MXPA99008951A - Method for enhancing expression of a foreign or endogenous gene product in plants - Google Patents

Abstract

The present invention provides a method for enhancing the expression of genes in plants by supplying a virally encoded booster sequence comprising the 5'proximal region of the potyvirus genome to the plant. The booster sequence enhances the expression of foreign genes or endogenous plant genes in plants by employing any known methodology of expressing introduced genes in plants. The booster sequence may be used to enhance expression of any gene, including foreign genes or endogenous plant genes, introduced by means of stable transformation into the genome of the plant or introduced by expression from a plant viral expression vector.

Description

METHOD TO INCREASE THE EXPRESSION OF A GENE OR ENDOGENOUS GENE PRODUCT IN PLANTS Field of the Invention The present invention is directed to the field of the production of plant gene products. Specifically, the invention relates to methods for increasing the expression of either foreign or endogenous genes introduced into plants.

Background of the Invention For the purposes of this description, the term "gene" or "genes" is used to mean nucleic acid sequences (including both RNA or DNA) that encode the genetic information for the synthesis of a complete RNA, a complete protein, any portions of such a complete RNA or of the complete protein. Genes that are not part of a particular plant genome are referred to as "foreign genes" and the genes that are a part of a particular plant genome are referred to as "endogenous genes". The term "gene products" refers to RNAs or proteins that are encoded by the gene. The foreign gene products are RNA or proteins encoded by foreign genes and the "endogenous gene products" are RNA proteins encoded by endogenous genes.

It has been known for some time that plants can be used to express foreign gene products or to overexpress endogenous gene products, through the introduction of the foreign or endogenous gene into the plant through the use of various biotechnological methods. In a biotechnological approach the gene that codes for the product of interest is introduced into the plant genome under the control of a promoter sequence that is functional in the plant resulting in the transcription of the gene to produce the messenger RN, followed by several events of RNA processing, the output of the RNA from the nucleus and the translation of the messenger RNA to produce the encoded protein. This approach has been exploited by agricultural and industrial interests to provide a ready and relatively cheap source of a variety of beneficial gene products.

In some cases the gene products serve s function in the plant from which they are expressed. U natural insecticide that. It confers resistance to insects and commercially available transgenic harvest plants is an example. In other cases, the gene product of interest can be extracted from the plant and serves its function to other parts. For example, potentially valuable proteins, such as antibodies, can be expressed in plants. Such production methods are seen as a marked improvement over the use of animal tissue for such production.

Several years after the first series of publications detailing the methods for introducing foreign genes or additional copies of endogenous genes in plants, an unexplained phenomenon was reported. Plants containing an additional copy of an endogenous plant gene not only failed to exhibit the hope for increased accumulation of the gene product, but also repressed the expression of the endogenous gene, effectively eliminating the expression of the endogenous gene product. . This phenomenon was mentioned as "cosuppression" since the expression of both the endogenous gene and the transgene were suppressed. The incidence of cosuppression in transformed plants containing extra copies of an endogenous gene is high. Up to 90% of independently transformed petunia plants containing an introduced chalcone synthase gene showed some variation in petal color, which is indicative of the co-suppression of this gene. It has been postulated that this same type of suppression of gene expression can occur when a particular messenger RNA sequence is expressed at high levels. This may explain the generally low levels of expression of genes introduced into plants.

Whatever the explanation, inactivation of gene expression by co-suppression is a problem in cases where high levels of expression of an introduced gene or expression of an endogenous gene are desirable. Therefore, the use of transgenic plants to express introduced genes has been limited due to this general restriction on the expression of high level gene in the plant cells.

Another approach to expressing foreign or endogenous gene products in plants is the use of plant viruses as vectors to express foreign genes in an appropriate host plant. An example of a viral vector for Expressing a foreign gene is described in U.S. Patent Nos. 5,316,931 and 5,589,367, both naming Donson and others as inventors. Both of these patents are incorporated herein in their entirety by reference. These patents provide viral nucleic acids of recombinant plant and recombinant virus that are stable for the maintenance and transcription or expression of non-native (foreign) nucleic acid sequences and which are capable of transcribing or expressing systemically the foreign sequences in the host plant. Others have also tried to use several viral base vectors to express genes that are not native to the virus. For example, Takamatsu and others described the use of tobacco mosaic virus ("TMV") as a vector to express encephaliña in the "Production of Encephaliña in Tobacco Protoplasts Using Vector RNA from Tobacco Mosaic Virus" 269 FEBS Lett. , 73-76 (1990). In 1993, Hamamoto and others described the production of an angiotensin-I converting enzyme inhibitor peptide from vector TMV RNA in "A New Vector of Mosaic Virus for Tobacco and its Use for the Systemic Production of or Angiotensin Conversion Inhibitor- I in Tobacco and Transgenic Tomato ", 11 Bio / Tecnología, 930-932 (1993). Kumagai et al. Described using a tobamovirus as a viral vector to produce an HIV-inhibitor, a-Triclosantin in "High-Level and Rapid Expression of Biologically Active a-Triclosantin in Transfected Plants Via a Viral Vector-RNA", 90 Proc. Nati Acad. Sci, USA, 427-430 (1993).

Other examples of the use of viral vectors for expressing foreign gene products by various methods are known to those skilled in the art. Generally, viral vectors of plants suitable for the expression of foreign genes must be self-replicating, capable of systemic infection in a host, and stable. In addition, they must be able to contain the nucleic acid sequences that are foreign to the native virus that forms the vector.

Although the use of plant viruses to express foreign gene products generally allows the expression of products at a higher level than that obtained from genes stably introduced into the plant genome, current methods of expressing viral vector genes suffer of several practical limitations. The virus is frequently weakened when a foreign gene is cloned into it. When a foreign gene sequence (one not foreign to the virus vector) and introduced into a virus, the virus is weakened and the weakened virus does not produce its products so easily. In addition to the weakening of viral gene expression, the virus is unable to duplicate itself and move as efficiently through the host plant as wild-type parental viruses can. In addition, viruses that carry foreign genes tend to become unstable and frequently suppress the inserted genes to duplicate the viruses. These tendencies are discussed by Dolia et al., In "Marking of Duplication of Plant Potiviruses and Movement by the Insertion of ß-Glucoronidase in the Viral Protein", Proc. Nati Acad. Sci, USA, 10208-10212 (1992) and Chapman et al., "Papa X virus as a vector for gene expression in plants, 2 El Diario de las Plantas, 549-55 (1992).

It has also been known for some time that in plants infected with more than one virus at the same time two coinfection viruses can interact synergistically to cause a more severe disease in the plant than any virus alone. In many cases it has been shown that the increase in severity of the host's symptoms correlates with an increase in the accumulation of a synergistic pa virus. For example, it is known that a synergistic disease is caused by the interaction of potato virus ("PVX") and potato Y virus ("PVY"). The PVX in such diseased plant synergistically accumulates at a higher level than in the individually infected plants and eventually causes the systemically infected leaves to die first of the double infected plant. Infection with either PVX PVY alone in the same plant has little or no effect at all.

