JP2008505622A - Biologically safe transient protein expression in plants - Google Patents

Abstract

  A method for producing a target protein by expressing the target protein from a target sequence in a plant or a plant leaf: a) Agrobacterium containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA A sequence encoding a replicon derived from a plant virus by transfecting the plant or plant leaf by infiltrating the plant or plant leaf in the presence of a complementing factor, is a sequence necessary for the replicon function of the replicon, And A) containing the target sequence to be expressed from the replicon, and b) optionally isolating the target protein from the plant or plant leaf infiltrated in step (a), wherein the Agrobacterium strain is complemented A first genetic modification that makes transfection of T-DNA into organisms poor in the absence of factors There has been provided, the method.

Description

  The present invention relates to a method for producing biologically safe recombinant proteins in plants using transient expression based on plant viral vectors delivered to target plants by Agrobacterium. The present invention also relates to a method for expressing a target sequence in a target host plant or plant leaf. The present invention describes the use of Agrobacterium-mediated delivery of viral vectors that provide high yields of large scale production of the protein of interest in the whole plant or plant part. The methods of the present invention control the transformation into undesirable organisms while limiting transient expression by Agrobacterium-mediated delivery to the host plant. Furthermore, the present invention relates to a biologically safe Agrobacterium for expressing a target sequence in a plant or plant leaf. The present invention provides high yields of industrial protein production in plants with increased biological safety.

BACKGROUND OF THE INVENTION Plant biotechnology currently relies on two approaches for delivering and expressing foreign genes in plants: stable transformation and transient expression. The latter can be constructed based on agroinfiltration or viral infection (for review see Fischer et al., 1999, Biotechnol. Appl. Biochem., 30, 113-116). Transient expression is achieved by agroinfiltrating plant tissues with a standard expression cassette that drives expression of the gene of interest under the control of a constitutive, eg, 35S promoter (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA, 96, 11128-11133), or on a large scale, can be achieved by transfecting a plant with a viral vector. Agroinvasion usually does not provide high yields, but protein expression levels can be increased up to 50-fold when combined with a post-transcriptional gene silencing (PTGS) suppressor such as p19 or HcPro (Vonet et al., 2003). Plant J., 33, 549-556). Still far below the biological limits achievable with viral expression systems (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). A description of a bioreactor based on the use of agroinfiltration to transiently express a recombinant protein of interest in plant tissue is provided in US Pat. No. 6,740,526. However, this patent does not describe how to improve the yield of the protein to be expressed and is not superior to other methods known in the art (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA., 96, 11128-11133). A more powerful approach for transient expression is the use of viral vectors. TMV-based expression of the reporter gene (GFP or DsRed) can reach the biological yield limit of the system and has been shown to produce several mg of recombinant protein per gram of fresh leaf biomass. (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). The relative yield of such a system can reach up to 80% of the TSP, greatly facilitating downstream processing and reducing costs. Such a high relative yield is possible due to the blocking of host protein biosynthesis induced by the virus.

  Viral vectors can provide higher expression levels than conventional vectors used for agroinvasion (for review: Porta & Lomonossoff, 1996, Mol. Biotechnol., 5, 209-221; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol. , 240, 81-94), a powerful tool for functional genomics studies (Dalmay et al., 2000, Plant Cell, 12, 369-379; Ratcliff et al., 2001, Plant J., 25, 237-). 245; Escobar et al., 2003, Plant Cell, 15, 1507-1523). Numerous publications and patents in the art describe systems based on DNA and RNA viral vectors (Kumagai et al., 1994, Proc. Natl. Acad. Sci. USA, 90, 427-430; Mallory et al. , 2002, Nature Biotechnol. 20, 622-625; Mor et al., 2003, Biotechnol. Bioeng., 81, 430-437; US5316931; WO02 / 097080; WO98 / 54342). Existing viral vector systems are usually limited to a narrow host range in terms of maximum performance, and even the level of expression of such vectors in the most suitable host is well below the biological upper limit of the system. Yes. An important issue with virus-based systems is how to deliver viral replicons to plant cells. The most widely applied delivery method for large scale production (eg, simultaneous production in many plants on a farm or greenhouse) is the use of infectious copies of RNA viral vectors (Kumagai et al., 1995, Proc. Natl. Acad). Sci. USA, 92, 1679-1683). Since recombinant RNA viral vectors have a relatively high tendency to lose heterologous inserts during the replication cycle, this method requires transcription of the DNA template in vitro and is therefore inefficient and expensive.

  The transient pathway is very fast but is very limited due to the low infectivity of the virus, the inability to transfect most of the plants, and gene size limitations. Use of agroinfiltration to deliver infectious viral vectors to plant cells (Liu & Lomonossof, 2002, J. Virol. Methods, 105, 343-348), or viral vector components from viral vector components delivered by Agrobacterium There are publications that describe assembly (Marrilonnet et al., 2004, Proc. Natl. Acad. Sci. USA, 101, 6852-6857). However, these publications do not address the issue of efficient synchronization of viral replicons in each cell of agro-infiltrated plant tissue, and further vector propagation is not related to viral translocation and osmotic migration. Depends on ability. This transition requires a relatively long period of time, and viral vectors usually provide the highest possible vector yield approximately 10-14 days after infection. This is unacceptable for the production of recombinant proteins that interfere with plant intracellular processes, particularly highly cytotoxic proteins such as restriction enzymes, proteases, non-specific nucleases, and many pharmaceutical proteins. Conversely, standard vectors delivered by agroinvasion reach the highest possible expression level 3-4 days after agrodelivery, but the yield provided by such vectors is unacceptably low. Thus, in spite of providing expression of the gene of interest in virtually all cells of agro-infiltrated plant tissue, it is caused by a less efficient standard transcription vector driven by a constitutive promoter to express the protein of interest. Face problems. In contrast, agro-delivered viral vectors can provide high expression levels, but rarely initiate replication and thus provide expression in most cells, with only a small fraction (agro infiltration) Less than 1% of total tissue). As a result, while such virus-based vectors require a significant (3-4 times) long period of time to provide expression, the productivity of the system is theoretically especially in the case of cytotoxic proteins. Still lower than productivity. This does not provide synchronized expression in agro-infiltrated tissues, thus affecting yield and is a significant disadvantage for using viral vectors in transient expression systems, especially when expressing cytotoxic genes. It is. Also, the infected plant host excludes the tissue from the production process because it contains no viral vectors in the majority of the tissue. Furthermore, the time required to achieve the highest possible transmission (and expression level) of viral vectors for infected plants is 3-4 times longer compared to standard agroinfiltration protocols. Moreover, any of the described systems for transient expression is an important factor in industrial scale protein production, including the use of genetically engineered organisms, a problem that increases the biological safety of the system. Did not work on.

  Despite numerous publications in the field, including patented technology, there are still sufficient efficiency and yield for commercial high-yield production, mainly for two main reasons. There is no large-scale production system based on a working virus: First, a transient plant virus-based expression system is usually restricted to a specific host and is susceptible to environmental factors, so large-scale culture Cannot be suitable. Moreover, because these systems are usually limited to a certain part of the plant host, most plant biomass is excluded from the production process, resulting in the relative yield of recombinant product per unit plant biomass being transduced. Minimizing to a level comparable to that achievable by conventional transcriptional promoters in transgenic plants; second, by creating a transgenic plant host with a viral replicon precursor stably integrated into each cell. Attempts to expand the production system based on, in particular, work lower than the standard for replicons in such positions, “leakage” of the gene of interest to be expressed from the replicon, and lack of an efficient switch system in the vector Does not provide a solution. Although some progress has been achieved in PVX-based vectors by using PTGS silencing suppressors as triggers for RNA replicon formation (Mallory et al., 2002, Nature Biotechnol., 20, 622-625), However, there is no solution that provides efficient control of the switch that induces viral vector replication (PTGS suppressor), so it remains far from practical value. However, this system provided an expression level of the GUS gene that reached 3% of the total soluble protein (TSP), the best known so far in this type of system, but still under the control of a strong promoter. No better than conventional transgene expression systems. Other inducible systems (Mori et al., 2001, Plant J., 27, 79-86) based on the plant tri-segment RNA virus, brom mosaic virus (BMV), produced very low yields of the target protein (3 ˜4 μg / g fresh weight). This is comparable to the yield provided by a standard transcription promoter.

  The low level of expression achieved so far using plant expression systems is the main reason why these systems rarely compete with other expression systems such as bacterial, fungal, or insect cell expression systems. is there. The low expression level makes the downstream costs for the isolation and purification of proteins in a huge background of plant material very high. Therefore, as the yield of the target protein or target product per unit plant biomass increases, the downstream processing cost decreases rapidly.

Currently, large-scale transient expression systems for plants with sufficiently high yield and efficiency to compete in the market with other large-scale expression systems such as bacterial, fungal, or insect cell expression systems are Absent. The expression of such plants will need to meet the following criteria as well as possible:
(I) high yield, including expression of the protein of interest in as many plant tissues and as many cells of the tissue as possible;
(Ii) To prevent the detrimental effect of protein expression on plant cell survival, expression of the target protein or target product must be initiated simultaneously in all plant cells of the treated plant or plant tissue.

Typically, the target protein or target product accumulates to a certain point in each cell that produces the product or protein. However, the decomposition process often proceeds during accumulation, and the yield or quality of the target protein or target product tends to decrease. Therefore, after the expression switch is turned on, there is an optimal time to harvest the target product or target protein. To make the overall process efficient and profitable, this optimal time point should be reached simultaneously in all tissues or cells of the plant and in all plants of the selected lot;
(Iii) The system incorporates improved biosafety, and Agrobacterium used for agroinfiltration will have at least one of the following characteristics: low or zero survival in an open environment Infectivity to non-target organisms or non-target plants is low or zero.

  Thus, the object of the present invention can be extended to large-scale applications, with high yields of proteins to be expressed, and at the same time, less likely to transfect or transform non-target organisms with foreign DNA. It is to provide a safe method for expressing proteins in plant systems.

General description of the invention This object is achieved by a method of producing a target protein by transient expression of the target protein from the target sequence in the target plant or the target plant leaves, which method comprises:
A plant or a plant leaf is infiltrated with an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA in the presence or absence of a complementing factor. Including transfection,
The sequence encoding the replicon is
(I) a sequence necessary for a replicon function of a replicon derived from a plant virus, and (ii) a target sequence to be expressed,
And Agrobacterium strains are provided with a first genetic modification that makes transfection of T-DNA into plants, including plants or plant leaves, poor in the absence of a complement factor.

Furthermore, the present invention provides
(I) an Agrobacterium strain as defined above, and (ii) a plant or seed thereof encoding a complementing factor capable of complementing the defects of the Agrobacterium strain defined above,
A kit part is provided.

Furthermore, the present invention provides
(I) an Agrobacterium strain as defined above, and (ii) a second Agrobacterium strain encoding a complementing factor capable of complementing the defects of the Agrobacterium strain defined above,
A kit part is provided.

Furthermore, the present invention provides:
(I) having a first genetic modification that, in the absence of a complementing factor, impairs bacterial function required for introduction of T-DNA into a plant or plant leaf cell;
(Ii) poor conjugation capacity for plasmid transfer to other bacteria, and (iii) auxotrophic for the major metabolites required for Agrobacterium growth;
Agrobacterium of the genus Agrobacterium for the method of the present invention is provided.

  According to the general principles of nature, improving one characteristic tends to negatively affect the other, and vice versa, making it difficult to simultaneously improve two or more opposing characteristics of a system. is there. In a plant-based protein expression system, achieving high expression levels of the protein to be expressed and simultaneously achieving high biological safety means that high expression efficiency increases system selectivity. This is difficult because it tends to be low and thus biological safety tends to be low. For example, if an Agrobacterium strain used to infect a plant to achieve a high rate expression system is highly infectious, the Agrobacterium strain is likely to be transfected into a non-target plant and the recombinant T -There is a tendency for DNA to propagate into the environment, but this must be avoided. On the other hand, if the biological safety is improved by reducing the infectivity of the Agrobacterium strain, the transfection and expression efficiency in the target plant will be poor. Thus, biological safety and expression efficiency are such conflicting characteristics.

  The need for plant-based protein expression systems is even more complex when the system needs to be adapted for large-scale applications, ie, co-expression in the leaves of many plants or many plants. On the other hand, biological safety in large-scale applications is more important because it increases the likelihood that the transgenic organism will expand into an open environment. On the other hand, high protein expression efficiency and high yield are essential in large scale applications to achieve economically competitive methods.

The present invention provides for the first time a means for producing a large scale, highly efficient and biologically safe method for producing a protein of interest.
In the method of the present invention, the target protein is transiently expressed from the target sequence in a plant or plant leaf. Transient expression is achieved by transient transfection of plants or plant leaves. The term “transient transfection” means that the introduction of a heterologous DNA sequence is performed without selecting transfected cells to stably integrate the heterologous DNA sequence into the plant chromosome. The target sequence encodes the target protein. The target sequence is introduced into a plant or plant leaf using an Agrobacterium strain capable of introducing foreign DNA into a plant cell. The target sequence is heterologous to the plant virus from which the replicon is derived. That is, the method of the present invention does not include the case where a plant or plant leaf is transformed with a wild type plant virus. DNA sequences are heterogeneous because they contain heterologous target sequences. The target sequence is preferably not unique to the Agrobacterium strain.

  Plants or plant leaves are transiently transfected with Agrobacterium strains. Plants or plant leaves need to be transfected so that many cells and many tissues of the plant are transfected in one working step. This can best be achieved by agroinfiltration, but other methods that allow transfection of the main parts of the plant or plant leaf can also be applied. Therefore, it is preferable to transiently transfect plants or plant leaves by infiltrating the plant or plant leaves with an Agrobacterium strain. Infiltration is an important element of the present invention because it significantly contributes to the efficiency of the system. The higher the efficiency, the more cells of the plant or plant cells express the protein of interest and the better the onset of expression in various tissues and cells. Invasion allows transformation of many cells of a plant or plant leaf in a single work step, so that the point at which expression of the protein of interest proceeds in various plant tissues and in various parts of the plant is substantial. Tune in. Furthermore, infiltration substantially increases the likelihood that a significant percentage of plants or plant leaf cells will be transformed. Thus, the possibility of remote distance transfer of the replicon can further increase the overall efficiency, but transfection and expression are not dependent on remote distance transfer of the replicon in the plant or plant leaf.

  The method of the present invention is designed for large-scale application to plants, i.e. to be performed in parallel on many plants. In the method of the present invention, preferably at least 5, more preferably at least 10, more preferably at least 30, even more preferably at least 100, most preferably at least 1000 plants are transfected in parallel with Agrobacterium strains.

  When the method of the invention is carried out on the whole plant, transfection (preferably infiltration) is carried out on several parts of the plant. For example, transfect or infiltrate most leaves. It is preferred to transfect or infiltrate all leaves. More preferably, all leaves and stems of the plant are transfected or infiltrated. All tissues subsequently harvested to isolate the protein of interest should be transfected or infiltrated. Although expression and harvest in the roots can further increase the overall efficiency of the system, it is usually not necessary to infiltrate the roots. The whole plant, or at least the top part of its soil, can be infiltrated by immersing the plant upside down in a suspension of an Agrobacterium strain, applying a vacuum, and then quickly releasing the vacuum. This procedure can be scaled up and applied to many plants in sequence or simultaneously.

  When the method of the present invention is performed on plant leaves, infiltration is performed on one or more regions of the leaves. Preferably, the leaves are sprayed or dipped in a suspension of an Agrobacterium strain, preferably after which Agrobacterium cells are pressed or absorbed into the leaf tissue. The leaves of the plant used according to the present invention are preferably attached to the plant. The method of the present invention is preferably not performed on plant tissue culture.

  Invasion or Agroinvasion can be defined as a transfection method using Agrobacterium suspension, and pressure difference is used to press Agrobacterium against plant tissues (cell gap).

  Alternatively, as is done to deliver virus particles (Pogue et al., 2002, Annu. Rev. Phytopathol., 40, 45-74) to deploy a mixture of Agrobacterium and abrasive to plants Agrobacterium can be delivered to plant tissue using a high pressure spray device.

  The Agrobacterium strain is provided with a first genetic modification that makes T-DNA poorly transfected into the organism in the absence of complementing factors. This is an essential element of the present invention for improving biological safety. This organism is an organism in which transfection with heterologous DNA sequences is undesirable ("non-target organism"). Non-target organisms are, inter alia, plants and microorganisms such as bacteria or fungi. In the absence of a complement factor, the plant species of the invention is not a non-target organism. In the absence of complementing factors, Agrobacterium strains are not capable of infecting or transducing such non-target organisms and are preferably not possible.

  Agrobacterium strains, plants (or plant leaves), and complement factors are selected to allow transfection and / or expression of the target sequence upon infection of the plant by the Agrobacterium strain, while non-targeting Organism transfection and / or expression is unlikely. The Agrobacterium strain deficiency, preferably inability, with respect to transfection of non-target organisms is achieved by genetic modification, referred to hereinafter as the first genetic modification of the Agrobacterium strain.

