WO2006005166A1

WO2006005166A1 - Viral expression of recombinant proteins in plants

Info

Publication number: WO2006005166A1
Application number: PCT/CA2005/001061
Authority: WO
Inventors: Jean-François LALIBERTE; Armand Seguin; Zarha Agharbaoui; Chantal Beauchemin; Arianne Tremblay
Original assignee: Inrs - Institut Armand-Frappier
Priority date: 2004-07-09
Filing date: 2005-07-08
Publication date: 2006-01-19
Also published as: WO2006005166B1

Abstract

A method for expressing a protein of interest in a plant is provided. The method involves providing a plant comprising both an inactivated viral replicon that comprises a nucleotide sequence encoding the protein of interest and a nucleotide sequence encoding a replicon reactivating protein that is operatively linked to an inducible promoter, and inducing the inducible promoter within the plant to produce the replicon reactivating protein. The replicon reactivating protein reactivates the inactivated viral replicon, and allows replication of the inactivated viral replicon. This results in the expressing the protein of interest within the plant.

Description

VIRAL EXPRESSION OF RECOMBINANT PROTEINS IN PLANTS

FIELD OF INVENTION

[0001] The present invention relates to protein production in plants. More ^% specifically, the present invention relates to viral expression of recombinant proteins in plants.

BACKGROUND OF THE INVENTION

[0002] Production of pharmaceutical proteins in plants has many practical, economic and safety advantages compared with more conventional systems. Consequently, the use of plants for large-scale protein synthesis is gaining wider acceptance. An important element for economic viability of this process is the concentration of the desired protein in the plant at the time of harvest. Approaches used for large-scale recombinant protein expression in plants include generation of stable transgenic plants and virus-derived expression vectors. Proteins of medical, diagnostic or novel agricultural interests have been produced using both methods.

[0003] Most virus vectors that have been developed are derived from positive-sense

RNA viruses. The first generation of virus vectors were designed as wild-type viruses, and modified to carry and express a gene of interest. These vectors were essentially fully functional viruses that, despite their modification, retained infectivity, had the ability to move systemically within their host, and produced infectious viral particles. For example, WO 99/02718 describes a turnip mosaic virus (TuMV) vector. These viral vectors have a high copy number of replicating virus genomes per cell, resulting in potentially high expression for an introduced gene of interest. Furthermore, they are relatively easy to introduce into plants, and they have a short interval time between inoculation and harvesting the protein of interest (for example, 2-3 weeks). However, a first-generation virus vector is generally suitable only for a limited number of plants, those that support replication and systemic spread of the virus. There is no spatial or temporal control over the expression of the gene of interest, rather the process is asynchronous as infection proceeds at different speeds in different parts of a plant. It is also difficult to use first-generation virus vectors on a large-scale basis. Another significant limitation is the low genetic stability of the first-generation virus vectors.

- l - [0004] Viral replication involves the synthesis from the positive-sense viral genome of negative-sense RNA, which then serves as template for the synthesis of multiple copies of genomic RNAs. These steps are catalyzed by an RNA-dependent RNA polymerase (RdRP) complex made up of viral and host proteins. The RdRP has the tendency of switching templates during genome amplification. At each replication cycle, a population of viral RNAs slightly different in size from the parent molecule is produced but only the best-fit molecules for replication are selected. The introduced gene of interest is progressively deleted after several viral replication cycles as it is of no utility, and in some cases may be detrimental to the virus.

[0005] In second-generation virus vectors, the stability of the vector has been addressed by stable integration of the engineered virus genome in the plant chromosome in the form of DNA-encoded viral replicon. For example, a replicon under the control of the CaMV 35S promoter has been reported. However, release of the replicon from the plant genome is uncontrolled (Mori, Kaido, et al, 1993, FEBS Lett. 336(1): 171-174; Angell & Baulcombe 1997, EMBO Journal 16(12): 3675-

3684), and expression levels are low, a result of post-transcriptional gene silencing (PTGS).

[0006] Crossing the replicon-containing plants with plants that express a suppressor of silencing has been suggested. For example, the potato virus X amplicon-plus system involves transgenic lines that encode a potato virus X vector carrying a gene of interest, accompanied by a second transgene coding for HC-Pro of Tobacco etch virus, a strong inhibitor of gene silencing. These dual transgenic plants had approximately 40-fold higher GUS activity than a conventional transgenic line that expressed GUS from a CaMV 35S promoter (Mallory, Parks, et al. 2002, Nature Biotechnology 20: 622-625; Anandalakshmi, Pruss, et al. 1998, Proc Natl Acad Sci U S A. 95(22):

13079-13084). WO 2003/104449 describes an expression system in which a first plant is transformed with the polymerase-coding gene from Cucumber Mosaic Virus, and a second plant is transformed with a transgene encoding the cucumoviral RNA3 in the minus-sense orientation. By crossing the two plants, selected Fl progeny contained both viral RNAs so that the replication of the RNA3 occurred, leading to the amplification of the RNA of interest. The resulting protein accumulation reached 5% of total soluble proteins. [0007] However, these two systems do not allow controlled, inducible, expression of the gene of interest. Since the DNA copy of the replicon is not transcriptionally silent, the constant synthesis of infectious viral transcripts may not result in the generation of normal transgenic plants. This approach is also not appropriate for the expression of genes that encode proteins that are toxic to plant cells.

[0008] There is a need for a regulated system to overcome this problem in the form of a threshold mechanism in the system to prevent unwanted virus replication in the non- induced state of the plant.

[0009] US 6,454,254 describes a method using Cre/loxP to improve the efficiency of gene manipulation within a chromosome. A specific DNA recombinase, Cre, derived from bacteriophage Pl of E. coli, recognizes a specific nucleotide sequence (loxP site) and processes DNA strand cleavage, strand exchange and ligation of each DNA strand within this site (for example see Hoess et al., Proc. Natl. Acad. ScL, 81: 1026-1029, 1984). If two loxP sites positioned in the same direction are present within the same DNA molecule, the DNA sequence between them is excised to form circular molecule product (DNA excision reaction). If the two loxP sites are positioned in different DNA molecules, and one of the DNA molecules is circular DNA, the circular DNA is inserted into the other DNA molecule at the loxP site (insertion reaction). These DNA recombination reactions are known to function in procaryotic and eucaryotic cells, including animal cells and for animal viruses. US 6,632,980 teaches a binary viral expression system utilizing Cre/LoxP. Expression of Cre recombinase results in excision of a blocker fragment from an inactive replicon, thereby activating replicon replication. The replicon may include a target gene that comprises a regulatory region.