These synergistic effects have also been shown as a result of the PVX interaction with at least three other members of the mototo group of mottled tobacco vein ("TVMV"), tobacco-tainted virus ("TEV") virus. mottled pepper ("PepMoV"). Such mixed PVX / potyvirus infections of a tabac host plant result in a dramatic increase in PVX particle accumulation (up to tenfold) without a corresponding increase or decrease in the accumulation of the potyvirus particles. These mixed PVX / potiviral infections also result in a dramatic increase in disease symptoms in the double infected plant.

The initial step in the present discovery of the viral enhancer sequence was the finding that the syndrom of potiviral synergistic disease / PVX, characterized by increases in the severity of the symptom in the accumulation of pathogenic PV does not require infection with both viruses. This was reported by Vanee et al. In "Potiviral Synergistic Disease / Potato X Virus Mediated Potiviral Sequences 5 'Ceranas in Transgenic Tobacco", Virology 206, 583-590 (1995) The synergistic disease is mimicked in plants expressing sol a subgame of the Genomic potiviral RNA and infected singularly with PVX. The potiviral region was shown to mediate the synergistic disease comprising the 5'-proximal 2780 nucleotides of genomic RNA, including the 5'-untranslated region (5'-UTR) and the region encoding the potiviral gene products Pl, and the component- proteinase (HC-Pro) and a part of P3 This described potiviral region is referred to herein as "Pl / HC-Pro sequence".

Therefore, Vanee et al. (1995) identified a determinant disease carried by the potyvirus genome (Pl / HC-Pro sequence), and this determinant of disease was shown to mediate the well-known synergistic PVX / potivira enferemedad. Even though the mechanism by which this medium potiviral sequence is potiviral synergistic / PVX disease was unknown, it is speculated that it involves a direct and specific interaction of the RNA sequence Pl / HC-Pro the potiviral gene products encoded with the genomic RNA. the duplicating proteins of the interacting PVX pathogen Even though the Pl / HC-Pro potiviral sequence was found to increase the accumulation of the PVX viral structural gene (coat protein) and the PVX viral particle accumulation, this increased accumulation was thought to be it was specific for the native PVX genes expressed from the native PVX genome. In addition, the increased accumulation of PV coating protein and PVX virus particles was closely correlated with the undesirable detrimental increase perceived in the symptoms of the disease.

Even though PVX and other viruses, such as cauliflower mosaic, geminiviruses and TMV, have been used as viral vectors to express foreign gene products, such vectors have not been completely successful as mentioned above. Even though the gene products have been produced through the expression of viral vectors, the utility of such vectors is limited by the instability of the inserted sequences and the failure of the viral vector to duplicate it efficiently. The expression of the gene products of the genes stably introduced into the plant genome also suffers from the limitations as mentioned above.

Therefore, it would be beneficial if the methods of preparing plant gene products could be developed to allow the increased expression of the ge introduced from the stably transformed plants and the increased expression of genes introduced into a plant through a viral vector of plants. , while s retains the stability of the introduced sequence to ensure the accumulation of the gene product. The present invention learned about some of the deficiencies in the above gene product expression methods by using a particular increment sequence obtained from a potyvirus.

Synthesis of the Invention It is an object of the present invention to provide methods for increasing the expression of gene product in plants.

Another object of the present invention is to provide methods for increasing the expression of endogenous and foreign gene products introduced into plants.

A further object of the present invention is to provide processes using a particular improved sequence to increase the expression of a product of g introduced into a plant.

These and other objects are achieved by providing a method for increasing the expression of the gene in plant by supplying a virally encoded improved sequence comprising the 5 'region near the genome of potyvirus, which may include the region encoded for the component. -proteinase assistant (HC-Pro), and a small part of P3. Some part of the sequence may be expressed either individually or fused to other sequences a modified version of the sequence enhancer to a related sequence of another virus or any part of the modified version or the related sequence already expressed individually or merged to another sequence. The sequence improves the expression of foreign genes or endogenous plant genes that are introduced to the plant through the use of any methodology. An example of such known methods includes the expression of the gene (s) introduced from one or more copies of a gene stably incorporated into the plant genome. Another example of such known methods is the expression of genes introduced through viral plant expression vectors.

The process of increasing the expression of ge can be carried out in several ways. For example, the enhancer sequence can be provided to the plant in a variety of ways, this can be provided by infection with a virus that expresses the enhancer sequence as a native viral gene product during its natural vine cycle. Alternatively, the enhancer sequence can be introduced through the use of a transgenic host plant that expresses the enhancer sequence as an introduced gene. The enhancer sequence can also be introduced using the same viral expression vector used to express the endogenous or foreign introduced gene of interest. A transient expression system can be used to temporarily express the enhancer sequence or a co-infection system of two components can be used where the two viruses are required for the successful increase of gene expression. In the two-component co-infection system, one virus expresses the incrementing sequence while the other virus expresses the inserted ge of interest.

In addition, the enhancer sequence can be used to increase the expression of any introduced gene including foreign genes or endogenous plant genes. The genes that are to be increased must be introduced into the plant in a variety of ways. Foreign or endogenous gene can be introduced by means of a stable transformation in the genome of the plant employing any of the known technologies used for this process. Alternatively, the foreign or endogenous gene may be introduced using any viral expression vector of the plant.

More specifically, the present invention involves a method of expressing a foreign gene or an endogenous plant gene that has been introduced into a plant material, which includes plant cells, plant protoplasts, or whole plants, wherein the enhancement comprises supplying an enhancer sequence comprising a portion of the proximal 5 'region of the genome of a protivirus and plant material so that the expression of said foreign gene or of the endogenous plant gene is increased. The plant gene may be a foreign gene that does not occur naturally in plant matter before being introduced there, or an endogenous plant gene that was occurring naturally in the plant material before being introduced as an additional copy or additional copy of the endogenous gene. The 5 'supplied region can comprise the coding region for Pl a proteinase auxiliary component (HC-Pro) and a small part of P3 and the part of the 5' close region can be expressed independently or can be fused to other sequences.

The foreign or endogenous gene can be introduced into the plant through a viral expression vector with the increasing sequence being supplied by the expression from the same viral vector; it can be introduced through a viral expression vector with the enhancer sequence provided by the expression of one or more DNA copies of the enhancer sequence stably incorporated in the plant genom; or introduced via a viral expression vector with the enhancer sequence being expressed from a transient expression system containing one or more DNA copies of said enhancer sequence. A two component viral system can be used with a viral component expressing the enhancer sequence and the other viral component expressing the ge introduced. The introduced gene may be a foreign gene or an endogenous plant gene introduced through a viral expression vector with the enhancer sequence being supplied by co-infection with a potyvirus expressing the native incrementing sequence encoded by that potyvirus; introduced through a viral expression vector with the enhancer sequence being supplied by coinfection with a potyvirus expressing a non-native version of said enhancer sequence; or introduced through a viral expression vector having the gene fused to the structural ge of said viral expression vector.

