MXPA01002374A - Binary viral expression system in plants - Google Patents

Abstract

The invention relates to plant transgene expression systems comprising chromosomally-integrated components that are individually heritable. The systems are useful for the regulated expression of foreign genes through gene amplification in plant tissue.

Description

SYSTEM OF VIRAL EXPRESSION, BINARY IN PLANTS FIELD OF THE INVENTION This. application claims the benefit of the Provisional Application, North American No. 60 / 150,255 filed on August 23, 1999 and the Application Provisional, North American No. 60 / 130,086, filed on April 20, 1999 and the Provisional Application, No. 60 / 101,558, filed September 23, 1998. The present invention relates to the field of molecular biology and the genetic transformation of plants with foreign gene fragments. More particularly, the invention relates to a binary expression system useful for conditionally expressing transgenes in plants.

BACKGROUND OF THE INVENTION Two serious technical problems surround transgenic plants. First, the expression of transgenes in plants achieves only low and inconsistent levels. These poor expression levels are attributed in part to chromosomal, random integration ("position effects") and partly to a general lack of expression dependent on the number of copies of REF: 127145 genes. It is expected that episomal vectors overcome these problems. In contrast to plants, microbes can achieve high level expression through the episomal vectors (plasmid) because these vectors can be maintained by selection. Although plant viruses have been used as episomal expression vectors, their use has been restricted to transient expression due to lack of selection and / or cellular toxicity (U.S. Patent No. 4,855,237, WO 9534668). Accordingly, non-specific expression for transgenes in unwanted cells and tissues hinders transgenic work in plants. This is important where the objective is to produce high levels of phytotoxic materials in transgenic plants. The conditional expression of transgenes will allow the economic production of desired chemical substances, monomers and polymers at levels that are likely to be phytotoxic for plant growth by restricting their production for tissue production of transgenic plants either just before or after harvesting. . Therefore, the lack of a conditional, commercially usable expression system and the difficulty in achieving high-level, reliable expression both limit the development of transgene expression in plants.

Plant viruses Viruses are infectious agents with relatively simple organization and unique modes of duplication. A given plant virus can contain either RNA or DNA, and can be either single or double stranded.

RNA Plant Viruses The plant viruses of double-stranded RNA include the diminutive rice virus (RDV) and the wound tumor virus (WTV, for its acronym in English). Plant viruses of single-chain RNA include tobacco mosaic virus (TMV) and potato X virus (PVX), turnip yellow mosaic virus (TYMV, by its abbreviations in English), the virus necrosis of the rice (RNV, by its abbreviations in English) and the virus of mosaic of bromine (BMV, by its abbreviations in English). The RNA in the single-stranded RNA viruses can be either a plus (+) or minus (-) chain. Although many plant viruses have RNA genomes, the organization of genetic information differs between groups (the main groupings designated as monopartite, bipartite and tripartite). The genome of most plant RNA viruses, monopartite is a single chain molecule in the (+) direction. There are at least 11 major groups of viruses with this type of genome. Examples of this type of virus are TMV and PVX. At least six major groups of plant RNA viruses have a bipartite genome. In these, the genome usually consists of two molecules of single-stranded RNA, in the (+) sense, distinct, covered with an outer coating, protein in separate particles. Both RNAs are required for infectivity. The cowpea mosaic virus (CPMW) is an example of a bipartite plant virus. A third major group, which contains at least six major types of plant viruses, is tripartite, with three single-stranded RNA molecules, in the (+) sense. Each chain is covered with an outer, protein coating separately, and all three are required for infectivity. An example of a plant virus, tripartite, is the alfalfa mosaic virus (AMV, for its acronym in English). Many plant viruses also have smaller subgenomic mRNAs that are synthesized to amplify a specific genetic product.

Plant DNA viruses Plant viruses with a double-stranded DNA genome include cauliflower mosaic virus (CaMV). Plant viruses with single-stranded DNA genomes include geminiviruses and, more specifically, include the China-African Melon Mosaic Virus (ACMV), Tomato Golden Mosaic Virus (TGMV). acronyms in English) and Virus in Stretch Marks (MSV, for its acronym in English). Geminiviruses are subdivided on the basis of whether they infect monocotyledonous or dicotyledonous plants and whether their insect vector is a hopper or a small homopterous insect. The geminiviruses of subgroup 1 are transmitted by the skips and infect the monocotyledonous plants (for example, the Tiny Wheat Virus): the geminiviruses of subgroup II are transmitted by the skips and infect the dicotyledonous plants (for example, the Virus of the Part Top Curly Beet); and the geminiviruses of subgroup III are transmitted by small homopterous insects and infect dicotyledonous plants (eg, Tomato Golden Mosaic Virus, TGMV and Chinese, African Melon Mosaic Virus, ACMV).

The geminiviruses of subgroup I and II have an individual genome (monopartite). The geminiviruses of subgroup III have a bipartite genome. For example, the geminiviruses of subgroup III TGMV and ACMV consist of two genomes of single-stranded, circular DNA, A and B of approximately 2.8 kB in size each. The A genome and the DNA B genome of a given subgroup III virus have little sequence similarity, except for a common, almost identical region of approximately 200 bp. While both the A genome of DNA and the B genome of DNA are required for infection, only the DNA A genome is necessary and sufficient for duplication and the DNA B genome encodes the functions required for the movement of the virus through of the infected plant. In both the TGMV and the ACMV, the DNA genome A contains four open reading structures (ORFs) that are expressed in a bidirectional manner and are ordered 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 the ACMV and to the left (L) or to the right (R) in the TGMV. In this way, the ORFs AL1, AL2, AL3 and AR1 of the TGMV are homologous for AC1, AC2, AC3 and AVI, respectively, of the ACMV. Three main transcripts in the DNA genome A of the ACMV have been identified and these map a map for the AVI and ACl ORFs, separately and the AC2 / AC3 ORFs together. There is experimental evidence for the function of these ORFs. In this way, in the ACMV the ACl encodes a duplication protein that is essential and sufficient for duplication; AC2 is required for transactivation of the coat protein gene, AC3 encodes a protein that is not essential for duplication but increases the accumulation of viral DNA; and AVI is the gene for the coating protein. Except for the essential viral duplication protein (encoded by the ACl and AL1 in the ACMV and the TGMV, respectively), the duplication of the geminiviruses depends on the duplication of the host and the transcription machinery. Although geminiviruses are single-stranded DNA plant viruses, they are duplicated by the double-stranded DNA intermediate when the circle duplication is put into operation. The contribution of the AC2 coding sequence when removed from the context of the ACMV genome and expressed from a potato X virus (PVX) based on the vector has been investigated in Hong et al. (Transactivation of dianthin transgene expression by African cassava mosaic virus AC2; Virology 228 (2); 383-387 (1997)). Using a vector of PVX to express the AC2 protein in plan ta, it was found that the expression of AC2 can induce necrosis in transgenic plants. While both the DNA genome A and the DNA genome B are required for infection, only the A genome of DNA is necessary and sufficient for the duplication and the DNA B genome encodes the functions required for the movement of the virus through the infected plant. This has been examined in detail by Hayes and collaborators (Replication of tomato golden mosaic virus DNA in transgenic plants expressing open reading frames ORFs .of DNA A -Requirement of ORF AL2 for production of single-stranded DNA; Nucl ei c Acids Res. 17 (24): 10213-10222). It was determined that transgenic plants for the ORF of the TGMV AL1 are capable of supporting the duplication of the double-stranded forms of the B-genome of DNA, but the ORF AL2 is required in the addition to produce the B-gen of single-stranded DNA.

Viruses as Expression Vectors The construction of plant viruses. for introducing and expressing foreign non-viral genes in plants has proven to be successful (U.S. Patent No. 4,855,237, WO 9534668). When the virus is a DNA virus, the constructions can be made for the virus itself. Alternatively, first the virus can be cloned into a bacterial plasmid by the ease in the construction of the viral vector, desired with the foreign DNA. If the virus is an RNA virus, the virus is generally cloned as a cDNA and inserted into a plasmid. The DNA plasmid is then used to make all constructions. The RNA virus is then produced by transcribing the viral sequence of the plasmid and translating the viral genes to produce the coating protein (s) which cover the viral RNA. The RNA viral genome cDNA can be cloned behind a heterologous plant promoter. This chimeric gene, called an "amplicon", can be introduced into a plant cell and can be used to transcribe viral RNA that can be duplicated autonomously [Sablowski et al. (1995) Proc. Na ti. Acad. Sci. USA vol. 92, pages 6901-6905]. Geminiviruses have many advantages such as plant expression vectors, potentials. These include 1) duplication at high copy numbers, 2) small well-characterized genomes, 3) assembly on nucleosomes, and 4) duplication and nuclear transcription.

The DNA genome A component of these viruses is capable of autonomous duplication in plant cells in the absence of DNA genome B. The vectors in which the ORF of the coat protein has been replaced by a heterologous coding sequence have been developed and the heterologous coding sequence has been expressed from the coat protein promoter [Hayes et al., Stability and expression of bacterial genes. in replicating geminivirus vectors in plants. Nuclei c Acids Res. 17: 2391-403 (1989); Hayes et al., Gene amplification and expression in plants by a replicating geminivirus vector. Na tuze (London) 334: 179-82 (1988)]. A vector system based on geminivirus to obtain the controlled expression of a nucleic acid fragment of interest has been reported (patent WO9419477). This vector system makes it possible for the transformed plants having cells, tissues or parts to exhibit an altered genotype and / or to express modified phenotypic properties. A transfected plant cell is produced by contacting a plant cell with the recombinant geminivirus transfer vector. Limited methods have been developed for the induction of transgene expression. Patent WO9525801 discloses a replicon or viral duplicating section whose construction makes it possible for the replicon or duplicating section to be non-coding in the absence of a virus or viral components. Conversely, in the presence of a virus or viral components, the replicon or duplicating section is activated by the viral replicase which converts the DNA in a negative, non-coding sense into RNA in the positive sense, thereby allowing gene expression ( is). This allows plants to gain resistance to viral infection, since the activation of a resistance response is directly linked to the replication of the virus. In addition, as viral duplication increases, the resistance response also increases. Larger-than-full-length copies of the TGMV genomes A and B of the non-cultured type were transformed into petunia [Rogers et al., Tomato golden mosaic virus A component DNA replicates autonomously in transgenic plants. Cel l (Cambridge, Mass.) 45: 593-600 (1986)]. Duplication was reported in the primary transformants and in some of the inbred progeny consistent with their Mendelian inherence, indicating that the original copy integrated chromosomally, not the replicon or duplicating section, it is inherited. This suggests that gametophytic and / or developmental seed tissues lack the capacity to withstand duplication. The report did not show if the virus doubled in seed tissue, not germinating. The prior art shows that geminiviruses are not transmitted in the seed in nature [Goodman, R.M. (1981) Geminivirus. J. Gen. I saw role. vol. 54, pages 9-21]. In this way, there was no evidence that these could be duplicated in the gametophytic tissue or the seed of development. DNA genome A of the Tomato Mosaic Virus (TMV) was modified by replacing its coding sequence for the coat protein with that of the reporter genes NPT II or GUS or with that of the 35S: NPT II gene and a larger copy that the full length of the modified viruses was transformed into tobacco [Hayes et al., Stability and expression of bacterial genes in replicating geminivirus vectors in plants. Nu cl ei c Acids Res. 17: 2391-403 (1989); Hayes et al., Gene amplification and expression in plants by a replicating geminivirus vector. Na ture (London) 334: 179-82 (1988)] The leaves of transgenic plants showed that the high levels of the reporter enzymes were dependent on the number of gene copies. However, the duplication of the vector and the expression of reporter genes were not reported in the seed and the genetic stability of the vector in transgenic plants in subsequent generations was not reported. Similarly, the use of China-African Melon Mosaic Virus (ACMV) has not been reported and it is not known that ACMV DNA or duplication protein (s) can be stably maintained in plants progeny and if it can be duplicated in seed tissues. In one report, a chimeric gene (in which the constitutive plant promoter, 35S, was fused to the TGMV sequence containing the ORFs AL1, AL2 and AL3) was transformed into Ni cotiana ben thamiana. The different transgenic lines did not show significant uniformity in the expression levels of 35S genes: ALl-3 as well as in their ability to complement viral duplication [Hanley-Bowdoin et al., Functional expression of the leftward open reading frames of the A component of tomato golden mosaic virus in transgenic tobaceous plants. Plan t Cel l 1: 1057-67 (1989)]. In another report, the chimeric genes (in which the plant promoter, constitutive, 35S, was fused to the coding sequence of the duplicating protein of TGMV AL1) were transformed into tobacco. The expression of the TGMV duplication protein in the primary transformants supported the duplication of a mutant A genome lacking the duplication protein. [Hanley-Bowdoin et al., Expression of functional replication protein from tomato golden mosaic virus in transgenic tobaceous plants. Proc Na ti. Acad. Sci. E. U. A 87: 1446-50 (1990)]. However, the publication neither reported the genetic stability of the chimeric gene of the duplication protein through subsequent generations nor its ability to support viral duplication in the seed tissue. In another report, the chimeric genes (in which the plant promoter, constitutive, 35S, was fused separately to the coding sequences of the TGMV duplication proteins AL1, AL2 and AL3) were transformed into tobacco [Hayes et al. collaborators, Replication of tomato golden mosaic virus DNA B in transgenic plants expressing open reading frames (ORFs) of DNA A: requirement of ORF AL2 for production of single-stranded DNA. Nucl ei c Acids Res. 17: 10213-22 (1989)]. The duplication protein of TGMV was expressed in the progeny but the genetic stability of the chimeric gene of the duplication protein was not reported through subsequent generations. In addition, it was not reported whether the transgenic plants will support duplication in the seed tissue.

In another description, Rogers et al. (EP 221044) demonstrated the expression of foreign proteins in 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 transformed into the plant tissue. Rogers et al. Did not report tissue-specific expression of the foreign protein and were indifferent to the genetic stability of the transformation plasmid. All viral vectors reported have a major disadvantage. It was not shown either that these were stably maintained in transgenic plants and / or were not practically useful. Thus, despite intensive efforts to develop viral, plant and virus vectors, recombinant vectors based on commercially useful plant viruses that are heritable and capable of episomal duplication and expression in desired tissue (s) have not been developed. (s) of the host plant, transgenic without the need for the infection of each generation. In fact, the duplication of plant viruses is expected to be harmful to the growth and development of plant cells. For example, when the larger than full-length copy of the TGMV genome A is entered into a tenth of the plant cell as many transgenic plants are obtained that when the B genome is used or when the control transformations are made [Rodgers et al. , Tomato golden mosaic virus A component DBA replicates autonomously in transgenic plants. Cell (Cambridge, MA) 45: 593-600 (1986)]. The authors suggest that this may be due to the expression of a gene in the DNA A of the TGMV. In addition, the crude extract of the plants that express double copies of both the TGMV and B TGMV genomes A are unable to infect Ni co tiana ben thamiana plants. This is consistent with having a low virus titer. In this way, transgenic plants that are regenerated could be selected for low level expression of a viral gene product, toxic and the low level of viral duplication or are turned off by the host. This is also consistent with the authors' finding that relatively few cells initiate virus release, a conclusion based on their observation that most tissues remain viable and non-symptomatic. Similarly, poor duplication in transgenic plants containing 35S: duplication protein in other reports suggests that the plants are either selected by the poor expression of the duplication protein (presumably due to its toxicity), or that the profiles of Specific expression for the tissue of the duplication gene are different from those of viral duplication. To date, there are no known recombinant vectors based on plant viruses that are heritable and capable of episomal duplication and the expression of foreign proteins in the target tissue (s) of a host plant, transgenic without the need for infection in each generation. The use of viral vectors to restrict genes has been reported [[Covey, S. N. et al. (1997) Na t ure (London) 385: 781-782; Kurnagai et al. (1995) Proc. Na ti. Aca d. Sci. (E.U.A.) 92: 1679-1683; Kjemtrup, S. et al. (1998) Plan t J. 14: 91-100; Ratcliff, F. et al. (1997) Sci ence (Washington, DC) 276: 1558-1560; Ruiz, M. T. et al. (1998) Pl an t Cel l 10: 937-946; Baulcombe, D.C. and Angeli, S.M. (1998) Virus amplicons of gene silencing in transgenic plants, International Application of PCT WO 9836083]. In a report it was concluded that the duplication of viral RNA is a powerful trigger for gene restriction (Baulcombe, DC and Angelí, SM Consistent gene silencing in transgenic plants expressing a replicating potato virus X RNA. EMBO 16 (12): 3675- 3684 (19.97)). It was suggested that the restriction of genes mediated by amplicons may provide a new important strategy for the consistent activation of gene restriction in transgenic plants. An additional study by Pruss and collaborators (Plant Cell 9: 959-868 (1997)) teaches that the Pl / HC Pro sequence of tobacco erosion or corrosion virus (TEV) acts as a general pathogenicity enhancer during co-infection by three different groups: potexvirus (PVX), tobamovirus (TMV) and cucumovirus (CMV)). Although the mechanism of the interaction is not yet well understood, the fact that the expression of the Pl / HC Pro sequence alters the induction of the disease by each of these heterologous, unrelated viruses suggests that this affects a common step in the Viral infection. Additionally, it is known that the central domain of HC Pro is also involved in the duplication of potyvirus RNA. Although these reports where viral vectors are used for gene restriction their use in transgenic plants under the control of site-specific recombination, conditional or regulated has not been reported so far. In the absence of such regulation, the current viral gene restriction is functioning constitutively. This could be harmful to a plant and suppresses viral restriction, transgenic for non-essential genes. Although the use of restriction-suppressor genes has been shown to overcome the restriction of transgenic genes, its use in the prevention of transgene restriction in viral episomes has not yet been demonstrated. The viral vectors, transgenic for the production of foreign proteins and / or the restriction of genes differ from the infection of viral vectors in not requiring a systemic movement. The use of viral transgenes, constitutively expressed for viral resistance, has been reported. However, the conditional expression of such transgenes, preferably through the conditional activation of the replicon or duplicating section, i.e. with viral infection, is likely to provide more effective control. The conditional or regulated expression has been reported in plants [see De Veylder, L. et al., Pl an t Cell Physiol. 38: 568-577 (1997); Gratz, C, Annu. Rev. Pl an t Physiol. Plan t Mol. Biol. 48: 89-108 (1997); Hansen, G. and collaborators, Mol. Gen Genet 254: 337-343 (1997); Jepson, I., International PCT Application (1997) WO 9706269 Al: Jepson, I. et al. PCT International Application (1997) WO 9711189 A2, and other references within this application]. However, when subjected to a test to convincingly for non-specific, basic expression, very few have been strictly specific [Odell, J. T. et al., Plan t Physi ol. 91994) 106: 447-458; van der Geest et al., Plan t Physiol (1995), 109 (4), 1151-58; Ma, and collaborators, Aus t. J. Pl an t Physi ol. (1998), 25 (1), 53-59; Czako et al Mol. Gen Gene t. (1992), 235 (1), 33-40]: Such promoters are not suitable for some applications, such as the use of transgenes to express new phytotoxic proteins, enzymes that lead to the biosynthesis of phytotoxic products and / or the restriction of genes. Dirks et al. (International PCT Application (1998) WO 9828431) teach an Arabidops i s promoter (AtDMCl) that can be used in the specific expression for meiosis of a heterologous sequence of genes in transgenic plants. The additional modalities of their invention allow the removal of any unwanted DNA sequence during the first cycle of sexual reproduction in a transgenic plant (using a Cre / lox system) and the specific transcription for the meiosis of a cytotoxic gene and death of meiotic cells (for the production of epomiéticas seeds or for the detection of apomiéticos mutants). These teachings are limited in that the conditional or regulated expression is necessarily linked to meiosis. Plant-specific recombinations have been reported in plants (Odell et al., Pl an t Physiol. 106: 447-458 (1994)).; Odell et al., International PCT Application (1991) WO 9109957; Surin et al., International PCT Application (1997) WO 9737012; Dirks et al., International Application of the PCT (1998) WO 9828431) and the reduction in the ability of Cre-mediated recombination by Lox P mutant sites and their use in increasing the frequency of integration based on Cre-lox [Albert and collaborators, Pl an t J. 7: 649-59 (1995); Araki et al., Nucl ei c Acids Res. 25: 868-872 (1997)]. However, the use of mutant sites to increase Cre-mediated recombination of specificity in conjunction with chimeric Cre genes under the control of regulated, available promoters has not been demonstrated. Thus, there is a need for a site-specific recombination system, appropriately stringent for site-specific, conditional, commercially attractive recombination.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides a viral, transgenic, binary expression system comprising: (i) an inactive, chromosomally integrated duplicating replicon or section comprising a) viral action elements required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase; and (ii) a chimeric, chromosomally integrated transactivation gene comprising a regulated, plant promoter operably linked to a site-specific recombinase coding sequence; wherein the expression of the chimeric transactivation gene in cells containing the inactive replicon or duplicating section results in site-specific recombination, replication replication of the replicon or duplicating section, and increased expression of the target gene. The invention further maintains that the inactive, duplicating replicon or section is derived from a geminivirus or a single-stranded RNA virus. Additionally, the invention holds that the regulated plant promoter may be tissue-specific, constitutive or inducible and the site-specific sequences, of the non-cultured or mutant type, responsive to a site-specific recombinase, the sequences specific to the site. site may be lox sequences, responsive to the Cre recombinase protein. The invention further provides a method for alternating the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a plant with the present viral expression system; and (ii) cultivate the vegetable seed, transformed under conditions where the protein is expressed. Additionally, the invention provides a method for altering the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a first plant with a duplicating replicon or section, inactive to form a first primary transformant, the replicon or duplicating section comprising: a) viral elements of action required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase, (ii) transforming a second plant with a chimeric transactivation gene to form a second primary transformant comprising a regulated, plant promoter operably linked to a sequence of site-specific recombinase coding; (iii) cultivate the first and second primary transformants where the progeny of both seeds are obtained; and (iv) crossing the progeny of the first and second transformants where the target gene is expressed.