  The first genetic modification preferably makes the function of the Agrobacterium strain necessary for introduction of T-DNA into a plant or plant leaf cell poor. This can be accomplished by deleting or disrupting the Agrobacterium gene that encodes a function required for transfection of the target organism. Such a function is encoded inter alia on a Ti-plasmid, for example a Ti-plasmid having T-DNA to be introduced into a plant or plant leaf cell. Examples of such genes are the Agrobacterium genes VirE2, GALLS, and VirF.

  In other embodiments, the first genetic modification renders the Agrobacterium strain auxotrophic, thereby making its growth dependent on exogenously added metabolites. In the absence of externally added metabolites, Agrobacterium strains cannot transfect organisms with T-DNA.

  In a further embodiment, as a first genetic modification, the function of the Agrobacterium strain required for transfection of the organism is placed under the control of a chemically regulated heterologous promoter. Examples of such genes are the Agrobacterium genes VirE2, GALLS, and VirF, which can be placed, for example, under the control of the lac promoter. In such embodiments, the complementing factor is a small molecule compound that can regulate a chemically regulated promoter. In the case of the lac promoter, the complement factor can be IPTG or lactose. Thus, Agrobacterium suspensions applied to plants or plant leaves allow small molecule compounds such as IPTG or lactose to allow plant infection and preferably introduction of T-DNA into plant cells. Can be contained.

  In other embodiments, the first genetic modification renders the Agrobacterium strain conditional lethal and the complementing factor is the major metabolite of the Agrobacterium strain that is necessary for the survival of the Agrobacterium strain.

  The above aspects can be combined. For example, if the Agrobacterium strain is deficient in the function required for the introduction of T-DNA into a plant or plant leaf cell, to further increase the biological safety of the system and / or Or it can be conditionally lethal.

  The complement factor provides a function that has been compromised by the first genetic modification. In the presence of the complementing factor, the transfection of T-DNA into the plant or plant leaf cells by the defective Agrobacterium strain is restored. The complement factor can be a small molecule compound or, depending on the first genetic modification, a polymeric compound such as a protein or a nucleic acid encoding the protein. If the Agrobacterium strain is auxotrophic, the complementing factor allows the Agrobacterium strain to grow and infect. If the Agrobacterium strain has the defective VirE2, GALLS, and VirF genes as the first genetic modification, the complementing factors are VirE2, GALLS, or VirF, respectively, or encode them. If the Agrobacterium strain is under the control of a chemically regulated promoter, the complementing factor can be a small molecule compound capable of regulating, preferably inducible, a chemically regulated promoter.

  Supplementary factors need to be provided to fulfill their purpose. If the cofactor is a small molecule compound, it can be present in an aqueous suspension of an Agrobacterium strain used for plant or plant leaf infiltration. If the cofactor is a protein, it can also be added to this aqueous suspension. However, it is preferred that a complementing factor that is a protein (eg, VirE2, GALLS, or VirF) is encoded by DNA and provided to be present in the plant or plant leaf upon infiltration. In one embodiment, a plant or plant leaf is infiltrated with a second Agrobacterium strain that can introduce a complement factor or DNA encoding the plant into the plant or plant leaf. The second Agrobacterium strain is preferably an Agrobacterium strain that does not have the first genetic modification and does not have the heterologous DNA sequence of the present invention. The Agrobacterium strain and the second Agrobacterium strain of the present invention can be used as a mixture when infiltrating plants or plant leaves. If a small amount of such a mixture (preferably included) escapes the environment for carrying out the method of the present invention, a mixture of such Agrobacterium strains will quickly increase the likelihood that the two strains will be in close proximity to each other. Diluted quickly to lower. Thus, non-target organisms will not touch two Agrobacterium strains simultaneously, and therefore neither artificial nucleic acids nor foreign nucleic acids (eg, heterologous DNA sequences) will transform such non-target organisms.

  In other more preferred embodiments, the complement factor is stably or transiently expressed in the plant or plant leaf. Stable expression of the complement factor is preferred. In this aspect, the plant is provided with a genetic modification encoding a complement factor (hereinafter referred to as “second genetic modification” in the claims). Since the Agrobacterium strain exclusively transforms plants with the second genetic modification, a high degree of biological safety can be achieved in this way. The likelihood that Agrobacterium strains will transform undesirable organisms or plants is very low because these organisms lack the second genetic modification.

  The biological safety of the methods described so far can be further improved in various ways. The sequence portion encoding the replicon can encode a replicon with a poor function that allows propagation of the replicon in the plant or plant leaf. The function can be a function for remote distance transfer or cell-to-cell transfer of a replicon, such as a virus coat protein or a virus transfer protein, respectively. Preferably, the replicon lacks at least the possibility of remote distance transfer, thereby avoiding the propagation of the replicon to other plants. Deletion of the possibility of distant migration can in principle be inconvenient for protein expression efficiency. However, the system of the present invention overcomes this disadvantage by other elements of the present invention, for example, by infiltrating a plant or a major part of a plant leaf, and / or by the improved methods of RNA replicon formation described below. Can be complemented.

  Other approaches to further improve biological safety are the use of Agrobacterium strains for transfection of plants expressing the tumor suppressor active protein Osa (REF) and a second to provide the virE2 gene. Of Agrobacterium strains. Furthermore, the Agrobacterium strain can have a genetically modified cell number sensing system or pathogenicity-inducing regulatory system to reduce T-DNA transmission to non-target organisms.

  The biological safety of the method of the present invention can also be improved so that Agrobacterium strains have further genetic modifications that make plasmid DNA poorly transmitted to other bacteria. Conjugative transmission can be achieved by disabling the function of at least one of the following Agrobacterium genes: oriT, traG, and traF.

  The sequence portion of the heterologous DNA sequence may encode a DNA replicon or an RNA replicon. An RNA replicon is preferred, and the following description mostly relates to the sequence portion encoding the RNA replicon.

  The sequence portion encoding the replicon contains a sequence necessary for replicon formation and a target sequence to be expressed from the replicon. The target sequence to be expressed typically encodes the target protein and can contain regulatory sequences for translating the target protein from the replicon or from the subgenomic RNA of the RNA replicon.

  When the replicon is an RNA replicon, the sequence necessary for the RNA replicon function of replicon (i) corresponds in particular to the sequence of the RNA virus when the former is the latter DNA copy (eg cDNA). In the case of a DNA replicon, the sequence relating to the replicon function provides a DNA replicon having the function of replicating in the cell nucleus; the DNA replicon may be formed from the heterologous DNA sequence by replication using the heterologous DNA sequence or part thereof as a template. it can. In the case of an RNA replicon, the sequence for replicon formation provides an RNA replicon that has the function of replicating in the cytoplasm. The sequence for RNA replicon function typically encodes one or more proteins involved in replication, such as RNA-dependent RNA polymerase (RdRp, replicase). The sequence for the replicon function may further be or encode a function necessary for expression of a replicase such as a subgenomic promoter, transcription enhancer, or translational enhancer. Proteins such as translocation proteins or coat proteins that are involved in cell-to-cell or osmotic propagation of RNA viruses in plants can be present in heterologous DNA sequences but have no replicon function. In any case, the sequence portion encoding the replicon does not encode a wild type plant virus. The sequence for the replicon function is preferably derived from the sequence of the plant RNA virus since plant RNA viruses are an easily accessible source for replicon function. In the case of an RNA replicon, “derived from” means that the sequence for the replicon function is a DNA copy of the corresponding sequence of the RNA virus, and the DNA copy is incorporated into a heterologous DNA sequence contained in the cell nucleus. “Derived” means that the sequence related to the replicon function need not be an exact DNA copy of the corresponding RNA sequence of the RNA virus, but can exhibit a function-conservative difference as described below. Including. Since the differences are conservative, the sequence for the replicon function preferably encodes a protein capable of performing the replicon function, as is the case for RNA viruses.

  In embodiments where the replicon is based on a tobamovirus such as tobacco mosaic virus, the replicon contains or encodes RdRp. This replicon can further encode a translocation protein. This replicon preferably does not encode or contain a coat protein (CP).

  In a further preferred embodiment, the biological safety of the method of the invention is further improved by using a highly diluted suspension of Agrobacterium strain. This aspect not only reduces the potential for Agrobacterium strain cells to spread to the environment, but also reduces the exposure and stress of plants or plant leaves when infected with an Agrobacterium strain that is a pathogen for the plant. Improves the efficiency of the method of the invention. The inventors have surprisingly found that within certain limits, the efficiency of the process increases with decreasing concentration of the Agrobacterium suspension used in step (a) of the present invention. In particular, the ability of the replicon produced in plant cells to move between cells increases with decreasing concentrations of these Agrobacterium suspensions. The reason for this unexpected phenomenon has not yet been identified. This phenomenon is due to the plant's response to Agrobacterium infection, and it is speculated that this reaction does not occur (or occurs to a lesser extent) at low Agrobacterium concentrations.

  In this preferred embodiment, a cell suspension of an Agrobacterium strain with an OD (optical density) of 600 nm of at least 25 times, preferably at least 100 times, more preferably at least 250 times, most preferably at least 1000 times. A plant or plant leaf is infiltrated with a cell suspension of an Agrobacterium strain at an Agrobacterium cell concentration that can be obtained by diluting to a low concentration. Such dilution can be achieved by using an agglomeration with a calculated OD value at 600 nm of at most 0.04, preferably at most 0.01, more preferably at most 0.004, most preferably at most 0.001. This results in a bacterial suspension.

  Next, when the replicon is an RNA replicon, an important aspect is described that significantly increases expression efficiency. In this embodiment, the sequence related to the replicon function exhibits a function-conservative difference from the plant RNA virus sequence at a selected position of the plant RNA virus sequence, and the difference is compared to an RNA replicon that does not exhibit the difference. Thus, the frequency of replicon formation is increased. The difference causes an increased frequency of replicon formation in plant cells once the overall process is switched on (see below). The causal relationship between increased replicon frequency and differences is experimentally determined by comparing the frequency of replicon formation between sequences with replicon functions that have differences and sequences with replicon functions that have no differences. Can be tested. Such experimental comparison can be performed, for example, by counting protoplasts expressing the target sequence, as described in the Examples. A target sequence encoding a reporter protein such as easily detectable green fluorescent protein (GFP) is preferably used for this purpose. Further, as described below, it is also preferable to make an experimental comparison with an RNA replicon that cannot be transmitted between cells.

  Function-conservative differences are introduced into sequences for replicon function at selected positions in the plant RNA virus sequence. The selected position is located within a sequence relating to the replicon function of the plant RNA virus, which is responsible for the low probability that the RNA replicon transcribed in the nucleus will appear in the cytoplasm as a functional replicon. Such a selected position has a high A / T (U) content, ie a high A content and / or a high T content (high U content at the RNA level) or a potential splicing site, ie a nuclear splicing device. It is preferable to have a sequence portion recognized as a splicing site. The selected position can be identified in the RNA virus on which the RNA replicon is based by analyzing the RNA profile of the RNA virus, as exemplified below. Furthermore, the selected position can be identified experimentally by analyzing RNA formed in plant cells after transfection with a heterologous DNA encoding an RNA replicon that does not exhibit differences according to the present invention. This experimental analysis can be performed by RT-PCR, preferably in conjunction with sequencing of the RT-PCR product. In this way, unwanted splicing products can be identified that exhibit splicing events that disrupt the RNA replicon. Furthermore, the exact site of unwanted splicing can be identified and corrected by introducing function-conservative differences at selected locations.

  Function-conservative differences increase RNA replicon formation frequency by suppressing the deleterious effects of selected positions on RNA replicon formation frequency. A function-conservative difference can include a decrease in the high A / U content in the RNA replicon by reducing the high A / T content in the sequence related to the replicon function of the sequence encoding the RNA replicon. High A / U content can be reduced by at least partial deletions or at least partial replacements with G / C bases (eg, using degeneracy of the genetic code) as long as the differences are function-conservative. . Furthermore, potential splicing sites adjacent to A / U rich regions of plant RNA virus-derived sequences can be removed. Such function-conserving differences can be introduced at one, preferably several selected locations.

  A preferred functionally conservative difference is the insertion of one or more introns near or into the A / U-rich position of the sequence derived from the plant RNA virus sequence, most preferably a nuclear intron, or a nuclear intron. Insertion of one or more sequences capable of forming Surprisingly, it has been discovered that introduction of introns at or near the A / U-rich location results in increased frequency of RNA replicon formation. Several introns can be introduced and are exemplified herein for varying numbers of inserted introns. The effect of more than one intron is cumulative. Furthermore, intron insertion can be combined with other function-conservative differences at other selected locations.

  FIG. 11 shows an example of introduction of a sequence capable of forming a nuclear intron, although it is within the target sequence to be expressed. In the example of FIG. 11, an intron is formed from two half-introns by inversion catalyzed by a recombinase of a portion of the heterologous DNA sequence. This principle can also be applied to sequences relating to the replicon function of RNA replicons. In embodiments where two different RNA replicons are formed in the same cell, recombination between two different replicons can form one intron from two half-introns present on different replicons. Furthermore, RNA replicons can be formed by recombination between two replicon precursors, both of which are not replicons. Again, one intron can be assembled from two half-introns derived from different replicon precursors.

  A heterologous DNA sequence having a sequence portion that encodes an RNA replicon is operably linked to or capable of being linked to a transcription promoter since transcription is a prerequisite for RNA replicon formation. In order to achieve transient expression of the target sequence in all tissues infiltrated with agro, the transcription promoter is preferably a constitutive promoter. Examples of constitutive promoters are known in the art.

  In one aspect of the invention, the sequence encoding an RNA replicon has one or more segments that together encode the RNA replicon. That is, the RNA replicon is not encoded by one continuous DNA. In fact, the RNA replicon is encoded discontinuously by two or more segments so that the segments can be present on the same T-DNA, preferably adjacent to each other. Second, RNA replicon formation may require rearrangement of segments, eg, by recombination. Recombinases for recombination can be provided by additional Agrobacterium strains or by genetically engineered plant hosts, thus allowing transient expression to remain in the plant host. By way of example, a portion of a sequence for replicon function (eg, a portion of a sequence encoding a replicase) can be present in a heterologous DNA sequence in an inverted orientation relative to other portions of such sequence. The inversion portion can be adjacent to the recombination site. Second, because a replicon function cannot be provided (eg, the transcript does not encode a functional replicase), the transcript of the heterologous DNA will not be a replicon. By providing a site-specific recombinase that recognizes the recombination site, one of the segments is inverted so that the replicon is encoded continuously. In this aspect, by providing the recombinase, it functions as a switch for switching to replicon formation and expression of the target sequence (see further below) and can contribute to a high level of biological safety.

  Alternatively, one of the segments can be present on the Agrobacterium T-DNA and the other can be integrated into the plant chromosome. Second, the formation of an RNA replicon will require transcription of both segments and trans-splicing of both transcripts to assemble the RNA replicon. This embodiment can be used to rapidly isolate segments that together encode an RNA replicon in a progeny plant or cell, as described in detail in PCT / EP03 / 02986, and the biological safety of the system Can contribute to sex.

  The method of the present invention involves transient transfection of a plant or plant leaf with the heterologous DNA sequence of the present invention (step (a)). The term “transient transfection” means introducing heterologous DNA without selecting for transformed transgenic cells that have the heterologous DNA stably integrated into the plant chromosome. Transient transfection usually provides for transient replication and / or transient expression of a gene encoded by a heterologous DNA sequence. The transfection of the present invention causes expression of the target sequence. Expression usually occurs spontaneously after providing a heterologous DNA sequence to the plant or plant leaf by agroinfiltration. Various methods of causing or switching on can be used as needed. When using a recombinase to switch on the method of the present invention, the recombinase can be provided transiently to the plant or plant leaf, and the provision can act as an expression switch for the protein of interest. Such a recombinase is preferably stably encoded in plant cells and is capable of expressing the recombinase under the control of a constitutive or stress-inducible promoter. By inducing recombinase expression by inducing a promoter, expression of the target sequence can be caused.

  Step (b) of the method of the present invention comprises isolating the protein of interest from the plant or plant leaf transfected, preferably infiltrated in step (a). Isolation of the target protein preferably includes homogenization of the plant or plant leaf containing the expressed target protein. Various methods for isolating proteins from plants are described in PCT / EP02 / 09605, incorporated herein, and the literature cited therein.

  The present invention can in principle be applied to any plant in which infectious DNA or RNA viruses exist and agroinfiltration providing transient expression is effective. Preferred plants are tobacco species such as Bensamiana tobacco and tobacco; preferred plant species other than tobacco species are petunia, rape, mustard, cress, arugula, mustard, strawberry, spinach, red aza, lettuce, sunflower, and cucumber It is.

  Suitable plant / DNA or RNA virus pairs can be derived from the list of DNA and RNA viruses shown below. In particular, because of the very high rate of RNA replicon formation according to the present invention, plant species specificity of plant viruses is hardly noticeable when practicing the present invention. Similarly, the present invention can be used with RNA replicons based on any RNA virus. RNA viruses will usually have a selected location that deploys outside the cell nucleus of the host plant and renders the replicon based on such viruses ineffective when the replicon is produced in the cell nucleus. The present invention can be applied to all RNA viruses, although the level of improvement can vary between different plant RNA viruses. The most preferred plant RNA viruses on which the present invention can be based are potex viruses such as tobamoviruses, in particular tobacco mosaic virus, and potato X virus. In the case of tobacco mosaic virus, it will usually be a coat protein replaced by the target sequence to be expressed. The transition protein can also be removed or replaced by the sequence of interest to be expressed. Preferably, however, the RNA replico derived from tobacco mosaic virus encodes a translocation protein and the coat protein needs to be replaced by the target sequence.