In addition, the viral gene or the endogenous plant gene can be introduced into the plant genome through any stable transformation mode of one or more copies of the introduced DNA, with the enhancer sequence being supplied before, during or after the introducing the foreign gene of the endogenous plant gene through stable transformation procedures so that it increases either the expression of the introduced gene product or the number of the transformant plants that express the introduced gene product. In this aspect, the enhancer sequence can be delivered through the expression of one more DNA copies of the incrementing sequence stably incorporated into the plant genome before, during after the transformation of the plant material with the introduced gene product. .

Brief Description of the Figures A complete and enabling description of the present invention, including the best mode thereof for one with ordinary skill in the art is set forth herein particularly in the remainder of the description including reference to the accompanying Figures in which: Figure 1 shows the increased pathogenesis of CMV and TMV in transgenic tobacco expressing the potiviral sequence Pl / HC-Pro.

Figures 1 (A) and 1 (C) show control tobacco plants which do not express the enhancer sequence and are infected, either with CMV (1 (A)) or TM (KO).

Figures 1 (B) and 1 (D) show the TEV-transformed line U-6 plants expressing the near 5 'region of the TEV genome and infected with either CMV (1 (B)) or TMV (1 (D) ).

Figure 2 is a diagram of the Pl / HC-Pr region of the TEV genome showing the location of the mutation insert within the Pl and HC-Pro coding regions, with the locations of the three resulting amino acid insertions within the mutant protein and the capacities of the transgene mutants to induce a synergistic disease are indicated below the diagram and the suppressed HC Pro coding sequence of the TEV-2del being indicated by dotting.

Figure 3 is a diagram of the PV vectors used to express TEV sequences.

Figure 3 (A) is a diagram of the PVX viral genomic RNAs carrying the nearby 5 'TEV sequences with PVX-5'TEV having nucleotides 146 to 2674 of TE encoding Pl, HC-Pro and part of P3, the PVX- HC carrying the region encoding the HC-Pro to the appropriate start and stop codons of translation, and the PVX-noHC being the same as PVX-HC except that the initial AUG has been changed to ACG.

Figure 3 (B) shows a protein gel blot showing the level of HC-Pro in infected N. benthamian leaves (line 1) or in leaves systematically infected with either PVX-5 'TEV (line 2), PVX -HC (line 3), PVX-noHC (line 4) or TEV (line 5).

Figure 3 (C) shows a Northern blot of RA isolated from leaves as given for the protein gel blot of Figure 3 (B) and hybridized with a radioactive probe specific for the TEV HC-Pro sequence.

Figure 4 shows the kinetics of RNA accumulation known as PVX (-) altered by the expression of the TEV Pl / HC-Pro sequence, so that the level of RNA accumulation is increased and also showing blot d gel analysis RNA of (+) and of (-) viral know ANRs of tabac protoplasts infected with PVX-5 'TEV (upper row), PVX-HC (medium hiler) or PVX-noHC (lower row) at 24 hours (line 1) ), 4 hours (line 2) or 72 hours (line 3) after inoculation where the left panel is the viral ANR of sepa (-) and right panel is the viral RNA of sepa (+). In this Figure, total RNA has been isolated from the protoplasts, fractionated by denaturing electrophoresis of agarose gel, transferred to the nylon membrane, and hybridized with specific radioactive probes for either PVX (+ right panel) or the sepa (-) RNA (left panel) and the blot regions containing genomic length ANRs are shown.

Figure 5 shows the relative levels of RNA d know PVX (-) at several moments after the infection of the protoplasts with PVX-5'TEV, PVX-HC, or PVX-noHC, with the values of 48 and 72 hours being normalized to the time point of 24 hours and each point of time being the average of 2 independent experiments.

Figure 6 shows the time course of the luciferase activity in protoplasts infected with the PVX reporter virus, with the PVX reporter viruses expressing the TEV Pl / HC-Pro sequence (5 'TEV) or a mutant version of the sequence [5]. 'TEV (K)] that encodes an altered HC-Pro that fails is to sustain synergism.

Detailed Description of the Preferred Incorporation Other objects, features and aspects of the present invention are discussed in more detail below. It will be understood by one of ordinary skill in the art that the present discussion is of a description of the exemplary embodiments only and that they are not intended as limiting the broader aspects of the present invention, with the broader aspects being involved. in the example construction.

The present invention employs a virally encoded enhancer sequence comprising the 5 'close region of the potyvirus genome, or some part or the encoded form thereof, supplied to a method for expressing a gene product in a plant. The enhancer sequence increases the expression of the gene product and allows the product to accumulate within the plant.

Contrary to the above beliefs, the present invention has revealed that the Pl / HC-Pro potiviral sequence is not merely the specific disease determinant for the PVX-potiviral synergism. Instead, the Pl / HC-Pro potiviral sequence functions in a general form increase the accumulation and pathogenesis of a wide range of plant viruses.

The present invention has revealed that the Pl / HC-Pro sequence not only increases the expression of a viral gene native to its native viral genome, as shown by Vanee et al. (1995) for the expression of a PVX coating protein, but which also increases the expression of foreign genes contained in a viral plant expression vector.

The present invention has also revealed that the enhancing action of the Pl / HC-Pro sequence can be separated from its detrimental effects on the plant. Experiments using genetically modified or fused versions of the Pl / HC-Pro potiviral sequence have identified regions that are required for the desirable enhancing activity of the sequence and the regions that are required for the detrimental disease-promoting action of the sequence.

Even when the two identified regions overlap, it is possible to modify the sequence so that the increasing action occurs without causing the symptoms of detrimental disease. Therefore, the invention provides a method for increasing the expression of any foreign or endogenous gene of a viral expression vector, by using the increasing action of the sequence in the absence of the detrimental effects on the host plant when such symptoms of disease consider undesirable for the intended purpose.

The present invention has further revealed that the effects of the Pl / HC-Pro potiviral sequence affect the expression of genes that have been introduced to the plant through the stable incorporation into the plant genome. Thus, the incremental sequence can reverse or inhibit the effects of the natural plant cosuppression system and also increase the expression of endogenous foreign plant genes introduced by stable incorporation into the plant genome.

Examples 1-4 To determine whether the Pl / HC.Pro sequence is involved in synergistic diseases other than PVX / potiviral synergism, two other plant viruses, tobacco mosaic virus ("TMV") and cucumber mosaic virus ("CMV"). ), were employees. Both TMV and CMV have the ability to infect tobacco and it is known to interact synergistically with a potivirus.

The transgenic tobacco plants expressing the Pl / HC-Pro sequence [transgenic line U-6B, described by Carrington, J.C., Freed, D.D. and Oh, CS, "Expression of Potyral Polyiproteins in Transgenic Plants Reveals the Required Proteolytic Activities for Full Processing", 9 EMBO J., 1347-1353 (1990) and Vanee, VB, Berger, PH, Carrington, JC, Hunt, AG and Shi, XM, "Potentiary Near Mediaeval 5 'Sequences to Potato X Virus / Potiviral Synergistic Disease in Transgenic Tobacco" 206, Virology, 583-590 (1995)] and tobacco control plants of the same line that do not expressed this sequence were inoculated with TMV with CMV. The development of the symptom was monitored. After infection with either TMV or CMV, the transgenic line U-6 developed symptoms which were dramatically different and much more severe than those in the control plants as shown in Figure 1. Even when both TMV and CMV induced Relatively mild symptoms in contro tobacco plants (Figure 1A and 1C, respectively) both viruses were lethal in the transgenic line U-6B, resulting in the death of the plant after several weeks (Figures IB and ID, respectively).