In an alternative embodiment the invention provides a method for altering the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a plant with a duplicating, inactive replicon or section, the replicon or duplicating section comprising: a) cis-acting viral elements required for viral replication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase, (ii) infecting the transformant with a virus containing a transactivation, chimeric gene comprising a regulated, plant promoter operably linked to a coding sequence of site-specific recombinase, transactivation; wherein the expression of the transactivation, chimeric gene in cells containing the replicon or duplicating section, inactive results in site-specific recombination, activation of duplication of the replicon or duplicating section, and increased expression of the target gene. In another embodiment, the invention provides a transgenic, binary expression system comprising an inactive transgene and a chimeric transactivation gene, the inactive transgene comprising: (i) regulatory elements of the transcription of action ci s attached in an inoperable manner to the coding sequence or functional RNA; and (ii) site-specific sequences responsive to a site-specific recombinase.; the transactivation, chimeric gene comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence of transactivation, wherein the expression of the transactivation, chimeric gene in cells containing the inactive transgene gives resulting in an operable linkage of the regulatory elements of the action transcript to the coding sequence or functional RNA through site-specific recombination and increased expression of the target gene.

In an alternate embodiment, the invention provides a transgenic, binary expression system comprising: (i) a chromosomally integrated blocking fragment, linked by site-specific sequences responsive to a site-specific recombinase; and (ii) an inhibitory, inactive, chromosomally integrated restriction transgene; wherein the expression of the site-specific recombinase results in site-specific recombination that activates the restriction-suppressor gene. Additionally, the invention provides a transgenic viral expression system comprising: (i) a chromosomally integrated geminivirus proreplicon comprising: a) cis-acting viral elements required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) side sequences that make possible the excision of the elements of a) and b), wherein the prorreplicon lacks a functional duplication gene for episomal duplication; (ii) a chimeric, chromosomally integrated, trans-acting duplication gene comprising a regulated, plant promoter operably linked to a viral duplication protein coding sequence of geminivirus; and (iii) a dimer of the genome B of the geminivirus; wherein the expression of the trans action duplication gene in cells containing the prorreplicon results in the duplication of the prorreplicon and the B genome, and the increased expression of the target gene. A further objective of the invention is to provide a transgenic geminivirus expression system comprising: (i) an inactive, chromosomally integrated replicon comprising: a) viral elements of cyclic action required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) sequence specific to the site responsive to a site-specific recombinase; (ii) a chromosomally integrated, chimeric transactivation gene comprising a regulated, plant promoter operably linked to a site-specific recombinase coding sequence; (iii) a dimer of a geminivirus B genome; wherein the expression of the chimeric transactivation gene in cells containing the inactive replicon results in site-specific recombination, B-gen replicon activation and increased expression of the target gene. Still another object of the invention is to provide a method for increasing viral resistance in a plant comprising: (i) transforming a first plant with an inactive replicon to form a first primary transformant, the inactive replicon comprising: a) viral elements of cis action required for viral duplication; b) viral sequences homologous to the infective virus capable of conferring resistance dependent on homology; c) site-specific sequences responsive to a site-specific recombinase; (ii) transforming a second plant with a chimeric transactivation gene to form a second primary transformant comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence of transactivation; (iii) cultivate the first and second primary transformants where the progeny of both seeds is obtained; Y (iv) crossing the progeny of the first and second transformants where the viral sequences homologous to the infective virus are expressed, transmitting the viral resistance to the plant. The present invention is useful in transgenic plants for the controlled duplication of replicons and the expression of transgenes with or without duplication of replicons. Both components of the system are chromosomally integrated and are hereditary independently. A component is an inactive replicon that is unable to replicate episomally unless an in trans transactivation protein is provided. The second component is a trans activation gene, chimeric in which the coding sequence of a transactivation protein is placed under the control of a tissue-specific or developmental and / or inducible promoter. The transactivation protein can be either a viral duplication protein or a site-specific recombination protein. When it is a viral duplication protein (s), the inactive replicon is of the prorreplicon type lacking a functional duplication protein (s) and can not be duplicated episomally unless (n) the (s) in trans duplication protein (s). When it is a site-specific recombinase, it can mediate site-specific recombination that involves the site-specific sequence (s), congested in the inactive replicon to turn it into an active one. capable of autonomous duplication or ci s. The two systems involving a replicon can be used independently or in combination. The site-specific recombination system can also be applied to the transactivation of an inactive transgene with or without the involvement of episomal duplication.

The different components of the invention are hereditary independently and can be introduced together in a transgenic plant or brought with ease by crossing the transgenic plants that carry the separated components, such as by the method to produce corn seed with high content of TopCross oil [ U.S. Patent No. 5,704,160]. Methods for making the expression cassettes and methods for using them to produce transformed plant cells having an altered genotype and / or phenotype are also provided.

BRIEF DESCRIPTION OF THE FIGURES AND DESCRIPTIONS OF SEQUENCES Figure 1 illustrates the division and regulation of the expression of a replicon and the generation of an active transgene from an inactive replicon containing site-specific sequences responsive to a site-specific recombinase. Figure 2 illustrates the division and regulation of the expression of a replicon and the generation of an active transgene from an inactive replicon containing site-specific sequences responsive to a site-specific recombinase where a sequence specific to the site is in the 5 'non-coding transcribed sequence and the other is in an inverted orientation in the promoter. Figure 3 illustrates the division and regulation of the expression of a replicon containing a transcription arrest fragment inserted between the site-specific sequences where duplication is mediated by a site-specific recombinase. Figure 4 illustrates the cleavage and activation of a prorreplicon by means of the expression of a chimeric transcription duplication gene. The following sequence descriptions and sequence listings appended thereto comply with the rules governing descriptions of nucleotide and / or amino acid sequences in the patent applications set forth in 37 C.F.R. § 1,821 - 1,825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures - the Sequence Rules ") and are consistent with the World Intellectual Property organization (WIPO) Standard ST2.5 (1998) and the sequence listing requirements of EPO and PCT (Rules 5.2 and 49.5 (a-bis), and Section 208 and Annex C of the Instructions of the Administration) . The Sequence Descriptions contain the one-letter code for the nucleotide sequence characters and the three-letter codes for the amino acids as defined in accordance with the IUPAC-IYUB standards described in Nuclei c Acids Res. 13: 3021-3030 (1985) and in the Biochemi cal Journal 219: 345-373 (1984). Sequences 1-42 are given in the present application, all corresponding to the primers used in the gene amplification.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a binary expression system that utilizes various genetic elements of plant viruses of DNA or RNA, regulated promoters and / or site-specific recombination systems. The expression system is useful for episomal, conditional duplication, expression of transgenes with or without episomal duplication, suppression of host genes induced by the virus, and viral resistance. These replicons may be either capable or incapable of cell-to-cell or systemic movement. The applicant solved the problems established by methods that provide a two-component expression system, at least one of which is chromosomally integrated.

In another method, the expression system comprises an inactive replicon and a site-specific, transactivation, chimeric, regulated recombinase gene. The inactive replicon comprises site-specific recombination sequences, non-cultured type or mutants and is unable to duplicate either because it can not be split from the chromosome (in the case of the DNA replicon) and / or because one or more viral genes can not be transcribed appropriately (in the case of both the DNA replicon and RNA virus amplicon). The site-specific recombinase, of transactivation mediates the site-specific recombination between the site-specific sequences, the uncultivated type and / or mutants within or around the inactive replicon that renders the inactive replicon active and makes duplication possible. These replicons may be either capable or incapable of cell-to-cell or systemic movement. In this way, site-specific recombination mediates the rearrangement of DNA (division or inversion) in the chromosome which results in either the cleavage of a DNA replicon or RNA amplicon and / or the proper transcription of one or more genes that lead to the release and autonomous replication (ie, ci s) of the replicon (Figures 1, 2). Figure 1 shows a scheme for the regulated transactivation of an inactive replicon or transgene by the division of DNA mediated by a site-specific recombination such as, for example, Cre-lox. The open triangle represents a lox P site of the non-cultivated or mutant type. The DNA genome A and C can be the promoter and the untranslated region ORF / 3 ', respectively, of a transgene or these can be any DNA. The DNA genome B can be a replicon and / or a Transcription Detention Fragment. When the construct is a geminivirus replicon inserted between the promoter (solid box) and the ORF (open box) of its duplication gene, the duplication gene is inactive. When the replicon also serves as a Transcription Detention Fragment, its insertion inactivates the transgene and with site-specific recombination, both doubling and chromosomal transgene genes become active and the latter can be a reporter for the division of the replicon. . Similarly, Figure 2 is a schematic illustrating the transactivation of an inactive replicon (amplicon) by DNA inversion mediated by site-specific recombination, such as Cre-lox. The Lox sequences are represented by the arrows above the amplicon. The open arrow represents the replicon. The open reading structures in the replicon are then represented by the arrows on the amplicon. TATA and TSS are the TATA table and the start site of the Transcription for the plant promoter. The ATAT and SST are the TATA and TSS sites, respectively, in the reverse order. The Ml, M2, M3 are the three movement proteins, RdRP is the RNA-dependent RNA polymerase. The PR is the coating protein and the triangles are the promoters of the duplicated PR. Pro 'and 3' poly A are regions containing the promoter and the 3 'polyadenylation signal. Alternatively, the applicant has developed a method for the transactivation of inactive transgenes by the site-specific recombination system without the use of replicons (Figure 3). Figure 3 presents a scheme for the transactivation of an inactive replicon (amplicon) by dividing DNA from a Transcription Detention Fragment mediated by site-specific recombination, such as Cre-lox. The Transcription Detention Fragment is represented by the filled box. The open arrow represents the replicon. The open reading structures in the replicon are represented later, the amplicon by the arrows. The Lox sequences are represented by the arrows above the amplicon. TATA and TSS are the TATA table and the start site of the Transcription (initiation) for the plant promoter. Ml, M2, M3, RdRP, PR is the coating protein. The Pro 'and 3' poly A are as described in Figure 2. In another embodiment, the expression system comprises a prorreplicon and a duplication, transactivation, chimeric, regulated gene. A prorreplicon contains the cis-acting viral sequences required for duplication but is incapable of episomal duplication in plant cells because this lacks an essential duplication (s) functional gene (s) for duplication . The transactivation gene expresses the missing viral duplication protein in the xprorreplicon and allows the prorreplicon to double in trans (Figure 4). Figure 4 illustrates a scheme for the duplication of transactivation? of an inactive replicon (prorreplicon) in trans. The regulated expression of a duplication gene, chimerically, chromosomally integrated will result in the duplicative and chromosomally releasing of the replicon from a chromosomally integrated original copy of the prorreplicon. Plant cells containing an inactive replicon duplicate the replicon episomally only in the presence of a site-specific recombinase.

In this manner, regulated expression of a chimeric, site-specific recombinase gene in these cells results in regulated duplication of the replicon and amplification of the target gene. While the individual elements of the invention are hereditary, the gene expression system may be hereditary or limited to the progeny of the crosses that genetically combine the two elements. Thus, in some applications the expression of transgenes or target genes will be restricted to the progeny of the crosses, such as in the method to produce the corn seed with high content of TopCross® oil. Using the present system, the Applicant has demonstrated that: (i) the tissue of the soybean seed and the corn seed will support the duplication of the geminivirus; (ii) the expression system will effect the expression of foreign genes in tobacco; (iii) the PVX amplicons can be duplicated in the development of the soybean seed; and (iv) both the geminivirus and the PVX virus can be activated to duplicate by the Cre-lox recombination system. The present invention advances the technique by providing vectors of plant viruses a) which are stably maintained in the chromosome of transgenic plants; b) whose duplication is controlled by the regulated expression of a site-specific recombinase; and c) which may contain nucleic acid sequences encoding foreign proteins that can be expressed in the transgenic plant for the production of foreign proteins or to restrict plant, host genes. The present invention also advances the art by providing a conditional, high-level expression method of transgenes using a site-specific, regulated recombination system using site-specific sequences, mutants and the regulated expression of the specific recombinase for the place . The following terms and definitions should be used to fully understand the specification and the claims. The "gene" refers to a fragment of nucleic acid that expresses mRNA, functional RNA, or specific protein, including regulatory sequences. The term "native gene" refers to the gene as it is found in nature. The term "chimeric gene" refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences that encode portions of naturally unbound proteins, or ) parts of promoters that do not bind naturally. Accordingly, a chimeric gene can comprise regulatory sequences and coding sequences that are derived from different sources or comprise regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature. A "transgene" refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes can include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism.