Preferred combinations of plants and viruses from which the RNA replicon is derived are: tobacco species and tobamovirus, tobacco species and tobacco mosaic virus, and the like.
Among DNA viruses, the most preferred viral vector can be constructed with geminivirus. A description of a viral expression vector based on Bean Golden Mosaic Virus (BGMV) is provided in Example 15.

  The main application of the present invention is the production of the target protein in plants or plant leaves. When the method of the invention is carried out on plants, plants that do not enter the human or animal food chain, such as tobacco species, are preferred. The whole plant or plant part after agroinfiltration will be trapped in a closed environment, such as a greenhouse or specially designed chamber, for the incubation time necessary to provide the desired level of expression.

  The efficiency of the present invention is such as to achieve a new aspect of the plant expression system. The expression levels achievable with the present invention are such that downstream processing costs (including separation and purification of the protein of interest) are low enough to make the method of the present invention competitive with other large-scale expression systems. In the prior art expression system using stably transformed plants, the replicon is produced in a small fraction of cells, so the expression level is low even when using a virus based vector. Replicons that propagate through plants cannot correct this problem, especially because of the slow propagation to remote distances. Therefore, the expression does not proceed uniformly in the plant, and the target protein is already degraded in some parts of the plant, but protein expression has not started in others. Furthermore, transient expression systems based on prior art transfection or agroinfiltration do not provide high expression levels or high biological safety in one production system. The present invention uniformly induces expression in whole plants or isolated plant parts (eg leaves) via agro-mediated delivery of competent plant hosts. A small fraction of cells that do not produce replicons can be rapidly infected with replicons from neighboring cells. The present invention provides the first high-yield plant expression system that can be used on a large scale. The present invention also provides high biological safety by limiting transient expression systems to specifically engineered competent plant hosts, thereby establishing expression in non-competent (non-target) organisms. Reduce the biological safety of the system.

Preferred Embodiments of the Invention A method for producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
Transfecting a plant or plant leaf by infiltrating the plant or plant leaf in the presence of a complementing factor with an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding an RNA replicon in T-DNA Including
The sequence encoding the replicon is
(-) A sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (-) a target sequence to be expressed,
Containing
Agrobacterium strains are provided with a first genetic modification that makes transfection of T-DNA into organisms poor in the absence of complementing factors.

A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
A plant or plant leaf is preferably infiltrated into a plant or plant leaf in the presence of a complementing factor with an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding an RNA replicon in T-DNA. Including transfection,
The sequence encoding the replicon is
(-) A sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (-) a target sequence to be expressed,
Containing
The Agrobacterium strain has been provided with a first genetic modification that makes T-DNA transfection into organisms poor in the absence of the complement factor, and the plant or plant leaf encodes the complement factor. Having a second genetic modification that expresses it in the plant or plant leaf.

A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
RNA having a cell concentration corresponding to a calculated optical density at 600 nm of at most 0.04, preferably at most 0.01, more preferably at most 0.004, most preferably at most 0.001 Plant or plant leaf by infiltrating the plant or plant leaf in the presence of a complementing factor with a cell suspension of an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA Transfecting, and
The sequence encoding the replicon is
(-) A sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (-) a target sequence to be expressed,
Containing
The Agrobacterium strain is provided with a first genetic modification that renders the transfection of T-DNA into an organism poor in the absence of a complement factor, and the sequence required for replicon function is selected at the selected position. Thus, a function-conservative difference from a plant RNA virus is exhibited, and the difference increases the frequency of replicon formation compared to an RNA replicon that does not exhibit the difference.

Further preferred embodiments are defined in the claims.
DETAILED DESCRIPTION OF THE INVENTION We have developed a highly active synthetic template for delivering RNA viral vectors as DNA precursors using soil bacteria and an effective vector, Agrobacterium. . Herein, this improved “Agro infection” method can be used to initiate transient gene amplification and high level expression simultaneously in virtually all mature leaves of a plant, and such transfection The route can be implemented inexpensively on an industrial scale. Target plant host organisms in a manner that limits production to genetically engineered “competent” plant hosts and can limit the ability of Agrobacterium to infect other plants or microorganisms that are not part of the production process It also addresses biological safety issues by genetically manipulating Agrobacterium and / or plant viruses. This technology combines the advantages of three biological systems: virus rate and expression level / yield, Agrobacterium transfection efficiency and osmotic delivery, and plant post-translational capacity and low production costs. The proposed method enables an industrial production pathway that does not require stable genetic modification of plants with heterologous nucleic acids encoding the target product, is safer and more controlled, and is compatible with the recent industrial base.

  The present invention describes a transient expression system for high-yield large-scale production of a protein of interest using Agrobacterium-mediated delivery of a viral replicon or its precursor. The replicon can express the target sequence, the system has built-in biological safety, and at least two of the three major components of the system (genetically engineered plant host, Agrobacterium, replicon) To be interdependent.

  Surprisingly, such interdependencies caused by partial delegation of functions from one of the system's main components to the other make the entire method more effective without adversely affecting system efficiency. It has been discovered that it can be well controlled and increase biological safety. The basic principle of the present invention is shown in FIG. A large-scale protein production method based on agroinfiltration of a viral replicon precursor is a transgenic host plant that complements Agrobacterium and / or replicon used for agroinvasion when performing functions necessary for expression of the target protein. Can be implemented. One example of such complementation for RNA replicons is described in Example 4. In this example, the entire plant was transiently transformed with a heterologous DNA sequence encoding an RNA replicon lacking a functional transfer protein (MP) (FIG. 5). This replicon cannot move between cells, and as a result it cannot provide high yields in plant hosts that do not have an expression cassette (pICH10745, FIG. 5) with viral MP.

This specification for increasing the efficiency of RNA release from the nucleus to the cytoplasm when the functions required for activation or manipulation of viral replicons or transient transfection are provided in trans by plants or other Agrobacterium strains When using the criteria described in, there is no negative effect on the expression efficiency. By incorporating plant introns into certain regions encoding viral RNA replicons, or by removing or replacing potential introns within sequences for replicon function, the efficiency of the replicon in the host plant can be dramatically added (at least 10 ² times). Such an increase in efficiency was reflected in at least one easily measurable parameter: the relative proportion of cells exhibiting vector replication, i.e. an increase in the frequency of replicon formation. Such optimization of the initiation of RNA replicon formation results in the ability to simultaneously switch on the expression switch of the sequence of interest substantially throughout the plant, dramatically increasing the yield of the protein of interest in a shorter time than the unmodified vector. Examples 6-14 of the present invention describe an improved vector approach to increase the efficiency of RNA viral vector release from the nucleus to the cytoplasm. A detailed description of viral vector improvements has recently been published by Marylonnet and colleagues (2005, Nature Biotechnol., 23, 718-723). This improvement is an important aspect of the present invention because it allows functions to be efficiently exchanged between RNA replicons, plant hosts, and Agrobacterium. Interestingly, such improved vectors based on TMV work efficiently in plant species other than tobacco, such as petunia, rape, mustard, and cress, arugula, and mustard. The most expressed non-solanum species are strawberry, spinach and red akaza. Compositae such as lettuce (especially seedlings) and sunflowers and cucumbers of the family Cucurbitaceae can be used successfully with several plant species from different plant families, preferably after further optimization of the method. Show.

  Publications on increased nuclear transgene expression by incorporation of introns into the coding region of recombinant DNA (Mascarenhas et al., 1990, Plant Mol. Biol., 15, 913-920; Bourdon et al., 2001, EMBO Reports, 2, 394-398; Rose, AB., 2002, RNA, 8, 1444-1453; US Pat. No. 5,955,330), the prior art includes the incorporation of introns into viral RNA replicon precursors. There is no implied indication that it will have a positive effect on the frequency of formation and the expression level of the target sequence from the replicon. This effect is surprising considering that nuclear mRNA transcription and viral RNA replication occur in different intracellular compartments. Even when a cDNA copy of the RNA replicon is placed in the nucleus, only the first copy of the viral replicon precursor is produced in the nucleus and then amplified in the cytoplasm under conditions different from those in the nucleus. The prior art describes the use of introns to prevent the “leaking” expression of viral genes in E. coli during cloning with wild-type viral cDNA (Johansen, IE, (1996, Proc. Natl. Acad. Sci. USA, 93, 12400-12405; Yang et al., 1998, Arch. Virol., 143, 2443-2451; Lopez-Moya & Garcia, 2000, Virus Res., 68, 99-107). . There is no suggestion that intron inclusion can increase the frequency of replicon formation from viral cDNA clones.

  The present invention provides a method for radically increasing the frequency of formation of RNA virus-derived replicons derived from transcription of a DNA precursor and designed for expression of a target sequence. This method overcomes the limitations of expression systems based on existing viral vectors, such as the size limitations of heterologous sequences to be expressed and the high degree of vector instability. Furthermore, this method provides better biological safety and allows the design of leak test controls for transgene expression (zero expression level in non-induced state). This is because such a design is an integrated part of the strategy for designing RNA virus-derived replicons. By providing high-frequency formation of RNA virus-derived replicons, the approach described herein enables the sequence of interest in whole plants or parts of plants such as leaves that contain heterologous DNA sequences encoding RNA replicons. Rapid onset of expression. By practicing the present invention, the performance of substantially all replicons derived from plant RNA viruses designed for the expression of heterologous target sequences via agroinfiltration of plants or plant leaves is determined by the frequency of replicon formation. Can be significantly improved by a dramatic rise in

DNA and RNA viruses belonging to various taxonomic groups are suitable for the construction of RNA replicons according to the present invention. A list of DNA and RNA viruses to which the present invention can be applied is presented below. A quoted taxon name (not italic) indicates that this taxon does not have an internationally recognized name on ICTV. The species name (common name) is shown in standard form. Viruses not officially assigned to a genus or family are indicated:
DNA virus:
Circular double-stranded DNA virus: family: caulimovirus family, genus: Badonauirusu genus, type species: Commelina macular virus, Genus: caulimovirus genus, type species: cauliflower mosaic virus, Genus: "SbCMV-like virus", a reference species: soybeans Retrograde macula virus, genus: “CsVMV-like virus”, reference species: cassava vein mosaic virus, genus: “RTBV-like virus”, reference species: rice tungro bacilliform virus, genus: “petunia vein permeability-like virus”, reference species : Petunia vein permeability virus ;
Circular single-stranded DNA viruses: family: geminiviridae, Genus: Masutore genus (subgroup I geminiviruses), type species: maize streak virus, Genus: Kurt genus (Subgroup II geminiviruses), type species: Beat curly top virus, genus: begomovirus genus (subgroup III geminivirus), reference species: bean golden mosaic virus;
RNA virus:
Single-stranded RNA virus: family: bromo virus family, the genus: alpha mode genus, type species: alfalfa mosaic virus, Genus: Ira Le genus, type species: tobacco streak virus, Genus: bromo genus, type species: Blom Mosaic virus, genus: genus Cucumovirus, reference species: cucumber mosaic virus;
Family: Cross-terrorism virus family, the genus: Cross-terrorism genus, type species: beat macular virus, Genus: Clinic genus, type species: Lettuce infectious macular virus, family: Como virus family, the genus: Como genus, type species : cowpea mosaic virus, genus: file server virus genus, type species: broad bean gangrene virus 1, genus: Nepouirusu genus, type species: tobacco ringspot virus;
Family: Potiviridae , Genus: Potivirus genus , Reference species: Potato virus Y, Genus: Rimovirus genus , Reference species: Rigosa mosaic virus , Genus: Bimovirus genus , Reference species: Barley stripe dwarf virus;
Family: cough virus family, the genus: cough genus, type species: parsnip macular virus, Genus: Wa squid genus, type species: rice tungro spherical virus, family: ton ugly virus family, the genus: Carmo genus, type species: carnation mottle virus, genus: Diane Seo genus, type species: carnation Waten virus, genus: macro mode genus, type species: maize yellow mottle virus, genus: Necro genus, type species: tobacco necrosis virus, genus: Tonbusu Virus genus , reference species: tomato bushy hatching virus, genus unclassified single-stranded RNA virus, genus: capirovirus genus , reference species: apple stem grooving virus;
Genus: Carlyle virus genus , Reference species: Carnation latent virus, Genus: Enamovirus genus , Reference species: Pea fold leaf mosaic virus,
Genus: phlovirus genus , reference species: soil infectious wheat mosaic virus, genus: hordei virus genus , reference species: barley spotted mosaic virus, genus: idaeovirus genus , reference species: raspberry bushy dwarf virus;
Genus: luteoviruses genus type species: barley Ki萎virus, Genus: Mara Fi genus, type species: maize rayado fino virus, Genus: potexvirus genus type species: potato virus X; Genus: sobemoviruses genus reference species : Southern bean mosaic virus, Genus: Tenui virus genus , Reference species: Rice stripe virus,
Genus: Tobamovirus genus , reference species: tobacco mosaic virus,
Genus: Tobravirus , Reference species: Tobacco stem gangrene virus,
Genus: Trichovirus genus , reference species: apple chlorotic leaf spot virus, genus: Timovirus genus , reference species: turnip yellow mosaic virus, genus: Ambra virus genus , reference species: carrot mottle virus; minus ssRNA virus: eyes: mononegative virus eyes, family: Rhabdoviridae, genus: site Love de genus, type species: lettuce necrotic macular disease virus, the genus: nucleocapsid Rhabdoviral genus, type species: potato yellow Fusarium virus;
Minus single-stranded RNA virus: Family: Bunyaviridae, Genus: Tospovirus , Reference species: Tomato yellow gangrene virus;
Double-stranded RNA virus: Family: Partiviridae , Genus: Alpha cryptovirus genus , Reference species: White clover latent virus 1, Genus: Beta cryptovirus genus , Reference species: White clover latent virus 2, Family: Reoviridae , genus : Fiji virus genus , reference species: Fiji disease virus, genus: phytoreovirus genus , reference species: wound tumor virus, genus: Orizavirus genus , reference species: rice rugged stunt virus;
Uncategorized virus:
Genome: Single-stranded RNA, Species: Garlic virus A, B, C, D, Species: Grape vein fleck virus, Species: Corn white line mosaic virus, Species: Olive latent virus 2, Species: Urumiamelon virus, Species: Pelargonium Zonate spot virus.

  The general principle of the present invention is shown in FIG. Many different approaches can be used to implement the present invention. These approaches can be based on the use of Agrobacterium mutants that cannot provide transient expression, and the mutants can be complemented in trans by a transgenic host plant or a second Agrobacterium strain.

  In order to increase the biological safety of the system, the present invention prevents Agrobacterium infection in susceptible plants, thereby increasing control over transient expression methods. There is comprehensive research on the mechanism of Agrobacterium T-DNA transfer to plant hosts, which can be successfully applied to the present invention.

  One possible approach is to explicitly limit Agrobacterium strains with heterologous DNA sequences encoding viral replicons to genetically engineered plant hosts. The osa gene of plasmid pSA encodes a tumor suppressor protein that suppresses transient expression by suppressing the virE2 protein necessary for T-DNA transport, and further suppresses integration into plant nuclear DNA (Lee, LY et al., 1999, J. Bacteriol., 181, 186-196). However, this suppression can be reversed by mixing Agrobacterium expressing the osa gene with Agrobacterium having wild-type virE2 but no osa gene, for example by complementing the mutant phenotype with trans. it can. Similar results can be achieved by creating transgenic plants that express virE2. T-DNA is transported to plant cells as a single-stranded intermediate with VirD2 covalently linked to its 5 ′ end, but Agrobacterium mutants lacking virE2 are not carcinogenic, so virE2 Is an important component of this method. The oncogenicity of such mutants can be restored by inoculating a transgenic plant expressing virE2 (Citovsky, V. et al., 1992, Science 256, 1802-1805). In addition, there are completely acetosyringone-dependent Agrobacterium mutants. In Example 5 of the present invention, the ability of an Agrobacterium strain having a heterologous DNA sequence of the present invention to depend on a genetically engineered plant lodging or wild type Agrobacterium to complement the virE2 protein in trans The expression method is described. Agrobacterium lysogenes GALLS protein can also complement the function of VirE2 (Hodges et al., 2004, J. Bacteriol. 186, 3065-3077). Such a system significantly improves the control of heterologous DNA propagation and increases the biological safety of the system. As described in this example, no expression of the target protein (GFP) was actually detected after agroinfiltration of wild-type N. benthamiana tobacco plants by Agrobacterium lacking virE2. However, when GFP is efficiently infiltrated into a complemented host plant that expresses virE2 or when Agrobacterium is agroinfiltrated in a mixture with an Agrobacterium strain having this first genetic modification, GFP is efficient. Expressed. In fact, the efficiency of T-DNA delivery by Agrobacterium lacking virE2 was reduced by about 10,000-fold compared to wild-type Agrobacterium and mutant Agrobacterium supplemented with trans (Examples). 5, see FIG.