As in the previously known PVX / potiviral interaction, the accumulation level of both the TMV and the CMV pathogens was increased by the presence of the Pl / HC-Pro potiviral sequence. This result supports the conclusion that the Pl / HC-Pro potiviral sequence acts in a general manner, improving the pathogenesis and accumulation of a wide range of viruses.

The Pl / HC-Pro sequence alters the host response to heterologous plant viruses from at least three different groups (PVX, potexvirus, TMV, tobamovirus and CMV, cucomovirus). The fact that the expression of this potiviral sequence alters the disease process for each of these unrelated heterologous viruses indicates that the sequence affects a step in the infection process that is common for all these viruses. Each of these viruses is capable of interacting in a mixed infection with a member of the potivirus group of plant viruses to induce synergistic diseases which occur in a diverse evolutionary range of the host plants and involve interactions with a large number of host groups. viral plant. The Pl / HC-Pro potiviral sequence mediates the increased pathogenesis and accumulation of the virus in each of these synergies. Thus, the use of the presently described enhancer sequence, (which is described herein to comprise the Pl / HC-Pro potiviral sequence or any modified part or version of that sequence) is applicable to a broad range of viral groups of host plants.

Because the expression of the Pl / HC Pro sequence affects a wide range of heterologous viruses, it was deduced that the mechanism for these effects may involve an indirect interaction through a host factor common to all tobacco infections rather than a direct interaction with three different viral gene or RNA products. Two different indirect mechanisms can explain the transactivation of viral duplication by Pl / HC-Pro. The TEV sequence can increase the activity or availability of a positive regulator of viral duplication that affects both the TEV and the heterologous viru. The stimulation of a virus by a host factor induced by another virus has been shown in infections mixed with human cytomegalovirus (HCMV) and human immunodeficiency virus-1 (HIV-1), where human cytomegalovirus can induce the expression of the virus. NF-Kappa host transcription factor d, which then activates the doubling of the human immunodeficiency virus- (Chinnandurai, "A Modification of the Increasing Activity of the Human Immunodeficiency Virus by Agent Heterologists: A Minirevisión "101 Gene, 165-170 (1991); Kim others," Essential Role of NF-kappa B in the Transactivation of the Long Terminal Repetition of Human Immunodeficiency Virus by Human Cytomegalovirus 1E1 Protein "77 J. Gen Virol., 83-91 (1996) Alternatively, the Pl / HC-Pr sequence may interfere with the activity or availability of a negative regulator of viral duplication, perhaps part of a host defense system that normally eliminates viral accumulation. Because HC-Pro increases the accumulation in a wide range of viruses and acts at the level of viral reduplication, the putative host defense system will necessarily be general in nature and will act at the single cell level. The requirement has been proposed as the underlying mode of action in RNA resistance mediated by RNA in transgenic plants.

(Baulcombe, "Mechanisms of Resistance Derived from Pathogen Virus in Transgenic Plants "8 Plant Cell, 1833-1844 (nineteen ninety six); Lindbo et al., "Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for the Regulation of Gene Expression and Resistance to the Virus" 5 Plant Cell, 1749-1759 (1993); Muller et al., "Resistenci Dependent of Homology: Resistance of Transgenic Virus and Plants Related to Silencing of Homologically Dependent Gene" 7 Plant Cell, 1001-1013 (1995); Smith, "Resistance of Transgenic Plant Virus Mediated through Non-Transferrable Perception RNAs: Expression, Regulation and Destination of Non-Essential RNAs" 6 Plant Cell, 5266-5271 (1994). In this model, a target system is activated by the high-level expression of a transgene that contains viral sequences. Once activated, the system rapidly destroys the specific viral RNA target, whether the RNA is expressed from the transgene or from a temperate viral RNA during viral duplication (English and other "Suppression of Virus Accumulation in Transgenic Plants Exhibiting the Silencing of Nuclear Genes ", 8 Cell d Plant 179-188 (1996), Goodwin et al.," Biochemical Genetic Dissection of Virus Resistance Mediated Transgenic RNA "8 Plant Cell, 95-105 (1996), Mueller et al. 1995). The cellular system itself is also thought to be involved in the silencing of the post-transcriptional gene (cosuppression) of non-viral transgenes in plants (Baulcombe (1996); d Carvalho Niebel et al., "Post-Transcriptional Cosuppression of ß 1,3-Glucanase no affects the Accumulation of Nuclear Transgen RNA, "7 Plant Cell, 347-358 (1995); Ingelbrecht others," Post-transcriptional Silencing of Transgenes d Reporter in Tobacco is Correlac ione with DNA Methylation ", 9 Proc. Nati Acad. Sci. USA, 10502-10506 (1994). The experimental data below indicate that Pl / HC-Pro increases the expression of genes introduced into plants by interfering with the induction of this cosuppression path and thereby allowing the messenger RNA of introduced genes to accumulate at high levels, resulting in a increased accumulation of encoded gene product.

As discussed above, the potiviral enhancer sequence has been identified as the Pl / HC-Pro sequence. This region is initially expressed as a polyprotein and subsequently processed by the proteolytic activities of both Pl and HC-Pro to produce the mature viral proteins. Both HC-Pro and Pl are multifunctional proteins. Pl has proteinase activity that unbinds the potiviral protein, creating the carboxy-terminus of PL and amino-terminus of HC-Pro [Verchot et al., "The Protein 35-kDa d N-Term of the Potiviral Polyprotein functions as a third proteinase coding-virus "185 Virology, 527-535 (1999). P also functions in trans as an accessory factor for l genome duplication (Verchot and Carrington," Evidence that Potivirus Pl Proteinase functions as an Accessory Factor for l Amplification of Genome "69 J. Virol 3668-3674 (1995) and has RNA binding activity (Brantley and Hunt," The N-terminal Protein of the Polyprotein encoded by the Potivirus Tobacco Manchad Virus is an RNA binding protein ", 74 J. Gen. Virol., 1157-1162 (1993)).

The HC-Pro has at least three functional domains: an amino-terminal domain required for afid transmission, a central domain involved in pathogenicity, RNA duplication and leaf-to-leaf movement of viruses through foem, and a carboxy domain. -terminal required for the autoproteolytic processing of the HC-Pro carboxy-terminus "Maia and others," Potiviral HC-Pro: A Multifunctional Protein ", 77 J. Gen. Virol. 1335-1341 (1996).) The central domain of HC Pro is of particular interest because it is involved in the regulation of both the pathogenicity and the RNA duplication of potyvirus and these are the characteristics that are altered in the heterologous virus during synergism.

