division, this shows that the undivided cells, completely or partially differentiated, are capable of supporting the replication of the geminivirus. In another embodiment of the expression system, the viral coat protein gene was replaced with a non-viral 1.2 Kb gene. The applicant introduces more than one full-length CP mutant with a 1.2 k target gene insertion in place of the coat protein in the tobacco leaf discs in a binary vector by the agrobacterium-mediated transformation. The Southern analysis of the regenerated shots showed the replication of the modified ACMV-A genome in the shots without suckers, but not in shots of shoots. Thus, the CP mutant can tolerate at least one 1.2 kb non-viral DNA insertion. Taking together the ability of the CP + REP mutant to replicate in trans, it is reasonable to expect that at least a 1.85 kb non-viral DNA can be inserted. Applicants also obtain genetically stable transgenic tobacco plants that contain more than one chromosomally integrated full length of the CP mutant. The retransformation of the discs of the leaves of these plants with a chimeric replicase gene results in the excision or cutting and replication of the replicon. Applicants also introduce into the tobacco via the transformation mediated by a groba cteri um, native or chimeric ACMV A sequences expressing reading structures open to the left containing ORFs ACl, AC2 and AC3 under the control of their native promoter or other promoters that they are expressed during the regeneration of the plant. However, the Souther analysis of transgenic plants showed that these chimeric genes are lost or suppressed. Thus, indistinctly, in the case of TGMV, tobacco plants transformed with wild-type ACMV DNA A or with full-length copies larger than the cover protein replacement vector, showed that the viral sequences were deleted. in the leaves of transgenic plants in shoots. This observation is consistent with the toxicity of the expression of the replication protein or proteins, since the viral sequences with the replication protein partially suppressed can be stably maintained. In addition, when chimeric genes for protein expression or replication proteins are introduced into tobacco plants, they are similarly suppressed in the leaves of transgenic shoot plants. Thus, the protein or replication proteins are detrimental to the regeneration of the plant. The applicant has shown that the expression of replicase is detrimental to plants during transformation / regeneration thereof. The applicant has shown 1) that a wide range of non-dividing tissues (mature tobacco leaf, soybean and corn embryos, and corn suspension cultures), are capable of supporting the replication of the geminivirus after the introduction of the geminivirus by the bombardment and 2) that the stably transformed shoots without shoots also allow the replication of the geminivirus. The successful use of plant viral vectors will depend on the expression of the extremely regulating replication protein (and replicon replication) in the terminally differentiated non-dividing cells.

EXAMPLES The present invention is further defined in the following Examples. These Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt to the various conditions and uses.

GENERAL METHOD The standard recombinant DNA and molecular cloning techniques, used in the Examples, are well known in the art and are described by Sambrook, J., Fritsch, E.F. and Maniatis, T.

Mol ecul ar Cl oning: A Labora t ory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor (1989) (Maniatis) and by T.J. Silhavy, M.L. Bennan, and L. W. Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1984) and by Ausbel, F. M. et al., Current Protocols in Molecular Biology, pub. By Greene Publishing Assoc. and Wiley-Interscience (1987). The digestions, phosphorylations, ligations and transformations of the restriction enzyme are as described in Sambrook et al., S upra. Restriction enzymes were obtained from New England Biolabs (Boston, MA), GIBCO / BRL (Gaithersburg, MD), or Promega (Madison, Wl). The thermally stable DNA polymerase was obtained from Perkin Elmer (Branchburg, NJ). The growth medium was obtained from GIBCO / BRL (Gaithersburg, MD). The LBA4404 strains of Agrobac teri um t umefasci ens were obtained from Dr. R. Schilperoot, Leiden [Hoekema et al. (1983) Na t ure vol. 303, pp. 179-180].

Transformation Protocols Biological transformations were made essentially as described in U.S. Pat. No. 4,945,050, incorporated herein by reference. The golden particles (1 mm in diameter) were covered with DNA using the following technique. Ten ug of plasmid DNAs were added to 50 mL of a suspension of golden particles (60 mg per mL). Calcium chloride (50 uL of a 2.5 M solution) and free base spermidine (20 mL of a 1.0 M solution) were added to the particles. The suspension was vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 seconds at 15,000 rpm) and the supernatant is removed. The particles are suspended in 200 mL of absolute ethanol, centrifuged again and the supernatant is removed. The methanol rinsing is performed again and the particles are resuspended in a final volume of 30 uL of ethanol. An aliquot (5 mL) of the gold particles coated with DNA can be placed in the center of an air disc (Bio-Rad Labs, 861 Ridgeview Dr, Medina, OH). The particles are then accelerated in the corn tissue, with a PDS-1000 / He (Bio-Rad Labs, Medina, OH), using a helium pressure of 1000 psi, an open distance of 0.5 cm, and an air distance of 1.0 cm Where the transformations of Agroba ct eri um were made, the process encompassed was essentially done as described by Park et al. (1995) J. Plant Bi ol. Vol. 38 (4), pp. 365-71.