The "coding sequence" refers to a DNA or RNA sequence that encodes a specific amino acid sequence and 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 coding sequence that specifies the initiation and chain termination, respectively, of protein synthesis ( translation of mRNA). A "functional RNA" refers to an RNA in antisense, ribozyme, or other RNA that is not translated. "Regulatory sequences" and "appropriate regulatory sequences" each refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence transcription, RNA processing or stability, or translation of the associated coding sequence . Regulatory sequences include enhancers, promoters, leader translation sequences, introns and polyadenylation signal sequences. These include the natural and synthetic sequences as well as the sequences which can be a combination of synthetic and natural sequences. As noted above, the term "appropriate regulatory sequences" is not limited to promoters, however, some suitable regulatory sequences useful in the present invention will include, but not be limited to, plant promoters, constituents, promoters. specific for plant tissue, specific promoters for plant development, plant promoters, inducible and viral promoters. The "non-coding sequence 5 '" refers to a nucleotide sequence located 5' (upstream) to the coding sequence. The sequence is present in the fully processed mRNA upstream of the initiation codon and can affect the processing of the primary transcript to the mRNA, the stability of the mRNA or the translation efficiency. (Turner et al., Mol ecula r Bi o technol ogy 3: 225 (1995)). The "non-coding sequence 3 '" refers to nucleotide sequences located 3' (downstream) to a coding sequence and include polyadenylation signal sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or the expression of genes. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid extensions to the 3 'end of the mRNA precursor. The use of different 3 'non-coding sequences is exemplified by Igelbrecht et al., Plan t Cel l 1: 671-680, (1989). The "promoter" refers to a nucleotide sequence, usually upstream (5 ') to its coding sequence, which controls the expression of the coding sequence by providing recognition for the RNA polymerase and other factors required for the proper transcription. The "promoter" includes a minimal promoter which is a short DNA sequence comprised of a TATA box and the other sequences that serve to specify the transcription initiation site, to which regulatory elements are added to control expression. The "promoter" also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a functional RNA coding sequence. This type of promoter sequence consists of the upstream elements near and more distant from the center, the latter elements frequently 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 inverted) and is capable of operating even when it is moved either upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind proteins that bind DNA, specific to the sequence that mediate their effects. The promoters can be derived in their entirety from a native gene, or they can be composed of different elements derived from different promoters found in nature, or they can even be comprised of segments of synthetic DNA. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of the initiation of transcription in response to physiological or developmental conditions. "Constitutive expression" refers to expression using a constitutive or regulated promoter. "Regulated expression" and "conditional" refers to the expression controlled by the regulated promoter. The "constitutive promoter" refers to the promoters that direct the expression of genes in all tissues and at all times. The "regulated promoter" refers to promoters that direct gene expression non-constitutively but in a temporally and / or spatially regulated manner and include both inducible and tissue-specific promoters. This includes the natural and synthetic sequences as well as the sequences which can be a combination of synthetic and natural sequences. Different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. New promoters of various useful types in plant cells are constantly being discovered; Numerous examples can be found in the compilation by Okamuro et al., Bi ochemis try of Plan ts 15: 1-82, 1989. Since in most cases the exact boundaries of regulatory sequences have not been fully defined, the fragments of different lengths of DNA may have identical promoter activity. Typical, regulated promoters useful in plants include but are not limited to promoters inducible by safener, promoters derived from the tetracycline-inducible system, promoters derived from salicylate-inducible systems, (Gatz, C, Curr. Biotechnol. (1996), 7 (2), 168-72) promoters derived from alcohol-inducible systems, promoters derived from the glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and derived promoters. of the inducible systems by ecdysonomes (Martínez et al, Inducible Gene Expression Plants (1999), 23-41) Editor (s): Reynolds, Paul HS Publisher: CABI Publishing, Wallingford, UK, Thompson et al., Mol. Cell. Biol. (1992), 12 (3), 1043-53). The "tissue-specific 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 embryo or cotyledonous plants) or cell-specific types (such as leaf parenchyma or seed storage cells). These may also include promoters that are temporarily regulated, such as in early or late embryogenesis, during the maturation of fruits in the development of seeds or fruit, in a completely differentiated leaf or in the beginning of old age. The term "non-specific expression" refers to the constitutive expression or the basic expression ('permeable), of low level in unwanted cells or tissues of a' regulated promoter '. The "inducible promoter" refers to those regulated promoters that can be activated in one or more cell types by an external stimulus, such as a chemical, light, hormone, stress, or a pathogenic agent. "Operably linked" refers to the association of the nucleic acid sequences in a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked to a coding sequence or functional RNA when it is capable of affecting the expression of that coding sequence or that functional RNA (i.e., that the coding sequence or the functional RNA is under the transcriptional control of the promoter). The coding sequences can be operably linked to the regulatory sequences in a sense or antisense orientation. "Expression" refers to the transcription and stable accumulation of sense (mRNA) or functional RNA. The expression also refers to the production of protein. The "altered levels" refer to the level of expression in transgenic organisms that differ from that of normal or non-transformed organisms. "Overexpression" refers to the level of expression in transgenic organisms that exceeds the expression levels in normal or untransformed organisms. An "antisense inhibition" refers to the production of transcripts of antisense RNA capable of suppressing the expression of the protein of an endogenous gene or a transgene. The "co-expression" and "transchange" each refers to the production of transcripts of RNA in sense capable of suppressing the expression of the identical or substantially similar transgene or endogenous genes (U.S. Patent No. 5,231,020). "Gene restriction" refers to the suppression dependent on the homology of viral genes, transgenes or nuclear genes, endogenous. The gene restriction can be transcriptional, when the deletion is due to decreased transcription of the affected genes, or post-transcriptional, when the deletion is due to an increased disorder (degradation) of RNA species homologous to the affected genes [see English and collaborators (1996) Plan t Cell 8: 179-188]. The suppression of genes includes the suppression of genes induced by the virus [see Teresa Ruiz et al., (1998) Plan t cell 10: 937-946]. The "restriction suppressor" gene refers to a gene whose expression leads to the counteracting of gene restriction and the increased expression of restricted genes. Restriction suppressor genes may be of plant, non-plant or viral origin. Examples include, but are not limited to, HC-Pro, Pl-HC-Pro and 2b proteins. Other examples include one or more genes in the B genome of the TGMV. "Homologue to" refers to the similarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Calculations of such homology are provided either by the hybridization of DNA-DNA or DNA-RNA under conditions of severity 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 "amplicon" refers to a chimeric gene in which the cDNA of an RNA virus is operably linked to the regulatory sequences of plants such that the primary transcript is the chain "positive" of the RNA virus. The "viral, binary expression system" describes the expression system comprised of two elements, at least one of which is chromosomally integrated. The first element is an inactive replicon which may contain a target gene whose expression is desired in a plant or plant cell. The second element is comprised of a regulated promoter operably linked to a transactivation gene. The first element may be a prorreplicon or it may be an inactive replicon. The inactive replicon or prorreplicon and a chimeric transactivation gene, which function together, will effect the duplication of the replicon and the expression of a target gene in a plant in a regulated manner. Both elements of the system can be chromosomally integrated and can be hereditary independently. The stimulation of the regulated promoter that drives the transactivation gene releases the replicon of the chromosome and its subsequent episomal duplication. The release may be the physical division of the chromosome replicon that involves site-specific recombination, a duplicative release of an original, chromosomal copy of a prorreplicon in the presence of the duplication protein, or the transcriptional release of an original, chromosomal copy of an amplicon. The "viral, transgenic, binary duplication system" refers to a duplication system comprised of two chromosomally integrated elements. The first element may be a prorreplicon or it may be an inactive replicon which lacks an objective gene encoding a foreign protein. The second element is comprised of a regulated promoter operably linked to a site-specific recombinase gene. The inactive replicon and a chimeric site-specific recombinase gene, which functions together, will duplicate the replicon in a plant in a regulated manner. This system is useful where the duplication of the virus is desired in a regulated manner but where the expression of foreign genes is not sought. For example, regulated expression of the virus may be useful in conferring a resistance to viral infection to a plant. The "transgene activation system" refers to the expression system comprised of an inactive transgene and a site-specific recombinase gene., chimeric, working together, to effect the expression of transgenes in a regulated manner. The specificity of the recombination will be determined by the specificity of the regulated promoters as well as the use of site-specific sequences, non-cultured type or mutants. Both elements of the system can be chromosomally integrated and independently inherited. These site-specific sequences are well known in the art, see for example the Cre-Lox system (Sauer, B., U.S. Patent No. 4,959,317) as well as the specific recombination system for the FLP / FRT site ( Lyznik et al., Nucl ei c Acids Res. (1993), 21 (4), 969-75). The "target gene" refers to a gene in the replicon that expresses the target, desired coding sequence, the functional RNA, or the protein. The target gene is not essential for duplication of the replicon. Additionally, the target genes can comprise non-viral, native genes inserted into a non-native organism, or chimeric genes and will be under the control of appropriate regulatory sequences. In this way, regulatory sequences in the target gene can come from any source, including viruses. 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 the viral, native genes. Typical target genes include but are not limited to genes that encode a structural protein, a storage protein of seeds, a protein that transmits resistance to herbicides, and a protein that transmits resistance to insects. The proteins encoded by the target genes are known as "foreign proteins." The expression of an objective gene in a plant will typically produce a characteristic, altered, vegetable quality. The term "characteristic, altered plant quality" means any phenotypic or genotypic change in a transgenic plant relative to the host plant of the non-cultivated or non-transgenic type.

The "Transcription Detention Fragment" refers to nucleotide sequences that contain one or more regulatory signals, such as polyadenylation signal sequences, capable of transcription termination. Examples include the 3 'non-regulatory regions of genes coding for nopaline tape and the small subunit of ribulose bisphosphate carboxylase. The "Translation Stopping Fragment" refers to nucleotide sequences that contain one or more regulatory signals, such as one or more termination codons in all three structures, capable of terminating translation. Insertion of the translation stop fragment adjacent or near the initiation codon at the 5 'end of the coding sequence will result in non-translation or inappropriate translation. Splitting the translation stop fragment by site-specific recombination will leave a sequence specific to the site in the coding sequence that does not interfere with the appropriate translation using the initiation codon. The "blocking fragment" refers to a DNA fragment that is flanked by site-specific sequences that can block the proper transcription and / or translation of a coding sequence which results in an inactive transgene. When the blocking fragment contains the sequences of polyadenylation signals and other sequences that encode the regulatory signals capable of terminating transcription, it can block the transcription of a coding sequence when it is placed in the 5 'untranslated region, ie between the start site of the transcript and the ORF. When inserted into the coding sequence, a blocking fragment can block inappropriate translation by disrupting its open reading structure. The reordering of DNA by site-specific recombination can restore transcription and / or appropriate translatability. For example, cleavage of the blocking fragment by site-specific recombination leaves behind a site-specific sequence that allows proper transcription and / or translatability. A Fragment of Stopping the Transcript or the Translation will be considered a blockage fragment. The terms "in cis" and "in trans" refers to the presence of DNA elements, such as the viral origin of duplication and the protein gene (.s) of duplication, in the same DNA molecule or different molecules of DNA. DNA, respectively. The terms "action sequence ci s" and "action element ci s" refers to the DNA or RNA sequence, whose function requires that they be in the same molecule. An example of a cis-acting sequence in the replicon is the origin of viral duplication. The terms "trans action sequence" and "trans action element" refer to DNA or RNA sequences, whose function does not require that they be in the same molecule. Examples of the trans action sequence is the duplication gene (ACl or AL1 in the geminivirus ACMV or TGMV, respectively), which can work in duplication without being in the replicon. The "cis-acting viral sequences" refer to the viral sequences necessary for viral duplication (such as the duplication origin) and in orientation ci s. The "transactivation gene" refers to a gene that encodes a transactivation protein. This can encode a viral replication protein (s) or a site-specific replicase. This may be a natural gene, for example, a viral duplication gene, or a chimeric gene, for example, when the regulatory sequences of plants are operably linked to the open reading structure of a site-specific recombinase or a viral duplication protein. The "transactivation genes" can be chromosomally integrated or they can be expressed transiently. The "episome" and the "replicon" refer to a DNA or RNA virus or a vector that undergoes episomal duplication in plant cells. This contains the cis-acting viral sequences, such as the duplication origin, necessary for duplication. This may or may not contain the trans-acting viral sequences necessary for duplication, such as the viral duplication genes (for example, the ACl and AL1 genes in the geminivirus ACMV and TGMV, respectively). This may or may not contain a target gene for expression in the host plant. The "inactive replicon" refers to a defective replicon of the duplication that contains the cis-acting viral sequences, such as the duplication origin, necessary for duplication but is defective in the duplication because it lacks either a gene viral, functional necessary for duplication and / or the ability to be released from the chromosome due to its ordering of DNA that involves recombination sequences specific to the site. Consequently, an inactive replicon can duplicate episomally only when it is supplied with the essential in trans duplication protein, as in the case of the geminivirus prorreplicon, or when its nonfunctional duplication gene becomes functional by site-specific recombination. or without release of the active replicon DNA from the chromosome. The "activation of replicon duplication" refers to the process in which an inactive replicon becomes active for episomal duplication. The "replicon with complete lox (floxed) sites" refers to a replicon flanked by sequences specific for the tandem site or one after another (i.e., directly repeated). The replicon may be a full-length copy of an amplicon of DNA virus or RNA virus. The replicon divides like the DNA after site-specific recombination. "Episomal duplication" and "replicon duplication" refer to the duplication of DNA or RNA viruses or replicons derived from viruses that are not chromosomally integrated. This requires the presence of the viral duplication protein (s) essential for duplication, is independent of chromosomal duplication, and results in the production of multiple copies of viruses or replicons per copy of the host genome. "Autonomic" or "cis" duplication refers to the duplication of a replicon that contains all the ci s and trans action sequences (such as the duplication gene) required for duplication. The "origin of duplication" refers to a sequence of cis-action duplication essential for viral or episomal duplication. "Prorreplicon" refers to an inactive replicon that is comprised of viral sequences of action required for duplication, and side sequences that make it possible to release the replicon from it. It is integrated into a bacterial plasmid or a plant chromosome, host and may contain a target gene. Prorreplicon lacks a gene that codes for a duplication protein essential for duplication. Therefore, it is unable to subject episomal duplication in the absence of the duplication protein. Its duplication requires both the release of the integration and the presence of the essential in trans duplication gene. The liberation of integration can be initiated in different ways. For example, the prorreplicon may be present as a tandem duplication or one after another, partial or complete, such that a full length replicon sequence is flanked by the virus sequences and such that the duplicated viral sequence includes the origin of the viral duplication. In this way, in this case, the prorreplicon serves as an original copy from which the replicons can be divided by duplicative release in the presence of the duplication protein (s) [Bisaro, David. Recombination in geminiviruses: Mechanisms for maintaining genome size and generating genomic diversity. Homologous Recomb. Gene Silencing Plants (1994), 219-70. Editor (s): Paszkowski, Jerzy, Publisher: Kluwer, Dordrecht. Germany] . Alternatively, prorreplicon can be divided by site-specific recombination between sequences that are next to it in the presence of an appropriate site-specific recombinase (as described in site-specific recombination systems). , such as Cre-lox and FLP / FRT systems, Odell et al., Use of site-specific recombination systems in plants Homologous Recomb Gene Silencing Plants (1994), 219-70 Editor (s): Paszkowski, Jerzy , Publisher: Kluwer, Dordrecht, Germany). In the case of the prorreplicons of the RNA virus, the sequences of the amplicon that are the side of the inactive replicon, which include the regulatory sequences, allow the generation of the replicon as transcripts of RNA that can be duplicated in trans in the presence of the protein of duplication. These regulatory sequences may be for constitutive or regulated expression. The "viral duplication protein" and "replicase" refers to the viral protein essential for viral duplication. This can be provided in trans to the replicon to support its duplication. Examples include the viral duplication proteins encoded by the ACl and AL1 genes in the geminivirus ACMV and TGMV, respectively. Some viruses have only one duplication protein: others may have more than one. The "duplication gene" refers to a gene that encodes a viral duplication protein. In addition to the ORF of the duplication protein, the duplication gene can also contain other overlapping or non-overlapping ORF (s) as found in viral sequences in nature. While not essential for duplication, these additional ORFs may increase the duplication and / or accumulation of viral DNA. Examples of these additional ORFs are AC3 and AL3 in the geminivirus ACMV and TGMV, respectively.

The "trans action, chimeric duplication gene" refers to either a duplication gene in which the coding sequence of a duplication protein is under the control of a different regulated, virally replicating, native, plant promoter. or a viral duplication gene, native, modified, for example, in which a specific sequence (s) of the site were inserted in the 5 'transcript but the untranslated region. These chimeric genes also include the insertion of the known sites of binding of the duplication proteins between the promoter and the transcription initiation site that attenuate the transcription of the viral duplication protein gene. "Chromosomically integrated" refers to the integration of a foreign gene or a DNA construct into the host DNA by covalent bonds. Where genes are not "chromosomally integrated" they can be "transiently expressed". Transient expression of a gene refers to the expression of a gene that is not integrated into the host chromosome but functions independently, either as part of a replication plasmid autonomously or as an expression cassette, for example, or as part of another system biological, such as a virus.

The "production tissue" refers to the mature, harvestable tissue consisting of terminally differentiated, non-divided cells. This excludes growing, young tissue consisting of germ line cells, meristematic and not completely differentiated. The "germ line cells" refer to cells that are destined to be gametes and whose genetic material is hereditary. "Trans activation" refers to the change of gene expression or replicon duplication by the expression of another (regulatory) gene in trans. "Transformation" refers to the transfer of a foreign gene into the genome of a host organism. Examples of plant transformation methods include transformation mediated by agrobacteria (De Blaere et al. (1987) Me th. Enzymol. 143: 277) and accelerated particle transformation technology or "gene gun" (Klein et al. (1987) Na ture (London) 327: 70-73: U.S. Patent No. 4,945,050). The terms "transformed", "transformant" and "Transgenic" refers to plants or calluses that have gone through the transformation process and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not passed through the transformation process. "Transiently transformed" refers to cells in which transgenes and foreign DNA have been introduced (for example, by methods such as agrobacterial-mediated transformation or biolistic bombardment), but not selected for stable maintenance. "Stably transformed" refers to cells that have been selected and regenerated in selection media after transformation. "Transient expression" refers to expression in cells in which the virus or transgene is introduced by viral infection or by methods such as agrobacteria-mediated transformation, electroporation or biolistic bombardment, but not selected for stable maintenance. "Genetically stable" and "hereditary" refers to chromosomally integrated genetic elements that are stably maintained in the plant and are stably inherited by the progeny through successive generations. The "primary transformant" and the "TO generation" refers to transgenic plants that are of the same genetic generation as the tissue which was transformed initially (ie, that has not passed through meiosis and fertilization since the transformation ). The "secondary transformants" and the "generations Tl, T2, T3, etcetera" refers to transgenic plants derived from the primary transformants through one or more meiotic and fertilization cycles. These can be derived by the self-fertilization of the primary or secondary transformants or the crossings of the primary or secondary transformants with other transformed or non-transformed plants. The "uncultivated type" refers to the gene, virus or normal organism found in nature without any known mutation. "Genome" refers to the genetic material, complete of an organism. The term "dimer" when used with reference to the genome B of the geminivirus refers to at least one copy one after another, partial or complete of the B genome. As used herein, "dimer" therefore refers to a tandem dimer or one after another, partial or complete of a genome of geminivirus, such that an individual replicon is flanked by the viral sequences of cis action, including the duplication origin, necessary for viral duplication. These geminivirus dimers can serve as original copies from which the replicons can be divided by duplicative release in the presence of the in trans duplication protein (Bisaro, David.) Recombination in geminiviruses: Mechanisms for maintaining genome size and generating genomic diversity. Homologous Recomb Gene Silencing Plants (1994), 219-70 Editor (s): Paszkowski, Jerzy, Publisher: Kluwer, Dordrecht, Germany). The "TopCross high-oil corn seed method" refers to a commercial method for making hybrid corn seeds in the field, as described, for example, in U.S. Patent No. 5,704,160. The invention provides a two-component expression system in transgenic plants. Both components are chromosomally integrated and, in this way, they are stably maintained by themselves. In one embodiment of the invention, a component is an inactive replicon carrying the site-specific sequence (s) that are incapable of duplicating themselves. The second component is a chimeric site-specific recombinase gene in which the coding sequence of a site-specific recombinase is operably linked to a regulated promoter. Expression of the recombinase under the appropriate stimulus will result in recombination between the site-specific sequences, of the non-cultured type or mutants, cognate in or around the inactive replicon which will activate the release of the replicon and / or the duplication of the replicon. . In yet another embodiment of the invention, a component is an inactive transgene carrying the site-specific sequence (s) and the second component is a chimeric transactivation gene in which the coding sequence of a site-specific recombinase is operably linked to a regulated promoter. Expression of the recombinase under an appropriate stimulus will result in recombination between the site-specific sequences, of the non-cultured type or mutants, cognate into or around the inactive transgene that will activate the expression of transgenes, without involving viral duplication. In this way, duplication of the replicon and / or expression of transgenes can be chosen as target for plant cells, specific by controlling the expression of duplication protein or recombinase for those cells. Plants will be more sensitive to cellular toxicity and / or the deleterious effect of duplication of the replicon and / or expression of the transgene or the duplication gene in early stages of plant growth and differentiation involving division and differentiation of plants. cells In this way, the control of such expression completely or largely for terminally differentiated, non-dividing cells will reduce the deleterious effect of duplication of the replicon on the growth and development of the plants. Examples of these terminally differentiated cells are those in the production tissue and include, but are not limited to, the storage cells of the seeds and the root tissue and the mature leaf cells. This invention provides a transgenic, regulated expression system. Since the components of this system are transformed in a stable manner, this invention solves the problem of episomal instability through cell divisions., since the episomes are unstable in the absence of selection. When recombination between the sequences specific for the site in an inactive replicon or the inactive transgene activates its duplication or expression of transgenes, respectively, the system will be hereditary unless the site-specific recombination involves the division of DNA into cells of the germ line. The replicon will be autonomous in the cells, if the necessary viral movement protein (s) are not expressed in the cells. This is the case using only the A of the geminivirus DNA or in the use of the PVX with a mutation in a movement protein. The replicon will be extended cell-to-cell systematically, if the necessary viral movement protein (s) are also expressed in the cell. Transgenic plants with different constructions will be selected and regenerated in plants in tissue culture by methods known to one skilled in the art and referred to above. The ability of a chimeric site-specific recombinase gene to transactivate to activate an inactive replicon on the plant chromosome in the duplication by means of site-specific recombination will be tested after one of the following methods . 1. infect the transgenic plants that carry the inactive replicon with viruses that carry the Cre gene, 2. cross the plants where one of the origin contains the chimeric Cre gene, correctly regulated and the other the inactive replicon. 3. make two strains of agrobacteria containing binary vectors with different selectable plant markers, one containing a chimeric Cre gene under the control of an appropriately regulated promoter and the other the inactive replicon. The two components can be introduced into plants together by co-transformation or by sequential transformations. The duplication in tissue of transgenic plants will be monitored by reporter gene expression or viral nucleic acid analysis by treatment with Southern blotting paper in the case of DNA viruses and by treatment with Northern blotting paper in the case of the RNA viruses.