EXAMPLES 5-7 In this example, the effect of mutations in this potiviral sequence on the induction of synergistic disease was examined. Three lines of transgenic tobacco expressing the TEV Pl / HC-Pro sequence with mutations within the Pl coding region were not impaired in their ability to mediate synergism when infected with PVX. In contrast, two of three transgenic lines with mutations in the HC-Pro coding region were unable to induce synergistic increases in either a symptom severity or PVX accumulation. The loss of increment function was associated with mutations within the region encoding the central domain of HC-Pro, while the ability to induce synergism was retained in a transgenic line expressing HC Pro with an alteration in the "finger domain". -zinc "amino terminal. The location of the various mutations and their effect on synergism are shown in Figure 2. In addition, 16 of 2 alanine scanning mutations affecting residue clusters loaded within the TEV HC-Pr coding sequence retained the ability to induce synergism . In addition, in co-inoculation experiments, a TEV mutant (TEV-del2 lacking the sequence coding for the zinc-finger domains of HC-Pro (Figure 2) induced a typical synergistic response and interaction with PVX.The deleted version of the sequence HC-Pr carried by (TEV-del2) was also merged with the partial sequencing of the GUS gene.

These results indicate that the TE Pl / HC-Pro sequence encoding the central domain of HC-Pro is required for both the increased sequence capacity and the increased pathogenicity conferred by the sequence. These results also indicate that the zinc-ded domain comprising the first 66 amino acid receipts of HC-Pr are indispensable for both the function of increase and detrimental increase in disease symptoms. Furthermore, the results indicate that many regions of the Pl / HC Pro sequence can be modified without affecting either the ability to increase or pathogenicity. The enhancer sequence can be fully functional as a function with another sequence (such as the GUS sequence as in the TEV-del2 mutant).

Finally, the results of this example show that the enhancer sequence is active for the expression of the enhancer gene in a variety of modified forms or as a fusion of protein with other proteins.

The present invention furthermore involves and increases the expression of a gene product in a plant through the use of a claimed incrementing sequence. Given that the increment sequence has been shown to contain several mutations and / or deletions fused to another sequence, it is functional for this purpose, the claimed incremental sequence includes the Pl / HC-Pro sequence of any potiviru or closely related groups of viruses, including the non-afid-born potyvir type viru or any modified form of this sequence including forms that have been mutated deleted or fused to other sequences.

The following examples describe a case example where the domains of the Pl / HC-Pr sequence required for pathogenicity were identified using deletions from the region.

EXAMPLES 8-10 The Pl / HC-Pro region of the expressed TEV genome in transgenic plants U-6B consists of 2670 nucleotides that include the 5'-UTR and the coding region for the mature viral proteins Pl and HC-Pro, as well as a part of P3 . The proteins are initially expressed as a polyprotein, then the autoproteolytic Pl and HC-Pr activities are processed by both. In order to determine if any or only a part of this sequence is required for synergism, the ability of the PVX to be used as a vector to express foreign gene was used to create the PVX / TEV synergism in a system in which the PVX itself expresses the Pl / HC-Pro sequence. This system was then used to determine the minimum TE sequence required for the induction of synergism.

Three different PVX vectors were constructed by cloning several TEV sequences in a modified version (Sriskanda, VS, Pruss, G., Ge, X., and Vence, VB, "A Sequence of Eight Nucleotides in the Potato Virus X 3 '- UTR s Requires both the Host Protein Agglutination and Viral Multiplication "70 J.Virol., 5266-5271 (1996)) of the PVX cDNA clone pTXS infections [Kavanaugh et al.," Molecular Analysis of a Resistance Breaking Strain of Virus of Papa X, "189 Virology, 609-617 (1992)]. These are shown schematically in Figure 3A. In these constructions the expression of the TEV insert is under the control of the authentic PVX coat protein subgenomic promoter, and the expression of the coat protein is under the control of a subgenomic repeating protein subgenomic promoter. The vector PVX-5 'TEV carries the coding region of the Pl / HC-Pro sequence of TEV (nucleotides 146-2674). The PVX-HC carries only the region encoding HC-Pro (nucleotides 1057-2433), with a start codon followed by GCC added at the 5 'end and a stop codon at the 3' end of the insert so that the protein Mature HC Pro (with two additional amino terminal residues) does without proteolytic processing. PVX-noHC carries the same TEV sequences as PVX-HC except that the translation start site was mutated from AUG to ACG.

These three designed viruses were used to infect Nicotiana benthamiana plants in order to test their ability to cause increased pathogenicity. The Western analysis indicated that the HC-Pro accumulated in the leaves was systematically infected by either PVX-5'TEV or PVX-HC (Figure 3B, lines 2 and 3, respectively), but, as expected, was not detectable in leaves infected with PVX-noHC (figure 3B, line 4). Even though the PVX-noHC did not express the HC-Pro gene at the protein level, it did not suppress the sequence of the genomic RNA as shown by the Nothern RNA analysis of systematically infected leaves. A single genomic RNA was detected using specific hybridization probes either for the TE sequence HC-Pro (figure 3C, line 4) or for PV strain genomic RNA (+) data not shown). Plants infected with PVX-noH exhibited mild symptoms. In contrast, the infection of plants with either viruses expressing the HC-Pr gene product (PVX-5 'TEV or PVX-HC) initially caused visual clarity followed by necrosis of leaves systematically affected the day. after inoculation and usually killed the plant. Together, these results indicate that the expression of the HC-Pro gene product but not the RNA sequence itself is sufficient to induce the increase in PVX pathogenicity. In addition, both Pl and P3 coding sequences are dispensable for the detrimental increase in disease symptoms induced by the Pl / HC-Pro sequence.

EXAMPLES 11-13 To determine if the potiviral sequences required for increased PVX pathogenicity were also required for increased sequence characteristics, the PVX vectors described above were used to examine the effect of TEV Pl / HC-Pro expression on the accumulation syn Strain RNA PVX (+) and (-) in tobacco protoplasts. The protoplasts were inoculated by mediant electroporation with transcripts of the three designed PV cDNAs (Figure 3A, PVX-5'TEV, PVX-HC and PVX-noHC), and the accumulation of (+) and (-) RNA strain was tested by analogy. Northern at various times after inoculation. The syn- thetic accumulation of RNA (-) strain in protoplast infected with PVX-noHC were similar to those previ- ously mentioned for the parent PVX strain (Srinskanda et al., 1996) with the RNA level of (-) strain reaching 24 hours after inoculation and then declining to approximately 0.2 times peak level over the next 48 hours as shown by Northern blot in Figure 4 (left side, bottom row) graphically in Figure 5. In protoplasts infected with PVX- HC, the level of RNA strain (-) declined only slightly during the same period at a level of approximately 0.8 times the peak 24 hours (Figure 4, left middle row, Figure 5), the most important result, however, was obtained and protoplasts infected with PVX-5'TEV. In this case, the level d RNA strain PVX (-) increased over the period of 72 full hours (figure 4, upper row left side) and at 72 hours after inoculation, the level of RNA strain (-) was 3.6 higher at the 24-hour level (figure 5). The accumulation of AR strain (+) in protoplasts infected with PVX-5'TEV and PVX-HC was also prolonged compared to that in cells infected with PVX-noHC (figure 4, right-hand side, compare mean upper row with lower row ). However, as also true in infections mixed with TEV. And PVX and in the transgenic plant system (Vanee et al., 1995), the effect of the TEV sequence on RNA accumulation of strain (+) was dramatically less than the effect on RNA accumulation of strain (-). The main effect of the TEV expression Pl / HC-Pro is to prolong the accumulation of the PVX (-) strain RNA.