Expression and Transformation Protocol Transgenic plants with different constructs were selected and regenerated in plants in tissue culture by methods known to one skilled in the art and referenced above. The ability of a chimeric trans-acting replication gene to replicate the replicon from the proreplicon on the chromosome of in trans plants will be tested by one of the following methods. 1. plant crosses, where one parent contained the chimeric replication gene, and the other the proreplicon. : 2. the co-transformation of tobacco discs with two types of a terba groa, one containing a binary vector with a selective plant marker (such as the resistance to the fos thotricin) and the proreplicon and another containing a binary vector with a different plant selective marker (such as resistance to kanamycin) and the chimeric replication gene. The discs of the transformed leaves will be selected, and will regenerate in the presence of both selection agents, or 3. the transformation of tobacco with an agrobacterium class that contains a binary vector with a selective plant marker, obtaining a regenerated transgenic plant and re-transforming it with another agrobac t eri a class that contains a binary -vector with a different selective plant marker. Thus, the first binary vector may contain either the proreplicon or the chimeric replication gene. The binary vector for retransformation will contain the complementary component. Tissue replication of transgenic plants will be monitored by Southern analysis of genomic DNA (either undigested or after digestion with restriction enzymes) that will distinguish the gene replicon from the chromosomal chimeric replication protein and the proreplicon by size. Alternatively, replication can be detected by the expression of the reporter gene. For example, increased expression of GUS reporter genes in the replicon will be assayed by the assay or staining of the GUS enzyme and increased expression of the 10 kD corn storage protein will be detected by Western blotting using specific antibodies to the 10 kD protein EXAMPLE 1 Constructs of Tomato Golden Mosaic Virus (TGMV) Partial Dimer of TGMV with a replication protein of native or wild type (plasmid pBE651) Plasmid pCSTA [Von Arnim et al. (1992) Virl ogy vol. 186 (1), pp. 286-293] was obtained from John Stanley (John Innes Center, Norwich, United Kingdom). It consists of a single, cloned complete TGMV DNA, which is unique to the Eco Rl site within the Eco Rl site of ufT derived pUC19 from the plasmid. The 1922 bp between its Neo I and Sal I sites containing the 3 'ends of the ORF ALl replication protein and the coat protein gene were deleted by restriction digestion, filled, and auto ligated to result in plasmid pGV650. Then, the Eco Rl insert fragment of plasmid pCSTA (containing the complete TGMV-A genome) was cloned into the Eco Rl site in pGV650 to provide plasmid pGV651. Thus, plasmid pGV651 consists of an intact TGMV-A genome and one duplication after another of a TGMV-A sequence of 570 bp including, a sequence ori TGMV of 206 bp and a sequence ORF AL1 5 'adjacent to the origin of replication ( ori), so that a TGMV-A genome can be made from pGV61 in plant cells, either by replication release or homologous recombination. Plasmid pGV651 can be replicated in a plant cell when introduced by biolistic bombardment. The Hind III fragment of pGV651 carrying the TGMV dimer with the native or wild type ALF ORF was cloned into pBinl9 [Frish et al., (1995) Plan t. Mol. Biol. Vol. 27 (2), pp. 405-409], such that an AC1 gene is transcribed away from the Nos: NPT II gene. The resulting binary plasmid pBE651 was introduced into tobacco plants via strain LBA4404 of Agroba c teri um t umefa ci ens.

TGMV Partial Dimer with wild-type replication protein or native GUS reporter (plasmid pBE671) The unique BstBl site in plasmid pGV651 was converted to Not I, followed by BstBl digestion, packed reaction, and ligation to Not I linkers (New England Biolabs catalog number 1125) to result in pGV652. Then, a Not I-Sac 1401 bp fragment (containing the coat protein gene in plasmid pGV652) was replaced with a Not I-Sac I fragment of 2545 bp from plasmid pGV662 (see below) which contains the GUS [Jefferson et al., The use of the b-glucuronidase gene of Escheri chia coli as a gene fusion marker for studies of gene expression in higher plants. Bi och em. Soc. Trans. (1987), vol. 15 (1), pp. 17-18] to provide plasmid pGV671. In plasmid pGV671, the US expression is under the control of the coat protein promoter. Plasmid pGV671 can be replicated when introduced into plant cells by biolistic bombardment. The Hind III fragment carrying the dimer will be cloned into a binary vector pBinl9 to result in pBE671, and transferred to the plants as known to one skilled in the art.

Proreplicon TGMV without the GUS reporter gene (plasmid PGV654) Plasmid pGV654 was made from plasmid pGV652 (see the description above) by deleting a 895 bp region between Bam Hl-Eco Rl, which includes 709 bp from the 3 'end of the ORF AL1 and the 5' regions of ORFs AL2 and AL3 after restriction digestion, filling and ligation. When introduced into plant cells by biolistic bombardment, the proreplicon in plasmid pGV654 is not able to replicate without the gene encoding the i n trans replication protein.

Proreplicones TGMV with the reporter gene GUS (plasmids pGV662 and pGV672) Three different TGMV proreplicons with the GUS reporter gene will be introduced into plant cells. The gene of replication differs in the mutation class. Two (pgV62 and pGV672) were elaborated by deletions of different sizes and one was made by introducing the mutation of the structure change. The Not I-Nco I fragment of 696 bp in the plasmid pGV654 (containing the largest envelope protein gene) was replaced with a Not I-Nco I fragment of 1875 bp containing the GUS ORF (the Neo I site). includes the initiation codon) to provide plasmid pGV661. Since the Neo I sites are current below the start codon of the envelope protein, a shell protein promoter modified with a Ncol in the untranslated region, is made by nucleic acid amplification using primers [(See U.S. Patent 4,683,195; 4,683,202; 4,965,188 for the polymerase chain reaction (PCR), 5 '-CGTCCGGATCCAATTCTCCCCATACAAGAGTATCT-3' [SEQ ID NO: 1] and 5'-GTCGACCCATGGTTAAAGA CCACGAAACGCA GT [SEQ ID NO: 2] in the plasmid pCSTA for 40 cycles of 60 ° C alignment 1 '; 90 ° C, 2 '. A Bam Hl + Ncol fragment of 663 bp, derived from the PCR product and containing the ori, and the coat protein promoter without some envelope protein coding sequence, was used to replace the Bg fragment. II-Nco I of 698 bp of plasmid pGV661 to result in plasmid pGV662.