Specific Recombination for the Site for Conditional Expression of Transgenes The use of regulated promoters for development or chemically induced for the conditional expression of transgenes is usually limited either by their insufficient resistance in the 'fully activated' stage or, more frequently, by its non-specific, basic (ie 'permeable') expression in the 'deactivated' stage, depending on the application. one can increase both the level and the specificity of the conditional expression by placing the coding sequence of the gene of interest under the control of a constitutive or regulated promoter, strong for expression in the production tissue in such a way that the gene is inactive transcriptionally unless subjected to a site-specific recombination through the conditional expression of the site-specific recombinase, cognate. In this way, the conditional expression of the gene of interest is now dependent on the conditional expression of the recombinase. In this way, the determinants for high level expression and for specificity are separated and one can then focus on the non-specific, basic (ie, 'permeable') expression of the recombinase. Since the levels of the recombinase enzyme required are not expected to be high, various 'specific' promoters may be used that might otherwise be too weak to express the gene of interest. In addition, since site-specific recombination depends on a threshold level of the recombinase, there may be a tolerance for permeable transcription resulting in sub-threshold levels of the recombinase. In addition, the increased 'tissue selectivity' for the regulated, available promoters is provided by decreasing the efficiency of Cre-mediated recombination of the non-cultured type, which raises the recombinase threshold required by using either a mutant site for recombination site-specific and / or a mutant recombinase that are not skilled at recombination. These mutants are well known, at least for the Cre-lox system. Applicants have shown that when safener-inducible Cre expression is used to activate the expression of a transgene (35S: luciferase), the use of a lox mutant site (lox72) and a lox P site of the non-cultured type in the activation The Cre-mediated transgene reduces the basic activity of the promoter compared to the use of both lox p sites of the non-cultivated type. The non-specificity of recombinase expression can be further reduced (ie, its specificity, of expression further increased) by other post-transcriptional approaches that include: 1. using a recombinant, chimeric gene that is poorly translated (such as having a non-ideal context sequence around the initiation codon following the Kozak rule or having short, additional ORFs in the 5 'untranslated region as in the yeast mRNA GCN4 or having 3' UTR sequences that make the mRNA unstable as is described by Pamela Green (Department of Biochemistry, Michigan State University, East Lansing, MI 48824-1312, USA) 2. use a mutant recombinase that has less cellular stability (ie, shorter shelf life). be made by adding the PEST sequences [Sekhar et al., Jr. Receptor Signa l Transduct ion Res. 18 (2-3), 113-132 (1998)]. Once a system is developed in a given crop, it can be easily adapted for the conditional expression of a variety of genes of characteristic qualities, target with or without replicon involvement.

In addition, duplication of the replicon is expected to achieve the expression of the high level of target genes through the amplification of genes that is hereditary. In addition, high-level transcription of these vectors can be used for gene restriction by inhibition in antisense or co-suppression. The invention further includes new constructs of recombinant viruses that include the transfer vectors and methods for making and using them. When they are used to transform a plant cell, the vectors provide a transgenic plant capable of level expression, regulated high despite the amplification of genes. This regulated expression could be in response to a particular stimulus, such as the stage of development, the injury of the plant (for example, by attack by insects or pathogens), an environmental stress (such as heat or high salinity), or chemical substances that induce specific promoters. Plants in which particular tissues and / or parts of plants have a new or altered phenotype can be produced by the subject method. Constructs include vectors, expression cassettes and binary plasmids depending on the proposed use of a particular construct. Two basic DNA constructs are required which can be combined in a variety of ways to transform a plant cell and to obtain a transgenic plant. For the agrobacteria-mediated transformation, the inactive replicon and the chimeric duplication 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 constructions can be combined by crossing two transgenic lines that contain one or another construction. The termination region used in the target gene in the inactive replicon as well as in the chimeric duplication protein gene will be selected primarily 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 can occur naturally or can be completely or partially synthetic. Suitable termination regions are available from the Ti plasmid of A. tumefa hundreds, such as octopine synthase and nopaline synthase termination regions or genes for β-phaseolin, the chemically inducible lant gene. pIN (Hershey et al., Isolation and characterization of cDNA clones for RNA species induced by substituted benzensulfonamides in corn, Plan t Mol. Biol. (1991), 17 (4), 679-90; U.S. Patent No. 5, 364, 780).

The Constructions: In one aspect of the invention, a new system for duplicating transactivation of plant viruses is developed using a site-specific recombination system. The system has the advantage of better tolerance of the basic, non-specific expression (ie, permeability) of 'regulated' promoters and provides more rigorous control of transactivation. In addition, when a site-specific recombination is developed, suitably regulated, it can be applied generically to the activation of the inactive replicons of different viruses as well as for the expression of transactivation of transgenes without replicon. The non-specific expression (ie, permeability) of some regulated, available promoters that express a site-specific recombinase (such as a Cre recombinase) will be more easily tolerated by plants since the recombinase has to reach a threshold level before to be able to carry out recombination [(Araki et al., Target integration of DNA using mutant lox sites in embryonic stem cells.) Nucl ei c Acids Res. 25: 868-872 (1997).] The 'specificity' of the promoters can be increased additionally by increasing the threshold level of the recombinase required by using any known mutant recombinase protein, as described for Cre [Abremski, K., et al., Properties of a mutant Cre protein that alters the topological linkage of recombination products J. Mol. Biol., 202: 59-66 (1988); Wierzbicki et al., A mutational analysis of the bacteriophage Pl recombinase Cre., J. Mol. Biol. 195: 785-94 (19 87) and / or sequences specific to the site, mutants, such as the l or P sites [Albert et al., Site-Specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Pl an t J. 7: 659-59 (1995)], which make the recombination less skillful than its site-specific recombination system, of the non-cultured type and, thus, requires the highest level of the recombinase.

In this system, the construction of the inactive replicon contains site-specific sequences of the non-cultured type or mutants within or adjacent to the replicon. Recombination between site-specific recognition sequences makes the replicon active and activates replicon duplication. When the sequences specific to the site are directly oriented (ie, they are one after another), site-specific recombination will result in DNA cleavage between site-specific sequences (Figures 1 and 3). When these are in an inverted orientation (ie, in head-to-head or tail-to-tail orientation), site-specific recombination will result in the inversion of the DNA between site-specific sequences (Figure 2). In another embodiment (Figure 1), the construction of the inactive replicon comprises an individual copy of the replicon (either a geminivirus replicon or an RNA virus amplicon) flanked by site-specific sequences, one after another and integrated into the chromosome. In this integrated state, the replicon is inactive and unable to duplicate. Site-specific recombination will divide the individual copy of the replicon containing an individual site-specific sequence that is capable of duplication. When the replicon is inserted between the sequences specific for the site at a site that is between the transcription start site and the open reading frame (ie, at 5 'transcript but the untranslated region of the duplication gene), the duplication gene is not functional and recombination specific for the site also constitutes the duplication gene, functional (Figure 2). When the RNA amplicon is inserted between the promoter and its transcription start site, the RNA replicon is not transcribed and site-specific recombination divides the amplicon from the chromosome as well as reconstitutes a functional amplicon. In any case, the site-specific recombination separates the replicon from the chromosome, which is preferable when the gene restriction induced by the virus is avoided. When the replicon is flanked by two lox sites in tandem or one after another, it is referred to as a 'replicon with the complete lox (floxed) sites'. The replicon with the complete lox (floxed) sites can be integrated into a reporter gene such that it serves as a Transcription Detention Fragment and blocks the proper transcription of the reporter gene. Site-specific recombination will divide the replicon and reconstitute a functional reporter gene. This reporter gene will be useful in the development of plant selection for an appropriately regulated Cre-lox activation system. It is preferable that a Transcription Detention Fragment be inserted near the amplicon with the complete lox sites to prevent inadvertent transcription of the duplication gene from the sequences adjacent to the amplicons with the complete lox sites, such as the DNA of the plant. context in transgenic plants. However, it is not required that the insertion of the replicon with the complete lox sites in a gene or a Transcription Detention Fragment be inserted, since the duplication gene is not expressed in its integrated state. The inactivation of the duplication gene in the inactive replicon is important when its expression is harmful for the development of the plants. As noted, gene restriction is a major obstacle in the expression of plant transgenes and the present expression and duplication systems can be modified to address this problem. Recently, it was shown that the determinants of the pathogenicity of Pl-HC-Pro and HC-Pro, the polypeptides of the Tobacco Corrosion Virus and the protein 2b of the Cucumber Mosaic Virus suppress the restriction of genes in transgene plants and / or RNA virus genes [Anandalakshmi, R., Pruss, GJ, Ge, X., Marathe, R., Mallory, AC, Smith. T. H., Vanee V. B.; A viral suppressor of gene silencing in plants. Proc. Na ti. Acad. Sci. E. U. A 95: 13079-13084 (1998); Brigneti, G., Voinnet, 0., Li, W.-X., Ji. L.-H., Ding. S.-W. Baulcombe, D. C; Viral pathogenicity determinants are suppressors of transgene silencing in Ni co tiana ben thamiana. EMBO J. 17: 6739-6746 (1998); Carrington, J. C, Whitham, S. A .; Viral invasion and host defense: strategies and counter-strategies. Curr. Opin. Plan t Bi ol. 1: 336-34 (1998); Vanee, V.B., Pruss G. J., Carrington, J., Martin, L., Dawson, W.O .; Potyvirus booster sequence and helper component proteinase for enhancing expression of a foreign or endogenous gene product in plants. International Application of the PCT, WO 9844097 (1998)]. However, the constitutive expression of such restriction suppressors in transgenic plants will be harmful to plants, since they will constitutively suppress the restriction of genes, a fundamental process for plants. This harmful effect is especially strong in conjunction with the viral vectors, since the above restriction suppressors also increase viral pathogenicity. In fact, the plants infected with the X-Virus of Potato containing HC-Pro showed severe symptoms, necrosis and atrophy, while those infected with the protein 2b became highly necrotic and died in 3 weeks [Brigneti, G., Voinnet , 0., Li, W.-X., Ji. L.-H., Ding, S.-W., Baulcombe, D. C: Viral pathogenicity determinants are suppressors of transgene silencing in Ni cotiana ben thamiana EMBO J. 17: 6739-6746 (1998)]. Therefore, the localized and / or regulated expression of these restriction suppressors in the production tissue of transgenic crops will be critically important for their practical application in the increase of the viral vector duplication and / or for the expression of highest level, especially the production of foreign proteins. The applicant has observed that the B genome of TGMV can also suppress the gene restriction induced by the virus. For example, when the leaves of the transgenic tobacco plants, transformed with T-DNA containing the TGMV genome A with the complete lox sites in which the ORF of the coating protein was replaced with the GUS ORF, were bombarded with the 35S gene: Cre and a dimer of the B genome of the TGMV shows the significantly higher expression of the GUS than those transformed with the 35S gene: Cre alone. In addition, the co-bombardment of the Nicotiana benthamiana of the non-cultivated type with PVX-GFP and the B genome of the TGMV resulted in the longer persistence of GFP activity than when bombarded with PVX-GFP alone. The applicant has discovered that infection of the Nicotiana benthamiana plant of the non-cultured type with the TGMV carrying the GFP resulted in the expression of the GFP that was not weakened or restricted for at least 2 months. This infection was achieved by the biolistic co-bombardment of two plasmids, collectively referred to herein as 'TGMV-GFP dimers', one containing a partial dimer of TGMV-A-GFP, in which the ORF of the coating protein is replaced with that of the brightest (mutant) form of GFP, and one containing a partial dimer of TGMV-B of the non-cultivated type. In addition, the Applicant has discovered that the expression of TGMV-GFP was similarly persistent in plants that were restricted by the expression of PVX-GFP. For this, the transgenic plant of N. ben thamiana, designated 714B-LL1, containing an inactive amplicon of the PVX-GFP of RNA virus was used. These transgenic plants did not express any GFP because the inactive form of PVX-GFP is unable to duplicate, unless subjected to site-specific recombination, mediated by Cre-lox. When line 714B-LL1 was bombarded with the 35S: Cre gene, this results in the activation of the PVX-GFP duplication and the expression of GFP that is restricted in about 2 weeks. When this restricted 714B-LL1 plant was infected with the 'TGMV-GFP' dimers, the GFP expression of TGMV-GFP, which is distinguishable from that in PVX-GFP by its brighter fluorescence, persisted as long as the control was not transformed . In addition, when line 714B LL-1 was co-bombarded with 35S: Cre and the 'TGMV-GFP' dimers, the GFP expression of TGMV-GFP persisted while the control did not transform further and beyond the time that the GFP of the PVX-GFP was restricted. Since expression of a foreign gene in TGMV in the presence of TGMV-B is persistent and not restricted over time, TGMV-B can be used to increase high-level expression by suppressing the restriction of the present transgenes in the viral vectors. It is anticipated that all geminiviruses have this restriction-suppressor activity, either with the monopartite or bipartite genome. It is likely that this persistent expression of the foreign gene in the geminivirus results is derived from the movement of geminivirus. The suppressor activity of the genome B genome restriction can be used in different ways. The genome B of the TGMV can be transformed into the chromosome of the host plant by one skilled in the art and can be combined with the cloned prorreplicon or the A genome with the complete lox sites. For example, the B genome of TGMV may be present in its entirety as a partial dimer in the chromosome or its duplication and expression may also be under activation controlled by site-specific recombination. When presented as a dimer, it can suppress the restriction with or without its duplication. For the former, the duplication can be directly transactivated by the expression of the duplication protein (s) under the control of a regulated promoter or indirectly by the activation of an inactive genome A by means of site-specific recombination. . As an alternative to the use of the complete genome B, one could identify the restriction-suppressor gene in the B genome and use it to increase the expression of foreign genes. Since TGMV-B has only two large ORFs, BL1 and BR1, which encode viral movement proteins, one skilled in the art can easily identify which ORF (s) is a restriction suppressor. For example, the leaves can be co-bombarded with the 35S dimer: Cre and TGMV-B with the mutant BR1 (or the PVX chimeric gene: BL1) or the 35S-Cre dimer and TGMV-D with the mutant BL1. (or the PVX chimeric gene: BR1) and the relative expression of the measured GUS expression. The restriction suppressor gene, identified later, can be used to increase the expression of transgenes. Regulated expression of restriction-suppressor genes can be achieved by placing them under the control of appropriately regulated promoters, or preferentially, by activation regulated by site-specific recombination. Thus, in one embodiment of the invention, the chimeric, restriction-suppressor genes will be activated by Cre. However, it would be more desirable to have the activation of both the viral expression system and the expression of restriction-suppressor genes under common control to ensure viral, simultaneous duplication and suppression of gene restriction for viral level duplication. high and to produce high levels of foreign proteins. Thus, in another embodiment of this invention, the conditional viral duplication system will incorporate a conditional expression of a restriction-suppressor gene. For example, with reference to Figure 1, element A could be a plant promoter, such as the 35S promoter, element B could be an amplicon derived from the inactivated RNA virus that also serves as a fragment of Stop transcription and / or of the translation of element C, and element C is the ORF of the restriction-suppressor gene, such as PI-HC-Pro, HC-Pro, or protein 2b (as described above) and the region does not translated 3 '. Site-regulated recombination will at the same time activate the cleavage and duplication of the viral replicon of RNA and the expression of the restriction-suppressor gene under the control of the promoter in element A. Similarly, with reference to FIG. 1, element A could be a plant promoter, such as the 35S promoter, element B could be an inactive geminivirus-derived replicon that also serves as a Stop fragment of transcription and / or translation of element C, and element C is the ORF of the genome B restriction suppressor gene of the TGMV and the 3 'untranslated region. Regulated site-specific recombination will activate both the division and duplication of the viral replicon of the geminivirus and the expression of the geminivirus restriction-suppressor gene under the control of the promoter in element A. Alternatively, the suppressor gene of the restriction can be expressed as a target gene in an inactive replicon. For example, the PVX amplicons, inactive with the lox sites as described above (Figures 2 and 3) will also contain a restriction-suppressor gene under the control of the viral promoter in addition to the target gene of interest. The target gene of interest and the restriction suppressor gene in the virus replicon could be present either as tandem genes under the control of the viral PR promoter, duplicated or as a fusion of N or C-terminal proteins with the target protein, as described by [Anandalakshrni, R., Pruss, G.J., Ge, X., Marathe, R., Mallory, A. C, Smith, T. H., Vanee, V. B .; A viral supressor of gene silencing in plants. Proc. Na ti. Acad.

Sci. E. U. A 95: 13079-13084 (1998)]. In transgenic plants ,. such virus-based vectors may or may not be capable of systemic diffusion. For example, in replicons based on geminivirus, the ORF of the coating protein can be replaced by that of a restriction-suppressor gene. The size limitation of the insert in the replicons can be circumvented by having 2 replicons, one that carries the restriction suppressor gene and the other a target gene of interest. In another embodiment, the transcription of a (one) gene (genes) of essential replication (s) of a replicon is blocked by a Fragment of Stopped Transcription flanked by sites specific for the tandem site and the specific recombination for the site divides the Transcription Detention Fragment leaving behind a sequence specific to the site, individual that allows the transcription of the previously blocked gene and the release and duplication of the subsequent replicon. For example, in TGMV and ACMV viruses, the Transcription Stopping Fragment flanked by sites specific for the tandem site can be inserted into the 5 'transcript but the untranslated region of the duplication gene, ACl (eg, in the Mfe I site) in a viral dimer. For the RNA virus amplicons, the lox site was inserted between the TATA box of the promoter and the transcription start site (Figure 3). In another embodiment, a region in or around a replicon is inverted by the site-specific sequences to disrupt the replicon genome or the RNA virus amplicon. The inverted region can be completely within the genome of the replicon which results in the disruption of the viral genome. Site-specific recombination restores the organization of the replicon, including the amplicon (except for the specific site sequence (s), residual (s)) that allows duplication. Alternatively * the inversion may be in part of the replicon and / or a regulatory, plant sequence of an amplicon that disrupts the proper transcription of the (essential) gep (genes) of duplication. Site-specific recombination restores proper transcription that allows for the release and duplication of the replicon. The sequences specific to the site and its recombined recombinase enzymes can be from any site-specific, natural recombination system. Well-known examples include the Cre-lox, FLP / FRT, R / RS, Gin / gix systems. These are described in Odell et al. Use of site-specific recombination systems in plants. Homologous Recomb. Gene Silencing Plants (1994), 219-70. Editor (s): Paszkowski, Jerzy, Publisher: Kluwer, Dordrecht, Germany). In one embodiment of the invention (Figure 4), the construction of the basic, inactive replicon is the prrereplicon, which, in the case of a geminivirus replicon, is preferably present as a tandem, partial or complete dimer in the T-DNA, such that an individual replicon is flanked by the cis-acting viral sequences necessary for viral duplication, including the origin of duplication.

These dimers of the geminivirus can serve as an original copy from which the replicons can be divided by duplicative release (Bisaro, David, Recombination in geminiviruses: Mechanisms for maintaining genome size and generating genomic diversity, Homologous Recomb. Gene Silencing Plants (1994). 219-70, Editor (s): Paszkowski, Jerzy, Publisher: Kluwer, Dordrecht, Germany) in the presence of the in trans duplication protein. The preferred source of prorreplicon sequences is a geminivirus (such as ACMV and TGMV) in which the essential duplication gene (e.g., AC1) becomes non-functional due to the mutation (in addition, the rearrangement, or a partial or complete deletion of the nucleotide sequences). The mutation can be in the non-coding sequence, such as the promoter, and / or it can be in the coding sequence of the duplication protein to result in either one or more altered amino acids in the duplication protein or a change in the structure. Preferably, the mutation is a change mutation of the structure at or near the initiation codon of the duplication protein so that a truncated duplication protein is not yet made. More preferably, the entire duplication gene is deleted from the pro-repelicon such that there is no homology between the transactivation duplication gene and the replicon in order to prevent restriction on the basis of the virus-induced homology of the transactivation duplication gene. during duplication of the replicon. In addition, the prrereplicon preferentially has the majority or the entire gene of the coating protein deleted and replaced by a restriction site for the cloning of the target gene. In this embodiment, the other basic construction is a chimeric trans action duplication gene consisting of a regulated, plant promoter operably linked to the duplication protein coding sequence. For the ACMV and TGMV geminivirus, the duplication proteins are encoded by the ACl and AL1 ORFs, respectively. Preferably, the ORFs AC2 and AC3 are included with the ORF ACl in the ACMV and the ORFs AL2 and AL3 are included with the ORF AL1 in the TGMV. In the case of the prorreplicons of the RNA virus, the sequences of amplicons flanking the inactive replicon, which includes the regulatory sequences, allow the generation of the replicon as transcripts of RNA that can be duplicated in trans in the presence of the duplication protein. . These regulatory sequences may be for constitutive or regulated expression. Preferably, the promoter used in these amplicons will be a weak promoter in order to minimize the gene restriction induced by the virus [Ruiz et al., (1998) Pl an t Cel l, Vol. 19, pages 937-946]. Duplicating proteins from single-stranded RNA viruses (such as RNA-dependent DNA polymerases) are also included when they can support in trans viral duplication (eg, Bromic Mosaic Virus (BMV)). Site-specific recombination can reconstitute a viral replicase gene, functionally and transactivate the duplication of the replicon, which in turn provides the in trans duplication protein for duplication of the prorreplicon. The skilled person appreciates that the present expression systems (which involve the inactive replicon) can be used to effect the regulated duplication in the absence of a target gene. In this situation, the expression of foreign genes is not the goal. Instead, the regulation of viral duplication is sought. This system can be useful, for example, where viral, regulated duplication will confer viral resistance to. the transgenic plant.