Therefore, unlike the detrimental synergistic increment of the PVX pathogenicity, which requires only expression of HC-Pro, the potentiating effect of the potentiation sequence requires the complete Pl / HC-Pro sequence.

EXAMPLES 14-16 Additional experiments were carried out to define the Pl / HC-Pro regions required for the function of pathogenicity and increase. The specific regions of the TEV Pl / HC-Pro sequence were mutated and tested for their ability to elicit severe disease symptoms when expressed from vecotr PVX by infecting N. benthamiana plants described above and by their ability to increase viral application. and the accumulation using kinetic analysis of (+) and (-) strain RNAs in tobacco protoplasts. At least one mutation within the region coding for the carboxylmethyl HC-Pro increased the duplication and accumulation of the PVX RNA per failed to induce increased symptoms in tobacco plants. This mutation was a point mutation which resulted in a single amino acid change within the active site of the HC-Pro proteinase domain.

These results indicate that the incrementing sequence can be modified so that its beneficial increase characteristics are exploited in the absence of detrimental influence on the symptoms of plant disease.

The introduction of the claimed enhancer sequence into the presence of the system containing a foreign gene an endogenous plant gene can be achieved by means of several different known methods. For example, the incrementing sequence can be introduced into the vector itself and then the viral vector carrying the incrementing sequence introduced into the host plant. Alternatively, a transgenic host plant can be used to express the enhancer sequence. In this method, the transgenic host plant that produces the incrementing sequence is infected with the viral vector. Another method for introduction is known as "co-infection", which involves combining in the host plant a virus that is producing the enhancer sequence and a virus that is producing the foreign gene or the endogenous gene of interest. In addition, a two-component co-infection system can be employed. In this process, two defective viruses are co-inoculated in the host plant. One virus will eventually express the incrementing sequence and the other will express the endogenous alien gene of interest. Until these viruses are combined, no viable virus doubles. However, in the combination the viruses create the synergistic increase in the expression of ge mentioned here. Finally, a transient expression system can be used. In this process, a plasmid and introduced into the plant cell. The plasmid duplicates and expresses the enhancer sequence in a transient fashion but is not stably incorporated into the host plant genome as with the transgenic plants described herein. From a broad standpoint, the present invention is not limited to any type of method of introduction d increaser.

An exemplary expression method employing a gene product introduced through a viral vector in which the same viral vector provides the enhancer sequence will now be described. The techniques that have been developed to construct the viral vectors containing nucleic acid sequences of endogenous or foreign plant are documented. Such methods include those described in U.S. Patent No. 5,589,367 which has already been incorporated herein by reference, as well as U.S. Patent No. 4,855,237 which is also incorporated herein by reference. reference. Any of the methods described in those patents or various other methods of constructing viral vectors with foreign genes in this will satisfy the requirements for use in the method claimed herein using viral expression vectors to express the foreign gene products.

EXAMPLES 17-18 The following are examples of the use of a viral expression vector with the present incrementing sequence to achieve an increased expression of the foreign gene products. Two virale PVX vectors were constructed. In these vectors, the luciferase gene was the foreign gene of interest and was cloned into the PVX vector to replace the PVX coat protein gene. Luciferase was expressed from the subgenomic promoter of coating protein. The TEV enhancer sequence was also expressed from the same PVX vector. In one vector, a functional enhancer sequence was expressed together with luciferase (PVX-5 'TEVluc) In the other PVX vector, a non-functional mutated version of the enhancer sequence was expressed together with the lucifera [PVX-5'TEV (K ) luc. The K mutation eliminated the increasing capacity of the increment sequence.

To test whether the expression of the foreign gene product has been increased or prolonged in the presence of the functional enhancer sequence, the protoplasts were inoculated with transcripts of the two viruses, PVX-5'TEVluc PVX-5'TEV (K) luc, and luciferase activity was tested at different times during post-inoculation.

In the protoplasts infected with PVX 5'TEV (K) luc, the luciferase activity increased exponentially for approximately 20 hours after the inoculation and then began to level off (FIG. 6). In contrast, the luciferase activity in the protoplasts infected with PVX-5'TEVluc continued to increase until approximately 9 hours after inoculation, reaching a peak level of more than 100 times higher than that in cells infected with PVX 5 'EV (K ) luc. These results indicate that the ability of the Pl / HC-Pro enhancer sequence to increase the expression of a foreign gene in plant cells.

The ability of the enhancer sequence to increase the expression of the foreign gene luciferase was tested on tobacco protoplasts rather than on whole plants because the particular PVX vector lacked the coating protein, and therefore, could not move inside the plant. However, this particular game of circumstance should not be considered as limiting, but rather as an example demonstrating the ability of the enhancer sequence to increase the expression of a foreign gene product in a plant cell when the gene is introduced on a vector of viruses Obviously this would apply to whole plants as well as to the protoplasts tested.

More specifically, the present example involves the use of an infectious cDNA clone of the UK-3 strain of PV obtained from Dr. David Baulcombe of the Sainsbury Institute and Norwich, United Kingdom. This clone served as the basis for the production of infectious PVX cDNA viral vectors carrying foreign gene products. The PVX PVX-5'TEVluc reporter virus was constructed using PCR to amplify the luciferase coding region of pTXS-luc, a PVX vector described in Sriskanda others, (1996). The amplified fragment comprising the coding region of luciferase was digested with Sal I and Xho I cloned into the Xho I site of the PVX vector carrying the incrementing sequence under the control of the subgenomic motor protein coat and lacking the majority of the protein coding sequence. of PVX coating. The "K" mutation was introduced by replacing a fragment of internal Spe within the HC-Pro coding region of pTXSd'TEVlu with the corresponding fragment of TEV cDNA containing a nine nucleotide insert that introduced a Neo I site and resulted in the insertion of the triplet amino acid Thr-Met-Al immediately after amino acid 426 of the TE polyprotein expressed.

The capped transcripts of PVX-5'TEVluc and PVX-5'TEV (K) were synthesized from two infectious PVX cDNAs described above using the Promeg Ribomax T7 transcription kit with the reduced rGTP concentration at 3.75 mM and the addition of 3.75 M. of analogous CAP distributed by New Englan Biolabs. Protoplasts were prepared in the NT-1 suspension culture cells in the logarithmic phase of culture and were cultured with viral transcripts exactly as s previously described in Sriskanda et al. (1996). The luciferase activity of the protoplasts infected with either PVX-5'TEVlu or PVX-5'TEV (K) luc was assayed as previously described by Sriskanda et al. (1996), using the Promega luciferase assay kit.