Another deletion of the replication gene was done for the deletion of 772 bp between the Xmn I site in ORF ALl and the Swa I site in ORF AL3. For this, plasmid pGV671 was digested with Swa I and partial Xmn I and then ligated again. The plasmids pGV662 and pGV672 differ from their wild type or native counterpart, the plasmid pGV671, which has deletions in ORFs AL1, AL2 and AL3 of 709 bp, and 433 bp, respectively. The Hind III fragment of plasmid pGV662 was isolated and cloned into the Hind III site of pBinl9 and OGV674 to provide binary plasmids pBE662 and pBE675, respectively. The Hind II fragment of pGV672 was cloned into pGV674 to provide pBE672. The binary vector pGV674 was developed by the replacement of the Bsu 361-Cla I fragment in pBinl9 (carrying the nopaline synthase promoter: npt Urgen chimeric nopaline synthase 3 ', which confers resistance to kanamycin in plants) with a Bsu promoter 36 I -Cla (fragment carrying the nopaline synthase promoter: bar: nopaline 3 'chimeric synthase gene that confers resistance to the herbicide phosphonitricin). The binary plasmid pBE662 was introduced into the plants via the Agroba c teri um t umefa ci ens-mediated transformation LBA4404 known to a person skilled in the art. [Waden, Vector Systems for Agrobacterium-mediated Transformation in Metod Plants Biochem (1997), 106 (Molecular Biology), 85-102].

EXAMPLE 2 TGMV replication genes chimeric transplants 35S: TGMV replication gene (plasmid pGV653) Plasmid pGV653 was derived from the p35S-GFP plasmid plant vector (Promega Corp., 7113 Benhart Dr., Raleigh, NC) by replacing the first 462 bp of the upstream region (between Hind III and Acc I) of the 35S promoter with the Not I fragment and then Bam Hl-Sac I, containing the ORF of the green fluorescent protein (between the 35S promoter and the 3 'region of the nopaline synthase gene) with that of the TGMV sequence containing ORFs AL1 , AL2, and AL3. Finally, a Bam Hl site was introduced at position 29 [Hamilton et al., EMBO J. 3: 2197-205 (1984)] in the 5 'untranslated region (16 bp upstream of the ORF AL1) of the TGMV gene AL1 and 2976 bp of the Bam HI-Bam Hl-Sac I TGMV sequence was used to replace the Bam Hl-Sac I region to form pGV653.

Chemically inducible promoter in 2-2: TGMV replication gene (plasmid pBE665) The Mfe I and BstBl sites in plasmid pGV651 were converted to Spe I following the filling and ligation of the Spe I linker (New England Biolab, catalog No. 1987). The Sep I fragment of 1603 bp (containing the TGMV AL1, AL2 and OR3 AL3) was isolated and cloned between the 2-2 inducible promoter and the 3f region of the 2-1 gene in plasmid pGV664 to provide plasmid pGV665. Plasmid pGV664 was derived from plasmid pGV659 (see later description) by replacing its Not I-Spe I fragment (containing the 2-2 promoter and the ACMV replication protein gene), with that of a 2-promoter. 2 in which, the Neo I site was modified to the Spe I site. The latter was made by PCR in such a way that the Neo I site at its 3 'end was replaced by the Spe I site, the Spe I site at the 5 end The promoter was destroyed, and the Bam Hl site at the 5 'end was changed to Bgl II. The 2561 bp BglII-Asp718 fragment from pGV665 containing the 2-lN: T-Rep ORF: 3'gen chimeric 2-2 promoter was isolated from pGV665 and cloned into the binary vector pBinl9 cut with BamHI. -As718 to result in pBE665. The chimeric gene was introduced into tobacco plants, via Agroba c teri um t umefaci ens LBA4404. Shots of each transformation of Ni co ti ana ben thamiana with shoots either BA662 and BA665, sprouted and regenerated in plants. All plants lack some observable abnormal phenotype. The applicant obtains a positive plant by the presence of the transgene (determined by the Southern staining) of each of the transformations BA662 and BA665. These plants were either the same or pure or crossed with each of the other trials. The seeds from the reciprocal crosses were planted in seedbeds of the soil and allowed to germinate in growth chambers. Seedlings of progeny 48 and 72, from crosses BA662 X BA665 and BA665 X BA662, respectively, were transplanted in a grid nursery. The phenotypes of the plants were relatively normal, except that the seedlings varied in size and in some of the leaves, wrinkles or folds appeared as also observed in the germination plates of seeds. The four-leaf discs were punched out from a leaf on each of the plants and a pair of discs were placed in a well of two different 96-well plates containing agar with Murashige and Skoog salts, 30 g of sucrose and no hormones . The wells of one of the plates were flooded with 1 L of 30 mg / L of 2-CBSU for 30 minutes and then the liquid was removed without rinsing. One disk from each of the wells was tested by the GUS stain after 24 and 72 hours. Out of the 48 seedlings treated with BA662 x BA665, 3 showed GUS staining after 24 hours and 10 more after 72 hours. Out of 72 seedlings treated with BA665 x BA662, 8 showed GUS staining after 24 hours and 23 after 72 hours. Only the discs of the seedlings that expressed GUS more intense, showed traces of GUS activity without insurance treatment. This "weak" induction can be attributed to the ligations and growth in agar. The degree of dyeing did not vary only between the different positive GUS discs, but also through a disc. After 72 hours, there are not only more individuals that compare to positive staining at 24 hours, but some that stained after 24 hours of staining are also stronger after 72 hours. This indicates that the virus is replicating.