[0010] Mori, Fujihara, et al. (2001, Plant J. 27, 79-86) describe an inducible system based on the Brome Mosaic Virus where RNAl encoding one subunit of a two- component replicase complex is under the control of a tightly regulated, glucocorticoid-inducible promoter. The transcript of PvNAl functions as messenger RNA for the Ia protein, but is not capable of self-replication as it contains non-viral sequences at both 5' and 3' ends. These non-viral sequences perturb cis-acting sequences necessary for efficient RNA replication and RNA amplification does not take place under non-inducing conditions. The plants were also transgenic for a replicable, and engineered, BMV RNA3 derivative carrying the human gamma interferon (IFN) gene. When transgenic plants were treated with dexamethasone (a glucocorticoid), the Ia protein reached a threshold level that allowed trans replication of the RNA of interest to levels over 30 to 230 times higher than for CaMV 35S promoter-driven levels. However, IFN accumulation in the plants was rather low (3.2- 3.7 ng in 1 mg fresh weight), if one considers the high level of RNA that had been produced. This suggests that these transcripts may not have been efficiently translated. In addition, the use of a chemical-regulated promoter to control the expression of the replicon may not be applicable to all virus vectors as we have observed leaky expression associated with this promoter, which lead to the inability of regenerating transgenic plants for the replicon.

SUMMARY OF THE INVENTION

[0011] The present invention relates to viral expression of recombinant proteins in plants.

[0012] It is an object of the invention to provide an improved method for viral expression of recombinant proteins in plants.

According to the present invention there is provided a method for expressing one or more than one protein of interest in a plant comprising:

i) providing a plant comprising:

- an inactivated viral replicon, the inactivated viral replicon comprising one or more than one nucleotide sequence encoding the one or more than one protein of interest, the one or more than one nucleotide sequence is not operatively linked to a regulatory region; and

- a nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter;

ii) inducing the inducible promoter within the plant to produce the replicon reactivating protein, the replicon reactivating protein reactivates the inactivated viral replicon, thereby allowing replication of the inactivated viral replicon, and expressing the one or more than one protein of interest within the plant.

The present invention pertains to the method as just described, wherein in the step of providing (step i):

- the inactivated viral replicon is introduced into the plant by crossing an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, with a reporter plant comprising the inactivated viral replicon to produce the plant;

- the inactivated viral replicon is introduced into the plant by transforming an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, with a nucleotide construct comprising the inactivated viral replicon, to produce the plant; or

- the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter is introduced into the plant by transforming a reporter plant comprising the inactivated viral replicon, with a nucleotide construct comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, to produce the plant.

[0013] The present invention also includes the method as described above, wherein in the step of providing (step i), the inactivated viral replicon comprises one or more than one nucleic acid sequence that encodes one or more than one protein that produces the inactivated viral replicon. For example, the one or more than protein is required for viral replicon replication. Preferably, the one or more than one protein is an RNA-dependant RNA polymerase.

[0014] The present invention also pertains to the method as described above, wherein in the step of providing (step i), the replicon reactivating protein complements the one or more than one protein that produces the inactivated viral replicon. Preferably, the replicon reactivating protein is an RNA-dependant RNA polymerase.

[0015] The present invention also provides the method as described , wherein the one or more than one nucleotide sequence is an inactivating element flanked by recombinase-recognition sites, so that insertion of the inactivating element in a viral replicon disrupts replication of the viral replicon. Preferably, the recombinase- recognition sites are loxP. The replicon reactivating protein is a recombinase that recognizes the recombinase-recognition sites and removes the inactivating element thereby restoring replication of the viral replicon. Preferably, the recombinase is a Cre recombinase.

[0016] The present invention also provides for the method described above, wherein in the step of providing (step i), the inducible promoter is selected from the group consisting of a dexamethasone inducible promoter, an estrogen inducible promoter, a teracycline-inducible promoter , a steroid inducible promoter, an ethanol-inducible promoter, a cytokinin inducible promoter, an auxin inducible promoter, a Top 10 promoter, and XVE transactivator.

[0017] The present invention pertains to method for expressing one or more than one protein of interest in a plant comprising:

i) introducing an inactivated viral replicon comprising one or more than one nucleotide sequence encoding the one or more than one protein of interest into a first plant, the first plant comprising a nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter; or the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter is introduced into an alternate first plant, the alternate first plant comprising the inactivated viral replicon comprising a nucleic acid sequence encoding the protein of interest, to produce the plant;

ii) inducing the inducible promoter within the plant to produce the replicon reactivating protein, the replicon reactivating protein reactivating the inactivated viral replicon, thereby allowing replication of the inactivated viral replicon, and expressing the one or more than one protein of interest within the plant.

[0018] In the step of introducing (step i), the inactivated viral replicon may be introduced into the first plant by crossing the first plant with a second plant, the second plant comprising the inactivated viral replicon. Alternatively, the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter may be introduced into the alternate first plant by crossing the alternate first plant with a second plant, the second plant comprising the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter. The inactivated viral replicon may also introduced into the first plant by transforming the first plant with a nucleotide construct comprising the inactivated viral replicon, or the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter may be introduced into the first plant by transforming the first plant with the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter.

[0019] Advantages of the DNA-encoded reversibly inactivated viral replicon over traditional transgene insertion in the plant genome include:

1. cytoplasmic transcript level amplification;

2. independence from transcriptional capacity of weak promoters; and

3. lower probability of encountering positional effects.

[0020] Advantages of the DNA-encoded reversibly inactivated viral replicon over current virus-based vectors include:

- higher genetic stability;

- stringent expression control in the non-induced state by a threshold mechanism;

- inducible and tissue-specific expression;

- no need for systemic spread - not limited to plants susceptible to the chosen virus;

- limited pathogenic impact; and

- no need for virion production, decreasing the possibility of environmental release. [0021] This summary of the invention does not necessarily describe all features of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] These and other features of the invention will become more apparent from the following description in which reference is made to the appended drawings wherein:

[0023] FIGURE 1 shows a schematic representation of plasmidp35Tunos comprising gfp cDNA inserted between the Pl and HC-Pro-coding genes in TuMV DNA.

[0024] FIGURE 2 shows schematic representation of the TuMV genome comprising uidk. (GUS) inserted between Pl and HC-Pro coding genes, and gfp between the polymerase and capsid protein coding genes.

[0025] FIGURE 3 shows simultaneous expression of two proteins of interest using TuMV polyprotein construct of Figure 2. Western blot analysis of the expression of GUS, GFP and the viral coat protein (CP).

[0026] FIGURE 4 shows GFP expression in N. tabacum transgenic for TGV, Pol and TuMV/GFP/VΝΝ. Figure 4A shows GPF expression by confocal microscopy. Left panel: no dexamethasone treatment, right panel: in the presence of dexamethosone. Figure 4B shows Western blot analysis using an anti-GFP serum. IN: expression under dexamethasone inducing conditions, NI: non-inducing conditions, GFP: positive control.

[0027] FIGURE 5 shows a schematic representation of the construct p35Tunos/GF, and the remaining elements of the construct following processing by the Cre recombinase.

[0028] FIGURE βshows the result of an in vivo recombination event mediated by Cre on p35Tunos/GF following agroinfiltration (CP - coat protein; see Examples for details).