More preferably, RNA virus duplication can be transactivated by either a site-specific cleavage of a Transcription Detention fragment of the amplicon that allows the normal transcription required for viral cleavage (Figure 3). For example, the Transcriptional Detention fragment can be placed between the promoter and the viral cDNA, within the viral cDNA, or between the viral cDNA and the 3 'polyadenylation signal. Since there is a limited space between the TATA box and the transcription start site, the overlap part of the lox sequence with the TATA box is preferred along with the use of a site-specific sequence, suppressed, as lox D117 [Abremski et al., J. Mol. Bi ol. (1988) 202: 59-66]. Alternatively, the replicon can be activated by site-specific inversion between two site-specific sites, inverted to result in a functional amplicon (Figure 2). These two site-specific sites can be anywhere in the amplicon as long as they do not interfere with the replicon after inversion. For example, a sequence specific for the site may be in the transcribed sequence, not 5 'of the GFP or GUS gene and the other in an inverted orientation between the enhancer and the TATA box of the 35S promoter (Figure 2). When duplication replicons contain sequences homologous to chromosomal genes, it has been reported that the homologous gene is restricted [Ruiz et al., (1998) Plan t Cell, Vol. 19, pages 937-946]. When the promoter in the replicon is expressed constitutively and strongly (such as the 35S promoter), ultimately the duplication can be restricted. It has been reported that such a restriction confers resistance to infection by the virus. It has been shown that resistance to virus-dependent homology is due to restriction of post-transcriptional genes dependent on homology [see Mueller et al., (1995) The Plant Journal 7: 1001-1013]. In this way, a conditional transactivation of an inactive amplicon is expected to confer resistance to infection by homologous viruses. Since it has been reported that this gene restriction is dependent on the mRNA threshold, when gene restriction is not desired, the promoters in the amplicon (Figure 2 and 3) should preferably be a weak promoter such as the 35S promoter. minimum to reduce the risk of being restricted during duplication of the replicon.

The inactive replicons may also contain an (one) target gene (genes) that will duplicate and express at an increased level when the replicon is transactivated to duplicate. The coding sequence in these target genes is operably linked to 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 transit peptides, leader secretory sequences, or introns) may also be present in the prorreplicon and the replicon as desired. It is well known how to obtain and the use of these sequences is well known to those skilled in the art. The target gene can encode a polypeptide of interest (eg, 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 sequence with the preferred plant codons. Target genes can encode functional RNAs or foreign proteins. The foreign proteins will typically encode the non-viral proteins and the proteins that may be foreign to the plant hosts. Such foreign proteins will include, for example, enzymes for primary or secondary metabolism in plants, proteins that confer resistance to diseases or herbicides, commercially useful non-vegetable enzymes and proteins with desired properties useful in animal feeding or human feeding. Additionally, foreign proteins encoded by the target genes will include seed storage proteins with improved nutritional properties, such as 10 kD high-sulfur corn seed protein or high-sulfur zein proteins. Regulated expression of the viral duplication protein (s) is possible by placing the coding sequence of the duplication protein under the control of promoters that are tissue-specific, developmentally specific or inducible. The various regulated genes, specific for tissue and / or promoters, have been reported in plants. These include genes that encode seed storage proteins (such as napin, cruciferin, beta-conglycinin and phaseolin), oily consistency proteins or zein (such as oleosin), or genes involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP denaturase, and fatty acid denaturases (fad 2-1)), and other genes expressed during embryonic development (such as Bce4, see, eg, EP 255378 and Kridl et al., Seed. Scien ce Research (1991) 1: 209-219). Particularly useful for the specific expression for the seed is the pea vicilin promoter [Czako et al., Mol. Gen Gene t. (1992), 235 (1), 33-40]. Other useful promoters for expression in mature leaves are those that are changed at the beginning of old age, such as the SAG promoter from Arabidopsis [Gan et al., Inhibition of leaf senescence by autoregulated production of cytokinin, Science (Washington, DC) ( 1995), 270 (5244), 1986-8]. A class of specific promoters for fruit expressed in or during the synthesis through the development of the fruit, at least until the initiation of ripening, is discussed in U.S. Patent No. 4,943,674. The cDNA clones that are preferentially expressed in the cotton fiber have been isolated [John et al., Gene expression in cotton (Gossypi um hirsu tum L.) fiber: cloning of the mRNAs, Proc. Na ti. Acad. Sci. E. U. A (1992), 89 (13), 5769-73]. The tomato cDNA clones exhibiting differential expression during the development of the fruit have been isolated and characterized [Mansson et al., Mol. Gen Gene t. (1985). 200: 356-361; Slater et al., Plan t Mol. Biol. (1985) 5: 137-147]. The promoter for the polygalacturonase gene is active in the ripening of the fruit. The polygalacturonase gene is described in U.S. Patent No. 4,535,060 (filed August 13, 1985), U.S. Patent No. 4,769,061 (filed September 6, 1988), U.S. Patent No. 4,801,590 (filed January 31, 1989) and US Patent No. 5,107,065 (filed April 21, 1992). The mRNA of the mature plastid for psbA (one of the components of photosystem II) reaches its highest level later in fruit development, in contrast to the plastid ARNSm for other components of photosystem I and II which declines to undetectable levels in chromoplasts after the onset of maturation [Piechulla et al., Plan t Mol. Biol. (1986) 7: 367-376]. Recently, cDNA clones representing the genes apparently involved in the interactions of tomato pollen [McCormick et al., Toma to Bio technolgy (1987) Alan R. Liss, Inc. New York) and the pistil (Gasser et al. Plan t Cell (1989), 1: 15-24] have also been isolated and characterized.Other examples of tissue-specific promoters include those that direct expression in leaf cells after damage to the leaf (eg, mastication). of insects), in tubers (for example, the promoter of patatin genes), and in fiber cells (an example of a fiber cell protein regulated by development is E6 [John et al., Gene expression in cotton ( Gossypium hirsu tum L.) fiber, cloning of the mRNAs, Proc. Na ti.Acid Sci. E.U.A. (1992), 89 (13), 5769-73).] The E6 gene is more active in fiber, although low levels of transcripts are found in the leaf, the ovule and the The tissue specificity of some "tissue-specific" promoters may not be absolute and may be tested by one skilled in the art using the diphtheria toxin sequence. One can also achieve tissue-specific expression with the expression "permeable" by a combination of different tissue-specific promoters (Beals et al., (1997) Plan t Cell, vol.9, 1527-1545). Other tissue-specific promoters can be isolated by one skilled in the art (see U.S. Patent No. 5,589,379). Several inducible promoters ("gene changes") have been reported. Many are described in the review by Gatz [Curren t Opinion in Biotechnology, 1996, vol. 7, 168-172; Gatz, C. Chemical control of gene expression, Annu. Rev. Plan t Physiol. Plan t Mol. Biol. (1997), 48, 89-108]. These include the tetracycline repressor system, the C repressor system, copper inducible systems, salicylate inducible systems (such as the PRla system), glucocorticoid-inducible systems [Aoyama T. et al., NH Plan t Journal ( 1997) vol. 11: 605-612] and by ecdysonomas. Also included are benzene sulfonamide-inducible systems (U.S. Patent No. 5,364,780) and alcohol (WO 97/06269 and WO 97/06268) and the glutathione S-transferase promoters. Other studies have focused on inducibly regulated genes in response to environmental stress or stimuli such as increased salinity, drought, pathogens and injury. [Graham et al., J. Biol. Chem. (1985) 260: 6555-6560; Graham et al J. Biol. Chem. (1985) 206: 6561-6554] [Smith et al., Plan ta (1986) 168: 94-400]. The accumulation of a metallocarboxypeptidase inhibitory protein has been reported in the leaves of injured potato plants [Graham et al., Biochem Biophys Res. Comm (1981) 101: 1164-1170]. It has been reported that other plant genes are induced by methyl jasmonate, sonar, heat shock, anaerobic stress or herbicide safeners. Regulated expression of chimeric transactivation viral duplication protein can be further regulated by other genetic strategies. For example, gene activation mediated by Cre described by Odell et al [(1990) Mol. gen. Genet 113: 369-278]. In this manner, a DNA fragment containing the 3 'regulatory sequence linked by the lox sites between the promoter and the coding sequence of the duplication protein that blocks the expression of a duplicating, chimeric promoter gene can be eliminated by the division mediated by Cre and results in the expression of the trans action duplication gene. In this case, the chimeric Cre gene, the chimeric trans action duplication gene, or both may be under the control of inducible or tissue-specific or developmental promoters. A genetic strategy, alternative is the use of the tRNA suppressor gene. For example, regulated expression of a tRNA suppressor gene can conditionally control the expression of a coding sequence of the trans action duplication protein containing an appropriate termination codon as described by Ulmasov et al [(1997) Plan t Molecular Biology, vol. 35, pages 417-424]. Again, either the chimeric tRNA suppressor gene, the transaction duplication gene, chimeric, or both may be under the control of inducible or tissue-specific and developmental promoters. One skilled in the art recognizes that the level of expression and regulation of a transgene in a plant can vary significantly from line to line. In this way, one has to test several lines to find one with the desired level of expression and regulation. Once a line with the desired regulatory specificity of a chimeric Cre transgene was identified, it can be crossed with lines carrying different inactive replicons or a transgene inactive for activation. A variety of techniques are available and 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. tumefaciens or A. rhizogenes as the transformation agent, electroporation, particle acceleration, etc. [See for example, European patent No. 295959 and European patent No. 138341]. It is particularly preferred to use the vectors of the binary type 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, rapeseed, tobacco and rice [Pacciotti et al. (1985) Bio / Technolgy 3: 24 1; Byrne et al. (1987) Plan t Cell. Tissue and Organ Cul ture 8: 3 Sukhapinda et al. (1987) Plan t Mol. Biol. 8: 209-216; Lorz et al. (1985) Mol. gen gene t, 199: 178; Potrykus (1985) Mol. gen. gene t. 199: 183, Park et al., J. Plan t biol. (1995), 38 (4), 365-71; Hiei et al., Plan t J. (1994). 6: 271-282]. The use of T-DNA to transform plant cells has received extensive study and is widely described [European Patent No. 120516; Hoekema, In: The Binary Plant Vector System. Offset-drukkerij Kanters BV: Alblasserdam (1985), Chapter V, Knauf et al., Geneti c Analysis of Hos t Range Expression by Agrobacterium In: Molecular genetics of the Bacteria-Plant Interaction, Puhler, A. Ed., Springer- Verlag, New York, 1983, page 245; and An et al., EMBO J. (1985) 4: 277-284]. For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors as described in the examples. Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs [see European Patent No. 295959], electroporation techniques [see Fromm et al. (1986) Na ture (London) 319 : 791] or high-velocity ballistic bombardment with metal particles coated with nucleic acid constructs [see Kline et al. (1987) Na ture (London) 327: 70, and see U.S. Patent No. 4,945,050]. Once transformed, the cells can be regenerated by those skilled in the art. Of particular relevance are the methods recently described for transforming foreign genes into commercially important crops, such as rape seed [see De Block et al. (1989) Plan t Physiol. 91: 694-701], sunflower [Everett et al. (1987) Bio / Technology 5: 1201], soybean seed [McCabe et al. (1988) Bio / Technology 6: 923; Hinchee et al. (1988) Bio / Technology 6: 915; Chee et al., (1989) Plan t Physiol. 91: 1212-1218; Christou et al. (1989) Proc. Na ti. Acad. Sci USA 86: 7500-7504; European Patent No. 301749], rice [Hiei et al., Plan t J. (1994), 6: 271-282] and corn [Gordon-Kamm et al. (1990) Plan t Cell 2: 603-618; Fromm et al (1990) Biotechnology 8: 833-839]. The cells of transgenic plants are then placed in a selective medium, suitable for the selection of transgenic cells which are then cultured on calluses. The shoots are grown from the calluses and the seedlings are generated from the shoot by cultivation in a root medium. The various constructions will normally be linked to a marker for selection in plant cells. Conveniently, the label can be resistant to a bactericide (particularly an antibiotic such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, a herbicide or the like). The particular marker used will allow the selection of the transformed cells compared to the cells lacking the DNA which has been introduced. The components of the DNA constructs including the transcription cassettes of this invention can be prepared from the sequences which are either native (endogenous) or foreign (exogenous) to the host.

By "strange" it is understood that the sequence is not found in the host of the non-cultivated type in which the construction 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, Southern blot analysis can be performed using methods known to those skilled in the art. The replicons can be detected and quantified by treatment with Southern blotting paper, since these can be easily distinguished from the prorreplicon sequences by the use of appropriate restriction enzymes. The expression products of the transgenes can be detected in any of a variety of ways, depending on the nature of the product, and include the Western blotting treatment and the enzyme assay. A particularly useful way to quantify protein expression and to detect duplication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they can be grown to produce plant tissues or parts that have the desired phenotype. The plant tissue or parts of plants can be collected and / or the seed can be collected. The seed can serve as a source to grow additional plants with tissues or parts that have the desired characteristics. The present viral expression system has been used to demonstrate that (i) soybean and corn seed tissue will support duplication of geminivirus; (ii) Cre can mediate site-specific recombination in inactive replicons, transgenic and inactive transgenes and that this recombination leads to foreign protein expression and / or restriction of host genes, and (iii) that the Expression will effect the expression of foreign genes in tobacco.

EXAMPLES The present invention is further defined in the following Examples. These Examples, while indicating the preferred embodiments of the invention, are given by way of illustration only. From the foregoing 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 it to various stages. and conditions.

GENERAL METHODS The recombinant DNA, normal 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. Molecular Cloning: A Labora tory 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 Ausubel. F. M. et al., Current Protocols in Molecular Biology, Published by Greene Publishing Assoc. and Wiley-Interscience (1987). Restriction enzyme digestions, phosphorylations, ligatures and transformations were made as described in Sambrook, J. et al., Supra. Restriction enzymes were obtained from New England Biolabs (Boston, MA), GIBCO / BRL (Gaithersburg, MD), or Promega (Madison, Wl). Taq polymerase was obtained from Perkin Elmer (Branchburg, NJ). The growth media were obtained from GIBCO / BRL (Gaithersburg, MD).

The Agroba cterium tumefa hundreds strain LBA4404 was obtained from Dr. R. Schilperoot, Leiden [Hoekema et al., Na ture 303: 179-180, (1983)].

Transformation Protocols Biological transformations were essentially done as described in U.S. Patent No. 4,945,050. Briefly, gold particles (1 mm in diameter) were coated with DNA using the following technique. Ten ug of plasmid DNAs were added to 50 mL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 uL of a 2.5 M solution) and a spermidine free base (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 were centrifuged briefly (5 sec at 15,000 rpm) and the supernatant was removed. The particles were resuspended in 200 mL of pure ethanol, centrifuged again and the supernatant was removed. The rinsing with ethanol was performed again and the particles were 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 a flying disc (Bio-Rad Labs. 861 Ridgeview Dr, Medina, OH). The particles were then accelerated in the corn tissue with a PDS-1000 / He (Bio-Rad Labs, 861 Ridgeview Dr., Medina, OH), using a helium pressure of 70,216 kg / cm2 (1000 psi), to a opening distance of 0.5 cm and a flight distance of 1.0 cm. Where the transformations of Agrobacterium were made, the procedure was performed essentially as described by Park et al., J. Plan t Biol. (1995), 38 (4) 365-71.

EXAMPLE 1 Construction of Monomers of ACMV and TGMV with the Complete Sites lox PMHP35 was made by cloning the Xba fragment I of 35SCabb: Ata (Arab ALS) in the Xba I site of pTZ18R.

Introduction of the Lox P site of the non-cultured type between the Xho I and Sac I sites of the 35S promoter: Two lox P sites of the non-tandem cultivated type (ie directly repeated) were introduced between the Xho I and Sac I sites in the region 5 'transcribed, but not translated, from the 35S promoter: GUS: reporter gene, chimeric 3'nos such that the two Lox P sites were separated by an Eco Rl site. For this, an adapter A of Xho I-Eco Rl [made by priming pairs of primers GV 48 (SEQ ID NO: 1) and GV 49 (SEQ ID NO: 2)] and an adapter B of Eco Rl-Sac I [ made by priming pairs of primers GV 50 (SEQ ID NO: 3) and GV 51 (SEQ ID NO: 4)) were co-ligated in the digested plasmid of Xho I and Sac I carrying the 35S promoter: GUS: reporter gene, chimeric 3 'us. [SEQ ID NO: 1] 5 '-TCG AGA TAA CTT CGT ATA ATG TAT GCT ATA CGAAGT TAT G-3' (GV48) [SEQ ID NO: 2)] 5 '-AAT TCA TAA CTT CGT ATA GCA TAC ATT ATA CGA AGT TAT C-3 '(GV48) [SEQ ID NO: 3] 5' -AAT TCT ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TGA GCT-3 '(GV50) [SEQ ID NO: 4] 5' -CAT AAC TTC GTA TAG CAT ACA TTA TAC GAA GTT ATA G-31 (GV51) The resulting plasmid was designated pGV686 and the lox sites introduced were confirmed by sequence analysis. Subsequently, the poly A signal region of the nopaline ribbon (3'nos) [Nos. of Access of Genbank J01541 V00087] was replaced by that of the tape of octopine (3 'oes) [Nos. Genbank Accession V00088 and J01820] to produce plasmid pGV690.