Proreplicon replication confirmed by Souther's analysis, that all positive GUS plants were positive for either IN: Rep and proreplicon. Genomic DNA was isolated from the leaves of six plants that express GUS more highly (three of each from progeny BA662 x BA665 and BA665 x BA662) either without or with 2-CBSU treatment. For the trainer, the genomic DNA was isolated from freshly harvested leaves. Finally, the leaves were cut into strips, placed in agar, treated with 30 mg / L 2-CBSU for 30 minutes, and placed in agar for 3 days after the elimination of the insurer. No replicon was detected in the untreated leaves and a large increase in replicon copy number was detected in the treated leaves.

Seed-specific vicilin promoter: TGMV replication gene "Plasmid pGV656 (containing a Hind III fragment of 3086 bp contains a storage protein ORF of 10 kD chimeric maize ba or the control of the specific vicilin promoter was prepared the seed) . The chimeric gene consists of 1) a 2308 bp, operably linked vicinalin Hind III-Nco I promoter, isolated from plasmid pGA971 [Czako et al. (1992) Mol. Gen Genet Vol. 235 (1), pp. 33-40) and 2) the Sac I-Hind III fragment of 276 bp containing a 3 'untranslated region of the nopaline synthase promoter. The Sep I-Xba I fragment of 394 bo containing the majority of the 10 kD ORF was replaced by the Spe64 fragment of 1064 bp containing the ORFs of AC1-3. (This fragment was elaborated by the addition of the Spe I linker to the TGMV clone digested by Bst Bl, and Mfe I, described above). The 56 bp between the Neo I and Sep I sites were suppressed by PCR. The 4236 bp Hind III fragment (containing the chimeric ^ vicilin promoter: the ORF of the replication protein TMV: the nopaline synthase gene) was cloned into the Hind III site of the binary vector pBin 19. The resulting binary plasmid pBE679 was introduced in tobacco plants via the transformation mediated by Agroba cteri um t umefasci ens LBA4404, known to a person skilled in the art. The transgenic plants are in pots and will be crossed with transgenic plants that contain proreplicon BA662 (see above).

Promoter associated senescence SAGrgen of TGMV replication (plasmid pBE667) The Spe I fragment of 1.6 kB, containing the ORFs AL1, AL2, AL3 TGMV (previously described), was cloned into the Xba I site of plasmid pGV666, downstream of a promoter associated with senescence (SAG). PGV666 was derived from plasmid pSG516 [Gan et al. (1995) Sci ence (Washington, DC) vol. 270 (5244), pp. 1986-8] by replacing its Bst Bl-Sac I fragment (containing the 3 'end of the promoter and the ORF isopentyl transferase) with a BstB I-Sac I fragment (containing the modified SAG promoter by PCR) to replace the Neo I site with the Xba I / Sac I sites. The Spe1-Sac I fragment of 3801 bp (containing the SAG promoter: TGMV replication gene) was isolated from pGV667 and cloned into the pBHOl vector digested with Xba I-Sac I (Clontech Inc., 6500 Donlon Rd, Somis, CA) to provide the binary plasmid pBE667, such that the SAG promoter: the ACMV replication gene, are operably linked to the 3 'untranslated region of the nopaline synthase gene. The chimeric gene in pBE667 was introduced into the plants via the Agrobac teri um tummery-mediated transformation LBA4404, known to one skilled in the art.

EXAMPLE 3 Constructs of the African Yucca Mosaic Virus (ACMV) Partial ACMV dimer with the wild-type or native replication gene (plasmid pGV596) Plasmid pCLV012 (ATCC 45039), which contains DNA from the African Yucca Mosaic Virus [West Kenyan isolate 844, Stanley et al. (1983) Na t ure vol. 301, pp. 260-262] was linearized with Pml I and ligated to the Bam Hl linker (New England Biolab ca, No. 1071). After digestion Bam Hl, the 2 kB fragment containing the ORI and the ORFs AC1-3, was isolated and cloned into the Bam Hl site of pSK to provide pGV592 and pGV592R. These plasmids differ in the orientation of the insert: the promoter of the coat protein is close to the Sal site in the vector in pGV592 and distant in pGV592R. The BstBl site in the ACl gene in pGV592R was modified to Sac II, after restriction digestion, filling, and ligation of the Sac II linker. The Not I-Sac II fragment of 358 bp was isolated and cloned between unfolded Not I-Sac II pGV592, to provide pGV596a. The deletion of 140 bp between the Sba Bl and Not I sites in pGV596a contains the 3 'end of the residual ACM coat protein gene, after the Sna Bl disgestion, binding of the Not I linker (New England Biolabs, catalog no. 1125), Not I digestion, and auto-ligation provided pGV605. Both plasmids pGV596a and pGV605 can be replicated when introduced individually into plant cells by biolistic bombardment.

Proreplicon ACMV (plasmid pGV596D) Deletion of a 651 bp region between the Bgl II sites including the 3 'end of the ACl gene in Pgv596A, provided the proreplicon ACMV (plasmid pGV596D).