[0029] FIGURE 7 shows the result of an in vivo recombination event mediated by an estradiol-inducible expression of Cre on p35Tunos/GF following agroinfiltration. Lane 1: leaves agroinfiltrated with p35Tunos/GF and treated with ImM estradiol; Lanes 2 and 3: leaves agroinfiltrated with pXVE/Cre with p35Tunos/GF (CP - coat protein; see Examples for details).

[0030] FIGURE 8 shows the result of an in vivo recombination event mediated by Cre following agroinfiltration in Arabidopsis thaliana plants transgenic for Tunos/GF. Lane 1 : A. thaliana infected with TuMV (positive control of the immunoreaction);

Lanes 2: leaves were agroinfiltrated with p35S-GUS; Lanes 3-6, leaves agroinfiltrated with p35S-Cre (CP - coat protein; see Examples for details).

DETAILED DESCRIPTION

[0031] The present invention relates to protein production in plants. More specifically, the present invention relates to viral expression of recombinant proteins in plants.

[0032] The following description is of a preferred embodiment.

[0033] The present invention involves producing a protein of interest within a plant when the plant has been induced or is in an induced state, and preventing unwanted virus replication in a non-induced state. The control over expression of a protein of interest within a plant may be achieved by reversibly inactivating a replicon so that no replication can take place even if expression of the transcript is detected. A reactivating component is then supplied in trans in a regulated manner. Preferably, the reactivating component needs to reach a threshold level before it can act on the disabled virus vector, or inactivated replicon.

[0034] Inactivation of the replicon may be carried out, for example, by introducing a mutation in a gene of a DNA-encoded viral replicon so that enzymatic activity of the protein encoded by the gene is inactivated (reporter construct). The modified replicon (reporter construct), under the control of a promoter, for example a constitutive or regulated promoter, is then introduced in the genome of a plant using available transformation protocols. Another gene construct is concomitantly, or successively, introduced in the replicon-containing plants. This gene construct may code for the fully active protein and is under the control of a regulated or inducible promoter (activator construct). At the appropriate stage of growth, the promoter in the activator construct is induced and sufficient active protein is produced to frvmy-complement the mutation in the replicon (reporter construct), allowing virus replication to take place.

[0035] An alternate strategy involves reactivation of viral replication using a chemically regulated recombination event. In this method, an inactivating element, flanked by recombinase-recognition sites is introduced in a DNA-encoded viral replicon (reporter construct). The modified replicon, under the control of a promoter, for example a constitutive, tissue specific or regulated promoter, is then introduced in the genome of a plant using available transformation protocols. A second gene construct is concomitantly, or successively, introduced in the replicon-containing plants (activator construct). This second gene construct comprises a gene that codes for a recombinase, and that is under the control of a regulated or inducible promoter. At the appropriate stage of growth, the promoter in the activator construct is induced and sufficient active recombinase is produced to remove the inactivating element in the replicon (reporter construct), and permitting the replicon to become fully functional.

[0036] Therefore, the present invention provides a method for expressing one or more than one protein of interest in a plant comprising:

i) providing a plant comprising:

ii) inducing the inducible promoter within the plant to produce the replicon reactivating protein, the replicon reactivating protein reactivates the inactivated viral replicon, thereby allowing replication of the inactivated viral replicon, and expressing the one or more than one protein of interest within the plant. [0037] It has been observed that the protein of interest encoded by the nucleic acid sequence (target sequence) within the inactivated replicon, and in the absence of an operatively linked regulatory region, is expressed at high levels following replicon reactivation. As expression of the protein of interest takes place in the absence of a regulatory region operatively linked with the nucleic acid sequence (target gene), this indicates that an operatively linked regulatory region is not required.

[0038] There are several benefits with having the coding region of interest expressed along with the viral gene complement. By expressing the protein of interest as a polyprotein, stability of the protein is increased as it is less likely to be degraded by proteases. This is in part due to the fact that the protein of interest is expressed at the same levels as the viral proteins. Furthermore, as described in more detail below, the construct described herein may be used to express one, or more than one coding region of interest to produce one, or more than one protein of interest. If for example, two proteins are produced that are subunits of a larger protein, then they are produced in equimolar amounts facilitating mature protein formation. Additionally, the reporter constructs (inactivated viral replicon) described herein (e.g. Figure 2) may be used to increase expression of a protein of interest by inserting one or more than one copy of the nucleotide sequence encoding the protein within the replicon. For example two or more than two coding regions of interest, coding two or more than one proteins of interest, may be inserted within the inactivated viral replicon. It is therefore preferred that the nucleic acid sequence encoding the protein of interest within the reporter construct, is not operatively linked to, or does not comprise, a regulatory region.

[0039] The inactivated viral replicon and the nucleotide sequence encoding a replicon reactivating protein may be introduced into a plant via crossing appropriate plants together. For example, the inactivated viral replicon may be introduced into the plant by crossing an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein that is operatively linked to an inducible promoter, with a reporter plant comprising the inactivated viral replicon to produce the plant comprising both nucleotide sequences. If desired offspring with a desired trait may be further selected.

[0040] The inactivated viral replicon and the nucleotide sequence encoding a replicon reactivating protein may also be introduced into a plant by introducing the required nucleic acid sequences into the plant by transformation, using techniques as known in the art. For example, the inactivated viral replicon may be introduced into a plant by transforming an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, with a nucleotide construct comprising the inactivated viral replicon, and in doing so produce the plant. Alternatively, the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter may be introduced into the plant by transforming a reporter plant comprising the inactivated viral replicon, with a nucleotide construct comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, to produce the plant.

[0041] The inactivated viral replicon may comprise one or more than one nucleic acid sequence that encodes one or more than one protein that produces the inactivated viral replicon. The one or more than protein is preferably required for viral replicon replication and may include a protein within the replicon that is involved either directly or indirectly within replication of the replicon. Non limiting examples of a protein include an RNA-dependant RNA polymerase, a helicase, HC-pro, P3, VPg- Pro, or P 1. However, the protein that may be modified may vary depending upon the viral replicon selected as other proteins may be present in different replicons. Preferably, the protein is an RNA-dependant RNA polymerase.

[0042] As described in more detail below, a DNA copy of a reversibly inactivated,

Turnip mosaic virus (TuMV), and insertion of the inactivated TuMV replicon within the chromosomal DNA of a plant, for example but not limited to Arabidopsis thaliana is described. In this example, reactivation of viral replication may be obtained by initiating a chemical-regulated Cre/loxP recombination event.

[0043] Reactivation of viral replication is also demonstrated using a mutation complementation strategy involving an RNA-dependent RNA polymerase (RdRP) - coding gene.

[0044] Any viral replicon may be modified and used as described herein for the production of a protein of interest. A non-limiting example of such a replicon is the Turnip mosaic virus (TuMV). The use of TuMV as a viral replicon has several advantages when compared to other known replicons. However, other replicons may also exhibit these features and are considered within the scope of the present application. For example, TuMV expresses its RNA genome (~10-kb ) as a single polyprotein that is cleaved by viral proteases. Foreign genes may be inserted in frame within the polyprotein gene sequence and sites for efficient processing by the viral proteinases may be engineered on either side of the introduced protein or proteins. A foreign protein is synthesised as part of the viral polyprotein and is produced in equimolar amounts with all the viral proteins. The TuMV also offers at least two insertion points for foreign genes, thereby allowing simultaneous expression of one or more than one different proteins. This may be of interest for the expression of hetero- dimeric proteins, or for expressing a protein along with its chaperone for efficient conformation of the protein of interest.