  Approaches for designing systems with enhanced biological safety are not limited to trans-complementation of virE2 function. The virF gene, like virE2, can also be used to produce a plant host suitable for transient expression by complementing virF-deficient Agrobacterium (Regensburg-Twink, AJ. & Hooykaas, PJ., 1993). , Nature, 363, 69-71). Interestingly, the osa protein prevents both virE2 and virF protein secretion (Chen, L. et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 7545-7550). Alternatively, the Agrobacterium tumefaciens IncP-type or IncN-type conjugation transfer system can be introduced into a different bacterium, E. coli strain, which can transmit T-DNA in the presence of a safe Agrobacterium tumefaciens strain. (Pappas, KM. & Winans, SC., 2003, Appl. Envir. Microbiol., 69, 6731-6739). The latter is for designing a system with enhanced biological safety having a competent acceptor host and one or more bacterial donor strains (eg donor and helper strains) limited to the competent host. It shows many different possibilities. This approach significantly limits the potential for unwanted T-DNA transfer to other organisms.

  In addition to Agrobacterium's ability to transmit T-DNA to plant cells, Agrobacterium, like many other bacteria, transfers genetic material (usually extrachromosomal plasmid DNA) through conjugation with other bacteria. You can also The ability of such plasmid DNA (eg, binary vectors) to transfer from industrial Agrobacterium strains to other Agrobacterium strains (eg, wild-type Agrobacterium) jeopardizes control over transgene release to the environment Will. In many cases, such conjugation transfer involves many binary vectors that do not contain the structural elements necessary for conjugation transfer (eg, oriT, which represents the origin of transmission), and thus the binary plasmid from E. coli to the desired Agrobacterium strain. A binary vector having a T-DNA region having a transgene is recombined with a safety Ti plasmid, since it can be introduced into an Agrobacterium strain only by direct transformation, without a three-parental junction based on conjugation transfer. Only possible if. For a review on various binary vectors, see Helens, R .; Mullineaux, Ph. & Klee, H .; (2000, Trend Plant Sci., 5, 446-451). However, even if the binary vector does not contain oriT, there is still a possibility of recombination between the binary vector and the resident safety Ti plasmid. Such recombination can result in the formation of a plasmid containing a T-DNA region with a transgene so that the plasmid can be conjugatively transferred between different bacteria. Several genes are involved in conjugative transmission between Agrobacterium cells (Cook et al., 1997, J. Bacteriol., 179, 1291-1297; Beck et al., 1989, J. Bacteriol., 171, 5281-5289). ). However, the most essential regions (oriT, traG, traF) are all located on the resident Ti plasmid (Oger & Farland, 2002, J. Bacteriol., 184, 1121-1131; Farland, Hwang & Cook, 1996, J. Bacteriol). 178, 4233-4247; Li & Farland, 2000, J. Bacteriol., 182, 179-188). Removal of this region by homologous recombination will reduce bacterial conjugal transmission even when homologous recombination between the resident Ti plasmid and the binary vector occurs. The design of an Agrobacterium strain lacking a region involved in conjugation transmission should be easily performed according to the above references and the description provided in Example 16 of the present invention using standard molecular biological techniques. Can do. Example 16 shows that deletion of genes involved in conjugative plasmid transfer does not affect the efficiency of T-DNA transfer from Agrobacterium to plant cells (see also FIG. 20).

  A further approach to increase the expression efficiency of the expression system according to the invention is the use of host cell factors and Agrobacterium factors that limit Agrobacterium infection. Many such factors are described in the review on stable plant transfection mediated by Agrobacterium (Gelvin, SB., 2003, Trends Biotechnol., 21, 95-98; Gelvin, SB., 2003). Microbiol.Mol.Biol.Rev., 67, 16-37). For example, we have found that overexpression of the Arabidopsis gene VIP1 in transgenic tobacco plants makes it more significantly susceptible to transient and stable gene transfection by Agrobacterium (Tzfira, T., Vaidya, M. & Citovsky, V., 2002, Proc. Natl. Acad. Sci. USA, 99, 10435-10440). A factor that improves plant transfection efficiency may be constitutive expression of Agrobacterium virulence genes. Usually such genes are under the control of cascades that induce the expression of environmental factors or vir genes and can be switched on by phenolic compounds, sugars, pH changes. These factors, VirG, can cause VirA protein-mediated phosphorylation and then activate the promoters of other vir genes. Mutations caused by single amino acid substitutions within VirG can cause constitutive expression of other vir genes independent of VirA (Hansen, G., Das, A. & Chicago, MD., 1994, Proc Natl.Acad.Sci.USA, 91, 7603-7607). In this document, a transient increase in the number of focal points expressing GUS in tobacco and corn tissues was detected in a transient expression experiment with a GUS reporter gene using a mutant VirG strain of Agrobacterium. Similar effects were observed when multiple copies of wild-type VirG were used to promote transient expression efficiency in tobacco, but in fact no effect in corn. The authors suggest that such VirG mutant strains may be useful for transformation of cumbersome plant species. This means that they are also useful for the development of transient expression systems based on viral vectors for plant species that are usually difficult to transform. With or with various chemical factors such as phenolic compounds (Melchers et al., 1989, Mol. Microbiol., 3, 969-977), acetosyringone, or opain that stimulate T-DNA transmission into plant cells The use of an independent VirG mutant gene as described above is a useful approach to increase transient expression efficiency (Berthelot et al., 1998, Phytchemistry, 49, 1537-1548).

  Another factor that can significantly increase the biological safety of the transient expression system and method of the present invention is the use of an auxotrophic Agrobacterium strain. This is because such auxotrophic strains have a low probability of living in an open environment. Production of auxotrophic Agrobacterium strains by X-ray treatment is described in Dirks & Peters (US 6323396). The patent describes several such strains (methionine or cysteine-requiring strains, or histidine and adenine-requiring strains). One of the mutants that require methionine for growth lacks the function of homocysteine methyltransferase. Given the availability of the complete Agrobacterium genome sequence in the database, that of a well-characterized (sequenced) Agrobacterium strain replaces the less predictable results obtained by X-ray treatment. It is straightforward to make such variants using gene replacement by homologous recombination. For example, the Agrobacterium tumefaciens C58 genomic sequence (GeneBank accession number NC00305) contains the gene encoding 5-methyltetrahydropteroyl-triglutamate-homocysteine methyltransferase (gene ID 1135697). Create a mutant gene using available sequence information, deliver it to Agrobacterium, and send it to Agrobacterium. Can be cloned into a suicide vector containing the counter-selectable marker gene sacBR along with flanking sequences (Berger & Christie, 1993, J. Bacteriology, 175, 1723-1734). Many other variants can be made using the described approach and publicly available information on various auxotrophic Agrobacterium strains. Collens and colleagues (Biotechnol. Prog. 20 (2004), 890-896) created auxotrophic Agrobacterium mutants by insertional mutagenesis. Such mutant strains cannot grow in the absence of adenine, leucine, cysteine, or thiamine. Some of these mutants have reduced T-DNA delivery efficiency, while others (cys-32) have the ability to deliver T-DNA to plant cells that are even higher than wild-type Agrobacterium. Have The Agrobacterium genes that are expected to be affected are probably leuB, thiD and cysE and cysK. Of course, the selection of an auxotrophic strain will be based on its suitability for efficient T-DNA transfer into plant cells. Also, for higher biological safety levels (low frequency of reversion to wild type), creating auxotrophic strains (eg having two or more mutant genes) for two or more different nutrient supplies Can do. The production of auxotrophic Agrobacterium strains (DleuB and DpyrE) is described in Example 17.

  In addition to mutant strains of Agrobacterium that perform only the required functions (eg T-DNA transfer) and / or can survive only in the presence of trans-complementing factors (eg addition of virE2 protein, metabolic compounds) An approach based on an active biological containment (ABC) system of bacterial cells can be used. An example of such a system is described by Ronchel & Ramos (2001, Appl. Environ. Microbiol., 67, 2649-2656). In this document, the authors describe an ABC system designed to freely control the survival or death of bacterial populations through the induction of cell death in the absence of contaminants. This system is based on the use of a death gene such as a porin-inducible protein encoded by the E. coli gef gene. This gene is placed under the control of a lacI inducible promoter. LacI expression in this system is under the control of a promoter that depends on environmental signals. Similar schemes using other dying genes to design the ABC system have been described in numerous references (eg, Knudsen et al. (1995) Appl. Environ. Microbiol. 61, 985-991; Ronchel et al. (1998). Appl.Environ.Microbiol.64, 4904-4911; Torres et al. (2003) Microbiology 149, 3595-3601). Containment systems based on streptavidin have been described by Szafranski and colleagues (1997, Proc. Natl. Acad. Sci. USA 94, 1059-1063), and Kaplan and colleagues (1999, Biomol. Eng., 16, 135-140). ).

  Alternatively, functional inducible systems in bacterial cells can be used to control the expression of genes involved in specific functions (eg, T-DNA transfer or biosynthesis of metabolites such as amino acids). Indeed, instead of deleting the gene from the bacterial cell to create a mutant strain (eg, an auxotrophic mutant or a virE2 gene function deficient mutant), the gene can be placed under the control of a regulatable promoter. Expression of the gene of interest from such a regulatable promoter can be controlled through the application of complementing factors, which in this case can be small molecules such as tetracycline or IPTG. For example, various modifications of the bacterial regulatable system based on the well-described tet and lac repressors can be used to control the expression of the gene of interest in Agrobacterium cells.

  In addition to Agrobacterium, the present invention can be extended to other microorganisms that have been genetically engineered for T-DNA transfer into plant cells. For example, recently it has been shown that symbiotic bacterial species other than the genus Agrobacterium can be modified to mediate gene transfer to many diverse plants (Brotheratats et al. (2005) Nature, 433, 629- 633). These bacteria have become qualified for gene transfer by obtaining safe Ti-plasmids and binary vectors.

  Plant RNA viruses (exceptions are viroids—small non-coding RNAs that amplify in the plant cell nucleus. For review, see Diener, TO, 1999, Arch. Virol. Suppl., 15, 203-220; Flores, R .; 2001, CR Acad.Sci.III, 324, 943-952) is known not to occur in the cell nucleus but in the cytoplasm. Thus, RNA viral sequences resist nuclear RNA processing due to the presence of motifs involved in processing processes, including pre-mRNA, rRNA and tRNA precursors, including cytoplasmic transport. Not so adapted. Processing events such as 5 'end capping, splicing, 3' end generation, polyadenylation, degradation, base and sugar modifications, and editing (inside plastids and mitochondria) have been intensively studied. However, many elements of such events remain unclear. The most dramatic changes in pre-mRNA in the nucleus occur during pre-mRNA splicing, a process in which intervening RNA sequences (introns) are removed from the initial transcript and simultaneously exons are ligated. Splicing is mediated by splicesomes, a complex structure containing small ribonucleoprotein particles of uridilate rich nuclei. The splicesome performs the splicing reaction in two successive steps: 1st step—cleaving the 5 ′ splice site of the upstream exon / intron junction to form a lariat, 2nd step—3 ′ splice of the intron / downstream exon junction Cleavage the site to join upstream and downstream exons (for review: Kramer, A., 1996, Annu. Rew. Biochem., 65, 367-409; Simpson, GG. & Filipowicz, W. 1996, Plant. Mol. Biol., 32, 1-41). The 5 'and 3' splicing site dinucleotides (5 '/ GU; AG / 3') adjacent to the intron sequence are highly conserved in higher plants and abandon splicing activity at sites involved by single G substitutions Will do. Surprisingly, despite the high conservation of splicing sites between plants and animals, heterologous introns are usually not spliced or incorrectly spliced in plants (van Santen, VL. Et al.). 1987, Gene, 56, 253-265; Wiebauer, K., Herrero, JJ, Filipowicz, W. 1988, Mol. Cel. Biol., 8, 2042-2051). Given that plant viral RNAs were not under evolutionary pressure to resist nuclear RNA processing equipment, these RNAs were subject to such processing, including splicing, once placed in the nuclear environment. Is very likely. This situation is quite different from the RNA transcript encoded by the nuclear gene. This is because the latter transcript is evolutionarily adapted to retain functionality despite a series of RNA modifications occurring in the nucleus. However, such modifications can have dramatic consequences for viral RNA replicon formation. Re-engineering plant viral genes to create expression vectors for heterologous genes adds additional sequences that interact with RNA sequences of viral origin, further increasing the instability of RNA virus-based replicons and replication Will produce incomplete defective RNA. The present invention introduces an expression vector into a plant or plant cell as a DNA precursor, and when it provides transient expression or stable integration into plant chromosomal DNA, a modification that significantly increases the frequency of formation of a functional RNA replicon Is addressed by applying to the expression vector. Modification of virus-derived sequences may be the best way to increase the efficiency of RNA virus-based replicons. The modification may be direct (eg, within a sequence derived from an RNA virus) or indirect (eg, within a sequence derived from a non-viral source), but indirect modification may have a stabilizing effect on the sequence derived from the virus. In the present invention, since it is indispensable for increasing the efficiency of RNA replicon formation, the main focus is on alterations in RNA virus-derived sequences.

  Surprisingly, the first attempt to find evidence that a potentially problematic area exists was successful. And surprisingly, it was experimentally confirmed by unexpectedly finding an order of magnitude improvement. The RNA virus-derived sequence of the expression vector pICH8543 (Example 1, FIG. 2A) is transferred to the Netgene II server program (http://www.cbs.dtu.dk/services/NetGene2/) for the presence of potential introns and RNA splicing sites. Analysis using showed the presence of intron-like regions that could be spliced by the nuclear RNA processing equipment (see circled region in FIG. 6). Programs that can be used to identify potentially problematic regions (selected locations) within plant viral RNA sequences are exon / intron prediction programs for various organisms (http://genes.mit.edu). /GENSCAN.html) or a splicing signal prediction program (http://125.itba.mi.cnr.it/˜webgene/wwwspliceview.html).

  Considering that all existing programs are not ideal and are prone to errors, potentially problematic areas can also be determined experimentally. This is an advanced version called real-time PCR suitable for RT-PCR (Frohman, MA., 1989, Methods Enzymol., 218, 340-356) or for accurately quantifying the concentration of various transcripts. (Gibson et al., 1996, Genome Res., 6, 995-1001) can be performed by analyzing transcripts derived from DNA vectors that have been tested in the nuclear environment, preferably sequencing PCR amplified products. Accompany. The conservative differences of the present invention can be achieved, for example, by introducing silent mutations with substitutions from A / U rich regions (intron-like) to G / C rich regions (exon-like), for example, intron-like Replacing the sequence with exon-like sequence sequences dramatically changes the RNA profile (see circled region in FIG. 3). Plant introns, unlike exons, are usually rich in A / U (Csunk, C. et al., 1990, Nucl. Acid Res., 18, 5133-5141; Goodall & Filipowicz, 1989, Cell, 58, 473-483) There are exceptions. For example, in monocotyledonous plants, introns rich in G / C have been discovered (Goodall & Filipowicz, 1989, Cell, 58, 473-483; Goodall & Filipowicz, 1991, EMBO J., 10, 2635-2644). In practicing this aspect of the invention, the A / U-rich region includes not only a long section that is a sequence of at least 20 nucleotides in length with an A / U content of at least 55%, but also pure A Also included are shorter sections ("islands") of 6-19 nucleotides in a sequence sequence containing / U. A / U content in the context of the present invention means a sequence richer in A than U, or a sequence rich in only A, and conversely all sequences covered by this definition. Example 6 demonstrates that modification of the A / U rich region increases the number of cells expressing GFP by at least 10-fold. This is demonstrated in FIG. 10 by comparing the region agroinfiltrated with pICH15466 (modified vector, FIG. 2A) and the region agroinfiltrated with pICH14833 (control vector, FIG. 2A). Removal of the migration protein (MP) allows accurate counting of primary cultured cells with functional RNA replicons because no cell-to-cell migration from the site of initial infection to adjacent cells occurs. In Example 7, modifications were made to other U-rich intron-like regions that contain many potential splice sites (FIG. 6B) and cover the subgenomic promoter of the transition protein (MP) (FIG. 8, circles). Enclosed). This modification has a dramatic effect on increasing the frequency of replicon formation from the viral vector pICH15900. As demonstrated in the protoplast counting experiment (Example 7), there was an approximately 100-fold increase for the two tobacco species tested-Bensamiana tobacco and tobacco compared to the unmodified vector pICH14833 (corresponding to FIGS. 10A, B). See infiltration area). In general, by using the approach described in the present invention, the frequency of RNA replicon formation is increased approximately 300-fold, ie, the proportion of cells with functional replicon is about 0.2% (control vector) to 50% (modified vector). ) It was possible to increase more. This is not a limit and reaching 100% frequency is also considered very realistic.