ACMV proreplicons with the reporter gene (plasmids PGV614D and pGV616D) Plasmid pGV605 was modified by converting the Sac I site to Asp718 and by destroying the Bam Hl site near Sal I to form the plasmid pGV611. The chimeric 10 kD corn storage protein gene, under the control of either a specific seed or a constitutively expressed promoter, used as a reporter for the expression gene. The seed-specific chimeric gene was cloned as a Bam HI-Bgl II fragment of 1164 bo at the Bam Hl site of pGVdII to provide plasmid pGV612. It consists of a seed-specific phaseolin promoter of 383 bp, operably linked - (from -295 to +82 bp with respect to the transcriptional start site), 450 bp contains the ORO of lOkD, and the 331 bp contains 279 bp of the 3 'untranslated region of the nopaline synthase promoter. For the constitutive reporter gene, plasmid pGV611 was first modified to suppress the sites between the Not I and Bam Hl digestion by Bam Hl digestion, filling, addition of the Not I linker (New England Biolabs, catalog No. 1125), and the religation to provide plasmid pGV611N. Then, the constitutive chimeric gene was cloned as a Not I fragment of 1194 bp into the Not I site of pGV611N to provide plasmid pGV61. It consists of a constitutively expressed 35S promoter of 411 bp operably linked (from -400 to +11 bp with respect to the transcription start site), 460 bp containing the 10 kD ORF, and 323 bp containing 279 bp of the 3 'untranslated region of the nopaline synthase promoter. Deletion of a 651 bp region between the Bgl II sites that include the 3 'end of the ACl replication gene in pGV614D and pGV616D by providing ACMV proreplicons with the reporter gene, pGV614D and pVG616D, respectively. The plasmids pGV614D and pGV6161D were linearized with the Sal I enzyme and cloned into the Sal I site of the binary plasmid pZBLl and introduced into the tobacco plants via Agrobac teri um tumefaci ens LBA4404.

Chimeric ACMV replication genes: seed-specific phaseolin promoter: ACMV replication gene _ Plasmid pCLV012 (ATCC 45039) containing the ACMV DNA was modified by the creation of a Neo I site at the initiation codon of ACl ORF by PCR and by modification of the Pml 1 site to the 3 'end of the protein gene of the cover to the Xba I. site. The 1685 bp Neo I-Nco I-Xba I fragment, containing the AC1-3 ORFs, was used to label the seed-specific chimeric replication genes. The chimeric phaseolin: the replication gene, consists of a Bam HI-Nco I fragment of 384 bp operably linked, containing 374 bp of the seed-specific phaseolin promoter (from -292 to +82 bp with respect to the start site of transcription), 1685 bp containing the AC1-3 ORFs (see above), and 323 bp containing 279 bp of the 3 'untranslated region of the nopaline synthase promoter. The phaseolin: ORF ACMV ACl fusion (without the 3 'nopaline synthase promoter) will be isolated as a 2120 bp Bam Hl-Sac I fragment, and cloned into the Bam Hl-Sac I sites of the binary vector pBHOl (Contech Co. , 6500 Donlon Rd, Somis, CA), to provide the binary plasmid pBE625, such that the phaseolin: ORF ACl ACMV promoter is operably linked to the 3 'untranslated region of the nopaline synthase gene. The chimeric gene in pBE625 will be introduced into the plants via the Agrobac teri um t umefa ci ens-mediated transformation known to a person skilled in the art.

Vicilin-specific promoter of the seed replication of the ACMV gene Plasmid pGV656 containing a Hind III fragment containing an ORF of 10 kD storage protein of chimeric corn was prepared, under the control of the specific vicilin promoter of the seed. The chimeric gene consists of 1) an operably linked 2308 bp vicinin Hind III-Nco I promoter, isolated from plasmid pGA971 [Czako et al. (192) Mol. Gen Genet Vol. 235 (l) m pp 33-40] and 2) a Sac I-Hind III fragment of 276 bp, containing the 3 'untranslated region of the nopaline synthase promoter. The 450 bp Neo I-Xba I fragment containing the 10 kD ORF will be replaced by the 1685 bp Neo I-Ncol-Xba I fragment containing the AC1-3 ORFs (see the above description). The 4321 bp Hind III fragment containing the chimeric vicilin promoter: the ORF of the ACMV replication protein: the 3 'nopaline synthase gene will be cloned into the Hind III site of the binary vector pBinl9 and introduced into tobacco plants via the transformation mediated by Agroba c teri um t umefa ci ens LBA4404, known to a person skilled in the art.

Chemically inducible promoter in 2-2: ACMV replication gene (plasmid pBE659) Plasmid pCLV012 (ATCC 45039) containing the ACMV DNA was modified by the creation of a Neo I site at the initiation codon of ACl ORF by PCR and by conversion of the Sna Bl site to the 3 'end of the envelope protein gene [Stanley et al. (1983) Na t ure vol. 301, pp. 260-262 (1983)] to multiply the cloning sites and the sites Ba, Hl). The Neo I-Neo I-Bam fragment. Hl (containing the ACMV ACLV, AC2, and AC3 ORFs) was isolated and cloned between the Neo I and Bgl II fragment in the vector pIN2-1-2 (supra) H. Hershey's, providing the plasmid pGV659. Plasmid pGV659 consists of a Bam HI-Nco I fragment of 452 bp (sequence shown in Figure 4 of U.S. Patent No. 5,364,780), which contains the 2-2 promoter of the maize gene [Hershey et al., Isolation and characterization of cDNA clones for RNA species, induced by substituted benzenesulfonamides in corn. Pl an t Mol. Bi ol. (1991), 17 (4), 679-90)], followed by the Neo I-Neo I-Bam Hl fragment of 960 bp (see above), and a 496 bp Bgl II-Asp718 I fragment containing the region 3 'non-coding gene of maize 21 [Hershey et al., Isolation and Characterization of cloneas cDNA for RNA species induced by substituted benzensulfonamides in corn. Pl an t Mol. Bi ol. (1991), 17 (4), 679-690; U.S. Patent No. 5,364,780]. The Bam HI-Asp 7181 fragment of 2538 bp in pGV659 containing the chemically-inducible promoter operably linked in 2-2, ORFs AC1-C3, and the 247 bp of the 3'-untranslated region of the maize gene 2-1, were Isolate and clone at the Bam HI-Asp718 sites of the binary vector pBinl9 and the resulting binary vector, they will be introduced into the plants via the Agrobac-mediated transformation, which is known to a person skilled in the art.