[0045] By "operatively linked" it is meant that the particular sequences interact either directly or indirectly to carry out their intended function, such as mediation or modulation of gene expression. The interaction of operatively linked sequences may, for example, be mediated by proteins that in turn interact with the sequences. A transcriptional regulatory region and a sequence of interest are "operably linked" when the sequences are functionally connected so as to permit transcription of the sequence of interest to be mediated or modulated by the transcriptional regulatory region.

[0046] The present invention provides a chimeric gene construct containing a nucleic acid that encodes a protein of interest that is inserted within an inactivated viral replicon. Any exogenous gene can be used and manipulated according to the present invention to result in the expression of the exogenous gene. A coding region of interest may also include, but is not limited to, a gene that encodes a pharmaceutically active protein, for example growth factors, growth regulators, antibodies, antigens, their derivatives useful for immunization or vaccination and the like. Such proteins include, but are not limited to, interleukins, insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinations thereof, interferons, for example, interferon-α, interferon-β, interferon-τ, blood clotting factors, for example, Factor VEI, Factor DC, or tPA or combinations thereof. A coding region of interest may also encode an industrial enzyme, protein supplement, nutraceutical, or a value-added product for feed, food, or both feed and food use. Examples of such proteins include, but are not limited to proteases, oxidases, phytases, chitinases, invertases, lipases, cellulases, xylanases, enzymes involved in oil biosynthesis etc.

[0047] By "regulatory region" it is meant a nucleic acid sequence that has the property of controlling the expression of a nucleic acid sequence that is operably linked with the regulatory region. Such regulatory regions may include promoter or enhancer regions, and other regulatory elements recognized by one of skill in the art. By "promoter" it is meant the nucleotide sequences at the 5' end of a coding region, or fragment thereof that contain all the signals essential for the initiation of transcription and for the regulation of the rate of transcription.

[0048] A constitutive regulatory element directs the expression of a gene throughout the various parts of a plant and continuously throughout plant development. Examples of known constitutive regulatory elements include promoters associated with the CaMV 35S transcript (Odell et al., 1985, Nature, 313: 810-812), the rice actin 1 (Zhang et al, 1991, Plant Cell, 3: 1155-1165) and triosephosphate isomerase 1 (Xu et al, 1994, Plant Physiol. 106: 459-467) genes, the maize ubiquitin 1 gene

(Cornejo et al, 1993, Plant MoI. Biol. 29: 637-646), the Arabidopsis ubiquitin 1 and 6 genes (Holtorf et al, 1995, Plant MoI. Biol. 29: 637-646), tobacco t-CUP promoter (WO/99/67389; US 5,824,872), the HPL promoter (WO 02/50291), and the tobacco translational initiation factor 4A gene (Mandel et al, 1995 Plant MoI. Biol. 29: 995-1004).

[0049] An inducible regulatory element is one that is capable of directly or indirectly activating transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer the DNA sequences or genes will not be transcribed. Typically the protein factor that binds specifically to an inducible regulatory element to activate transcription may be present in an inactive form which is then directly or indirectly converted to the active form by the inducer. However, the protein factor may also be absent. The inducer can be a chemical agent such as a protein, metabolite, growth regulator, herbicide or phenolic compound or a physiological stress imposed directly by heat, cold, salt, or toxic elements or indirectly through the action of a pathogen or disease agent such as a virus. A plant cell containing an inducible regulatory element may be exposed to an inducer by externally applying the inducer to the cell or plant such as by spraying, watering, heating or similar methods. Inducible elements may be derived from either plant or non-plant genes (e.g. Gatz, C. and Lenk, I.R.P.,1998, Trends Plant Sci. 3, 352-358; which is incorporated by reference). Examples of potential inducible promoters include, but not limited to, the dexamethasone inducible promoter system developed by (Bohner, S., et al., 1999, Plant J 19, 87-95; which is incorporated herein by reference), an estrogen inducible promoter (Zuo J. et al., 2001, Nat Biotech 19:157-161; Zuo, J. et al., 2000, Plant J24, 265-73; which are incorporated herein by reference), a teracycline-inducible promoter (Gatz, C, 1997, Ann. Rev. Plant Physiol. Plant MoI. Biol. 48, 89-108; which is incorporated by reference), a steroid inducible promoter (Aoyama, T. and Chua, N.H.,1997, Plant J. 2, 397-404; which is incorporated by reference) an ethanol-inducible promoter (Salter, M.G., et al, 1998, Plant Journal 16, 127-132; Caddick, M.X., et al,1998, Nature Biotech. 16, 177-180, which are incorporated by reference) cytokinin inducible promoters from the IB6 and CKIl genes (Brandstatter, I. and Kieber, JJ., 1998, Plant

Cell 10, 1009-1019; Kakimoto, T., 1996, Science 274, 982-985; which are incorporated by reference) and the auxin inducible element from DR5 (Ulmasov, T., et al., 1997, Plant Cell 9, 1963-1971; which is incorporated by reference).

[0050] A chimeric gene construct of the present invention can further comprise a 3' untranslated region. A 3' untranslated region refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of effecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by effecting the addition of polyadenylic acid tracks to the 3' end of the mRNA precursor. Polyadenylation signals are commonly recognized by the presence of homology to the canonical form

5' AATAAA-3' although variations are not uncommon. Non-limiting examples of 3' regions are the 3' transcribed non-translated regions containing a polyadenylation signal of Agrobacterium tumor inducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene) and plant genes such as the soybean storage protein genes and the small subunit of the ribulose-1, 5-bisphosphate carboxylase (ssRUBISCO) gene.

The 3' untranslated region from the structural gene of the present construct can therefore be used to construct chimeric genes for expression in plants. [0051] To aid in identification of transformed plant cells, the constructs of this invention may be further manipulated to include plant selectable markers. Useful selectable markers include enzymes that provide for resistance to an antibiotic such as gentamycin, hygromycin, kanamycin, and the like. Similarly, enzymes providing for production of a compound identifiable by colour change such as GUS (β- glucuronidase), green fluorescent protein (GFP), or luminescence, such as luciferase are useful.

[0052] Also considered part of this invention are transgenic plants containing the nucleotide constructs as described herein. Such plants include, but are not limited to, corn, wheat, barley, oat, tobacco, Brassica, soybean, pea, alfalfa, potato, ginseng,

Arahidopsis. The preferred plant is alfalfa. Alfalfa possesses many favorable characteristics for the production of pharmaceuticals (see US 5,990,385; which is incorporated by reference). For example, alfalfa is a perennial plant, is easily propagated through stem cutting, and can be grown for many years in a greenhouse. Yields are high (25 kg/m²) and protein content reaches 20% of dry weight.