  Such a high rate of replicon formation opens the way for two or more different genes to be expressed from two different RNA replicons within the same plant cell, for example using a vector based on a plant RNA virus to co-express different genes. . Achieving synchronized release of two or more replicons simultaneously in the same cell is important for such co-expression because the “first-come-first-served” principle is particularly true for viral vectors. Because different viral vectors usually do not overlap in the transmission region, or because such overlap is small, neither osmotic or cell-to-cell transfer is helpful. A simple calculation demonstrates the importance of the technique described in the present invention to achieve co-expression of two target sequences from two replicons in the same plant cell. In the case of a non-optimized viral vector in which the frequency of functional replicon formation is 0.2% of all cells, the proportion of cells that co-express two genes from two different RNA replicons is 0.2 × 0.2 = 0. For constructs with 04% but an increased frequency of functional RNA replicon formation (50% or 1/2 of total cells), the percentage of cells is 0.5 × 0.5 = 0.25, ie 25 %, For example about 625 times higher. Using the highest performing vectors (eg, pICH16191, FIG. 10C), the percentage of cells with functional replicons can reach approximately 90% (FIG. 10C, upper right). This means that by using such a vector to express two different sequences of interest from two independent replicons, co-expression can occur in 80% of all cells. The possibility of further improving the technology and achieving 100% co-expression is considered very high.

  It is important to note that function-conservative differences in heterologous target sequences to be expressed from RNA replicons can be used to increase the frequency of RNA replicon formation, especially in combination with differences in sequences related to replicon function. . For example, modifications necessary for replicon formation and / or processing can be introduced into the target sequence.

  In another aspect of the present invention, the frequency of replicon formation was improved by inserting nuclear introns into sequences related to replicon function (Example 8). Intron incorporation into the coding region of viral RNA-dependent RNA polymerase (RdRP) (Examples 8 and 12) significantly increased the frequency of replicon formation from modified vectors (pICH15034, pICH15025, pICH15499 in FIGS. 2A, B) (at least 50 times) (Fig. 10A, B). The RNA profile of a vector containing six inserted introns from Arabidopsis thaliana is shown in FIG. In another example (Example 11), the insertion of an intron into the MP sequence increases the frequency of replicon formation by at least 100 times.

  Many nuclear introns can be used to practice the present invention. Examples of such introns include, but are not limited to, rice tpi Act1 and salT gene derived introns (Rethmeier et al., 1997, Plant J., 12, 895-899; Xu et al., 1994, Plant Physiol. , 100, 459-467; McElroy et al., 1990, Plant Cell, 2, 163-171); introns derived from maize Adh1, GapA1, actin and Bz1 genes (Callis et al., 1987, Genes Dev., 1). Donath et al., 1995, Plant Mol. Biol., 28, 667-676; Maas et al., 1991, Plant Mol. Biol., 16, 199-207; Ohn, supervised by K Moldave, “Advances in Nucleic Acid Research and Molecular Biology”, Volume 42, Academic Press, New York, pp. 229-257; Intron from Petunia Rubisco gene SSU301 (Dean et al., 1989). , Plant Cell, 1, 201-208); Arabidopsis A1 EF1α gene, UBQ10 gene, UBQ3 gene, intron derived from PAT1 gene (Curie et al., 1993, Mol. Gen. Genet. 228, 428-436; Norris et al., 1993, Plant Mol.Biol., 21, 895-906; Rose & Last, 1997, Plant J., 11, 455-464); The Synthetic introns can also be used in the present invention. The smallest usable introns or portions thereof can be limited to splice donor and acceptor sites, usually adjacent to internal intron sequences. Preferably the intron size should be at least 50 nucleotides, more preferably 100-200 nucleotides, but in practice the intron size is not limited. However, the size of the construct needs to be maintained to be suitable for genetic manipulation. The origin of the intron, its structure and size can be selected individually depending on the nature of the vector. Transient expression experiments can be used to test the efficiency of selected introns or corresponding intron moieties.

  The effect of the modification is cumulative. For example, when intron insertion is combined with modification of the MP subgenomic promoter, the frequency of replicon formation can be increased approximately 300 times (Example 9). Preferred regions for intron insertion to increase the frequency of RNA replicon formation are referred to herein as selected regions. Such positions can contain “intron-like” structures. This is confirmed by inserting an intron in the immediate vicinity of the problematic region, such as MP, actually the MP subgenomic promoter (Example 11). A 100-fold increase in replicon formation frequency was observed. Insertion of an intron into an “exon-like” region has no obvious effect like insertion into an intron-like region (Example 10).

  A variety of methods are used to generate transgenic plant hosts that have the surrogate function necessary for the generation of plant hosts that are eligible for transient expression of the target sequence, i.e., suitable for complementing the defective Agrobacterium strain of the present invention. be able to. By Ti-plasmid carried by Agrobacterium (US 5,591,616; US 4,940,838; US 5,464,763) or by particle gun or fine particle gun (US 05100792; EP00444882B1; EP00434616B1) Plant cells can be transformed with vectors. Other plant transformation methods such as microinjection (WO09 / 209696; WO09 / 400583A1; EP175966B1), electroporation (EP00564595B1; EP00290395B1; WO08 / 706614A1), or PEG-mediated transformation of protoplasts Can also be used. The choice of vector delivery method may depend on the plant species to be transformed. For example, particle guns are usually preferred for transformation of monocotyledonous plants, whereas for dicotyledonous plants, Agrobacterium-mediated transformation generally gives good results.

  In the examples below, Agrobacterium-mediated vector (heterologous DNA sequence) delivery to tobacco cells was used. However, the vector can be incorporated into plants according to standard techniques suitable for stable or transient transformation or transfection of the plant species of interest. Dicotyledonous transformation techniques are well known in the art and include techniques based on Agrobacterium and techniques that do not require Agrobacterium. Non-Agrobacterium techniques include direct uptake of foreign genetic material by protoplasts or cells. These techniques include PEG or electroporation mediated uptake, particle gun mediated delivery, and microinjection. Examples of these techniques are described in Paszkowski et al., EMBO J. et al. 3, 2717-2722 (1984); Potrykus et al., Mol. Gen. Genet. 199, 169-177 (1985); Reich et al., Biotechnology 4: 1001-1004 (1986); and Klein et al., Nature 327, 70-73 (1987). In either case, transformed cells are regenerated into complete plants using standard techniques.

  Agrobacterium-mediated transfection is a preferred technique for transfection of dicotyledons because of its high transfection efficiency and broad utility with many different species. Many crop species that can be routinely transformed by Agrobacterium include tobacco, tomato, sunflower, cotton, rape, potato, soybean, alfalfa, and poplar (EP 0 317 511 (cotta), EP No. 0 249 432 (tomato), WO 87/07299 (Brassica), US Pat. No. 4,795,855 (poplar)).

  In the examples of the present invention, Agro inoculation, which is an Agrobacterium-mediated delivery method of T-DNA for transient expression of a target gene, was used (Vaquero et al., 1999, Proc. Natl. Acad. Sci. USA, 96). 11128-11133). Agro inoculation is a very useful tool not only for small to medium recombinant protein production systems, but also as an element of vector optimization, to obtain rapid results using various variants of the construct Is possible.

To perform agroinfiltration, an overnight culture of Agrobacterium is usually set up as described in the prior art (Marillonnet et al., 2004, Proc. Natl. Acad. Sci. USA., 101, 6853- 6857). Typically, the optical density (OD) of an overnight culture reaches 3 to 3.5 units at a wavelength of 600 nm and is diluted 3 to 5 times before agroinfiltration to give 5-9 × 10 ⁹ colony forming units. (Turpen et al., 1993, J. Virol. Methods, 42, 227-240). The inventors have also found that 10 ² fold, 10 ³ fold, and 10 ⁴ fold dilutions are also very efficient, especially in combination with sequences related to replicon functions that have function-conservative differences as described herein I found it to work. Surprisingly, the vector in infiltrated tobacco leaves further improved its performance and gave a good yield of GFP with increasing dilution of transformed Agrobacterium. For example, a 10 ³ fold dilution gave better results than a 10 ² fold dilution. A 10 ² fold dilution yields a better GFP yield than a 10 fold dilution. A possible explanation for this phenomenon is the negative effect of highly concentrated Agrobacterium suspensions on viral vector functions, such as cell-to-cell migration, possibly as a result of the plant's response to high concentrations of pathogenic bacteria. is there. This phenomenon has particular value in large-scale industrial protein expression methods because it reduces the amount of Agrobacterium required for recombinant protein production via agroinvasion by at least an order of magnitude compared to prior art methods. is there.

  Example 13 shows a replicon DNA precursor based on inactivated viral RNA. The replicon is optimized according to the present invention. In addition, replicons contain structures that prevent expression of the target sequence when agro-delivered to non-competent plants, such as plants that do not contain an expression cassette that provides recombinase stably integrated into genomic DNA. Similar to the formation of a functional RNA replicon, expression can be induced by reversing part of the construct by site-specific recombination. This inversion can result in the formation of two introns and a collection of functional target sequences. The system described in Example 13 demonstrates how to achieve high biological safety standards using plant expression systems to express industrial or pharmaceutical proteins as well as viral vector optimization. .

  Recombinase transcription can be under the control of an inducible or other regulated (eg, developmentally regulated) promoter. Inducible promoters can be divided into two categories depending on their induction conditions: those induced by abiotic factors (temperature, light, chemical) and those induced by biological factors such as pathogen or pest attack. Is. Examples of the first category are heat-inducible (US05187287) and cold-inducible (US05847102) promoters, copper-inducible systems (Mett et al., 1993, Proc. Natl. Acad. Sci. USA, 90, 4567- 4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; US06063985), ethanol-inducible systems (Caddick et al., 1997). , Nature Biotech., 16, 177-180; WO 09/321334), and the tetracycline inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). That. One of the latest developments in the field of plant chemical inducible systems is a chimeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review of chemical inductive systems: see Zuo & Chua (2000, Current Opin. Biotechnol., 11, 146-151) and Padidam, M (2003, Curr. Opin. Plant Biol., 6, 169-177). . Other examples of inducible promoters are promoters that control the expression of plant pathogenicity-related (PR) genes. These promoters can direct plants to salicylic acid, a key component of the plant signaling pathway in response to pathogen attack, or other chemical compounds that can induce PR gene expression (benzo-1,2,3-thiadiazole or isoforms). It can be induced by treatment with nicotinic acid (US 05942662).

  The present invention is not limited to the TMV-based vectors described in the examples, but can also be applied to replicons based on other plant RNA viruses. Analysis of other plant viral RNA sequences (Example 14, FIG. 13, FIG. 14) shows selected positions similar to those described for TMV and the sequence of the pre-mRNA of the plant nuclear gene (FIG. 12). This is a powerful that can actually eliminate plant RNA virus-derived replicons by removing / substituting problematic regions and / or inserting nuclear introns using the approach described in the present invention. This is proof.

  The target proteins that can be expressed in plants or plant cells using the present invention, their fragments (functional or non-functional), and their artificial derivatives include, but are not limited to: : Starch modifying enzyme (starch synthase, starch phosphorylase, debranching enzyme, starch branching enzyme, starch branching enzyme II, granule-bound starch synthase), sucrose phosphate synthase, sucrose phosphorylase, polygalacturonase , Polyfructan sucrase, ADP glucose pyrophosphorylase, cyclodextrin glycosyltransferase, fructosyl transferase, glycogen synthase, pectin esterase, aprotinin, avidin, bacterial levansucrase, E. coli glgA protein, MAPK4 and phase Molecular species, nitrogen assimilation / metabolizing enzyme, glutamine synthase, plant osmotin, 2S albumin, thaumatin, site-specific recombinase / integrase (FLP, Cre, R recombinase, Int, SSVI integrase R, integrase φC31, or those Active fragment or mutant), oil modifying enzyme (such as fatty acid desaturase, elongase, etc.), isopentenyl transferase, Sca M5 (soybean calmodulin), beetle-type toxin or insecticidal active fragment, ubiquitin-conjugating enzyme (E2) fusion Enzymes that metabolize proteins, lipids, amino acids, sugars, nucleic acids, and polysaccharides, superoxide dismutase, inactive proenzyme-type proteases, plant protein toxins, modified fibers in fiber-producing plants, and shells derived from Bacillus thuringiensis Active toxins (Bt2 toxin, insecticidal crystal protein (ICP), CrylC toxin, δ endotoxin, poliopeptide toxin, protoxin, etc.), insect-specific toxin AaIT, cellulolytic enzyme, E1 cellulase from Acidothermus celluloticus, lignin modifying enzyme, Cinnamoyl alcohol dehydrogenase, trehalose-6-phosphate synthase, cytokinin metabolic pathway enzyme, HMG-CoA reductase, E. coli inorganic pyrophosphatase, seed storage protein, Erwinia herbicola lycopene synthase, ACC oxidase, pTOM36 Protein, phytase, ketohydrolase, acetoacetyl CoA reductase, PHB (polyhydroxybutanoate) synthase, polyhydroxyl alkenyl Enzyme involved in the synthesis of ate (PHA), acyl carrier protein, napin, EA9, non-higher plant phytoene synthase, protein encoded by pTOM5, ETR (ethylene receptor), plastid pyruvate phosphate dikinase, nematode Inducible transmembrane pore protein, trait that enhances photosynthesis or plastid function of plant cells, stilbene synthase, enzyme capable of hydroxylating phenol, catechol dioxygenase, catechol 2,3-dioxygenase, chloromuconate cycloisomerase, anthranil Acid synthase, Brassica AGL15 protein, fructose 1,6-biphosphatase (FBPase), AMV RNA3, PVY replicase, PLRV replicase, potyvirus coat protein, CMV co Protein, TMV coat protein, luteovirus replicase, MDMV messenger RNA, mutant geminivirus replicase, Umbellularia californica C12: 0 selective acyl-ACP thioesterase, plant C10 or C12: 0 selective acyl-ACP thioesterase, C14 : 0-selective acyl-ACP thioesterase (luxD), plant synthase factor A, plant synthase factor B, D6-desaturase, fatty acid biosynthesis and modification, eg enzymes in peroxisome β oxidation of fatty acids in plant cells Active protein, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, lipase, maize acetyl-CoA-carboxylase, etc .; 5-eno Pyruvylshikimate-3-phosphate synthase (EPSP), phosphinothricin acetyltransferase (BAR, PAT), CP4 protein, ACC deaminase, protein with post-translational cleavage site, DHPS gene conferring sulfonamide resistance, Bacterial nitrilase, 2,4-D monooxygenase, acetolactate synthase or acetohydroxy acid synthase (ALS, AHAS), polygalacturonase, Taq polymerase, bacterial nitrilase, restriction enzyme, methylase, DNA and RNA ligase, DNA and Many other enzymes from bacteria or phage, including RNA polymerases, reverse transcriptases, nucleases (DNA-degrading enzymes and RNA-degrading enzymes), phosphatases, transferases, etc.

  The present invention relates to commercially valuable and pharmaceutically important protein molecules, including industrial enzymes (cellulase, lipase, protease, phytase, etc.) and fibrous proteins (collagen, spider silk protein, etc.) Can be used for agriculture and refining. Healthy human or animal proteins can be expressed and purified using the approaches described in this invention. Examples of such target proteins include, inter alia, immune response proteins (monoclonal antibodies, single chain antibodies, T cell receptors, etc.), antigens including those derived from pathogenic microorganisms, colony stimulating factors, relaxin, somatotropin ( Polypeptide hormones including HGH) and proinsulin, cytokines and their receptors, interferons, growth factors and clotting factors, enzymatically active lysosomal enzymes, fibrinolytic polypeptides, blood clotting factors, trypsin, trypsinogen, a1- Function-conservative proteins such as antitrypsin (AAT), human serum albumin, glucocerebrosidase, natural cholera toxin B, and fusion, mutant and synthetic derivatives of the above proteins, thrombin, human gastric lipase, granulocyte-macrophages Colony stimulating factor GM-CMF), serpin, lactoferrin, lysozyme, oleosin, prothrombin, alpha-galactosidase.

The contents of European Patent Application No. 04016011.1 claiming priority in this patent application are hereby incorporated by reference in their entirety.
Examples The following examples are presented to illustrate the present invention. Modifications and modifications can be made as necessary.

Construction of RNA Vectors Based on TMV Brassicaceae Infectious Tobamovirus (cr-TMV; Dorokhov et al., 1994, FEBS Lett. 350, 5-8) and Turnip Leaf Vibrating Virus (TVCV; Lartey et al., 1994, Arch. Virol.138.287-298) was obtained from Prof. Tabekov at the University of Moscow, Russia. A viral vector containing the green fluorescent protein (GFP) gene was generated in several cloning steps. The resulting construct, pICH8543 (FIG. 2A), in order: Arabidopsis actin 2 promoter-derived 787 bp fragment (ACT2, references An et al., 1996, GenBank accession number AB026654, bp57962-58748), TVCV 5 ′ end (GenBank accession). No. BRU03387, bp1-5455), cr-TMV fragment (GenBank accession number Z29370, bp5457-5567, thymine 5606 is changed to cytosine to remove the start codon of coat protein CP), the sequence “taa tegata act cga g ”, Synthetic GFP (sGFP) gene, cr-TMV 3′-untranslated region (3′-NTR; GenBank accession number Z29370, bp 6078-6312), and finally nopaline synthase (Nos) ) Contains a terminator. The entire fragment was cloned between the left border (LB) and the right border (RB) of the T-DNA of pICBV10, a binary vector derived from Carb ^R pBIN19. Agrobacterium strain GV3101 was transformed with pICH8543 and infiltrated into the leaves of Bensamiana tobacco. The focal point of GFP fluorescence that appeared at 3 dpi grew and became confluent. Surprisingly, even though most cells in the infiltrated area eventually express GFP by viral replication and translocation, they initiate viral replication as detected by the many independent foci that express GFP Only a few fractions of cells were done. The limiting factor is not DNA delivery to plant cells because GFP gene is expressed in almost all cells in the invasion region by infiltrating the GFP gene under the control of 35S promoter into the leaf of Bensamiana tobacco (not shown) It became clear.