EXAMPLE 19 In this example, an exemplary expression method is described wherein the enhancer sequence delivered through the expression from a stably transformed host plant and a foreign gene is introduced through a vector vector. The infectious TMV clones carrying the fluorescent protein Green (GFP) were derived by cloning the GFP coding sequence into an Xho I site of an infectious cDNA TM called p30B, a derivative of an infectious pTB cDNA previously described. (Donson et al., "Systemic Expression of a Bacterial Gene Using a Viru Base Vector of Tobacco Mosaic", 88 Proc. Nati. Acad. Sci. USA, 7204-720 (1991)). The inserted GFP gene is expressed under TMV coat protein promoter control and is located just upstream of the Odontoglossum ring spot virus coat protein (ORSV) which replaces the TMV coating protein in this infectious cDNA and it is expressed from its own subgenomic promoter. The TMV vector was used to introduce the GFP gene into two classes of tobacco plants, a line of transgenic tobacco U-6B and tobacco plants of transformed vector-only transformed. The U-6B transgenic plants and the single-vector control plants were transgenic lines and Nicotiana tabacum cv Havana 425 and have been previously described e Carrington et al. (1990) and VAnce et al. (1995). The U-6B plants express the TEV enhancer sequence of a single AD copy stably incorporated in the tobacco genome.

The TMV-GFP vector was able to infect control tobacco plants and express GFP in the inoculated leaf. The viral vector can be moved systemically to the higher inoculated leaves as evidenced by the symptoms of virus infection in the form of mottle on the upper leaves. However, the level of GFP production in infected upper leaves was very low. In contrast, the TMV-GFP viral vector infection of the U-6B plants resulted in a high level of GFP expression in both the inoculated and superior non-inoculated leaves. Therefore, the enhancer sequence improved the expression of the foreign GFP gene when it was introduced through a viral vector. The increased mode of expression can be an increased duplication of the viral vector allowing the production of more viral RNAs expressing the introduced gene, improving the stability of the GFP gene inserted in the viral vector, the increased movement of the viral vector carrying the GFP gene to higher parts of the plant, or some combination of these factors.

EXAMPLE 20 The present example establishes an exemplary method for increasing the expression of the endogenous plant gene or of a foreign gene (or a part of a foreign or endogenous gene) that has been introduced into a plant as a function for a viral protein expressed from a viral vector. A viral vector expressing a foreign gene or an endogenous plant sequence as a function for the virus coat protein, such as the vector described in Sugiyama, Hamamoto, TAkemoto, Watanabe, Okada, "Systematic Production of Surface-Isolated Peptides d Particle of the Tobacco Mosaic Virus ", 359 FEBS Lett. , 247 250 (1995) is one such example. The viral vector can be used to infect a host of a transgenic plant which supplies the enhancer sequence through the expression from stably incorporated DNA copies of said incrementing sequence, for example the U 6B transgenic tobacco plants described herein. The expression of the foreign peptides fused to the viral coat protein can be improved.

Other gene product production vectors which could be applied to the sequence of increment currently described include those described by Hamamoto, Sugiyama, Nakagawa, Hashida, Matsunaga, Takemoto, Watanabe, Okada, "A Vector of New Tobacco Mosaic Virus and its Us for the Systematic Production of an Enzyme Inhibitor d Conversion of Angiotensin-I-In Transgenic Tobacco and Tomato ", 11 Bio / Technology 930-932 (1993); Takamatsu, Watanabe Yanagi, Meshi, Shiba, Okada, "Production of Encephalin and Tobacco Protoplasts Using the RNA Vector from the Tobacco Mosaic Virus", 269 FEBS Lett. 73-76 (1990).

EXAMPLES 21-26 It has been determined that the Pl / HC-Pr potiviral sequence interferes with the induction of a cellular inducing system involved in silencing the post-transcription gene (the phenomenon of co-suppression described above) d transgenes in higher plant systems. The model for this type of gene silencing states that the high level of expression of a transgene (or a viral RNA sequence) activates an RNA target system which then actively destroys the target specific RNA. The experiments described below exploit a resistance system of RNA-mediated viruses in which plants are silenced post-transcriptionally a gene are resistant to a virus carrying that gene to show the sequence Pl / HC-Pro interferes with some aspect of the co-suppression.

The particular system used in these experiments used a line of tobacco transformed with a non-translatable ß glucuronidase (GUS) gene (line 407). The 407 line plants were resistant to the TEV vector carrying the β-glucuronidase gene. However, these plants were susceptible to TEV carrying the green fluorescent protein (GFP) gene. The 407 line plants were individually crossed with three tobacco lines: untransformed tobacco, line 407 and TEV B line. TEV B plants expressed the TEV Pl / HC-Pro sequence (Verchot and Carrington, 1995) and are capable of mediate synergistic disease when infected with PVX as described here (example 5-7), figure 2.

The shoots of the three individual crosses were inoculated with TEV-GUS and TEV-GFP and examined for the expression of the reporter gene which is indicative of viral duplication. The shoots of the untransformed 407 cross and those of the 407/407 cross were susceptible to the control TEV-GFP virus, but resistant to TEV-GUS. In contrast, shoots of cross 407 / TEV B were susceptible to both viruses. This result shows that the expression of the Pl / HC-Pro sequence interferes with the induction or action of the RNA target system which involves RNA-mediated resistance and indicates that the described enhancer sequence can be used to increase the expression of genes introduced to the plant through any stable incorporation into the host genome or through a viral expression vector.

EXAMPLE 27 In order to determine whether the enhancer sequence will work to increase the expression of a stably incorporated transgene, the present example can be carried out. A plant is first transformed by any mode of stable transformation with an endogenous plant gene for the purpose of overexpressing that particular gene. A part of the transformants will be co-deleted for the introduced gene will fail to express the gene product. These cosuprimid plants are then crossed with a plant stably transformed with the incrementing sequence, such as the U-6B plants. The offshoot of the cross will express the endogenous gene introduced previously silenced (cosuprimido).

EXAMPLE 28 The following example describes the present method which is used to express high levels of an endogenous gene product. A plant stably transformed with one or more copies of the enhancer sequence such as the U-6 plants described herein was subsequently transformed with an additional copy or additional copies of an endogenous gene for the purpose of overexpressing that gene product. The transformants will exhibit high levels of expression of the introduced gene product and / or the reduced numbers of the cosupprimed sucker that fail to express the gene product.

Gen Product Extraction Methods As with the introduction of the enhancer sequence, any number of known extraction methods can be employed to remove the foreign gene product from the host plant for beneficial use in certain applications. This particular protein extraction method will vary depending on the nature of the foreign protein that is being expressed. If the foreign protein is expressed in the viral vector as a function with the viral layer protein, the foreign sequence will be part of the structure of the virus particle and will be isolated by known methods for the isolation of particles that are being used as a protein. vector. For example, if an a-Triclosantin is the particular product that is being isolated, the purification procedure described in Kuimagai et al. (1993) is one that can be used. In some cases, however, expression driven by enhancer sequence will result in desirable qualities for the host plant itself. Obviously, in these cases, the extraction of the expressed gene product or the products is not desired.