[0053] The constructs of the present invention can be introduced into plant cells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, micro-injection, whiskers, electroporation, etc. For reviews of such techniques see for example Weissbach and Weissbach, Methods for Plant Molecular Biology, Academy Press, New York VIII, pp. 421-463 (1988); Geierson and Corey, Plant Molecular

Biology, 2d Ed. (1988); and Miki and Iyer, Fundamentals of Gene Transfer in Plants. In Plant Metabolism, 2d Ed. DT. Dennis, DH Turpin, DD Lefebrve, DB Layzell (eds), Addison Wesly, Langmans Ltd. London, pp. 561-579 (1997), also see US 4,684,611, US 5,508,184, US 5,453,367 and US 5,231,019 (plant protoplast transformation), US 5,302,523, US 5,464,765, and WO9428148 (plant transformation using ceramic whiskers). For Λrabidospsis see Clough and Bent, 1998 (Plant J. 16, 735-743).

[0054] Methods of regenerating whole plants from plant cells are also known in the art. In general, transformed plant cells are cultured in an appropriate medium, which may contain selective agents such as antibiotics, where selectable markers are used to facilitate identification of transformed plant cells. Once callus forms, shoot formation can be encouraged by employing the appropriate plant hormones in accordance with known methods and the shoots transferred to rooting medium for regeneration of plants. The plants may then be used to establish repetitive generations, either from seeds or using vegetative propagation techniques.

[0055] The transcript level produced using the DNA-encoded reversibly inactivated viral replicon, as described in the present invention, when compared to traditional transgene insertion in the plant genome is greater. Furthermore, the DNA-encoded reversibly inactivated viral replicon, as described in the present invention exhibits higher genetic stability when compared to virus-based vectors of the prior art. Additional advantages of the of the expression system of the present invention include:

- stringent expression control in the non-induced state by a threshold mechanism,

- inducible and tissue-specific expression as desired,

- no need for systemic spread

- not limited to plants susceptible to the chosen virus;

- limited pathogenic impact; and

- no need for virion production, decreasing the possibility of environmental release.

[0056] The present invention will be further illustrated in the following example.

Example

[0057] Standard DNA recombinant techniques were used, unless specified.

Expression of one or more than one coding region of interest using TuMV

[0058] For the expression of a coding sequence of interest (a foreign sequence), the cDNA encoding green fluorescent protein, gfp cDNA, was inserted between the Pl and HC-Pro-coding genes in the TuMV DNA copy of the plasmid p35Tunos (Figure

1), yielding p35Tunos/GFP. PCR site-directed mutagenesis by the overlap extension method (Ho et al. (1989) Gene 77, 51-59) was used to introduce a Sadl restriction site near the beginning of the HC-Pro-coding sequence of TuMV, resulting in p35Tυnos/SacH. The 5' primer pair was:

5'-TACTCGGGAAGAGAGCACAGC-S' (SEQ ID NO: 1) with

5'-GTTCTGTTCTTCAGGTATCGT-3 ' (SEQ ID NO:2),

and the 3' primer pair was:

5'-CACTTTAGTGCCGCGGGAGCCAAC-S' (SEQ ID NO:3) with

5'-GTTGGCTCCCGCGGCACTAAAGTG-S' (SEQ ID NO:4).

Plasmid p35Tunos (Sanchez et al. (1998) Virus Research 55, 207-219) was used as template and amplification was performed with the Pwo DNA polymerase (Roche).

The two amplification products were mixed and subjected to a second round of PCR with primers:

5'-TACTCGGGAAGAGAGCACAGC-S' (SEQ ID NO: 1) and

5'-GTTGGCTCCCGCGGCACTAAAGTG-3 ΪSEO ID NO:4).

[0059] The 720 bp assembled amplification fragment was digested with Hpal and

Xhoϊ and ligated into similarly digested p35Tunos. The cDNA coding for GFP was amplified from pBin m-gfp5-ER (Haseloff et al. (1997) PNAS 94, 2122-2127) using the sense primer:

5 '-TTATCCTCCGCGGGAATGAGTAAAG-3 ' (SEQ ID NO:5), and the anti- sense primer:

5'-CCTCCGCGGCTGCCTGGTGATAGACACAAGCTTTGTATAGTTCATCCAT- 3' (SEQ ID NO:6).

The 720 bp amplification fragment was digested with SacTL and ligated into similarly digested p35Tunos/SacE. The resulting plasmid p35Tunos/nGFP leads to the appearance of symptoms characteristics of TuMV infection in Brassica perviridis following inoculation by particle bombardment. Systemic symptoms appeared 8-14 days post-inoculation (dpi), a delay of two days with respect to a wild-type TuMV infection (data not shown). The symptoms were similar to those that were observed with the wild-type virus. Leaves showed mosaic symptoms and deformation, and plants were stunted when compared to healthy plants. Virus infection was confirmed by immunoblot analysis using a rabbit serum against the TuMV CP (data not shown).

No significant virus yield difference between the wild-type virus and the recombinant virus form was observed.

[0060] To confirm expression of a coding region of interest using this expression system, green fluorescence derived from GFP production was determined. Green fluorescence was readily observed when plants infected with TuMV-GFP were illuminated with UV light. No fluorescence was observed in healthy leaves, or leaves infected with wild-type TuMV (data not shown).

[0061] Proteolytic processing of the foreign proteins from the TuMV polyprotein was monitored by Western blot analysis using specific antibodies recognizing GFP. GFP was detected with an estimated molecular weight of 30 kDa, which is the expected size if proper proteolytic processing had taken place. Expression and proteolytic processing was also confirmed when the uidA gene was inserted in lieu of gfp (data not shown).

[0062] An additional insertion point between the polymerase and the capsid protein- coding genes of TuMV was also tested. The construct comprised insertion of gfp between Pl and HC-Pro-coding genes and uidA between the polymerase and capsid protein-coding genes (Figure 2). PCR site-directed mutagenesis by the overlap extension method (Ho et al. (1989) Gene 77, 51-59) was used to introduce an NgoMN restriction site near the beginning of the CP-coding sequence, resulting in p35Tunos/NgoMTV. The 5' primer pair was:

5'-TTTATCACCAGGCCGGCGAAACGCTTG-S' (SEQ ID NO: 7) with

5'-AGCGGATAACAATTTCACACAGG-S' (SEQ ID NO:8) and the 3' primer pair was

5'-CCCAGTCACGACGTTGTAAAACG-S' (SEQ ID NO:9) with 5'-CAAGCGTTTCGCCGGCCTGGTGATAAA-S' (SEQ ID NO:10).

[0063] Plasmid pSSTunosΔSαcI was used as template - this plasmid was obtained by digestion of p35Tunos with Sad followed by re-ligation of the plasmid unto itself. Amplification was performed with the Pwo DNA polymerase (Roche). The two amplification products were mixed and subjected to a second round of PCR with primers:

5'-TTTATCACCAGGCCGGCGAAACGCTTG-S' (SEQ ID NO:7) and

5'-CAAGCGTTTCGCCGGCCTGGTGATAAA-S' (SEQ ID NO:10).