Promoter associated with the sequence SAG: Rep ACMV (plasmid pBE640) A Neo I-Nco I-Sac I fragment of 1737 bp containing the ACI-1 ACMV ORFs was cloned into the pSG516 plasmid digested Neo I-Sacl [Gan et al. (1995) Sci ence (Washington, D.C.), VOL. 270) 5244), PP. 1986-8] to provide plasmid pGV640. A SpeI-Sac I fragment of 3924 bp from pGV640 (containing the SAG promoter operably linked to the Rep ACMV ORF), will be isolated and cloned into the digested pBHOl Xba I-Sac I to provide the binary plasmid pBE767, such that the SAG: Rep ACMV promoter is operably linked to the 3 'untranslated region of the nopaline synthase gene. The chimeric gene in pBE676 will be introduced into plants via the transformation mediated by Agroba cteri um tumefa ci ens, known to a person skilled in the art.

EXAMPLE 4 Construction of modified Proreplicons The modified proreplicons with minimal ori, will be elaborated as follows: The sequence ori TGMV-A of 101 bp [positions 53 to 153, Orozco et al. (1998) Virol ogy vol. 242, pp. 346-356], will be isolated as a PCR product, using PCR primers Pl and P2 in the template DNA pGV662. Primer Pl will have a restriction site Not I adjacent to position 53 of the ori and primer P2 will have a Sal I site adjacent to position ori 153. After digestion Not I-Sal I, the PCR product will be cloned in the digested pGV662 Not I-Sal I. The resulting plasmid pGV662A, will have the ori and the mutant AL1 in pGV662, replaced with the minimum ori of 101 bp. A 192 bp sequence containing the CaMV polyadenylation sequence [positions 7440-7638, Gene Bank, accessions # V00140 J02046], will be isolated as a B PCR product, using PCR primers P3 and P4. The P3 primer will have an Xba I restriction site, adjacent to position 7440 and the P4 primer will have a Bam Hl I site, adjacent to position 7638. A 262 bp sequence containing the polyadenylation sequence of the nopaline synthase gene (positions 2068-2344, Gene Bank ACCESS No. J01541 V00067), will be isolated as a C PCR product, using PCR primers P5 and P6. The P5 choke will have a Not I restriction site, adjacent to position 1068 and the P6 primer will have a Bgl II site adjacent to position 2344. Bam Hl digested product B PCR, and Bgl II digested product CPR will be ligated and the resistant ligation product Bam Hl and Bgl II, will be subjected to PCR, using primers P3 and P6. The resulting 454 bp PCR product, comprising inverted polyadenylation (head-to-head) sequences, will be digested with Xba I and Not I and cloned between the Xba I and Not I sites, in the 3 'untranslated region, followed by the GUS ORF in plasmid pGV662A, such that in the resulting plasmid, pGV662b, the GUS transcript will be polyadenylated using the CaMV polyadenylation signal sequence and the transcription from the AL1 promoter in ori s will polyadenylate using the signal sequence of polyadenylation nos. A 275 bp sequence of TGMV [positions 53-228, Orozco et al. (1998) Vi rol ogy vol. 242, pp. 346-356] will contain the minimal TGMV-A ori sequence and the coat protein promoter, will be isolated as the E PCR fragment, using PCR primers P7 and P8 in plasmid pGV662 of the template DNA. Primer P7 will have a restriction site Sac I, adjacent to position 53, and primer P8, will have an Xho I-Nco I site, adjacent to position 228. After digestion with Sac I and Neo I, the 275 bp sequence containing the shell protein promoter and minimal ori, will be cloned into digested pGV662B Sac I-Nco I, to result in plasmid pGV662C. The sequence ori TGMV-A minimum of 101 bp [positions 53 to 153, Orozco et al. (1998) Vi rol ogy vol. 242, pp. 346-356], it will be isolated as a F PCR product, using PCR primers P9 and PIO in the template DNA pGV662. The P9 primer will have a Sac I restriction site adjacent to position 53 of the ori and the PIO primer will have a Bgl II site adjacent to the ori position 153. The phaseolin promoter will be isolated as a 323 bp G PCR product, using primers Pll and P12 in a plasmid previously described in the template DNA pGV614. Primer Pll has a restriction site Bam Hl adjacent to position -295 (site Bel I) and primer P12 has an Ncol site at position +20 (site Sea I) with respect to the initiator site of transcription of the phaseolin promoter [Bustos et. to the. (1991) EMBO J. Vol. 10, pp. 1469]. The Bgl II digested PCR product and the Bma Hl digested G PCR product will be ligated and the resistant ligation product Bam I and Bgl II will be subjected to PCR, using primers P9 and P12. The resulting 424 PCR fragment, containing the promoter phaseolin and ori, will be cloned into pGV662 after digestion Sac I-Noc I to result in pGV662D. PGV662C and pGV662D will be modified to pGV662, in which, the GUS target gene will be under the control of the shell protein promoter or the phaseolin promoter, respectively. The GUS sequence will be replaced by other target genes. These modified plasmids will be cloned as a Hind III fragment in a binary plasmid and used to transform transgenic plants as described above. The PCR primer sequences (with the underlined site introduced) used above are given below.