Insertion of TGMV monomer and ACMV between the lox sites in pGV690: A single copy of a modified ACMV (in which the majority of the coat protein gene was deleted) was isolated as an Mfe I fragment from plasmid pGV596 (patent WO 99/22003) and cloned into the EcoR I site of pGV690 to produce pGV691, such that the viral origin is adjacent to the 35S promoter. The ability of Cre to transactivate both GUS expression and viral duplication was first tested by co-bombarding viral genomes with the complete lox sites, pGV691, with the pNY102 plasmid containing the 35S: Chimeric Cre gene in the leaves of Ni cotiana taba cum var Xan thi and N. ben thamiana, as well as the bombardment of the leaves of Xan thi plants stably transformed with the gene 35S: Cre. Gus activity and replicon duplication were detected only in the presence of Cre, providing evidence that Gus is a good reporter for the division, that the duplication of the geminivirus can tolerate at least one lox site, and that the Cre expression of A chimeric, chromosomally integrated Cre gene can transactivate viral duplication. When pGV691, pGV596d (an ACMV proreplicon with a duplication protein, mutant described in PCT International Application WO 99/22003), and pNY102 were co-bombarded, trans duplication of the prorreplicon of pGV596d was observed as well as the duplication ci s of the replicon of pGV691. The Pvu II site in pGV691 was converted to Xma I using the NEB 1048 Sma I linker and then the Xma I-H3 fragment was cloned into pSK (Stratagene, 11011 North Torrey Pines Road La Jolla, CA 92037), Xma I-Hind III to produce pGV696. PGV699 was made by cloning a Pst I fragment of pGV697 containing a fragment of Transcriptional Detention consisting of 3 'untranslated regions in tandem of a small subunit of ribulose-1, 5-bisphosphate carboxylase and nopaline synthase genes in the Pst I site of pGV696 in the desired orientation to prevent inadvertent transcription of the viral duplication gene by a plant promoter within the T-DNA or adjacent to its insertion site. The Xma I-Hind III fragment of pGV699 was cloned into pBE673, a binary vector for bar selection (described in PCT International Patent Application WO 99/22003) to produce pBE704. To make a binary vector containing both the ACMV vector with the complete lox sites and a dimer of a defective mutant of the ACMV duplication, the Sac I-Xma I fragment of the pGV596d containing the mutant ACMV dimer was cloned into the vector binary of pBE673 Sac I-Xma I to make pBE695. Then the Xma I-H3 fragment of pGV699 was cloned into pBE695 to form pBE705. The constructs of pBE704 and pBE705 were introduced into N. ben thamiana and N. taba cum through a transformation mediated by agrobacteria as described above either alone or in the presence of the ACMV pro-pollicon pGV596d. Bombardment of transgenic plants with Cre showed the activation of GUS expression and replication of the ACMV replicon was observed in both transformants 704 and 705. In addition, duplication of the mutant ACMV replicon was observed in the transformants 705 confirming the data of the transient analysis. The results indicated that the duplication of the divided replicon was significantly higher in the absence than in the present of the prorreplicon. The GUS ORF in pGV690 was replaced with one carrying the Luc ORF of Promega pSP-luc + vector (2800 Woods Hollow Road madison, Wl 53711) using? Co I-Xba I to make pGV716. A single copy of the full-length genome of TGMV was isolated as a 2.6 kb Mfe I fragment from plasmid pTAl .3 (? Robertson,? Orth Carolina State University) was cloned into the Eco Rl site between the Lox P sites of pGV716 to result in the replicons of the TGMV "with the complete lox sites" in the plasmid pGV731, such that the viral origin is adjacent to the 35S promoter. The coat protein gene in pGV731 was replaced with the GUS ORF of pGV671 (PCT International Application WO 99/22003) using the Ndel / Sall to produce pGV733. The Bgl II to Hind III fragment of pGV733 was cloned into pBE673 cut with Bam HI / Hind III to produce pBE733, a binary vector of bars. A binary vector pBE736 was made which was identical to that in pBE733 except that one of the lox sites was changed from P of the non-cultivated type to lox 72 mutant [Albert et al., Plan t J. 7: 649-59 (1995)] . The replicons with the complete lox mutant sites were introduced into a binary vector and the modified binary vectors were introduced into a groba cterium tumefa hundreds and transformed into plants by agrobacterium mediated transformation. The progeny of the plants were collected and crossed with lines containing the correctly regulated Cre gene. The binary vectors pBE733 and pBE736 were transformed into agrobacteria and introduced into tobacco (Ni cotiana taba cum var. Xanthi) and leaf discs of Ni cotiana ben thamiana through agrobacteria-mediated transformation (using 25 ml of the agro-culture in OD A600). 1.0). After 3 days in the MS media, the discs were incubated for 6 weeks in the shoot medium (the MS media supplemented with 1 mg / ml claforane 1 ug / ml BAP, 0.1 ug / ml NAA) containing 10 and 6 ug / ml PPT (Sigma Chemical Co., 6050 Spruce St., St. Louis, MO 63103) for tobacco and benthamiana, respectively. Transformants of BE733 were confirmed for the transgene by Southern analysis and analyzed for duplication by Southern analysis and for GUS expression with bombardment of a pNY102 plasmid containing the 35S: Cre gene. The transgenic tobacco and N. ben thamiana containing the TGMV were obtained with the complete lox sites of pBE733. Bombardment of the transgenic leaf or plant with PVX: Cre and 35S: Cre gave the marked staining of the expected GUS for duplication. The Tl progeny seedlings of the N. ben thamiana 733 # 23 line were infected with PVC-Cre. This resulted in the expression of GUS in the infected leaves that persisted for up to 25 days without apparent restriction. These results confirm that the inactive replicon was hereditary and able to be activated by the division mediated by Cre.

EXAMPLE 2 Inactive Amplicons of PVX-GFP and PVX-GUS with the Lox Sites Plasmids pVX201, pTXS-GFP and TXGC3S.vec were obtained from Dr. D. Baulcombe (The Sainsbury Laboratory, John Innes Center, Norwich Research Park, Norwich Research Park, NR4 7UH, UK) pVX201 contains a clone of 35S: cDNA of PVX: 3'nos ('amplicon PVX'), the pTXS-GFP contains T7 promoter: PVX-GFP construct and the TXGC3S.vec contains the T7 promoter: PVX-GFP construct. The GFP and GUS ORFs were cloned after the promoter of the PVX coating protein. PGV680 containing PVX-GFP ('amplicon PVX-GFP') was made by replacing the Sac I-Avr II fragment of amplicon pVX201 with that of pTXS-GFP containing the ORF of GFP. PGV681 containing the PVX-GUS ('PVX-GFP amplicon') was made by replacing the Sac I-Avr II fragment of the pVX201 amplicon with that of the TXGC3S.vec containing the GUS ORF. A mutation of structure change in the open reading structure of RNA polymerase dependent viral RNA of pGV680 and pGV681 yielded plasmids pGV682 and pGV683, respectively. This mutation was made by restricting the DNAs of pGV680 and pGV681 with Age I, filling and relinking. The leaves detached from Ni cotiana ben thamiana were bombarded with the previous plasmids g the biolistic pistol. Analysis of the leaves 10-14 days after the bombardment showed that GFP fluorescence was detected in the leaves bombarded with pGV680 but not with pGV682 and that GUS staining was detected in the leaves bombarded with pGV681 but not with pGV683. This confirmed that reporter gene expression was dependent on an RNA polymerase driven by functional viral RNA (RdRP) and that reporter genes can be used to detect duplication. When pGV681 was bombarded into the cotyledons approximately 50-100 mg of zygotic soybean embryos, GUS staining detected duplication of the PVX vector 10-14 days after the bombardment. This showed that the PVX can be duplicated in the seed tissue and as diverse plant species as Glycine max and Ni cotiana ben thamiana.

Introduction of the Lox Mutants, Inverted Sites in PVX Amplicons: A lox mutant site (lox 43) was introduced by PCR into the PVX-GFP amplicon. For this, PCR products I and II were made g pGV681 as the template and primer pairs I of PCR [SEC ED No: 5 (GV70, upper primer) and SEQ ID No: 6 (GV71, primer lower)] and II (SEQ ID NO: 7 (GV73, upper primer) and SEQ ID NO: 8 (GV72, lower primer)], respectively. [SEQ ID NO: 5] 5 '-GCG GCA TGC GTC GAC ACA TGG TGG AGC ACG ACA-3 '(GV70) (SEQ ID No: 6) 5' -GCC GGG TAC CGA GAC GCG TCA TCC CTT ACG-3 '(GV71) [SEQ ID No: 7] 5' -GTC TCG GTA CCT ATA ATG TAT GCT ATA CGA AGT TAT ATA AGG AAG TTC ATT TCA-3 '(GV73), [SEQ ID NO: 8] 5' -TGA TCC GCG GGT TTC TTC TCA TGT-3 * (GV72). the PCR was digested with Sphl and Asp718 and PCR product II with Asp718 and Sac II to result in fragments of 369 bp and 464 bp, respectively. Plasmid pGV680 was digested with Sph I and Sac II and the 9792 bp vector fragment was ligated in a 3-way ligation with the two PCR fragments to produce the plasmid pGV701 containing the lox 43 mutant site between the As element -1 and the TATA box in the 35S promoter of the amplicon. A mutant frame site (lox 44) was inserted by ligating the adapter in the 5 'untranslated region to the GUS ORF in pGV681. For this, pGV681 was digested with Xma I and ligated into an adapter made by destemming the following 51-mer primers: [SEQ ID NO: 9] 5'- CCG GGA ATG CAT GCT ATA GCA TAC ATT ATA CGA AGT TAT TCG AAT TTA AAT -3 '[SEQ ID NO: 10] 5' -CCG GAT TTA AAT TCG AAT AAC TTC GTA TAA TGT ATG CTA TAG CAT GCA TTC -3 'After digestion with Swa I of the ligated DNA, the linear DNA it was isolated, bound again and transformed into E. col i to produce the plasmid pGV700. The insertion of lox 44 in the PVX-GUS amplicon produced an N-terminal extension of 31 amino acids from the GUS ORF. The lox 43 and lox 44 mutant sites in pGV700 and pGV701 were confirmed by DNA sequence analysis. The PVX-GFP and PVX-GUS amplicons with two inverted lox sites were made by combining the mutant lox sites, previous Thus, for the PVX-GUS, the Avr II-Sac I fragment, containing the GFP ORF, of pGV701 was replaced with that of pGV700, which carries the lox 44 and the ORF of the GUS, producing pGV702. For PVX-GFP, the 4432 bp Age I-Cla I fragment in pGV701 was replaced with the 4476 Pb Age I-BstBl fragment, which carries lox 44, from pGV700, producing pGV708 with an N-terminal extension of 23 amino acids of GFP. The detached leaves of Ni cotiana ben thamiana were bombarded with plasmids pGV700, pGV701, pGV702 and pGV708 using the biolistic gun. Based on the expression of the reporter genes in the leaves 10-14 days after the bombardment, duplication was observed in all four cases, although the level of duplication was lower in pGV708. These results show that the insertion of the lox sites in the plant promoter and / or in the 5 'intergenic sequence for the reporter genes does not affect the duplication. The poorer GFP fluorescence in pGV708 may be due to the N-terminal extension in the GFP protein. To obtain a non-functional amplicon, pGV702 and pGV708 were passed through the Cre-expressing bacteria or incubated with the purified Cre enzyme (Novagen, Madison Wl) to reverse the sequence between the inverted lox sites. However, no inversion was detected in any plasmid. This may be due to the poor investment efficiency with these lox mutant sites that were the most efficient in Cre-lox recombination.

Introduction of Lox P sites of the uncultivated type, invested in the PVX amplicons: A lox P site of the non-cultured type was cloned as an adapter in the Cía I site in the 5 'intergenic region for the GFP ORF in the plasmid pGV701 followed by digestion with Xma I, isolation of the linear vector and its self ligature to produce the plasmid pGV712. The adapter was primed by the primers GV78 (SEQ ID NO: ll) and GV77 (SEQ ID NO: 12) [SEQ ID NO: 11] 5 '-CGA TAA CTT CGT ATTA TAT GCT ATA CGA AGT TAT CCC GGG- 3 '(GV78) (SEQ ID NO: 12) 5' -CGC CCG GGA TAA CTT CGT ATA GCA TAC ATT ATA CGA AGT TAT-3 '(GV77) The lox 43 mutant site in the 35S promoter in pGV712 was replaced by a lox P site of the non-cultured type as follows: A PCR product was made on the DNA template of pGV712 using a higher primer GV74 (SEQ ID NO: 13) and a lower primer GV72, (SEQ ID NO: 8) [ SEQ ID NO: 13; 5'- GAT GAC GCG TAT AAC TTC GTA TAA TGT-3 '(GV74) The PCR product was digested with Mlu I and Sac II, and the resulting product was used to replace the Mlu I fragment -Sac II containing lox 44 in pGV712 to produce plasmid pGV713.PGV713 contains two loxP sites of the non-cultivated, inverted type.Incubation of pGV713 with Cre recombinase as suggested by the manufacturer (Novagen, Madison, Wl invest Did the DNA sequence between the lox sites? containing the RNA-dependent RNA polymerase to produce a non-functional amplicon in the plasmid pGV714. Plasmid pGV714 was purified after transformation of XLl cells of E. coli A lox P site of the non-cultured type was cloned as an adapter at the Cía I site in the 5 'intergenic region for the GUS ORF in the plasmid pGV702. For this, the plasmid pGV702 was linearized with Xma I, ligated to the annealing adapter of the primers GV80 [SEQ ID NO: 14] and GV81 [SEQ ID NO: 15]. [SEQ ID NO: 14) 5 '-CCG GGG ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA TTC GAA CAT TTA AAT 3' GV80 [SEQ ID No: 15] 5 '-CCG GAT TTA AAT GTT CGA ATA ACT TCG TAT AAT GTA TGC TAT ACG AAG TTA TCC -3 'GV81 The resulting plasmid, designated pGV702W, was used to construct the PVX-GUS with lox P sites of the non-cultivated, inverted type. For this, the Avr II-Sac I fragment, containing the GFP ORF, of pGV713 was replaced with that of pGV702W, which carries lox P and the ORF of GUS producing pGV708W.

The inability of non-functional amplicons, inverted to duplicate without undergoing Cre-mediated inversion to a functional amplicon, was confirmed by bombardment of the leaves of Ni co tiana ben thamiana. The plasmids pGV712 and pGV713 were duplicated and diffused in the bombarded leaves. This was clear from showing that the fluorescence of GFP was detected 10-14 days after the bombardment. However, plasmid pGV714 did not duplicate unless co-bombarded with plasmid pNY102 which expresses Cre under the control of a 35S promoter. The 3.01 kB BspEl-Sac I fragment containing the inverted non-functional amplicon of plasmid pGV714 was cloned into pBE674 of the cut with Xma I (compatible with BspEl) -Sac I, a binary vector with the selection of bars in the plants , as described above, to yield pBE714. The plasmid pBE714 was introduced by means of LBA4404 of agrobacteria in the plants of Ni cotiana ben thamiana of the non-cultivated type, as described above. The plants were selected in PPT 30 ug / ml of medium for phosphonitricin shoots and then 10 ug / ml of root medium. 10 transformants were obtained (714B #s E-5, E-6, G-1, M-4, M-6, M-7, Q-1, S-4, LL-1, UU-1). Southern analyzes showed that all were positive for the transgene except for M-7 and Q-1. All appeared phenotypically normal and without any viral symptoms. The ability of the transformants to be transactivated by Cre expression was tested using the infection with PVX expressing Cre. The leaves of five transgenic plants 714 B positive (LL-1, UU-1, E-5, M-4 and S4) were bombarded with the particle gun using 1 Wu of a plasmid DNA carrying the 35S: Cre chimeric gene to test the activation of the amplicon or they are gold only as a control. After approximately 5 days, the expression of GFP was observed in the leaves of three (HI-1, M-4 and S4) of the five transgenic plants bombarded with 35S: Cre but not with the control of gold alone. After approximately 12 days, the expression of GFP in the transgenic leaves bombarded with 35S: Cre decreased, suggesting that the restriction might be occurring, as is frequently observed with the infection of the leaves of the non-cultivated type with PVX-GFP. The levels of expression, diffusion and duration of GFP in the leaves activated with Cre were comparable to those of the PVX: GFP bombarded directly in the leaves of N. ben thamiana of the non-cultivated type. Five out of the eight seedlings of the Tl progeny of transgenic 714B LL-1 showed the expression of GFP with the bombardment with 35S: Cre. A similar bombardment of the leaves detached from 12 other progeny seedlings showed that 11 were positive for activation. In this way, the inactive virus is hereditary and the progeny capable of being activated with Cre. The levels of expression, diffusion and duration of GFP in Cre-activated seedlings were slightly variable among the individuals of the Tl progeny but were generally comparable to those of the PVX: GFP bombarded directly in the N. ben thamiana seedlings of the non-cultivated type . The transgenic lines 714B will be genetically combined with the chimeric Cre genes, correctly regulated, for example by crossing with the transgenic lines carrying such Cre genes.

EXAMPLE 3 Inactive, Divisional Pvx Amplicons To make a replicon of the AR virus? of the PVX flanked by tandem lox sites, two PCR products were made in pGV680: a 438 bp PCR product containing the TATA box ('minimal promoter') and the lox P site using the primer pairs GV85 [SEQ ID NO: 16] (with the Sph 1 site) -GV86 [SEQ ID NO: 171 (with the Not I site) and a 441 bp PCR product containing the mutant lox site (loxD117) [Abrenski] , K. and Hoess R. (1985) J. Mol. Biol. 184: 211-220] and the 5 'end of the PVX cDNA using the primer pairs GV87 [SEQ ID NO: 18] (with the Not I site) -GV88 [SEQ ID NO: 19] (with the Sac II site ). Then, the two PCR products were digested with Not I, ligated and used as a template for PCR using the primer pairs: the primers GV105 [SEQ ID NO: 20] and GV88 [SEQ ID NO: 19] to give the PCR product of 509 bp. The resulting PCR product was digested with Sph I and Sac II and the 509 bp fragment containing the TATA box ('minimal promoter') followed by the lox P sites of the non-cultured and mutant type, in tandem (D117) in front of the amplicon cDNA was isolated and cloned into pGV680 digested with Sph I-Sac II to give pGV720. Thus, pGV720 is a PVX-GFP amplicon with the minimal 35S promoter and the lox P and loxD117 sites in tandem between the TATA box and the transcription start site, the pGV720 did not duplicate efficiently when bombarded in N ben thamiana Therefore, it had to be replaced with the full-length promoter, which was isolated as a 438 bp PCR product using the PCR primers GV85 [SEC IN NO: 16] (with the Sph I site) and GV86 [SEQ ID NO: 17] (with the Not I site) in pGV680 and cloned into pGV720 digested with Sph I-Not I to result in pGV740. In this way, pGV740 is a PVX-GFP amplicon with the 35S promoter and the loxP and loxD117 sites in tandem between the TATA box and the transcription start site. PGV740 could duplicate when bombardment in N. ben thamiana even without a Cre expression gene, suggesting that the amplicon can have at least 66 bp between the TATA box and the 5 'end of the PVX cDNA. Although pGV740 can be easily inactivated by inserting a fragment of Transcriptional DETENTION into the Not I site as depicted in Figure 3, it was decided to make a divisional replicon that physically divides the RNA virus amplicon from the chromosome with specific recombination for the site. For this, the promoter at the end of the PVX cDNA was first moved by suppressing the promoter by ligation of an Xmal / Notl / Xmal adapter (primers GV157 [SEQ ID NO: 21] and GV158 [SEQ ID NO: 22] to the Xmal site of pGV740, followed by Not I digestion and ligation again This resulted in pGV760, which is an amplicon of PVX-GFP minus the promoter with a lox site D117, mutant upstream of the start site Then a 2u-trp fragment of yeast was isolated by PCR using the primers GV165 [SEQ ID NO: 23) and GV166 [SEQ ID NO: 24] and cloned by recombination around the Sph I site in the pGV760 of the cut with Sph I by transforming the vector and the target into the yeast. The DNA from the prototrophic yeast colonies for trp was isolated and transformed into E. coli The E. ampicillin resistant coli were confirmed to be E shuttle shuttle plasmid. coli-yeast desired, pGV774. A 351 promoter + lox P site of 391 bp was isolated by PCR using primers GV170 [SEQ ID NO: 25] and GV171 [SEQ ID NO: 26] in pGV740 with an Xma I site at the 3 'end of the site lox P and was cloned by recombination with yeast using poor 20 bp positions to the regions that are on the side of the Nar I site in pGV774. The resulting plasmid, pGV783, is an E-yeast shuttle vector. col i containing a divisional PVX-GFP amplicon, with complete lox sites flanked by tandem WT and lox D117 sites. As a divisional amplicon is represented by the element B with or without the elements A and / or C in Figure 1.