The assembled amplification fragment was digested with MwI and Apal and ligated into similarly digested p35Tunos. The cDNA coding for β-glucuronidase was amplified from pBI121 using the sense primer:

5'- CATCATAGCCGGCATGTTACGTCCTGTAGA -3'(SEQ ID NO:11) and the anti-sense primer,

5'. TTTTTTGCCGGCTTGATGGTATACGCATGCTTGTTTGCCTCCCTGC -3¹ (SEQ ID NO: 12).

[0064] The amplification fragment of 1.8 kbp was digested with NgoMTV and ligated into similarly digested p35Tunos/ NgoMTV, to yield p35Tunos/cGUS. Plasmid 35Tunos/nGFP was digested with Kpnl and Apal and the 8.2 kbp fragment was replaced with the 10 kbp KpnVApal fragment from p35Tunos/cGUS, to yield p35Tunos/nGFPcGUS.A similar construct comprising insertion of uidA between Pl and HC-Pro-coding genes and gfp between the polymerase and capsid protein-coding genes was also prepared.

[0065] Both of these constructs were infectious, and GUS as well as GFP were correctly expressed. As shown in Figure 3, expression of GFP, GUS and viral coat protein was detected using Western analysis. No expression was observed in non- infected tissue.

[0066] The use of construct as shown in Figure 2, that permits expression of one, or more than one coding region of interest to produce one, or more than one protein of interest, is of benefit when several subunits of a larger protein are required to be synthesized in equimolar amounts to facilitate mature protein formation. Alternatively, this type of construct may be used to increase expression of a protein of interest by inserting one or more than one copies of the nucleotide sequence encoding the protein within the replicon.

Complementation strategy

[0067] The p35Tunos/nGFP cassette (see above) was modified by changing the nucleotides encoding the "GDD" motif within the viral polymerase to those encoding VNN to produce p35Tunos/GFP/VNN (Reporter construct). PCR site-directed mutagenesis by the overlap extension method (Ho et al. (1989) Gene 77, 51-59) was used. The 5' primer pair was:

5'- TCCAAACCAAGAGCATAACGA-3' (SEQ K) NO: 13) with

5'-TAGCAGTAAATTGTTAACGTTGACGAA-S' (SEQ ID NO: 14)

and the 3' primer pair was:

5 'TTCGTCAACGTTAACAATTTACTGCTA-S ' (SEQ ID NO: 15) with

5'-ATTAACCCTCACTAAAG-S ' (SEQ ID NO: 16).

[0068] Plasmid pSKTunos/C/αl was used as template - this plasmid was obtained by ligation of the 2.9 kbp Clal fragment of p35Tunos cloned in similarly digested pSKBluescript. Amplification was performed with the Pwo DNA polymerase (Roche). The assembled amplification fragment was digested with Spel and Kpnϊ and ligated into similarly digested pSKTunos/CM, to yield pSKTunos/C/αl/VNN. This plasmid was digested with Clal and the 2.9 kbp fragment ligated into similarly digested p35Tunos/nGFP, to yield p35Tunos/nGFP/VNN. This vector cannot replicate by itself due to the presence of the mutant polymerase, but the mutation in this vector can be complemented by a functional polymerase provided in trans (Li &

Carrington (1995) Proc Natl Acad Sci U S A. 92, 457-461). The TuMV cassette, including the CaMV 35S promoter, was excised from p35Tunos/nGFP/VNN by Smal smάApal digestion and ligated into similarly digested pGreen0029 (Hellens et al. (2000) Plant MoI. Bio. 42, 819-832) , yielding pGreenTunos/GFP/VNN for tobacco transformation.

£θOW] ^*A second gene construct pTopPol (activator construct) was made in which the functional polymerase-coding gene is under the control of the inducible Top 10 promoter (Bohner et al. (1999) Plant J 19, 87-95). This promoter is turned on upon binding of the transcription factor TGV, which consists of the Tet operator receptor, the receptor domain of the rat glucocorticoid receptor and the activating domain of the herpes viral protein VPl 6. The transcription factor is activated upon application of dexamethasone. The plasmid pTopPol was made as follows. Plasmid p35TunosΔ/StuI was produced by digestion of p35Tunos with Hpal and BamΗl, polishing the ends with Klenow, and religating the plasmid unto itself, resulting in p35TunosΔ. The Stul restriction site was added by PCR site-directed mutagenesis by the overlap extension method (Ho et al. (1989) Gene 77, 51-59). The 5' primer pair was

5'-CCCAGTCACGACGTTGTAAAACG-S' (SEQ ID NO: 17) with

5'-ATGAAATGAAAGGCCTTATATAG-S' (SEQ IDN0:18) and the 3' primer pair was

5' CTATATAAGGCCTTTCATTTCAT-3' (SEQ ID NO: 19) with

5'-AGCGGATAACAATTTCACACAGG-S' (SEQ ID NO:20).

[0070] Plasmid p35Tunos was used as template and amplification was performed with the P wo DNA polymerase (Roche). The 1.7 kbp assembled amplification fragment was digested with .EcoRI and Pstl and ligated into similarly digested p35TunosΔ. The 5' non-translated region (5'NTR) of TuMV was amplified using the sense primer:

5'- CCCAGTCACGACGTTGTAAAACG-3' (SEQ ID NO: 21) and the anti-sense primer

5'-TACCCACCGATTCTGCTGGGCCATGGGGTTTGCTGTTGGTGA-S', (SEQ ID NO:22) using p35TunosΔ/StuI as template. The polymerase coding region was amplified using the sense primer

5'-TCACCAACAGCAAACCCCATGGCCCAGCAGAATCGGTGGATG-S' (SEQ ID NO:23) and the anti-sense primer

5'-TGCATCAAGCGCTGCAGCCTACTGGTGATA-S' (SEQ E⁾ NO:24)

using p35TunosΔEcoRV as template - this plasmid was obtained by digesting p35Tunos with EcoKV and re-ligation. The 5'NTR and polymerase amplification fragments were mixed together and asssembled together by PCR using the sense primer :

5'- CCCAGTCACGACGTTGTAAAACG-3' (SEQ E) NO: 25) and the anti-sense primer

5'-TGCATCAAGCGCTGCAGCCTACTGGTGATA-3'(SEQ TD NO:26).

[pOTP^The assembled product was digested with Smal and Pstl and ligated into similarly digested pSKBluescript. The resulting plasmid was digested with Stul and SaR and ligated with similarly digested pBinHygTF_mod (Bohner et al. (1999) Plant J

19, 87-95).Two types of transgenic plants were generated. In the first type, Nicotiana tabacum plants expressing TGV (Bohner et al. (1999) Plant J 19, 87-95) were transformed with the activator construct pTopPol comprising a functional polymerase coding sequence, and made transgenic for Pol (activator plants). Seeds were germinated on MS medium containing 30 mg/1 of hygromycin and 50 mg/1 of kanamycin and plantlets were grown in soil at 20 ⁰C and at a 16 hour photoperiod. At the 4-6 leaf stage, plants were watered with a 90 μM dexamethasone solution for five days, and leavess were sprayed with the same solution twice a day for three days. These transgenic plants had no RNA transcripts coding for the polymerase in non- induced conditions, but had considerable polymerase RNA levels upon application of dexamethasone on Northern blots (data not shown).