Pl: 3 '^ CTOCGGCCGCTCC ^ AAAGTGATATGAATTGGTAGTAAGGT-3' [SEQ ID NO: 3] P2: 5'-CGA = T ^ A2GCGCGGCCATCCGGTAAT-3 '[SEQ ID NO: 4] P3: S'-GCAGGATCCACpXKAA.TTTrrGGTTTTAGGA-y [SEQ ID NO: 5] P4: 5 * -GCATCTAGAAAATCACCAGTCTCTCTCTACA -3' [SEQ ID NO. : 63 P5: S'-GCTGCGGCCGCTGGAGTAAAGAAGGAGTG -3 '[SEQ ID NO: 7] P6: S ^ CCAGATCTAGTAACATAGAtGACACCG-3' [SEQ ID NO: 8] P7: S '^ GTGAGCTXrrCCAAAAGTTATATGAATTGGTAGTAAGsT-S' [SEQ ID NO: 9] P8: S'-CCTCGAGCCATGGTTTGAATTAAAGATCCACGAAA ^ '[SEQ ID NO: 10] P9: 5' ^ GT 3AGCTGCT: C ^ AAAGTTATATGAATTGGTAGTAAGGTAAGGT-3 '[SEQ ID NO: l 1] PÍO: 5'-GGTAGATCTGCGCGGCCATCCGGTAAT-3 '[SEQ ID NO: 12] P 11: y-CACGGATCCAGATCGCCGCGTC-3' [SEQ ID NO: 13] P12: S ^ CTtX GCeATGGACTCTGGATGGATGGATGATG-3 '[SEQ ID NO: 14] It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property.

SEQUENCE US < 110 > E. I. DU PONT DE NEMOURS AND COMPANY < 120 > SYSTEM OF BIRTH VIRAL EXPRESSION IN PLANTS < 130 > C -1127-A < 140 > < i4 i > < 150 > 60 / 063,504 < 15I > October 7, 1997 < € 1 0 > 14 < 170 > Mieroßoít Word Version 7.0A < 210 > 1 < 2I1 > 35 < 212 > 'DNA < 213 > Unknown < 22D > . < 223 > Description of unknown organism: primers < 400 > 1 egtepgga c? Iaattctccc ca c »* (ja5 tatct 35 < 210 > 2 < 211 > 35 < 212 > DNA < 213 > Unknown < 220 < 223 > "Description of unknown organism: primers <400> 2 c / tcgaccoa. Gcrttßa * g * t caccjaaßcg c * fcej-fc 35 < 210 3 < 211 > 41 < 212 > DNA < 213 > Unknown < 22t) > < 223 > Description of unknown organism: cebadares < 400 > 3 gctgocjgccg ccccaaaagt tatatgaatt ggt «gt * agtj t 41 < 210 > ? < 211 > 21 - < 212 > DNA < 213 > Unknown < 220 > < 223 > Description of unknown organism: primers < 400 > 4 cc »gtcg * cg e? Rcgrjcc? Tc cggtßat 2T < 210 > 5 < 211 > 29 < 23a2 > DNA < 213 > Unknown < 220 > < 223 > Description of unknown organism: cebadares < 400 > 5 g aggatcca. ctggattttg ttttagga 29 < 210 > 6 < 21l > 31 < 212 > "DNA <213> Unknown <220> <223> Description of unknown organism: <4O0> 6 primers gcatctagaa aateaccagt ctctctctac ß 31 < 21D > 7 < 211 > 29 < 212 > DNA 213 > Unknown < 220 > < 223 > Description of unknown organism: primers! < 400 > 7 gc gcggccg ctggagtaaa gaaggagtg 29 < 210 > S < 211 > 2T < 212 > DNA < 213 > Unknown < 220 > 223 > Description of unknown organism: primers < 400 > B geeugatc? ? taaeataga tgacaeeg 28 < 210 > 9 < 211 > 38 «í212 > . DNA < 213 > Unknown < 220 > < 223 > Descri | > of unknown organism: primers < 400 > 9 eg gagctct ccaaaa? Rcta tatgaattgg tag aagg 38 < 210 > 10 < 211 > 35 < 212 > DNA • 213 * Unknown < 220 > -, _. . - < 2ZZ > Description of unknown organism: primers < 400 > 10 cetegageca tggcftgaat taaagatoca egaaa 35 < 210 > 11 < 2Xi > 44 < 2I2 > DNA < 213 > _ Unknown 220 > < 223 > Descption of unknown organism: primers < 40Q > 11 ce tgagcrtct ccaaaagtta tatgaattgg tagtaaggta aggt 44 < 210 > 12 < 211 > 27 < 212 > DNA < 213 > Unknown «220 < 223 > D Dcyccrriipption of d-esoonocid organism: primers < 40 > 12 ggtagatctg cgcggccatc cggtaat 27 < 210 > 13 < 211 > 22 < 212 > DNA < 213 > Unknown < 220 > < 223 > Description of organi < 400 > 13 cacggatcca gatcgccgcg t 22 < 210 > 14 < 211 > 34 < 212 > DNA < 213 > Unknown < 220 > < 223 > Description of organi < 400 > 14 ~ ctgg atggatggat gatg 34

Claims (21)

1. A binary transgenic viral expression system for the replication and increase of the expression of an objective gene, characterized in that it comprises: a) a chromosomally integrated inheritable proreplicon, lacking a functional replication gene for autonomous episomal replication, and comprising: i) cis-acting viral elements required for viral replication; ii) an objective gene comprising at least one suitable regulatory sequence; and iii) flanking sequences that allow the excision or cleavage of the elements of (i) and (ii), and, b) a chromosomally integrated, inheritable chimeric trans-acting replication gene, comprising a regulated plant promoter, operably linked to a viral protein replication code sequence.