PVX Divisional Amplicon Defective Motion, Inactive: The gene for the coating protein in the divisional PVX-GFP amplicon in pGV783 was suppressed by digestion with Xho I and Sal I of pGV783 followed by ligation again to result in a defective amplicon of movement, pGV819. This mutant amplicon was isolated as an Xma I fragment and cloned into the Xma I site of the binary vector pBIN19 to result in pBE819. This was introduced into the tobacco plants by means of the transformation mediated by the agrobacteria.

Amplicon Divisional, Inactive of PVX with Double Reporters for the Expression of Extraneous Proteins To demonstrate that amplicons can be used to both express a foreign protein as well as to restrict an endogenous gene, pGV784 was made. This construct is a shuttle vector of yeast-i., Col i containing a divisional PVX-CP-GFP-PDS amplicon, with the complete lox sites with the WT and lox D117 sites. The 3.6 kb Avr II / Sac I band of pGV770 was cloned into the pGV783 cut with Avr II / Sac I. The pGV770 is the PVX-GFP-PDS-CP amplicon. This contains a chimera of the GFP ORF (740 bp) followed by a fragment of approximately 200 bp of the partial phytoene desaturase cDNA of N. ben thamiana. This was built using two-step PCR. First, the full-length ORF of the FGP was isolated by PCR in the plasmid pGV680 using primer pairs GV162 [SEQ ID? O: 27] / GV163 [SEQ ID? O: 28] and a phytoene desaturase from N. ben thamiana from 200 bp ordered in sequences was isolated by PCR using primer pairs GV133 [SEQ ID? O: 29] / GV109 [SEQ ID? O: 30] in plasmid pGV723 carrying a partial AD? C clone of desaturase from phytoene from N. ben thamiana [Ruiz et al. (1998) Pl an t Cell 10: 937-946], since the 3 'sequence of 18 bp of these primers is specific for the phytoene desaturase sequence, these sequences of pb can also be used by isolating the sequence directly by RT-PCR from the isolated ARβ of the N. ben thamiana leaf, by techniques well known to one skilled in the art. Then, these 2 fragments were ligated by Age I / Xma I sites introduced by the primers and reamplified by PCR using GV162 [SEQ ID: O: 27] and GV109 [SEQ ID? O: 30]. The complete, chimeric GFP-PDS fragment was digested with Cia I and Xho I and cloned into the Cía I-Sal I sites of pVX201 to result in pGV770. The 3.6 kb Avr Il / Sac I band of pGV770 was cloned into the pGV783 cut with Avr II / Sac I (see below) to result in plasmid pGV784. In this case, the divisional amplicon in pGV784 represents element B with elements A and C in Figure 1. This was isolated as an Xma I fragment of 8,387 kB and cloned into the Xma I site of the binary vector pBinl9 to give as a result, pBE784, which was introduced into tobacco by means of transformation mediated by agrobacteria. The primers referred to in the above discussion are given below: GV85 5 '-GAG GCA TGC CCG GGC AAC ATG GTG GAG CAC GAC A3' [SEQ ID NO: 16] GV86 5 '-TAT GCG GCC GCA TAA CTT CGT ATA GCA TAC ATT ATA CGA AGT TAT ATA GAG GAA GGG T-3 '[SEQ ID NO: 17] GV87 TCC TTG ATC CGC GGG TTT CTT CTC ATG T [SEQ ID NO: 18] GV88 5 '-TCC TTG ATC CGC GGG TTT CTT CTC ATG T-3' [SEQ ID NO: 19] GV105 5 '-CAC GCA TGC ACT ATC CTT CGC AAG ACC C-3' [SEQ ID NO: 20] GV157 5 '-CCG GGG CGG CCG CAT AC-3' [SEQ ID NO: 21] GV158 5'-CCG GGT ATG CGG CCG CC-3 '[SEQ ID NO: 22] GV165 5 '-ACC ATG ATT ACG CCA AGC TTA AGA AAA GGA GAG GGC CAA GA-3' [SEQ ID NO: 23] GV166 5 '-AGT TAT GCG GCC GCC CCG GGC ATA TGA TCC AAT ATC AAA GGA-3 '[SEQ ID NO: 24] GV170 5 '-GCG CAG CCT GAA TGG CGA ATG GCG CCC CAA AAA TAT CAA AGA TAC A-31 [SEQ ID NO: 25] GV171 5' -AAG GAG AAA ATA CCG CAT CAC CCG GGA TAA CTT CGT ATA GCA TAC A-3 '[SEQ ID NO: 26] GV162 (25-mer) 5 '-GCC AAT CGA TCA TGA GTA AAG GAG A-3' [SEQ ID NO: 27] GV163 (35-mer! 5 '-GCT AAC CGG TAG ACA TTT ATT TGT ATA GTT CAT CC-3' [SEQ ID NO: 28] GV133 (3 -mer; 5 '-GAA GTC GAC CGC GGG CAG ACT AAA CTC ACG AAT A-3' GV109 (30-mer: 5 '-GAA TTC TCG AGC CAT ATA TGG ACA TTT ATC-3' [SEQ ID NO: 30] EXAMPLE 4 Co-Activation of an Inactive Replicon of PVX and a Restriction Generator of Restriction Through Specific Recombination for the Site A restriction suppressor gene will be incorporated into the PVX amplicons containing lox by two methods. In one method, the ORF of a restriction suppressor will replace the target gene or ORF of the coating protein in amplicons depicted in Figures 1-3 such that the restriction suppressor is in the replicon. In the second method, the ORF of the constraint suppressor will replace the divisional reporter, such that it is represented by element C and the amplicon with the complete lox sites acting as a fragment of Stop transcription / translation, is represented by the Element B in Figure 1. In any case, the activation of the amplicon will also activate the expression of the restriction-suppressor gene to overcome the antiviral defense system of the host that involves the homology-dependent restriction and results in a higher duplication and a higher production of foreign proteins.

Co-Activation of an Inactive Replicon of the PVX (Inverted) and a Restriction-Suppressor Gene by Site-Specific Recombination pGV714 is a non-functional PVX-GFP amplicon with the inverted loxP sites, whose construction is described above. This was used to make the replacement protein vectors of the coating, pGV806 and pGV808. For pGV806, the ORF of the coat protein in pGV714 was replaced with that of the HC-Pro restriction suppressor (bases 1057-2433 of the tobacco corrosion virus genome, Genbank accession number M15239) or Pl-HC -Pro (bases 145-2433 of the tobacco corrosion virus genome, accession number of Genbank M15239) isolated from the Ptl-0059 plasmid (American Type Culture Collection, ATCC 45035). This cloning was done by homologous recombination in yeast [Hua, S. B. et al., (1997) Plasmid 38 (2): 91-6; Oldenburg, K. R. et al., (1997) Nucleic Acids Res 25 (2): 451-2 and Prado, F., et al., (1994) Curr Genet 1994 Feb; 25 (2): 180-3]. For this, first a fragment of the PCR containing the yeast selection marker (trp) and the yeast origin of 2 microns of the duplication was made by using the PCR primers P216 and P217 [SEQ ID Nos: 31 and 32],. Bases 25 of 5 'of these primers have homology on either side of Kas I in the E vector. col i of pGV714, such that the co-transformation of the PCR product into the yeast cells together with pGV714 linelized with Kas Y resulted in the cloning of the yeast fragment when repairing the opening (homologous recombination through the Kas site). I in the vector) which results in a shuttle vector of E. coli-yeast, pGV800. Then, the PCR products containing HC-Pro or Pl-HC-Pro were made by using the PCR primer pairs P233-P235 [SEQ ID NO: 33 and 35 respectively] and P234-P235 [SEQ ID NO. : 34 and 35 respectively], respectively, in pTL-0059. The 33 bases 5 '-terminals and the 30 bases 5' -terminals in P234 are homologous to the promoter of coating proteins, while the 31 bases 5 '-terminals in 235 are homologous to the 3' UTR of the protein ORF. coating. The cotransformation of HC-Pro or Pl-HC-Pro PCR products together with pGV800 linearized with Stu I resulted in opening repair (homologous recombination through the Stu I site in the ORF of the coating protein) and replacing the coding sequence of the coating protein with that of HC-Pro or Pl-HC-Pro to result in pGV806 and pGV808, respectively. These restriction suppressors replace the ORF of the coating protein in the amplicons represented by Figure 2. The primers referred to in the above discussion are given below: P216 (46-mer) 5-TGC GTA AGG AGA AAA TAC CGC ATC AAA GAA AAG GAG AGG GCC AAG A-3 '[SEQ ID NO: 31] The 25 bases 5' -terminals (underlined) are homologous to the vector pGV714 and the 21 bases of 3 'are homologous to the yeast fragment.

P217 (47-mer) 5 '-GCG CAG CCT GAA TGG CGA ATG GCG CCA TAT GAT CCA ATA TCA AAG GA-3' [SEQ ID NO: 32] The 25 bases 5'-terminal (underlined) are homologous to the vector pGV714 and the 22 bases of 3 'are homologous to the yeast fragment.

P233 (52 bp UP for HCP): 5 '-AAC GGT TAA GTT TCC ATT GAT ACT CGA AAG ATG AGC GAC AAA TCA ATC TCT GA-3"[SEQ ID NO: 33] The 33 bases 5 '-terminals (underlined) are the promoter of the PVX coating protein in pGV800 and the 20 bases of 3' are homologous to the termination 5 'of the coding sequence HC-Pro.

P234 (50 bp UP for Pl-HC-Pro): 5 '-AAC GGT TAA GTT TCC ATT GAT ACT CGA AAG ATG GCA CTG ATC TTT GGC AC3"[SEQ ID NO: 34] The 30 5'-terminal (underlined) bases are homologous to the PVX coat protein promoter in pGV800 and the 20 3 'bases are homologous to the 5' terminus. of the coding sequence Pl-HC-Pro.

P235 (53 bp of LP for HCP): 5 '-GGG GTA GGC GTC GGT TAT GTA GTA GTA GTT ATC CAA CAT TGT AAG TTT TCA TT-3"[SEQ ID NO: 35] The 31 bases 5 '-terminals (underlined) are homologous to 3'-UTR of the PVX coating protein sequence in pGV800 and the 22 bases of 3' they are homologous to the 3 'end of the HC-Pro coding sequence. These modified PVX cDNAs carrying GFP and the restriction suppressors without the coating protein will be used to transform tobacco plants by means of the transformation mediated by known agrobacteria. For example, the amplicon in pGV806 was isolated as a fragment of Bspe 1 and Xma I of d.3 kB and was cloned in pBHOl linearized with Xma I. With the Cre-mediated recombination, controlled, the co-activation of the viral duplication without systemic diffusion and of the suppressor of the restriction will increase the foreign protein, in this case the production of GFP.

Co-Activation of an Inactive, Divisional Replicon of the PVX and the Suppressor Gene of the Restriction Through the Recombination Specified for the Site The complete region between the lox sites containing the PVX cDNA and the 35S promoter of the plasmid pGV7d3 can be isolated and cloned between the lox sites, such as element B, with the elements A and / or C in the Figure 1. For this, the sequence of and around the lox sites before the division will be: atgATAACTTCGTATAGCATACATTATACGAAGTTAT [SEQ ID NO: 36] -applicon inactive of the PVX -TAANTAAATAACTTCGTATAGCATACATTATACGAAGTTAT [SEC ID NO: 37] Q; and after the division is: atgATAACTTCGTATAGCATACATTATACGAAGTTAT [SEC ID NO: 3d] Q; where the codon of the lower case is the initiation codon, the underlined sequences are the lox P site of the non-cultivated type flanking the inactive replicon, N is any base, and Q is the coding sequence of the restriction suppressor, such that it is in the structure for the initiation codon after division. In this way, the restriction suppressor is not translated unless the blocking fragment is divided to restore its proper reading structure. The lox sequence of the non-cultured type will also be replaced by the mutant sequences for conditional specificity, augmented while retaining this translation activation by methods known to one skilled in the art.

EXAMPLE 5 Co-Activation of an Inactive Geminivirus Replicon (with the complete lox sites) and a deletion gene of the Restriction Through Specific Recombination for Site: The Suppressor Gene is in the Replicon A restriction suppressor gene will be incorporated into the geminivirus vector with complete lox sites by two methods. The ORF of a constraint suppressor will replace either the GUS ORF or the ORF of luciferase in pGV733. In the oldest case, the suppressor of the restriction is in the replicon (as a B element with or without the elements A and / or C in the Figure 1. While in the last case, the replicon is outside, as the element C together with the elements A and B in the Figure 1, such that element B acts as a fragment of Detention cié the transcription / translation. An example of a translation stopping fragment is as described above for the PVX. In any case, the activation of the replicon will also activate the expression of the restriction suppressor gel to overcome the antiviral defense system of the host which involves the homology-dependent restriction and results in the duplication and production of higher foreign proteins.

EXAMPLE 6 TGMV Binary, with the complete Lox Sites with GFP gue Replace the Coating Protein A coating replacement vector of the TGMV was made with GFP. For this, a fragment of the PCR containing the yeast selection marker (trp) and yeast duplication origin of 2 microns was made by using the PCR primers P216 and P217 [SEQ ID NO: 31 and 32] and it was cloned in Kas I in the E vector. coli in pCSTA [Von Arnim, Albrechit; Stanley, John, Virology (1992), 186 (1), 286-93], obtained from Dr. John Stanley (John Innes Center, Norwich, UK) by repairing the aperture (homologous recombination through the Kas I site in the vector, as previously described) resulting in a shuttle vector of E. coli-yeast, pGV793. Then, a PCR product containing the 796 bp GFP ORF was made from the plasmid psmGFP [Davis, S.J. and Vierstra, R.D. (1998) Plan t Molecular Biology 36: 521-536] with primers P218 [SEQ ID NO: 39] and P219 [SEQ ID NO: 40] and cloned into pGV793 cut from Hpa I + BstBl by cloning yeast to result in pGV798. P218 is a 49 bp primer whose 29 bases of 5 'are homologous to the promoter of the coating protein and the 18 bp of 3' have homology to the 5 'end of the GFP ORF (except for an inequality of 2 bp) , whereas p219 is a 48-mer, whose 31 bases of 5 'are homologous to the 3' untranslated region of the coating protein and the 26 3 'bases are homologous to the 3' end of the GFP ORF. Finally, the coat protein of the Sac I-Nhe I fragment in pGV651, a dimer TGMV-A (International Application of PCT WO 99/22003) was replaced with that of pGV798 to make pGV802, a TGMV-A dimer with GFP which replaces the coating protein. When N. ben thamiana was co-bombarded with pGV802 and the TGMV-B dimer (obtained from Dr. D. Robertson,? Orth Carolina Estate University), the infected tissue expressed GFP which was persistent and was not restricted for at least 2 months. To make a replicon of TGMV-GFP with the complete lox sites, an individual copy of the replicon will be isolated as a partial Mfe I and will be cloned into the Eco Rl site of pGV690, as described above. The Bam Hl-Xho I fragment containing the GFP ORF of the resulting plasmid will be used to replace the GUS ORF in pBE733, cut with Bam Hl and Xho I. This binary vector will be transformed into plants by means of the transformation mediated by Agrobacteria alone or co-transformed with the plasmid pBE795. BE795 is a binary vector that contains the TGMV-B dimer. This was done by replacing the Sma I to Sal I sequence of pBIB, [Becker, D. (1990) Nucleic Acids Research ld: 203] with that of the BsrB I to Sal I fragment of the TGMV B dimer.

P218 (UP primer for GFP ORF) (49-mer) 5 '-AAA GTT ATA TAA AAC GAC ATG CGT TTC GTA CTA AGG AGA TAT AAC A-3' [SEQ ID NO: 39] P219 (LP for the GFP ORF) (48-mer) 5 '-AAT TTT ATT AAT TTG TTA TCG AAT CAT AAA TTA TTT GTA TTC TTC ATC-3 '[SEQ ID NO: 40] EXAMPLE 7 Transgenic Lines Expressing Chimeric, Regulated Cre Genes For the chimeric replicase genes the ORF of Cre was isolated as a Neo I-Xba Y fragment of 1.3 kB from a plasmid 35S: Cre and was used to replace the Neo I-Xba I fragment containing the 10 kD ORF in pGV656 and the Neo I-Xba I fragment containing the ACMV duplication protein in pGV659 to result in the plasmids pGV692 and pGV693, respectively. The Hind III fragment of pGV692 containing the Vc: Cre gene was cloned into the Hind III site of pBinl9 (GEN BANK ACCESS U09365) and pBE673 (described in the PCT International Application WO). 92/22003) to produce the binary plasmids pBE692 (with the kanamycin resistance gene, vegetable) and pBE692b (with the phosphotrichon resistance gene, vegetable), respectively. The Bam HI-Bam HI-Asp716 I partial fragment of pGV693 containing the IN: Cre gene was cloned into pBinl9 and pBE673 cut with Bam HI-Asp718 I to produce the binary plasmids pBE693 and pBE693b, respectively. Nicotiana ben thamiana and N. taba cum var Xan thi were transformed with BA692 and BA693 from agrobacterium containing the binary plasmids pBE692 and pBE693, respectively. These transgenic plants will be crossed with those that carry the viruses with the complete lox sites. An IN: Cre chimeric gene was modified to reduce its ability to be translated in order to tolerate a permeable Cre transcript. Since, it has been reported that upstream ORFs usually reduce, or in extreme cases prevent, downstream translation [Kozak, M. (1996) Mammalian Genome. 7, 563-74], pGV693 was linearized with the unique Neo I site at the start codon of the ORF of Cre and ligated an adapter made of primers P224 [SEQ ID NO: 41] and P225 [SEQ ID NO. : 42]. This resulted in the addition of the 39 bp sequence upstream of the translation initiation codon that includes the 21 bp ORF 18 bp upstream of the translation initiation codon. The modification in the resulting plasmid, pGV787, was confirmed by sequencing the DNA. The Bam Hl fragment from pGV7d7 was used to replace the corresponding region in pBE673 to produce the binary vector pBE787. This vector was tested in transgenic plants. The primers used in the above discussion are given below: P224 5 '-CAT GCG TGT CGC ATA CTA TTA CTA ATA GGC AGC GAG GAT-3' [SEQ ID NO: 41] P225 5 '-CAT GAT CCT CGC TGC CTA TTA GTA ATA GTA TGC GAC ACG-3' [SEQ ID NO: 42] Selection for correctly regulated Cre Expression and the use of Mux Lox Sites A system was developed to make possible the selection of transgenic lines that have correctly regulated Cre expression. The luciferase gene was selected as a non-destructive reporter, sensitive for division mediated by Cre. We selected a set of vectors with the complete lox sites, pGV751-754, which contain the basic cassette "promoter 35S-lox-gene NPT II-terminator rbcS 3'-terminator Nos 3'-lox-Luc". The expression of Cre would divide the "DETENTION" fragment containing the NPT II gene and the transcriptional terminator sequences between the lox sites, thus changing the expression of luciferase. In this way, the division of the NPT II gene during the selection will make the plants sensitive to the kanamycin selection. The co-transformation of these bar-resistant binary vectors bearing IN2: Cre (pBE692) or Vc: Cre (pBE693), as previously described, in the kanamycin + bar selection plates allowed the selection of transgenic lines where expression of Cre is not low enough to mediate recombination. Using the above selection method, the efficacy of the lox mutant sites was tested to improve the 'specificity' of the regulated promoters expressing Cre. It has been reported that various lox mutant sites have been reported as requiring more Cre protein to activate site-specific recombination [Albert et al., Plan t J. 7: 649-59 (1995)]. For example, the in vitro efficiency of mutant sites, sites lox 72, lox 78 and lox 65 were reported to be 12.5%, 5% and 2.5%, respectively, relative to lox P of the non-cultivated type. Plasmids pGV751, pGV752, pGV753 and pGV754 contain a lox site of the non-cultivated type and a second lox site which is lox P, lox 65, lox 72 and lox 78 of the non-cultivated type, respectively, in the previous cassette. PGV751 and pGV752 were selected for the initial test in transgenic plants. Constructs with complete lox sites were introduced into the binary vectors (later referred to with the prefix pBE) and transformed into tobacco plants (N. taba cum, cv. Xan thi) by transformation of leaf disc mediated by agrobacteria with pBE751 (kan) + pBE693b (bar), pBE751 (kan) + pBE692b (bar), pBE753 (kan) + pBE693b (bar) and pBER753 (kan) + pBE692b (bar). For induction by safener, the leaf discs of the primary transformants were flooded for 30 minutes in 30 ppm of recently made 2-CBSU [Hershey, HP and Stoner, TD (1991) Plan t Molecular Biol ogy 17: 676-690] and then placed in the flat, solid MS medium for 1-2 days before the test. The whole seedling or the leaf discs to be tested were sprayed with 5 mM beetle luciferin and then kept in the dark for 5 minutes before the formation of images for luciferase expression under a cold CCD camera.