[0072] The second type of Nicotiana tabacum plant (reporter plants) were transformed with the reporter construct pGreenTunos/GFP/VNN. [0073] These two types of lines were cross-fertilised (activator plant X reporter plant) and offspring were selected for triple transgenic plants expressing TGV, Pol and TuMV/GFP/VNN (product plants). Southern blots confirmed that such triple transgenic plants (product plants) were obtained.

[0074] Seeds were germinated on MS medium containing 30 mg/1 of hygromycin, 50 mg/1 of kanamycin and 5-10 mg/1 of gluphosinate. Plantlets were grown in soil at 2O⁰C and at a 16 hour photoperiod. At the 4-6 leaf stage, plants were watered with a 90 μM dexamethasone solution for five days, and leaves were sprayed with the same solution twice a day for three days. Ten days later, leaves were observed by confocal microscopy for GFP expression As shown in Figure 4a, transgenic plants that were not treated with dexamethasone showed a background level of GFP (left pannel). This background level of expression arises due to CaMV 35S-driven transcription of the TuMV transgene followed by translation of the transcripts. However, upon application of the chemical inducer, high expression of GFP was observed throughout the plants (right panel, Figure 4a), due to viral replication resulting from complementation of the mutant polymerase by the active polymerase. Western blot analysis (Figure 4b) using an anti-GFP serum confirmed GFP expression under dexamethasone inducing conditions (IN), while no GFP was detected under non- inducing conditions (NI). GFP is a positive control of the immunoreaction.

Reactivation strategy

[0075] Reactivation of viral replication using by a chemical-regulated Cre/foxP- mediated recombination event was also tested.

[0076] In this system, an inactivating element (blocker fragment) was inserted between the Pl and HC-Pro-coding genes in

to produce a disabled TuMV sequence (see Figure 5). The inactivating element was the uidA gene, which contained a translation stop codon, flanked by two loxP sites. The uidA cDNA (pBIlOl.l) was amplified with the sense primer

S'-ACTACCGCGGGCATAACTTCGTATAGCATACATTATACGAAGTTA TGGATGCTTATGTTACGTCCTGTAGAAAAC-S¹ (SEQ ID NO:27) and the anti- sense primer

5'-CGGTGATACGTACACTTTTCCCGGCAATAACAT-S (SEQ ID NO-.28).

The amplified fragment was cloned in pCR2.1 using the TA cloning kit of Invitrogen and the resulting plasmid was named pCR2.1/GFl. Similarly, (pBIlOl.l) was amplified with the sense primer

5'-AAAGTGTACGTATCACCGTTTGTGTGAACAACG-3^l (SEQ ID NO:29) and the antisens primer

5'- AGTCCGCGGCCCATAACTTCGTATAATGTATGCTATACGAAGTTAT

TCATTGTTTGCCTCCCTGCTGCGG-3' (SEQ ID NO:30).

The amplified fragment was cloned in pCR2.1 using the TA cloning kit of Invitrogen and the resulting plasmid was named pCR2.1/GF2. pCR2.1/GF2 was digested with SnάBl and EcoRl and the 1.4 kbp fragment was cloned into similarly digested pCR2.1/GFl, to yield pCR2.1/GF. PCR2.1/GF was digested with SacTL and cloned into similarly digested p35tunos/SacII (see above), to yield p35tunos/GF.

[0077] Studies using particle bombardment demonstrated that the modified viral construct containing the inactivating element (blocker fragment) had lost its infectivity (data not shown).

[0078] Using an in vitro recombination procedure, the /ox-flanked uid sequence was removed from the TuMV genome leaving a single loxP site, which does not disrupt the polyprotein open reading frame. p35Tunos/GF was digested with Smal and 250 ng was incubated with 1 unit of Cre (New England Biolab) for 30 min at 37°C. The plasmid was religated unto itself and the reaction mixture was used for E. coli transformation. Colonies harbouring plasmids that underwent recombination were selected (i.e. p35Tunos/loxP), and sequencing confirmed that recombination at the loxP sites has taken place. Following particle bombardment, the TuMV genome with one loxP site was as infectious as the wild-type virus. [0079] In vivo recombination was tested in N. benthamiana by agro-infiltration. For this study, a gene cassette, 35SCre, was made in which the Cre recombinase gene was under the control of the CaMV 35S promoter The Cre-coding cDΝA (p6X-GFP) (Zuo et al. (2001) Nat Biotechnol 19, 157-161) was amplified with the sense primer:

5'-TCGAGCTGAAGCTAGTCGACTCTAGCC-3¹ (SEQ ID NO:31) and the anti- sense primer:

5'-GATCTACCCCGCTCGAGGTCGAAGATCCT-S ' (SEQ ED NO:32).

The amplified fragment was cloned in pCR2.1 using the TA cloning kit of Invitrogen and the resulting plasmid was named pCR2.1/Crenos. pCR2.1/Crenos was digested with Kpnl andXbal and cloned into similarly digested pRT106 (Tδpfer et al. (1993)

Methods Enzymol. 217, 66-78), to yield pRT106/Cre. pRT106/Cre was digested with HindSR and cloned into similarly digested pGreen0029 (Hellens et al. (2000), to yield p35S-Cre. N. benthamiana leaves were infiltrated with A tumefaciens suspensions containing p35S-Cre and p35Tunos/GF. Infiltrated leaves of six individual plants were collected twelve days following infiltration and were analysed for virus production using a rabbit serum raised against TuMV CP.

[0080] Figure 6 shows that coat protein (CP) was detected in p35S-Cre with p35Tunos/GF-agroinfiltrated leaves (Cre + TuMV/GF), indicating that recombination took place. Leaves were also agroinfiltrated with p35Tunos (TuMV) as a positive control of the agroinfiltration procedure and of the immunoreaction.Νo recombination was observed with p35Tunos/GF (TuMV/GF) alone, or co-agroinfiltrated with p35S- GUS (TuMV/GF+GUS).