2. The binary transgenic viral expression system of claim 1, characterized in that the proreplicon and the transacting replication gene are independently derived from any geminivirus.

3. The binary transgenic viral expression system of claim 2, characterized by the geminivirus is selected from the group consisting of TGMV and ACMV.

4. The binary transgenic viral expression system of claim 1, characterized in that the promoter of the regulated plant is selected from the group consisting of specific tissue promoters, inducible promoters, and developmental state promoters.

5. The binary transgenic viral expression system of claim 4, characterized in that the promoter of the regulated plant is selected from the group consisting of promoters derived from genes from systems inducible by insurers, systems inducible by tetracycline, systems inducible by salicylate , alcohol-inducible systems, glucocorticoid-inducible systems, and ecdysoma-inducible systems.

6. The binary transgenic viral expression system of claim 1, characterized in that the transposable, chimeric, chromosomally integrated, inheritable, replication gene comprises at least one open reading structure, selected from the group consisting of AC1, AL1, AC2, AL2, AC3 and AL3.

7. The binary transgenic viral expression system of claim 1, characterized in that the target gene encoding a protein is selected from the group consisting of an enzyme, a structural protein, a storage protein of maize, a protein bearing resistance to the herbicide, and a protein that carries resistance to insects.

8. The binary transgenic viral expression system of claim 1, characterized in that the target gene encodes a functional AN7R whose expression produces an altered plant assay.

9. The binary transgenic viral expression system of claim 1, characterized in that at least one suitable regulatory sequence of the target gene is selected from the group consisting of constitutive promoters, promoters specific to the plant tissue, promoters specific to the development, inducible promoters and viral promoters.

10. The binary transgenic viral expression system of claim 9, characterized in that at least one regulatory sequence is selected from the group consisting of a viral coat protein promoter, the nopaline synthase promoter, the phaseolin promoter, and the promoter. of the cauliflower mosaic virus.

11. The binary transgenic viral expression system of claim 1, characterized in that the proreplicon optionally contains a DNA fragment encoding a transient peptide.

12. A method of altering the expression of an endogenous plant gene or a transgene in a plant, characterized in that it comprises: a) transformation of a plant with the binary transgenic viral expression system of claim 1, wherein the target gene encodes the less, a functional RNA having substantial similarity to a plant of endogenous gene or transgene of the plant; and b) growth of the transformed plant of a) under conditions where at least one functional RNA is expressed, the expression of lamellae a functional RNA, inhibits the gene of the endogenous plant substantially similar or to the transgene in a plant.

13. A method of altering the levels of a protein encoded by a target gene in a plant, characterized in that it comprises: a) transformation of a plant with the transgenic binary heritable viral expression system of claim i; and b) growth of the transformed plant under conditions where the protein is expressed.

14. The method of claim 13, characterized in that the target gene is in the sense orientation and the level of the expressed protein is increased.

15. A method of altering the levels of a protein encoded by a target gene in a plant, characterized in that it comprises: a) transformation of a first plant with a replicon to form a first primary transformant, the proreplicon lacks a functional replication gene for Autonomic episomal replication and comprises: i) cis-acting viral elements required for viral replication; ii) an objective gene comprising at least one suitable regulatory sequence; and iii) flanking sequences that allow excision or cleavage of the elements of (i) and (ii), and, b) transformation of a second plant with a chimeric trans-acting replication gene, to form a second primary transformant, comprising a regulated promoter operably linked to a viral replication protein coding sequence; c) growth of the first and second primary transformants, where the progeny of both plants are obtained; and d) crossing the progeny of the first and second primary transformants, to provide plants in which the target gene is expressed.

16. A binary transgenic viral expression system for replicon replication, characterized in that it comprises in a cell: (a) a chromosomally integrated inheritable proreplicon, lacking a functional replication gene, for an autonomous episomal replication and comprising cis-acting elements required for viral replication and flanking sequences for the excision or cleavage of the proreplicon from the chromosome, and (b) an inheritable chimeric trans-acting replication gene, comprising a regulated plant promoter, operably linked to a coding sequence of viral replication protein.

17. The transgenic viral replication system of claim 16, characterized in that the proreplicon and the trans-acting replication gene are independently derived from any geminivirus

18. The transgenic viral expression system of claim 17, characterized in that the geminivirus is selected from the group consisting of TGMV and ACMV.

19. The transgenic viral replication system of claim 16, characterized in that the promoter of the regulated plant is selected from the group consisting of specific tissue promoters.

20. The viral replication system of claim 19, characterized in that the promoter of the regulated plant is selected from the group of promoters derived from genes from inducible insurance systems, from systems inducible by tetracycline, from systems inducible by salicylate, from systems inducible by alcohol, of systems inducible by glucocorticoids, and of inducible systems of ecdysomes.

21. The viral replication system of claim 16, characterized in that the inheritable chimeric trans-acting replication gene, comprising at least one open reading structure is selected from the group consisting of AC1-3 of ACMV and AL1 -3 of TGMV . Prompücon: on op Gen ective regulated expression of a replication protein Replication of the Rep in trans: Target Gene FÍ2ura 1