In order to confirm this observation result, a viral vector construct containing a mutation in MP was prepared. This construct, called pICH14833, is similar to pICH8543, but differs by a 389 bp deletion in the MP gene upstream of the EcoRI site present in MP. The sequence of the NcoI-EcoRI fragment including this deletion is shown in the Appendix as SEQ ID NO: 1. The entire viral construct (ACT2 promoter to Nos terminator) was cloned between the left and right borders of the T-DNA of pICBV49, a pBIN19-derived Kan ^R binary vector. Because of the deletion in the MP, the replicon produced from this construct cannot translocate between cells, but is capable of autonomous replication within the cell. When MP is provided in trans from a constitutive promoter such as the cauliflower mosaic virus 35S promoter, cell-to-cell transition can be restored.

  To create an MP expression construct, the TVCV MP gene was amplified by PCR from cloned TVCV cDNA (GenBank accession number Z29370, bp 4802-5628) and subcloned into a binary vector under the control of the 35S promoter. The resulting construct called pICH10745 (not shown) and pICH14833 were transformed into Agrobacterium strain GV3101 and infiltrated into the leaves of Bensamiana tobacco. When infiltrated with pICH14833 alone, few GFP-expressing cells appeared in the infiltrated area. By counting the protoplasts made from the infiltrated area, it was found that only 1-3 protoplasts expressed GFP out of a total of 500 protoplasts (0.2-0.6%). The co-infiltration of pICH14833 and pICH10745 formed a focal point for GFP growth from each initial GFP-expressing cell. Eventually, due to cell-to-cell migration, a significant proportion of cells in the infiltrated area expressed GFP (FIG. 10A).

The cruciferous plant infectious tobamovirus (cr-TMV; Dorokhov et al., 1994, FEBS Lett. 350, 5-8) and turnip vein transilluminating virus (TVCV; Lartey et al., 1994, Arch.Virol.138, 287-298), and several additional viral vectors were constructed using the same cloned cDNA. A viral vector containing the green fluorescent protein (GFP) gene was generated in several cloning steps. The resulting construct, pICH16707 (FIG. 2C), in order: Arabidopsis actin 2 promoter-derived 787 bp fragment (ACT2, References An et al., 1996, GenBank accession number AB026654, bp57962-58748), TVCV 5 ′ end (GenBank accession) Number BRU03387, bp 1-5455), a fragment of cr-TMV (GenBank accession number Z29370, bp 5457-5679, thymine 5606 has been changed to cytosine to remove the start codon of coat protein CP), the sequence "taa tcg data act cgag ", synthetic GFP (sGFP) gene, cr-TMV 3'-untranslated region (3'-NTR; GenBank accession number Z29370, bp 6078-6312), and finally nopaline synthase ( Nos) contains a terminator. The complete fragment was cloned between the left border (LB) and the right border (RB) of the T-DNA of pICBV29, a binary vector derived from Kan ^R pBIN19. Agrobacterium strain GV3101 was transformed with pICH16707. Transformed Agrobacterium strains were grown overnight in LB medium until saturated. An overnight culture of 200 μl grown Agrobacterium was precipitated for 3 minutes at 8000 rpm in a microcentrifuge. The precipitate was resuspended in 1 ml of solution containing 10 mM MES (pH 5.5), 10 mM MgSO ₄ to an OD of approximately 0.7. The leaves of greenhouse-grown benthamiana tobacco plants were infiltrated with a syringe without a needle. The focus of GFP fluorescence appeared 3 days after infiltration. The fluorescence focus became confluent after a few days of growth.

  A large volume of infiltration solution (3 liters) with an Agrobacterium of OD 0.3-0.4 was prepared. The whole Bensamiana tobacco plant was inverted using a pot and dipped into the solution, placed in a drier and vacuumed for 1-2 minutes until enough solution had penetrated the leaves. The plants were then removed and placed in a greenhouse for regeneration. On the 3rd to 4th days after the infiltration, fluorescent spots appeared in the infiltrated area like an area infiltrated by hand with a syringe. The picture shows such a plant 10 days after infiltration (Figure 3).

  RNA viruses such as tobamovirus replicate in the cytoplasm and never enter the nucleus. Therefore, it deploys in an environment that is not exposed to nuclear pre-mRNA processing equipment. As a result, RNA replicon transcripts produced in the nucleus from artificial virus constructs cannot be recognized and processed correctly by the RNA processing apparatus. Moreover, viral vector-derived RNA replicons are very large: approximately 7,000 nucleotides for TMV-based replicons. There are very few plant genes of such size, and most of such genes have introns that facilitate pre-mRNA processing, transport from the nucleus, and improve the stability of the processed transcripts. contains. Therefore, it is hypothesized that alterations in pre-mRNA that improve the efficiency of accurate processing and transport of correctly processed transcripts from the nucleus to the cytoplasm will result in an increase in the number of cells that initiate viral replication. It was. Ultimately, it can be seen that there are two approaches that can be used to create RNA virus-based vectors that can initiate viral replication more efficiently after DNA delivery to the nucleus: (1) One approach is useless. Removal of sequence features that can induce processing events (such as alternative splicing events with potential splice sites, or premature termination events); (2) The second approach increases the amount of transcripts that are correctly processed The addition of introns that improve RNA transport from the nucleus to the cytoplasm and / or improve transcript stability.

Construction of an improved RNA vector based on TMV In order to increase the rate at which the target sequence is expressed in the invasion region, 16 Arabidopsis introns of size 91-443 nucleotides were added at positions 1-16 of the vector: (for the TVCV sequence Position, GenBank accession number BRU03387): 1, bp 209; 2, bp 423; 3, bp 828; 4, bp 1169; 5, bp 1378; 6, bp 1622; 7, bp 1844; 8, bp 2289; 9, bp 2944; 11, 12, bp 3672; 13, bp 3850; 15, bp 4299; 16, bp 4497; 17, bp 5099; 18, bp 5287; 19, bp 5444. The resulting construct pICH18711 (FIG. 2C) was used for vacuum infiltration of several N. benthamiana tobacco plants at various stages (17-35 days after sowing). GFP fluorescence appeared after 2 days and was very strong at 6 days after infiltration. The photograph shows the fourth day after infiltration (FIG. 4). GFP fluorescence covered the entire leaf area much faster (4-6 days after infiltration) than the unmodified vector pICH16707. A detailed description of the improved viral vectors has been published recently (Marillonnet et al., 2005, Nature Biotechnol., 23, 718-723).

  Instead of the whole plant, the branches (stems and leaves) of the benthamiana tobacco plant were vacuum infiltrated. The trunk was restored by placing the trunk base in a glass of water. GFP appeared in the leaves after 3 days, indicating that plant parts, not whole plants, could be used for expression of the target gene.

Expression of medicinal proteins using vacuum infiltration of viral vectors delivered by Agrobacterium Two proteins of pharmaceutical interest were cloned into the improved vector instead of GFP. The vector pICH17991 was obtained by cloning of the first protein human growth hormone (hGH) (sequence Genbank accession number NM — 000515) (FIG. 2C). The vector pICH19081 was obtained by cloning the second protein human interferon α (sequence Genbank accession number V00548) (FIG. 2C). In this construct, the first 17 amino acids of the interferon (LLVALLVLLSCKSSCSVG) have been replaced with the Arabidopsis calreticulin apoplast target sequence (matqrranpssllitvfsllvvvsaev). The construct was inserted into Agrobacterium strain GV3101 and used to infiltrate the entire plant. High levels of protein expression were obtained for both proteins. For pICH17991, high levels of expression of 1 mg hGH per gram of infiltrated leaf tissue were obtained even though protein toxicity resulted in cell death in the infiltrated area.

Invasion of entire plant by vector lacking MP A frameshift was made at the first AvrII site of MP in pICH17811 (FIG. 2C), resulting in construct pICH18722 (FIG. 5). This frameshift completely eliminates MP function, and invasion of this construct results in replication and expression only in individual cells. However, due to the presence of introns, many cells still express GFP.

  The MPC coding sequence of TVCV was amplified by PCR from the cloned TVCV cDNA (GenBank accession number Z29370, bp 4802-5628) and subcloned into a binary vector under the control of the 35S promoter to obtain plasmid pICH10745. Co-infiltration of pICH18722 and pICH10745 completely restores cell-to-cell migration at least during the transient expression of MP. Bensamiana tobacco was stably transformed with pICH10745. By infiltrating pICH18722 into a complete transformant expressing MP, invasion of wild type plants does not result in cell-to-cell transfer of the viral vector, but GFP expression is similar to when pICH18711 is infiltrated throughout the wild type plant. (See FIG. 16).

Complementation of mutant Agrobacterium to provide transient expression of the gene of interest a) Trans-complementation by a transgenic plant host expressing VirE2 The VirE2 gene was transferred to Agrobacterium tumefaciens C58 T-DNA (Gene Bank accession number) AE009437, b.p6368-8038) was amplified by PCR and cloned into the vector pICH10745 in which the TVCV MP gene was replaced. The resulting construct pICHVirE2 (FIG. 15) was transformed into Agrobacterium tumefaciens (GV3101) and Agrobacterium-mediated leaf disc transfer of Bensamiana tobacco plant using 50 mg / L kanamycin (Km) as a selective agent. The functional VirE2 gene-deleted Agrobacterium tumefaciens used for the transfection was fixed (Horsh et al., 1985, Science 227, 1229-1231). A VirE2 function-deficient Agrobacterium tumefaciens strain was transformed with the construct pICH18722 (FIG. 5) having a frameshift in MP. This deficiency was caused by the deletion of the VirE2 gene and was used for agroinfiltration experiments in wild type plants and plants transformed with pICH10745.

  Compare competent and wild type hosts by counting the number of GFP-expressing cells per leaf surface, or (more precisely) the frequency of initiation of virus replication using control and modified viral vectors Therefore, as shown in FIG. 10C, transient expression efficiency was compared by measuring and comparing the proportion of GFP expression protoplasts in competent and wild type hosts. Such relative frequency of T-DNA delivery that provides transient expression can be used as a measure of the biological safety level of the system in comparison to a system that has not established biological safety. .

  Many other Agrobacterium strains lacking VirE2 function, for example, strains having insertion mutations in VirE2 (Christie et al., 1988, J. Bacteriol., 170, 2659-2667) or plasmids having an osa gene that suppresses virE2 function Strains carrying pSA (Lee, LY et al., 1999, J. Bacteriol., 181, 186-196) can be used for this experiment. Furthermore, the availability of Agrobacterium genomic sequence information, including Ti plasmids (eg GeneBank accession number NC003065; NC001277) and chromosomal DNA (eg GeneBank accession numbers NC003063; NC003063), mutates functional genes by homologous recombination. All kinds of mutants can be created by substituting for body shape. The effect of the VirE2 gene on T-DNA transfer efficiency to plant cells was tested by comparing overnight cultures of various diluted VirE2-containing and VirE2-deficient Agrobacterium strains. Both Agrobacterium strains contain the pICH18711 viral vector within the T-DNA region of the binary vector (FIG. 2C). The result of such comparison is shown in FIG. It is clear from the photograph that the T-DNA transfer efficiency by the VirE2-deficient Agrobacterium strain is at least 10,000 fold lower than that of the functional VirE2 gene-containing Agrobacterium. Comparison of transient expression efficiency in leaves of competent (transformed with pICHVIRE2) and wild-type N. benthamiana tobacco plants, and very efficient of VirE2 gene function from transgenic plants transformed with pICHVirE2 Showed trans-complementation (FIG. 18). Experiments with different dilution series (not shown) demonstrated that the trans-complementing efficiency of VirE2 gene function from the transgenic host is comparable to the use of Agrobacterium strains with a functional VirE2 gene.

b) Trans complementation by mixing with wild type Agrobacterium strain expressing VirE2 By mixing a virE2 function deficient strain carrying pICH18722 with an Agrobacterium tumefaciens strain C58 which complements virE2 function in trans, The experiment described in the previous example was repeated except that complementation was achieved. Freshly inoculated cultures of both Agrobacterium strains were grown overnight until the optical density OD ₆₀₀ was 2.5-3.0 and mixed in equal amounts for infiltration of plant tissue. The final mixture for infiltration was diluted 50 times, 100 times, 1000 times, and 10,000 times. Plant tissue infiltration was performed with N. benthamiana tobacco and tobacco leaves. Similar experiments were performed using the ΔVirE2 strain (with functional MP) with pICH18711 binary. The result of this experiment is shown in FIG. Co-invasion of mutant strains (T-DNA +, ΔVirE2) with pICH18711 binary and wild-type strains (T-DNA-, VirE2) is more directly from T-DNA delivery by wild-type strains (T-DNA +, VirE2). However, it is clear that T-DNA delivery is just efficient.

Removal of intron-like sequences increases the frequency of viral RNA replicon formation in the cytoplasm Analysis of RNA replicon sequences from pICH4351 using the NetgeneII server program (http://www.cbs.dtu.dk/services/NetGene2/) As a result, we noticed several intron-like sequence features that could induce alternative splicing events. One such feature is the 0.6 kb uridine rich region at the beginning of RdRP (corresponding to nucleotides 827 to 1462 of GenBank accession number BRU03387) (FIG. 6A). In pICH14833, this region was replaced by a 54 mutated sequence that was different from the original sequence and mutated by PCR (sequence shown as SEQ ID NO: 2 in the Appendix, FIG. 7). A 52 nucleotide substitution was made to replace the T rich sequence with a more GC rich sequence. All nucleotide substitutions were made silent so as not to change the protein sequence of RdRP. This mutant fragment also contains two nucleotide substitutions (positions 829 and 1459; coordinates for GenBank accession number BRU03387) introduced to remove the putative potential splice donor and acceptor sites, respectively. To test the effects of these mutations, the resulting clone, pICH15466 (FIG. 2A), was agroinfiltrated into the leaves of Bensamiana tobacco with or without pICH10745 (transtranslocation protein). On the 8th day after infiltration, a 10-fold increase in the number of GFP-expressing cells was observed in the region infiltrated with pICH15466 (compared with pICH14833, FIG. 10). This suggests that removal of intron-like sequences from the viral replicon prevents unwanted alternative splicing events and initiates viral replication more efficiently. Co-infiltration of pICH15466 and pICH10745 results in cell-to-cell movement of the modified replicon at a similar rate as the unmodified replicon. This indicates that the modification of the RNA sequence did not affect the intercellular transfer of the viral vector.

Removal of intron-like sequences in the MP subgenomic promoter The second potentially problematic region corresponds to the MP subgenomic promoter (Figure 6B). This region is very T rich and very close to the intron sequence. As a result, many potential splice donor and acceptor sites are predicted in the flanking sequences by the intron prediction program. Unfortunately, this region cannot be easily modified without affecting subgenomic promoter function. It was decided to completely mutate the entire region regardless of the subgenomic promoter and provide MP in trans to compensate for the expected loss of MP expression. Since MP was not expressed from this construct, most of the MP sequence was also deleted except for the 3 ′ sequence containing the CP subgenomic promoter necessary to drive expression of the target gene. Therefore, the 383 bp fragment of pICH14833 (genBank accession number BRU03387, bp4584-5455) was replaced with the 297 bp mutant fragment. The resulting construct, pICH15900 (FIG. 2A), was agroinfiltrated into leaves of Bensamiana tobacco with or without pICH10745. Interestingly, a significant increase in the number of replication-initiating cells was detected compared to the leaf region infiltrated with pICH14833. By counting GFP-expressing protoplasts prepared from the infiltrated leaf region, it is estimated that this modification will increase the number of viral replication initiation cells by 80-100 fold compared to unmodified pICH14833. pICH15900 was co-infiltrated with pICH10745 (p35S-MP expression cassette), and an increase in GFP fluorescence due to cell-to-cell transfer was detected. However, this increase was very limited because so many cells had already expressed GFP even without cell-to-cell transfer. A 1000-fold dilution of the pICH15900-containing Agrobacterium suspension was co-infiltrated with an undiluted suspension of pICH10745-containing Agrobacterium to generate isolated GFP expression foci. The fluorescence focus was as bright and the same size as the control focus obtained with pICH14833. This indicates that modification of pICH15900 and trans-delivery of MP do not impair the functionality of the replicon with respect to replication levels, expression of target sequences, and cell-to-cell migration. The same construct (with or without pICH14933 and pICH15900, pICH10745) was co-infiltrated into tobacco leaves. The modification of pICH15900 increased the number of replication-initiating cells as compared to Bensamiana tobacco (compared to pICH14833).