[0081] The XVE-coding cDΝA under the control of the constitutive promoter G10-90 (ρ6X-GFP) (Zuo et al. (2001) Nat Biotechnol 19, 157-161) was amplified with the sense primer:

5¹-CCAATATATGCGGCCGCCCTGTCAAACACTGATAGTTTAAAC-3^l (SEQ ID NO:33) and the anti-sense primer:

5'-GCTTGTAAGCTTTTGGGATGTTTTACTCCTCATATT-S' (SEQ ID NO:34). T/CA2005/001061

The amplified fragment was cloned in pCR2.1 using the TA cloning kit of Invitrogen and the resulting plasmid was named pCR2.1-XVE. The Cre-coding cDNA is under the control of estradiol-inducible pLexAop.35Smin promoter (p6X-GFP) (Zuo et al. (2001) Nat Biotechnol 19, 157-161). This promoter is turned on upon binding of the transcription factor XVE, which consists of the LexA binding protein, the receptor domain of the estrogen receptor and the activating domain of the herpes viral protein VP 16. The transcription factor is activated upon application of estradiol. The cDNA was amplified with the sense primer:

5'-AAATCGATTCGCATTATCATCCCCTCGACGTA-S' (SEQ E) NO:35) and the anti-sense primer:

5'-GATCTACCCCGCTCGAGGTCGAAGATCCT-S' (SEQ ID NO:36).

The amplified fragment was cloned in pCR2.1 using the TA cloning kit of Invitrogen and the resulting plasmid was named pCR2.1-Cre. pCR2.1/XVE was digested with Spel and Notl and cloned into similarly digested pGreen0029 (Hellens et al. (2000), yielding pXVE. PCR2.1/Cre was digested with HindUl and Xhol and cloned into similarly digested pXVE, yielding pXVE/Cre.

[0082] N. benthamiana leaves were infiltrated with A. tumefaciens suspensions containing pXVE/Cre and p35Tunos/GF, and were infiltrated 48hr later with ImM estradiol. Infiltrated leaves of two individual plants were collected twelve days following infiltration and were analysed for virus production using a rabbit serum raised against TuMV CP.

[0083] Figure 7 shows that CP was detected in pXVE/Cre with p35Tunos/GF- agroinfiltrated leaves that had been treated with ImM estradiol (lanes 2 and 3), indicating that recombination took place under inducing conditions. Leaves agroinfiltrated with p35Tunos/GF alone (lane 1) did not undergo recombination.

[0084] Arabidopsis thaliana plants were transformed with pGreenTunos/GF, which was obtained by digestion of p35Tunos/GF withApal and Smal and ligation in similarly digested pGreenO179 (Hellens et al. (2000). Transgenic A thaliana leaves were infiltrated with A. tumefaciens suspensions containing p35S-Cre. Infiltrated leaves of four individual plants were collected twelve days following infiltration and were analysed for virus production using a rabbit serum raised against TuMV CP. Figure 8 shows that CP was detected in three of the above transgenic plants (lanes 3,4 and 6), indicating that recombination took place in transgenic A. thaliana. No recombination was observed when leaves were agroinfiltrated with p35 S-GUS (lane

2). A. thaliana infected with TuMV serves as a positive control of the immunoreaction (lane 1).

[0085] All citations are hereby incorporated by reference.

[0086] The present invention has been described with regard to one or more embodiments. However, it will be apparent to persons skilled in the art that a number of variations and modifications can be made without departing from the scope of the invention as defined in the claims.

Claims

WHAT IS CLAIMED IS:

1. A method for expressing one or more than one protein of interest in a plant comprising:

i) providing a plant comprising:

- an inactivated viral replicon, the inactivated viral replicon comprising

. one or more than one nucleotide sequence encoding the one or more than one protein of interest, the one or more than one nucleotide sequence is not operatively linked to a regulatory region; and

2. The method of claim I₅ wherein in the step of providing (step i), the inactivated viral replicon is introduced into the plant by crossing an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, with a reporter plant comprising the inactivated viral replicon to produce the plant.

3. The method of claim 1, wherein in the step of providing (step i), the inactivated viral replicon is introduced into the plant by transforming an activator plant comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, with a nucleotide construct comprising the inactivated viral replicon, to produce the plant.

4. The method of claim 1, wherein in the step of providing (step i), the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter is introduced into the plant by transforming a reporter plant comprising the inactivated viral replicon, with a nucleotide construct comprising the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter, to produce the plant.

5. The method of claim 1, wherein in the step of providing (step i), the inactivated viral replicon comprises two or more than two nucleic acid sequences that encodes two or more than two proteins that produces the inactivated viral replicon.

6. The method of claim 1, wherein the one or more than protein is required for viral replicon replication.

7. The method of claim 6, wherein the one or more than one protein is selected from the group consisting of an RNA-dependant RNA polymerase, a helicase, HC-Pro, P3, VPg-Pro and Pl.

8. The method of claim 7, wherein the one or more than one protein is RNA- dependant RNA polymerase.

9. The method of claim 5, wherein the replicon reactivating protein compliments the one or more than one protein that produces the inactivated viral replicon.

■ 10. The method of claim 9, wherein the replicon reactivating protein is selected from the group consisting of an RNA-dependant RNA polymerase, a helicase, HC-pro, P3 VPg-Pro and Pl.

11. The method of claim 10, wherein the replicon reactivating protein is RNA- dependant RNA polymerase.

12. The method of claim 5, wherein the one or more than one nucleotide sequence is an inactivating element flanked by recombinase-recognition sites, so that insertion of the inactivating element in a viral replicon disrupts replication of the viral replicon.

13. The method of claim 12, wherein the recombinase-recognition sites are loxP.

14. The method of claim 12, wherein the replicon reactivating protein is a recombinase that recognizes the recombinase-recognition sites and removes the inactivating element thereby restoring replication of the viral replicon.

15. The method of claim 14, wherein the recombinase is a Cre recombinase.

16. The method of claim 1, wherein in the step of providing (step i), the inducible promoter is selected from the group consisting of a dexamethasone inducible promoter, an estrogen inducible promoter, a teracycline-inducible promoter , a steroid inducible promoter, an ethanol-inducible promoter, a cytokinin inducible promoter, an auxin inducible promoter, a Top 10 promoter, and XVE transactivator.

17. The method of claim 1, wherein in the step of providing (step i), the inactivated viral replicon is a turnip mosaic virus replicon.

18. A method for expressing one or more than one protein of interest in a plant comprising:

i) introducing, an inactivated viral replicon comprising one or more than one nucleotide sequence encoding the one or more than one protein of interest into a first plant, the first plant comprising a nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter; or the nucleotide sequence encoding a replicon reactivating protein operatively linked to an inducible promoter is introduced into an alternate first plant, the alternate first plant comprising the inactivated viral replicon comprising a nucleic acid sequence encoding the protein of interest, to produce the plant;

19. The method of claim 18, wherein in the step of introducing (step i), the inactivated viral replicon is introduced into the first plant by crossing the first plant with a second plant, the second plant comprising the inactivated viral replicon.

20. The method of claim 18, wherein in the step of introducing (step i), the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an • inducible promoter is introduced into the alternate first plant by crossing the alternate first plant with a second plant, the second plant comprising the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter.

21. The method of claim 18, wherein in the step of introducing (step i), the inactivated viral replicon is introduced into the first plant by transforming the first plant with a nucleotide construct comprising the inactivated viral replicon.

22. The method of claim 18, wherein in the step of introducing (step i), the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter is introduced into the first plant by transforming the first plant with the nucleic acid sequence encoding a replicon reactivating protein operatively linked to an inducible promoter.