As described above, the 5'-potyviral sequence has been shown to mediate the increased pathogenesis and accumulation of heterologous viruses such as PVX, TMV, and CM. The present invention uses that sequence in a beneficial manner to increase the expression of the endogenous gene products of the plants and can be used to produce characteristics of beneficial plants conferred by the expressed introduced gene product or to produce beneficial gene products for the extraction of the plant such as similar drugs.

Many of the examples and procedures discussed herein in terms of the use of a viral expression vector to introduce genes from plants to genes or endogenous to the tobacco host plant. It will be understood, however, that the present invention is not limited, but any method of using the claimed incremental sequence is applied to increase the production of a ge product introduced through any plant vector or through expression from one or more than the DNA copies of a gestably incorporated in the plant genome. Although the preferred embodiments of the invention have been described using specific terms, devices and methods, such a description is for illustrative purposes only. The words used are words of description rather than limitation. It is understood that changes and variations can be made by those with ordinary skill in the art without departing from the spirit or scope of the present invention which is set forth in the following claims. In addition, it should be understood that the aspects of the various incorporations can be exchanged both in whole and in part.

Claims (21)

R E I V I N D I C A C I O N S

1. In a method of expressing a foreign gene or an endogenous plant gene that has been introduced into cells of plants, plant rotoplasts, or whole plants, the best comprising the supply of an enhancer sequence comprising a part of the nearby region 5 'of the genome of u potovirus to said plant cells, plant rotoplasts whole plants so that the said foreign gene expression of the endogenous plant gene is increased.

2. The method as claimed in clause 1, characterized in that said introduced gene is a foreign gene that is not occurring naturally in said cells of plants, rotoplasts of plants or whole plants before being introduced therein.

3. The method as claimed in clause 1, characterized in that said gene is an endogenous plant gene that was occurring naturally in said cells of plants, plant rotoplasts, or whole plants, before being introduced as an additional copy or copies of the endogenous gene.

4. The method as claimed in clause 1, characterized in that the portion of the supplied 5 'region includes the coding region for Pl, the auxiliary proteinase-component (HC-Pro) and a part of P3.

5. The method as claimed in clause 1, characterized in that the part of the region 5 'is independently expressed.

6. The method as claimed in clause 1, characterized in that the part of the 5 'region is operated to other sequences.

7. A method for improving the expression of a foreign gene or of an endogenous plant gene that has been introduced into a plant material such as plant cells, plant rotoplasts, or whole plants, by supplying an increasing sequence to said plant material, said enhancer sequence comprises the near region of the genome and a potyvirus, said 5 'region encompassing the coding region for Pl, the proteinase auxiliary component (HC-Pro) and a part of P3, or some part d this sequence expressed either independently or fused to other sequences, or a modified version of that sequence or a related sequence from another virus or any part of or a modified version of that related sequence expresses, either independently or as a fusion to another sequence .

8. The method as claimed in clause 7, characterized in that said introduced gene is a foreign gene or an endogenous plant gene introduced through a viral expression vector and said enhancer sequence is supplied by the expression of the same viral vector.

9. The method as claimed in clause 7, characterized in that said gene is a foreign gene or an endogenous plant gene introduced through a viral expression vector and said increasing sequence is supplied by the expression of one or more DNA copies. of said incrementing sequence stably incorporated within the plant's genome.

10. The method as claimed in clause 7, characterized in that it is an alien gene or an endogenous plant gene introduced through a viral vector and said incremental sequence expressed from a transient expression system containing one or more DNA copies of said incremental sequence.

11. The method as claimed in clause 7, characterized in that a two-component viral vector system is used with a viral component expressing said enhancer sequence and the other viral component expressing said introduced gene.

12. The method as claimed in clause 7, characterized in that said introduced gene is a foreign gene or an endogenous plant gene introduced through the vector of viral expression and said enhancer sequence and supplied by co-infection with a potyvirus. expresses the native encoding sequence encoded for is potivirus.

13. The method as claimed in clause 7, characterized in that said gene is a foreign gene or gene of endogenous plant introduced through a vector d viral expression and said incrementing sequence is supplied by co-infection with a potyvirus expressing a native version of said incremental sequence.

14. The method as claimed in clause 7, characterized in that said foreign gene or endogenous plant gene is introduced through a viral expression vector having said gene fused to the structural gene of said viral expression vector.

15. The method as claimed in clause 7, characterized in that said foreign gene or endogenous plant gene is introduced to said plant genome through any stable transformation mode of one or more DNA copy of said introduced gene, and said enhancer sequence supplied before the introduction of said foreign gene or endogenous plant gene through the stable transformation procedures and increases either the expression of the introduced gene product or the number or proportion of the transformant plants expressing said gene gene product introduced.

16. The method as claimed in clause 7, characterized in that said foreign gene or said gene endogenous plant is introduced to said plant genome through any stable transformation mode of one or more DNA copy of said gene introduced into the The plant genome and the dich increment sequence are supplied during the process of introducing said foreign gene or endogenous plant gene through the stable transformation procedures and increase either the expression of the introduced gene product or the number or proportion of the Transformant plants that express dich product of introduced gene.

17. The method as claimed in clause 7, characterized in that said foreign gene or the endogenous plant gene is introduced through said plant genome by means of any stable transformation mode of one more DNA copies of said gene introduced in of the plant genome and said enhancer sequence is supplied after the introduction of said foreign gene or endogenous plant gene through the stable transformation procedures or increases either the expression of the product of ge introduced or the number of the proportion of the transformant plants that express said introduced gene product.

18. The method as claimed in clause 15, characterized in that said incrementing sequence is supplied through the expression of one or more AD copies of the incrementing sequence stably incorporated within the plant genome.

19. The method as claimed in clause 16, characterized in that said enhancer sequence is supplied through the expression of one or more DNA copies of the incrementing sequence incorporated within the plant genome.

20. The method as claimed in clause 17, characterized in that said enhancer sequence is supplied through the expression of one or more DNA copies of the incrementing sequence stably incorporated within the plant genome.

21. A method for improving the expression of a foreign gene or an endogenous plant gene that has been introduced into a plant material such as plant cells, plant protoplasts, or whole plants by supplying an increasing sequence to said plant material and then extracting said foreign gene or endogenous plant gene from said plant material, said increased sequence comprises the proximal 5 'region of the genome of a potyvirus, said nearby 5' region comprising the coding region for Pl, and auxiliary component -proteinase (HC-pro) and a part of P3, some part of that expressed sequence, either independently or fused to other sequences, or a modified version of that sequence or a related sequence of ot virus or any part or a modified version of a related sequence expressed either independently or with a fusion to another sequence. E S U M E N The present invention provides a method for increasing the expression of genes in plants by supplying a virally encoded enhancer sequence comprising the close 5 'region of the genome potyvirus to the plant. The enhancer sequence increases the expression of foreign genes or endogenous plant genes in plants by employing any expression methodology by introducing genes into plants. The enhancer sequence can be used to increase the expression of any gene including foreign genes or endogenous plant genes introduced by means of transformation into the plant genome or introduced by a plant viral expression.