Intron addition increases the frequency of functional RNA replicon formation in the cytoplasm Next, it was tested whether intron addition to the sequence portion encoding the replicon would increase the frequency of replication initiation. Two constructs, pICH15025 and pICH15034 (FIG. 2A) were generated. Both contain three different Arabidopsis introns in two different regions of RdRP. pICH15025 was designed to contain an intron in the middle of RdRP. On the other hand, pICH15034 contains an intron upstream of the MP subgenomic promoter, which is the 3 ′ end of RdRP. Introns were amplified from Arabidopsis genomic DNA by PCR and incorporated into viral sequences by PCR using primers that overlap the expected intron / exon junction. The intron-containing fragment was subcloned into pICH14833 as an AvaI / HindIII fragment (Appendix SEQ ID NO: 4) to generate pICH15025, or was subcloned as a PstI / Ncol fragment (Appendix SEQ ID NO: 5) to generate pICH15034.

  The two constructs were agroinfiltrated into the leaves of Bensamiana tobacco and compared with pICH14833. The two constructs significantly increased the number of cells that initiated viral replication (FIG. 10A). This increase was estimated to be about a 50-fold improvement over pICH14833. In addition, the two constructs were co-infiltrated with the MP-expressing clone, and the cell-to-cell transition was found to be the same as the clone without intron. Two constructs were also tested in tobacco and observed the same improvement as Bensamiana tobacco (FIG. 10B).

  A third clone, pICH15499, containing all 6 introns was generated (FIGS. 9, 2B, 10A, 10B). This construct was tested in N. benthamiana and tobacco. Although this construct was more efficient than the individual construct with three introns, the improvement was not additive.

Intron addition and removal of intron-like sequences increase the frequency of functional RNA replicon formation in the cytoplasm Removal of intron-like features in one construct and addition of additional introns both types of modifications improve viral replication initiation It showed that it can contribute. The six introns of pICH15499 were subcloned into pICH15900 containing the mutated MP subgenomic promoter region. The resulting clone, pICH15860 (FIG. 2B), was infiltrated into the leaves of N. benthamiana tobacco and was significantly better than the respective parent clones in the range of approximately 50% to 90% of all protoplasts expressing GFP. It was found to work (FIG. 10). The highest performing construct contains introns in the RdRP region and the modified MP subgenomic promoter region (pICH16191, FIG. 10C). Compared to unmodified clones, this represents an 80-300 fold improvement. The construct was also co-infiltrated with the MP expression construct (pICH10745) and found that the modification did not damage cell-to-cell migration or replication.

Not all intron additions increase the appearance of functional RNA replicons in the cytoplasm Two different Arabidopsis introns were inserted at the beginning of RdRP, resulting in clone pICH15477 (FIG. 2B) (the sequence of this region is SEQ ID NO: 7 in the appendix). The sequence in this region already appears very “exon-like” (eg, rich in GC and lack of potential splice sites) before adding introns. This construct did not show any improvement in the initiation of viral replication. Thus, not every intron addition improves the viral vector. The location chosen for intron insertion or mutagenesis appears to be an important parameter. For example, all intron insertions or nucleotide substitutions made in the vicinity of problematic structures such as the MP subgenomic promoter have resulted in significant improvements, but intron insertion into sequences that are already “exon-like” Did not improve replication initiation.

Intron insertion into the MP sequence increases the frequency of virus replicon formation. A frameshift was created in the MP by first digesting with the restriction enzyme AvrII, filling and religation. Next, two introns were inserted into the MP. The resulting clone, pICH16422 (FIG. 2B), was infiltrated into the leaves of Bensamiana tobacco. An approximately 100-fold increase in the number of functional virus replicon-containing cells was detected.

Intron insertion into MP-containing vectors increases the frequency of viral replication initiation of autonomously functional clones. Subcloning into the KpnI / EcoRI fragment pICH8543 of pICH15499. The resulting clone 16700 (FIG. 2B) contains a complete viral vector with 6 introns in RdRP. When this clone was infiltrated into a leaf of Bensamiana tobacco, replication was efficiently initiated. This clone can also be transferred between cells without having to provide additional MP in trans.

Activation of agro-delivered inactive replicons in genetically engineered host plants It is also possible to introgress intron-containing viral vector precursors into transgenic plants. In order to avoid accidental infection of plants that are not destined to produce the target protein (for example, plants in an open environment), inactive clones can be produced in which a part of the vector is present in the antisense direction ( FIG. 11). By incorporating a recombination site and an intron sequence at the tip of the inverted fragment, it is possible to “invert” this fragment in the correct direction by using an appropriate recombinase. The recombination site will be completely eliminated from the replicon by splicing. Introns in the proreplicon efficiently initiate replication after recombination and transcription. In one embodiment, the recombination site is located within the gene of interest and downstream of the proreplicon. Such a configuration prevents gene expression prior to recombination. When recombination sites are localized in other regions of the proreplicon, such as in RdRP or upstream of the promoter, other configurations can be considered. Intron sequences at the recombination site not only have the advantage of completely removing the recombination site from the replicon, as described above, but also increase the efficiency of viral replication. Transgenic plant hosts that provide recombinase activity can be genetically engineered and used for agroinfiltration along with vectors containing inactive viral vector precursors. Viral vector precursors can only be activated in genetically engineered plant host cells.

Plant virus RNA sequences contain potentially unstable regions The RNA gene profile of the selected plant RNA virus, as well as that of the well-characterized plant gene (AtDMC1), can be obtained from the Netgene II server program (http: /// www.cbs.dtu.dk/services/NetGene2/). The RNA profile shown in FIG. 12 for AtDMC1 clearly reflects the presence of 14 introns previously identified by comparison of cDNA and genomic DNA sequences (circled). It is clear that the RNA profiles of the two plant viruses have regions that can cause problems with RNA stability when placed in the plant's nuclear environment (see FIGS. 13 and 14). The RNA profiles of several other representative plant RNA viruses (not shown) were analyzed, such as bromo mosaic virus, different TMV strains, and many others. All of these have potentially problematic areas that can reduce the efficiency of replicon formation based on plant RNA viruses when delivered to plant cells as DNA precursors.

Whole Plant Invasion with Geminivirus Vectors Expression vectors pICH4300, pICH5202 / 3, and pICH5170 (FIG. 15) based on Bean Golden Mosaic Virus (BGMV) isolate DSMZ PV-0094 (FIG. 15) are described in Example 10 and WO02 / 077246. 11 was prepared. The construct pICH5170 was used for stable transformation of N. benthamiana and tobacco plants as described in Example 5. The constructs pICH5202 / 3 and pICH5170 were immobilized on VirE2 function deficient Agrobacterium tumefaciens and Agrobacterium tumefaciens C58 strains. Strains carrying the immobilized viral replicon (pICH5202 / 3) were used for agroinfiltration of whole tobacco plants transformed with pICH5170. The transgenic plant host provided replication of a DNA replicon with GFP with a geminivirus origin of replication located in the so-called common region (CR). In the case of the Agrobacterium strain deficient in VirE2, Agro infiltration was performed together with non-transformed Agrobacterium tumefaciens C58. Alternatively, a benthamiana tobacco plant transformed with pICHVirE2 was crossed with a plant transformed with pICH5170. F1 progeny containing the T-DNA region from both constructs were selected by PCR and used for Agro infiltration with Agrobacterium function and pICH5202 / 3.

A strain with an immobilized viral replicon (pICH5170) was used for agroinfiltration of the entire tobacco plant.
The results of the above experiment showed that GFP was not expressed at all in the infiltrating plant when the VirE2 function-deficient strain was used without complementing the function with the transgenic host plant or VirE2-expressing Agrobacterium. However, expression levels in experiments requiring trans-complementation of VirE2 function did not show any significant difference from when the vector was immobilized on Agrobacterium tumefaciens C58.

Generation of Bacterial Plasmid Conjugative Transmission Deficient Mutants The tra and trb genes have been described to be involved in conjugative transmission of Ti plasmids (Farland et al., 1996, J. Bacteriol. 178, 4233-4247; Li et al., 1999, J. Bacteriol., 181, 5033-41). In order to delete these genes from the Ti plasmid of Agrobacterium tumefaciens strain C58, a vector containing flanking DNA regions was used for transformation and the deletion was introduced by two consecutive recombination events. The deletion vector is based on the vector pDNR-1r (Clontech) containing the sacB gene causing carbenicillin resistance as a positive selectable marker and sucrose intolerance as a negative selectable marker.

  To delete the tra gene (traG, traD, traC, oriT, traA, traF, traB, traH, traM), an approximately 700 bp flanking region was deleted from Agrobacterium C58 DNA with the oSM570 primer (5'-gaggatccaacgtttaggagaccag-3 ') + OSM571 primer (5'-ttggtcacgggtatacgcactactaaccatgcg-3') and oSM572 primer (5'-ttgggtcaccaccggttcccctttgttctcc-3 ') + oSM573 primer (5'-actgtcggg The PCR fragment was ligated after restriction treatment with BsaI, and a Bst11071 restriction site was created without introducing additional nucleotides; a BamHI site and an XbaI site were introduced at the outer ends using primers. Two PCR fragments were ligated to the vector pDNR-1r restricted with BamHI and XbaI. The resulting plasmid pICF12721 was used for Agrobacterium transformation by electroporation and was first selected with carbenicillin and then without antibiotics but in the presence of 5% sucrose and having a double recombination event Cells that did not contain were counterselected.

  To delete the trb gene (trbB, trbC, trbD, trbE, trbJ), an approximately 700 bp flanking region was extracted from Agrobacterium C58 DNA with oSM566 primer (5'-ctgaattcaggcaacccccgtggatagg-3 ') + oSM56' primer -Tcaccatgggtcacgcggcactcctg-3 ') and oSM568 primer (5'-tcaccatggccccagggccccggcgtgaac-3') + oSM569 primer (5'-actattagatgccgggcatcgagagtggtggg ') PCR fragment ligation created an NcoI restriction site without introducing additional nucleotides; an EcoRI site and an XbaI site were introduced at the outer ends with primers. The two PCR fragments were ligated into the vector pDNR-1r restricted with EcoRII and XbaI. The resulting plasmid pICF12711 was used for Agrobacterium transformation as described above.

To measure conjugative transmission between Agrobacterium, a selectable Ti plasmid showing constitutive transmission was constructed by replacing the accR gene encoding an opain binding repressor with a kanamycin resistance cassette. This is also based on the vector pDNR-1r, from Agrobacterium C58 DNA to oSM554 primer (5'-gagcttagctccgtccttcacctggggc-3gt), ') + OSM557 primer (5'-atgggcccttcgaacgcaattcctgttgc-3'), two adjacent regions amplified by PCR, and the XmaIII site between them, kanamycin resistance (oSM584 primers (5'-cctggggccgcgaacgggcctcac-3 ') Primer The 5'-ctacggccgctgacagctaaaacaattcatcc-3 ') Agrobacterium tumefaciens strain C58 in vector pICF12741 containing was amplified by PCR from the binary vector PICH18711) was used to prepare by transforming. Strains carrying the kanamycin resistant Ti plasmid together with acceptor strain GV3101 resistant to chromosomal rifampicin but without Ti plasmid at 28 ° C. for 2 hours on a nitrocellulose membrane, Piper & Farrand (1999, Appl Environ Microbiol., 65, 2798-2801 Incubation was carried out as described in). Transzygous individuals were selected on plates supplemented with rifampicin and kanamycin. In the case of Ti plasmid lacking ace but having intact tra and trb genes, the conjugation efficiency per input donor was approximately 3-4 × 10 ⁻⁴ transconjugation individuals. In the case of the tra and trb mutants, the conjugation efficiency was below the detection limit (<10 ⁻⁷ ).

  Bensamiana tobacco plants were tested for the effect of mutations on DNA transfer to plants by infiltration with bacteria containing the viral vector pICH18711 encoding GFP. Bacteria were used at various dilutions so that a single fluorescent spot could be observed on the leaves. DNA transfer efficiency to plants was comparable to bacteria without ace, tra, or trb deletions.

Preparation of auxotrophic mutants To produce an auxotrophic Agrobacterium strain, leuB gene encoding 3-isopropylmalate dehydrogenase involved in leucine biosynthesis and orotic acid involved in uridine biosynthesis PyrE encoding phosphoribosyltransferase was selected. The coding sequence of leuB overlaps 3 'of the hypothetical protein ATU2792 located in the reverse strand of the Agrobacterium chromosome and cannot be completely deleted. Thus, chromosome 1 bp 27938384-2793470 was selected to be deleted by joining the upstream sequence 775 bp and the downstream sequence 834 bp to the 5 ′ end of the leuB coding sequence via a naturally occurring BspHI restriction site. . A deletion was made in the target gene of Agrobacterium using the vector pDNR-1r (Clontech).

  As with leuB, catabolic complementation of the pyrE gene is not possible. Since the uracil phosphoribosyltransferase gene is present in Agrobacterium, uridine addition can be replaced by cheap uracil. Furthermore, pyrE deletion can be used as a positive selectable marker. Selection takes advantage of the fact that wild-type Agrobacterium is sensitive to the toxic uracil analog 5-fluoroorotic acid, but the pyrE mutant is naturally resistant.

  The start codon for pyrE is noted differently in different databases (bp 394734-395534, accession numbers NP_531105 and bp 394836-395534, accession number Q8UI98). However, homology to other pyrE sequences does not begin before bp 394836. Nevertheless, two deletion mutants were designed (deletions start at position 394760 or 394849 on chromosome 1 and both end at position 395531). The deletion-containing fragment was cloned into the EcoRI-BamHI site of the vector pDNR-1r, resulting in the vectors pICF1263 (ΔleuB bp2792384-2793470), pICF1264 (ΔpyrE bp394760-395531), and pICF1265 (ΔpyrE bp394948-3951) (FIG. 3). See).

  Agrobacterium strain GV3101 was transformed with plasmids pICF1263-3 (ΔleuB bp27932384-2793470), pICF1264-1 (ΔpyrE bp394760-395531), and pICF1265-10 (ΔpyrE bp394848-395531). A small amount of transformants (6 to 10 cfu / μg) was obtained on the second day of selection on a medium containing carbenicillin. Co-recombinants were determined by serial growth of selected clones in LB + Rifampicin solution and LB + Rifampicin + Saccharose solution. Agrobacterium mutants containing the desired deletion in the target gene were confirmed by PCR analysis of Agrobacterium DNA isolated from LB + Rifampicin + Saccharose cultures.

  Mutant strains 1263-3-3 and 1263-3-10 and wild type parent strain GV3101 were grown in LB rich medium and M9 minimal medium with or without the addition of 30 mg / l and 150 mg / l leucine. There was no difference in growth in the LB medium between the mutant strain and the wild type strain, but only the wild type strain was able to grow in the M9 medium. When leucine was added to M9 medium, both wild type and mutant strains were able to grow. Growth of various mutant strains in media with and without addition is shown in FIGS. Growth rate increased with increasing leucine concentration. Agrobacterium mutant 1263-3-3 was transformed with GFP plasmid pICH18711 and analyzed for infection efficiency in N. benthamiana tobacco.