Results of cotransformation of bar 751/693 and bar 753/693 Expression of luciferase was detected by a cold CCD camera of the transgenic lines, independent, untreated and treated with safener transformed with I?: Cre in the presence of the construction with the complete sites lox pBE751 (with the lox P site of the non-cultivated type) or pBE753 (with the lox 72 mutant). Table 1 shows that in relation to the lox P of the non-cultivated type, the lox 72 mutant reduces the number of lines that show the luciferase in the leaf discs at the time point zero. The lines that showed inducibility by the safener without environment were selected for further analysis. Leaf discs from these plants were incubated for 2 days with or without safener treatment. Table 2 shows that the expression of luciferase, a division reporter, is higher in the lines cotransformed with pBE751 than with pBR753.

TABLE 1 Expression of luciferase in leaf discs with or without treatment with safener TABLE 2 Relative luciferase expression in leaf discs with or without safener treatment after 2 days * Control 2 is a transgenic plant with bar 751/693. However, the luciferase in this transgenic plant is not induced either by injury or by treatment with safener.

LIST OF SEQUENCES < 110 > E. I. DuPont de Nemours and Company < 120 > Viral Expression System, Binary in Plants < 130 > CL-1127-C < 140 > < 141 > < 150 > 60 / 101,558 < 151 > 1998-09-23 < 160 > 42 < 170 > Microsoft Office 97 < 210 > 1 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 1 tcgagataac ttcgtataat gtatgctata cgaagttatg 40 < 210 > 2 < 211 > 40 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 2 aattcataac ttcgtatagc atacattata cgaagttatc 40 < 210 > 3 < 211 > 45 < 2 > 12 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 3 aattctataa cttcgtataa tgtatgctat acgaagttat g.agct 45 < 210 > 4 < 211 > . 37 < 212 > DNA < 213 > Sequence. Artificial < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 4 cataacttcg tatagcatac attatacgaa gttatag 37 < 210 > 5 < 211 > 33 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 5 gcggcatgcg tcgacacatg gtggagcacg here 33 < 210 > 6 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 6 gccgggtacc gagacgcgtc atcccttacg 30 < 210 > 7 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 7 gtctcggtac ctataatgta tgctatacga agttatataa ggaagttcat ttca 54 < 210 > 8 < 211 > 24 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 8 tgatccgcgg gtttcttctc atgt 24 < 210 > 9 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 9 ccgggaatgc atgctatagc atacattata cgaagttatt cgaatttaaa t 51 < 210 > 10 < 211 > 51 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 10 ccggatttaa attcgaataa cttcgtataa tgtatgctat agcatgcatt c 51 < 210 > 11 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 11 cgataacttc gtataatgta tgctatacga agttatcccg gg 42- < 210 > 12 < 211 > 42 < 212 > DNA < 213 > Sequence .Artificial < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 12 cgcccgggat aacttcgtat agcatacatt atacgaagtt at 42 < 210 > 13 < 211 > 27 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 13 gatgacgcgt ataacttcgt ataatgt 27 < 210 > 14 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 14 ccggggataa cttcgtatag catacattat acgaagttat tcgaacattt aaat 54 < 210 > 15 < 211 > 54 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 15 ccggatttaa atgttcgaat aacttcgtat aatgtatgct atacgaagtt atcc 54 < 210 > 16 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer * .íl < 400 > 16 gaggcatgcc cgggcaacat ggtggagcac gaca 34 < 210 > 17 < 211 > 58 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 17 tatgcggccg cataacttcg tatagcatac attatacgaa gttatataga ggaagggt 58 < 210 > 18 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 18 tccttgatcc gcgggtttct tctcatgt 28 < 210 > '19 < 211 > 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 19 tccttgatcc gcggcftttct tctcatgt 28 < 210 > 20 < 211 > - 28 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 20 cacgcatgca ctatccttcg caagaccc 28 < 210 > 21 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 21 ccggggcggc cgcatac 17 < 210 > 22 < 211 > 17 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 22 ccgggtatgc ggccgcc 17 < 210 > 23 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 23 accatgatta cgccaagctt aagaaaagga gagggccaag a 41 < 210 > 24 < 211 > 42 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 24 agttatgcgg ccgccccggg catatgatcc aatatcaaag ga 42 < 210 > 25 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 25 gcgcagcctg aatggcgaat ggcgccccaa aaatatcaaa gataca 46 < 210 > 26 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 26 aaggagaaaa tacc? Catca cccgggataa cttcgtatag cataca 46 < 210 > 27 < 211 > 25 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 27 gccaatcgat catgagtaaa ggaga 25 < 210 > 28 < 211 > 35 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 28 gctaaccggt agacatttat ttgtatagtt catee 35 < 210 > 29 < 211 > 34 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 29 gaagtcgacc gcgggcagac taaactcacg aata 34 < 210 > 30 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 30 gaattetega gccatatatg gacatttatc 30 < 210 > 31 < 211 > 46 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 31 tgcgtaagga? Aaaataccg catcaaagaa aaggagaggg ccaaga 46 < 210 > 32 < 211 > 47 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 32 gcgcagcctg aatggcgaat ggcgccatat gatecaatat caaagga 47 < 210 > 33 < 211 > 53 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 33 aacggttaag tttccattga tactcgaaag atgagcgaca aatcaatctc tga 53 < 210 > 34 < 211 > 50 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 34 aacggttaag tttccattga tactcgaaag atggcactga tctttggcac 50_ < 210 > 35 < 211 > 53 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 35 ggggtaggcg tcggttatgt agacgtagtt atccaacatt gtaagttttc att 53 < 210 > 36 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 36 atgataactt cgtatagcat acattatacg aagttat 37 < 210 > 37 < 211 > 41 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 37 taantaaata acttcgtata gcatacatta tacgaa tta t 41 < 210 > 38 < 211 > 37 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 38 atgataactt cgtatagcat acattatacg aagttat 31 < 210 > 39 < 211 > 49 < 212 > DNA < 213 > Sequence. Artificial < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 39 aaagttatat aaaéicgacat gcgtttcgta gatctaagga gatataaca 49 < 210 > 40 < 211 > 48 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 40 aattttatta atttgttatc gaatcataaa ttatttgtat agttcatc 48 < 210 > 41 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 41 catgcgtgtc gcatactatt actaataggc agcgaggat 39 < 210 > 42 < 211 > 39 < 212 > DNA < 213 > Artificial Sequence < 220 > < 223 > Description of the Artificial Sequence: Primer < 400 > 42 catgatcctc gctgcctatt agtaatagta tgcgacacg 39 It is noted that in relation to this date, the best method known to 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 claims is claimed as property.

Claims (41)

1. A transgenic, binary viral expression system, characterized in that it comprises: (i) an inactive, chromosomally integrated replicon comprising: a) viral elements of action required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase; and (ii) a chimeric, chromosomally integrated transactivation gene comprising a regulated, plant promoter operably linked to a site-specific recombinase coding sequence; wherein the expression of the transactivation, chimeric gene in cells containing the inactive replicon results in site-specific recombination, activation of replicon duplication, and increased expression of the target gene.

2. The viral expression system according to claim 1, characterized in that the site-specific sequences responsive to the recombinase are the lox sequences and the site-specific recombinase-encoding sequence encodes the Cre protein.

3. The transgenic viral expression system according to claim 1, characterized in that the inactive replicon is derived from the viruses selected from the group consisting of geminivirus and the single chain RNA viruses.

4. The viral expression system, transgenic according to claim 3, characterized in that the geminivirus is selected from the group consisting of Tomato Golden Mosaic Virus (TGMV) and the Chinese, African Melon Mosaic Virus (ACMV).

5. The transgenic viral expression system according to claim 3, characterized in that the single chain RNA virus is a potato X virus.

6. The viral expression system according to claim 1, characterized in that the regulated, plant promoter is selected from the group consisting of tissue-specific promoters, inducible promoters, and promoters specific for the developmental stage.

7. The viral expression system according to claim 6, characterized in that the regulated promoter is derived from genes selected from the group consisting of genes derived from a safener-inducible system, genes derived from the tetracycline-inducible system, genes derived from systems inducible by salicylate, genes derived from alcohol-inducible systems, genes derived from the glucocorticoid-inducible system, a gene derived from systems inducible by pathogens, and a gene derived from systems inducible by ecdysonomes.

8. The viral expression system according to claim 1, characterized in that the target gene encodes a protein selected from the group consisting of an enzyme, a structural protein, a seed storage protein, a protein that transmits resistance to herbicides and a protein that transmits resistance to insects.

9. The viral expression system according to claim 1, characterized in that the target gene encodes an RNA whose expression results in restriction of the gene dependent on the homology of an endogenous transgene or gene.

10. The viral expression system according to claim 1, characterized in that at least one regulatory sequence, suitable, linked to the target gene is selected from the group consisting of plant promoters, constituents, plant-specific promoters, plant-specific promoters , plant promoters, inducible and viral promoters.

11. The vial expression system according to claim 10, characterized by at least one suitable regulatory sequence is selected from the group consisting of a viral coat protein promoter, the nopaline tapeza promoter, the phaseolin promoter, and the promoter of the cauliflower mosaic virus.

12. The viral expression system according to claim 1, characterized in that the inactive replicon optionally contains a DNA fragment encoding a transient peptide.

13. The method for altering the levels of a protein encoded by a target gene in a plant, characterized in that it comprises: (i) transforming a plant with the viral expression system of claim 1; Y (ii) cultivate the vegetable seed, transformed under conditions where the protein is expressed.

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

15. The method according to claim 13, characterized in that the site-specific sequences responsive to the recombinase are lox mutant sequences that are not efficient for the Cre-lox recombination and the coding sequence of the site-specific recombinase encodes the protein of Cre.

16. A method for altering the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a first plant with an inactive replicon to form a first primary transformant, the inactive replicon comprising: a) viral elements of action cis required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase, (ii) transforming a second plant with a chimeric transactivation gene to form a second primary transformant comprising a regulated, plant promoter operably linked to a sequence Recombinase-specific coding for the transactivation site; (iii) cultivate the first and second primary transformants where the progeny of both seeds are obtained; and (iv) crossing the progeny of the first and second transformants where the target gene is expressed.

17. The transgenic, binary expression system according to claim 1, characterized in that the inactive, chromosomally integrated replicon is inserted into a reporter gene sequence such that when the replicon is divided the reporter gene is activated.

18. The transgenic, binary expression system according to claim 1, characterized in that a Transcription Detention Fragment is inserted into the inactive replicon.

19. The transgenic, binary viral expression system characterized by comprises: (i) an inactive, chromosomally integrated replicon, comprising: a) cis-acting viral elements required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase; and (ii) a transiently expressed chimeric transactivation gene comprising a plant or viral promoter operably linked to a site-specific recombinase-encoding sequence; wherein the expression of the chimeric transactivation gene in cells containing the inactive replicon results in site-specific recombination, activation of replicon duplication, and increased expression of the target gene.

20. A method for altering the levels of a protein encoded by a target gene in a plant, characterized in that it comprises: (i) transforming a plant with an inactive replicon, the inactive replicon comprising: a) cis-acting viral elements required for duplication viral; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase; (ii) infecting the transformant with a virus containing a transactivation, chimeric gene comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence of transactivation; wherein the expression of the transactivation, chimeric gene in cells containing the inactive replicon results in site-specific recombination, activation of replicon duplication, and increased expression of the target gene.

21. A transgenic, binary expression system, characterized in that it comprises an inactive transgene and a chimeric transactivation gene, the inactive transgene comprising: (i) cis-acting transcription regulatory elements operably linked to the coding sequence or RNA functional, and (ii) site-specific sequences responsive to a site-specific recombinase; the transactivation, chimeric gene comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence of transactivation, wherein the expression of the transactivation, chimeric gene in cells containing the inactive transgene gives as a result an operable linkage of the regulatory elements of the action transcript to the coding sequence of the functional RNA through site-specific recombination and increased expression of the target gel.

22. The transgenic, binary expression system according to claim 21, characterized in that the site-specific sequences responsive to the recombinase are the lox sequences.

23. The viral expression system according to claim 21, characterized in that the lox sequences are lox mutant sequences that are not efficient for Cre-lox recombination.

24. A viral, transgenic, binary duplication system characterized in that it comprises: (i) an inactive, chromosomally integrated replicon comprising cis-acting viral elements required for viral duplication and site-specific sequences responsive to a site-specific recombinase; and (ii) a chimeric transactivation gene, comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence; wherein the expression of the transactivation, chimeric gene in cells containing the inactive replicon results in site-specific recombination and activation of replicon duplication.

25. The transgenic viral duplication system according to claim 24, characterized in that the sequences specific for the site responsive to the recombinase are the lox sequences.

26. The transgenic viral duplication system according to claim 24, characterized in that the inactive replicon is derived from the viruses selected from the group consisting of geminivirus and the single chain RNA viruses.

27. The transgenic viral duplication system according to claim 26, characterized in that the geminivirus is selected from the group consisting of Tomato Golden Mosaic Virus (TGMV) and the Chinese, African Melon Mosaic Virus (ACMV).

28. The transgenic viral duplication system according to claim 26, characterized in that the single-stranded RNA virus is a potato X virus.

29. The viral duplication system according to claim 24, characterized in that the regulated, plant promoter is selected from the group consisting of tissue-specific promoters, inducible promoters, and promoters specific to the developmental stage.

30. The viral duplication system according to claim 29, characterized in that the regulated promoter is derived from genes selected from the group consisting of genes derived from a safener-inducible system, genes derived from the tetracycline-inducible system, genes derived from systems inducible by salicylate, genes derived from alcohol-inducible systems, genes derived from the glucocorticoid-inducible system, a gene derived from systems inducible by pathogens, and a gene derived from systems inducible by ecdysonomes.

31. A binary transgene expression system according to claim 21, characterized in that the inactive transgene is a restriction suppressor gene.

32. A transgenic, binary expression system, characterized in that it comprises: (i) a chromosomally integrated blocking fragment, linked by the sequences specific to the site responsive to a site-specific recombinase; and (ii) an inhibitory, inactive, chromosomally integrated restriction transgene; wherein the expression of a site-specific recombinase results in site-specific recombination that activates the restriction-suppressor gene.

33. The viral, transgenic, binary expression system according to claim 32, characterized in that the blocking fragment is in an inactive replicon comprising: (i) a target gene comprising at least one suitable regulatory sequence; and (ii) site-specific sequences responsive to a site-specific recombinase; wherein the expression of the site-specific recombinase results in site-specific recombination, and activation of both the replicon and the restriction-suppressor gene, and the increased expression of the target gene.

34. The transgenic, binary viral expression system according to claim 32, characterized in that the blocking fragment is an inactive replicon comprising the site-specific sequences responsive to a site-specific recombinase, wherein the expression of the recombinase site-specific recombination results in site-specific recombination, and activation of both the replicon and the restriction-suppressor gene.

35. The viral, transgenic, binary expression system according to claim 32, characterized in that the restriction-suppressor gene is selected from the group consisting of genes encoding the protein component of the helper component Pl (Pl-HC-Pro), the proteinase of the helper component (HC-Pro) and the cucumoviral protein 2b.

36. The viral, transgenic, binary expression system according to claim 32, characterized in that the restriction-suppressor gene is selected from the group consisting of genes encoding the geminivirus movement proteins BL1 or BR1.

37. A transgenic viral expression system, characterized in that it comprises: (i) a chromosomally integrated geminivirus proreplicon comprising: a) viral action elements required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) flanking sequences that make possible the division of the elements of a) and b), wherein the prrereplicon lacks a functional duplication gene for episomal duplication: (ii) a chromosomically integrated chimeric, trans action duplication gene comprising a regulated, plant promoter operably linked to a coding sequence of the viral duplication protein of the geminivirus; and (ii) a dimer of the genome B of the geminivirus; wherein the expression of the trans action duplication gene in the cells containing the prorreplicon results in the duplication of the prorreplicon and the B genome, and the increased expression of the target gene.

38. A method for altering the levels of a protein encoded by a target gene in a plant comprising: (i) transforming a plant with the viral expression system of claim 37; and (ii) growing the seed of the transformed plant under conditions where the protein is expressed.

39. A transgenic geminivirus expression system, characterized in that it comprises: (i) an inactive, chromosomally integrated replicon comprising: a) viral elements of action required for viral duplication; b) an objective gene comprising at least one suitable regulatory sequence; and c) site-specific sequences responsive to a site-specific recombinase; and (ii) a chimaeric, chromosomally integrated, transactivation gene comprising a regulated, plant promoter operably linked to a site-specific recombinase coding sequence.; (iii) a dimer of a genome B of the geminivirus; wherein the expression of the chimeric transactivation gene in cells containing the inactive replicon results in site-specific recombination, replicon activation and duplication of the B genome, and increased expression of the target gene.

40. A method for altering the levels of a protein encoded by a target gene in a plant, characterized in that it comprises: (i) transforming a plant with the viral expression system of claim 39; and (ii) growing the seed of the transformed plant under conditions where the protein is expressed.

41. A method for increasing the road resistance in a plant comprising: (i) transforming a first plant with an inactive replicon to form a first primary transformant, the inactive replicon comprising: a) Viral elements of action required for viral duplication; b) viral sequences homologous to the virus of infection capable of conferring resistance dependent on homology; c) site-specific sequences responsive to a site-specific recombinase; and (ii) transforming a second plant with a chimeric transactivation gene to form a second primary transformant comprising a regulated, plant promoter operably linked to a site-specific recombinase-encoding sequence of transactivation; (iii) cultivate the first and second primary transformants where the progeny of both seeds are obtained; Y (iv) crossing the progeny of the first and second transformants where the viral sequences homologous to the virus of infection are expressed, transmitting the viral resistance to the plant.