Appendix SEQ ID NO: 1 (NcoI-EcoRI fragment of pICH14833):

配列番号２（ｐＩＣＨ１５４６６の部分）：

SEQ ID NO: 2 (part of pICH15466):

配列番号３（ｐＩＣＨ１５９００の部分）：

SEQ ID NO: 3 (part of pICH15900):

配列番号４（ｐＩＣＨ１５０２５の部分）：

SEQ ID NO: 4 (part of pICH15025):

配列番号５（ｐＩＣＨ１５０３４の部分）：

SEQ ID NO: 5 (part of pICH15034):

配列番号６（ｐＩＣＨ１５４７７の断片）：

SEQ ID NO: 6 (fragment of pICH15477):

FIG. 1A represents the general principle of the present invention based on increased biological safety (A). FIG. 1B represents the general principle of the present invention based on an increase in the frequency of replicon formation based on RNA viruses (B). FIG. 2A schematically shows an unmodified and modified virus construct according to the present invention. Act2-Arabidopsis actin 2 gene promoter; RdRP-virus RNA-dependent RNA polymerase; MP-virus translocation protein; NTR-virus 3 'untranslated region; GFP-green fluorescent protein; interferon-human interferon alpha 2b; hGH-human growth Hormone; nos-nopaline synthase gene transcription termination region. FIG. 2B schematically shows an unmodified and modified virus construct according to the present invention. Act2-Arabidopsis actin 2 gene promoter; RdRP-virus RNA-dependent RNA polymerase; MP-virus translocation protein; NTR-virus 3 'untranslated region; GFP-green fluorescent protein; interferon-human interferon alpha 2b; hGH-human growth Hormone; nos-nopaline synthase gene transcription termination region. FIG. 2C schematically shows an unmodified and modified virus construct according to the present invention. Act2-Arabidopsis actin 2 gene promoter; RdRP-virus RNA-dependent RNA polymerase; MP-virus translocation protein; NTR-virus 3 'untranslated region; GFP-green fluorescent protein; interferon-human interferon alpha 2b; hGH-human growth Hormone; nos-nopaline synthase gene transcription termination region. FIG. 3 shows a benthamiana tobacco plant 10 days after agroinfiltration with vector pICH16707 under UV light. FIG. 4 shows 17 days (A), 22 days (B), 28 days (C), and 35 days (D) of N. benthamiana tobacco plants under UV light 4 days after agroinfiltration of vector pICH18711. Is shown. FIG. 5 schematically shows the T-DNA region of the constructs pICH18722 and pICH10745. Act2-Arabidopsis actin 2 gene promoter; RdRP-viral RNA-dependent RNA polymerase; MP-virus translocation protein; NTR-virus 3 'untranslated region; GFP-green fluorescent protein; transcription termination region of nosT-nopaline synthase gene; transcription promoter of nosP-nopaline synthase gene; transcription termination region of oscT-octopine synthase gene; coding sequence of NPTII-neomycin phosphotransferase II gene; transition protein of TVCV MP- turnip vein translucency virus. FIG. 6 shows the intron prediction profile of the transcription region of vector pICH8543. Nucleotide numbers are shown on the horizontal axis. The vertical axis indicates the probability that the corresponding sequence / sequence region is a coding sequence (code) or acts as a donor site (donor) or an acceptor site (acceptor). The circled area corresponds to the selected position where a function-conservative difference should be introduced. FIG. 6 shows the intron prediction profile of the transcription region of vector pICH8543. Nucleotide numbers are shown on the horizontal axis. The vertical axis indicates the probability that the corresponding sequence / sequence region is a coding sequence (code) or acts as a donor site (donor) or an acceptor site (acceptor). The circled area corresponds to the selected position where a function-conservative difference should be introduced. FIG. 7 shows the intron prediction profile of the transcription region of vector pICH15466 (the first half of the transcription region). The circled region has been modified as described in the present invention (comparison with FIG. 6A). FIG. 8 shows the intron prediction profile of the transcription region of pICH15900 (second half of the transcription region). The circled region has been modified as described in the present invention (comparison with FIG. 6B). FIG. 9 shows the intron prediction profile of the transcription region of pICH15499. The circled region corresponds to six inserted plant nucleus introns. FIG. 9 shows the intron prediction profile of the transcription region of pICH15499. The circled region corresponds to six inserted plant nucleus introns. FIG. 10 shows GFP expression after agroinfiltration of viral constructs in N. benthamiana tobacco and tobacco leaves. The vector identification number in each infiltration area is shown. A-Benthamiana tobacco, 8 days after agroinfiltration. FIG. 10 shows GFP expression after agroinfiltration of viral constructs in N. benthamiana tobacco and tobacco leaves. The vector identification number in each infiltration area is shown. B-Tobacco, 8 days after agro infiltration. FIG. 10 shows GFP expression after agroinfiltration of viral constructs in N. benthamiana tobacco and tobacco leaves. The vector identification number in each infiltration area is shown. C-Protoplast of N. benthamiana tobacco isolated 5 days after infiltration of agro. Many light spots in the right photograph show very high frequency replicon formation and GFP expression. FIG. 11 is a schematic representation of a replicon precursor based on an RNA virus designed according to the present invention, where the expression level of the target sequence (denoted as GFP, G) is zero in the non-induced state. T-transcription promoter; T-transcription termination region; SM-selectable marker gene; Ac2-Arabidopsis actin 2 gene promoter; RdRP-viral RNA-dependent RNA polymerase; MP-viral translocation protein; NTR-virus 3 'untranslated region . FIG. 12 shows the intron prediction profile of the Arabidopsis meiosis-specific gene AtDMC1 (GenBank accession number U76670) using the straight chain (+ strand). The intron coding region is circled. FIG. 13A shows the predicted profile of a potentially problematic region (circled) within the straight chain (+ strand) of the potato X virus (PVX) genome (GenBank accession number AF172259). FIG. 13B shows the predicted profile of potentially problematic regions (circled) within the straight chain (+ strand) of the potato X virus (PVX) genome (GenBank accession number AF172259). FIG. 14 shows the predicted profile of the linear (+ strand) potentially problematic region (circled) of alfalfa mosaic virus genomic RNA1 (GenBank accession number K02703). FIG. 14 shows the predicted profile of the linear (+ strand) potentially problematic region (circled) of alfalfa mosaic virus genomic RNA2 (GenBank accession number K02702). FIG. 14 shows the predicted profile of the linear (+ strand) potentially problematic region (circled) of alfalfa mosaic virus genomic RNA3 (GenBank accession number L00163). FIG. 15 shows a schematic representation of the T-DNA regions of the constructs pICHVirE2, pICH4300, pICH5202 / 3, and pICH5170. FIG. 16 shows the effect of trans-complementation from a transgenic plant host (N. benthamiana tobacco) on cell-to-cell transfer of a functional MP (translocation protein) -deficient viral vector. The upper label represents a plant and the lower label represents the vector used for agroinfiltration. The photograph on the left shows a typical leaf of a plant treated as shown. The photo on the right shows the entire transgenic plant. WT: wild type plant; pICH10745 transgenic: transgenic plant expressing TVCV transition protein (MP); pICH18722: invasion by a viral vector with poor cell-to-cell transfer; Plants are shown under UV light to detect GFP expression. FIG. 17 shows the effect of the VirE2 gene on T-DNA transfer efficiency from the binary vector pICH18711. VirE2: Agrobacterium leaf infiltrated with Agrobacterium containing a functional VirE2 gene; ΔvirE2: A leaf of Venthamiana tobacco infiltrated with a dysfunctional Agrobacterium; 10 ² to 10 ⁷ Numbers indicate dilution of Agrobacterium stock prior to infiltration (ΔVirE2 and VirE2 stocks are the same OD ₆₀₀ ). The arrows linking the condition to exert the same T-DNA transfer frequency, DerutaVirE2 strain 10 ^2-fold dilutions, the VirE2 strains corresponding to 10 ⁷ fold dilution. FIG. 18 shows wild type (WT) plants and transgenic (pICHVirE2) plants infiltrated with a ΔVirE2 Agrobacterium strain containing the pICH18711 binary vector. The photograph was taken under UV light at 10 dpi (days after infiltration). The same Agrobacterium overnight culture was diluted 10-fold for WT infiltration and 1000-fold for transgenic plant infiltration. FIG. 19 shows trans-complementation of VirE2 gene function by co-infiltration of mutant Agrobacterium (deleting functional virE2 gene) and wild-type Agrobacterium (maintaining VirE2 gene function) into wild type plants. Bensamiana tobacco leaves infiltrated with various combinations of mutant and wild type Agrobacterium are shown under UV light. The labels in the square represent individual types of Agrobacterium. A plus sign between the squares indicates that two types of Agrobacterium were co-infiltrated at the same location on the leaf. A + mark or a − sign in front of “T-DNA” indicates the presence or absence of T-DNA having GFP, respectively. The absence of functional VirE2 is indicated by Δ. FIG. 20 shows the results of a T-DNA transfer efficiency test from Agrobacterium lacking the ace operon. Agrobacterium strain GV3101 carrying the Ti plasmid pMP90 (control) or pTiC58Δacc (acc deletion) was transformed with the viral vector pICH18711 encoding GFP and various dilutions of the overnight culture were used for plant infiltration . Photographs were taken under UV light 7 days after inoculation. FIG. 21 represents a strategy for making Agrobacterium ΔleuB and ΔpyrE variants. FIG. 22 shows the growth rate of Agrobacterium strain 1264 (ΔpyrE) on various media with and without complementation. ΔpyrE mutant strains 1264-1-2 and 1264-1-8 grow only in media containing uracil. The numerical value on the vertical axis represents OD ₆₀₀ . FIG. 23 shows the growth rate of Agrobacterium strain 1263 (ΔleuB) on various media with and without complementation. ΔleuB mutant strains 1263-3-3 and 1263-3-3-10 are leucine-dependent, whereas wild-type parent strain GV3101 is not leucine-dependent. The numerical value on the vertical axis represents OD ₆₀₀ .

Claims (40)

A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Agrobacterium strain containing a heterologous DNA sequence having a sequence part encoding a replicon in T-DNA is transfected into a plant or a plant leaf in the presence or absence of a complementing factor to encode the replicon The array to be
(I) a sequence necessary for a replicon function of a replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
The method, wherein the Agrobacterium strain is provided with a first genetic modification that renders the transfection of T-DNA into an organism poor in the absence of a complementing factor.   The method according to claim 1, wherein the first genetic modification makes the function of the Agrobacterium strain necessary for introduction of T-DNA into the plant or plant leaf cell poor.   The method according to claim 1 or 2, wherein the complementing factor provides a function necessary for transfection of T-DNA into a plant or plant leaf cell by an Agrobacterium strain.   4. The method of claim 3, wherein the function required for transfection of T-DNA into a plant or plant leaf cell is provided by a gene or protein selected from the group consisting of VirE2, GALLS, and VirF.   As a first genetic modification, the function of the Agrobacterium strain required for transfection into the organism is placed under the control of a chemically regulated heterologous promoter; the complementing factor is chemically regulated The method according to any one of claims 1 to 4, which is a small molecule compound capable of regulating a promoter.   5. A method according to any one of claims 1 to 4, wherein the complementing factor is provided to the plant or plant leaf by a second Agrobacterium strain.   The method according to any one of claims 1 to 4, wherein the plant is provided with a second genetic modification that is a complement factor or encodes a complement factor.   9. Agrobacterium strain is auxotrophic and is provided to a plant or plant leaf in the presence of a major metabolite required for auxotrophic growth of the Agrobacterium strain. The method described in 1.   The method according to claim 1, wherein the first genetic modification makes the Agrobacterium strain auxotrophic and the complementing factor is a major metabolite of the Agrobacterium strain required for the auxotrophic growth of the Agrobacterium strain. .   The Agrobacterium strain is conditionally lethal, grows in the presence of metabolites necessary for the survival of the Agrobacterium strain, and / or is provided to the plant or plant leaf. The method according to claim 1.   2. The method of claim 1, wherein the first genetic modification renders the Agrobacterium strain conditional lethal and the complementing factor is the major metabolite of the Agrobacterium strain that is necessary for the survival of the Agrobacterium strain.   12. A method according to any one of claims 1 to 11, wherein the Agrobacterium strain has a further genetic modification that makes the conjugative transfer of the plasmid from or to Agrobacterium poor.   13. The method of claim 12, wherein poor conjugative transmission occurs by deleting a function of at least one of the following genes: oriT, traG, and traF.   14. The method according to any one of claims 1 to 13, wherein the sequence portion encoding the replicon encodes a replicon lacking a function necessary for propagation of the replicon in a plant or plant leaf.   15. The method of claim 14, wherein the sequence portion encodes a replicon that lacks a function necessary for replicon distance transfer or cell-to-cell transfer.   The method according to claim 14 or 15, wherein the function required for propagation of the replicon is provided to the plant or plant leaf by expressing the function from a nucleic acid sequence other than a heterologous DNA sequence in the plant or plant leaf.   The method according to any one of claims 1 to 16, wherein the Agrobacterium strain expresses a tumor suppressor active protein Osa.   The Agrobacterium strain has a genetically modified cell number sensing system or pathogenicity induction regulation system for reducing T-DNA transmission to a non-target organism according to any one of claims 1 to 17. The method described.   The complementing factor is provided by trans-splicing of two RNA sequences in a plant or plant leaf, and at most one of the two RNA sequences is encoded by an Agrobacterium strain. 2. The method according to item 1.   The method according to any one of claims 1 to 19, wherein the replicon is a DNA replicon.   20. The method according to any one of claims 1 to 19, wherein the replicon is an RNA replicon, and the sequence portion encoding the RNA replicon is operably linked to a transcription promoter.   The sequence required for the replicon function exhibits a function-conservative difference with the plant RNA virus at the selected position, and the difference increases the frequency of replicon formation compared to an RNA replicon that does not exhibit the difference. Item 22. The method according to Item 21.   23. The method of claim 22, wherein the function conservative difference comprises removal of a potential splicing site adjacent to the A / U rich region.   The function-conservative difference comprises the insertion of one or more nuclear introns or one or more sequences capable of forming a nuclear intron in or near the A / U-rich position of the sequence required for replicon formation. The method according to 22 or 23.   25. The method according to any one of claims 1 to 24, wherein the replicon has poor intercellular transfer in plants or plant leaves.   26. The method according to any one of claims 21 to 25, wherein the plant virus is a tobamovirus, preferably a tobacco mosaic virus.   27. The Agrobacterium strain is Agrobacterium tumefaciens or Agrobacterium rhizogenes, or other microorganisms engineered to contain an Agrobacterium-derived functional T-DNA transfer generator. The method according to any one of the above.   The method according to any one of claims 1 to 27, wherein the genetic modification is performed on a bacterial chromosome and / or a plasmid chromosome.   The plant or plant leaf in step (a) has a calculated optical density at 600 nm of at most 0.04, preferably at most 0.01, more preferably at most 0.004, most preferably at most Is infiltrated into a suspension of an Agrobacterium strain having a cell concentration of Agrobacterium strain equivalent to 0.001, and the calculated optical density is at least each of an Agrobacterium suspension having an OD of 600 nm of 1.0. 29. A method according to any one of the preceding claims, defined by a 25-fold dilution, preferably at least a 100-fold dilution, more preferably at least a 250-fold dilution, most preferably at least a 1000-fold dilution.   The plant or plant leaf belongs to one of the following species: Bensamiana tobacco, tobacco, petunia, rape, mustard, cress, arugula, mustard, strawberry, spinach, red aza, lettuce, sunflower, and cucumber Item 30. The method according to any one of Items 1 to 29.   The method according to any one of claims 1 to 30, wherein the transfection is performed by infiltrating a plant or a leaf of the plant. A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Transfecting an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA into a plant or a plant leaf in the presence or absence of a complementing factor, The encoding sequence is
(I) a sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
The Agrobacterium strain is provided with a first genetic modification that renders the transfection of T-DNA into an organism poor in the absence of a complement factor, and the sequence required for replicon function is selected at the selected position. Thus, the method exhibits a function-conservative difference from a plant RNA virus, and the difference increases the frequency of replicon formation compared to an RNA replicon that does not exhibit the difference. A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Transfecting an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA into a plant or a plant leaf in the presence or absence of a complementing factor, The encoding sequence is
(I) a sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
The Agrobacterium strain is provided with a first genetic modification that makes T-DNA transfection poor in organisms in the absence of complementing factors, and the sequence required for replicon function is one or more nuclear introns. Containing said process. A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Transfecting an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA into a plant or a plant leaf in the presence or absence of a complementing factor, The encoding sequence is
(I) a sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
Agrobacterium strains are provided with a first genetic modification that, in the absence of a complementing factor, impairs the function of the Agrobacterium strain necessary for the introduction of T-DNA into a plant or plant leaf cell. And the Agrobacterium strain has a further genetic modification that impairs the conjugative transfer of plasmid DNA to other bacteria. A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Transfecting an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA into a plant or a plant leaf in the presence or absence of a complementing factor, The encoding sequence is
(I) a sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
The Agrobacterium strain is provided with a first genetic modification that makes the Agrobacterium strain function defective for the introduction of T-DNA into the plant or plant leaf cells in the absence of a complement factor. And
Said method, wherein the Agrobacterium strain is auxotrophic for the major metabolites required for the auxotrophic growth of the Agrobacterium strain. A method of producing a target protein by expressing a target sequence in a plant or plant leaf, comprising:
(A) Transfecting an Agrobacterium strain containing a heterologous DNA sequence having a sequence portion encoding a replicon in T-DNA into a plant or a plant leaf in the presence or absence of a complementing factor, The encoding sequence is
(I) a sequence necessary for a replicon function of an RNA replicon derived from a plant virus, and (ii) a target sequence to be expressed,
Containing
(B) optionally, isolating the protein of interest from the plant or plant leaf infiltrated in step (a),
Including
Agrobacterium strains are provided with a first genetic modification that, in the absence of a complementing factor, impairs the function of the Agrobacterium strain necessary for the introduction of T-DNA into a plant or plant leaf cell. ,
Said method wherein the Agrobacterium strain is auxotrophic for the major metabolites required for the auxotrophic growth of the Agrobacterium strain, and the sequence required for replicon function contains one or more nuclear introns. (I) an Agrobacterium strain as defined in claim 1, and (ii) a plant encoding a complement factor capable of complementing the defects of the Agrobacterium strain defined in claim 1 or seeds thereof,
Including kit parts. (I) an Agrobacterium strain as defined in claim 1, and (ii) a second Agrobacterium strain encoding a complement factor capable of complementing the defects of the Agrobacterium strain defined in claim 1;
Including kit parts. (I) having a first genetic modification lacking a bacterial function necessary for introduction of T-DNA into a plant or plant leaf cell in the absence of a complement factor;
(Ii) lacks the ability to conjugate plasmid transfer to other bacteria, and (iii) is auxotrophic.
A bacterium belonging to the genus Agrobacterium, characterized by Agrobacterium contains a heterologous DNA sequence in the T-DNA having a sequence portion encoding a replicon, and the sequence encoding the replicon is:
(I) a sequence necessary for a replicon function of a plant replicon derived from a plant virus, and (ii) a target sequence to be expressed in a plant to which a bacterium is applied,
40. The bacterium of claim 39, comprising:

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