JP2002112771A - Rearranged replication enzyme originating from plant virus, plant virus retaining the same, and their use - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a plant virus having a rearranged replication enzyme imparting a rearranged intercellular mobility to a plant virus and a genome encoding the replication enzyme, and to provide usages of the replication enzyme and the plant virus for a transgenic plant having a strong resistance to virus, to obtain an attenuated virus having a high safety and its application to a plant used for the effective production of a useful material. SOLUTION: It is bound through the analysis of a chimera virus UR-he1 of the tobacco mosaic virus strains TMV-R and TMV-U1 that a replication enzyme of a tobacco mosaic virus participates not only in replication of a virus in a cell of a plant body but also in an intercellular migration of a virus. The plant virus retaining the replication enzyme and replication enzyme gene is expected for use in preparation of a plant having a virus resistance, preparation of an attenuated virus and application for production of a useful material in plants.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD [0001] The present invention relates to a modified replication enzyme derived from a plant virus, a plant virus having the replication enzyme, and use thereof. The replication enzyme and the plant virus are effective in improving virus resistance in plants and in producing substances in plants.

[0002]

2. Description of the Related Art Tobacco mosaic virus (TMV) is a typical plant virus whose genome is a single-stranded RNA of + strand. Four genes are encoded in the genome of tobacco mosaic virus. Two of them are replication enzymes, 126K.
183K protein consisting of a protein and its read-through, and is responsible for intracellular replication. The other is a translocation protein (MP) involved in cell-to-cell movement after infection of the plant, and the other is a coat protein (CP) essential for tissue-to-tissue movement after infection of the plant.

[0003] Infection of a plant with the tobacco mosaic virus is generally carried out in the following three stages. First, the viral genome is replicated in the invading single cell by the 183 kDa replication enzyme consisting of the 126 kDa replication enzyme and its read-through. The viral genome is then translocated to the neighboring cells of the infected cell through protoplasmic communication by a translocating protein downstream of the replication enzyme and spread throughout the infected leaf. Then
As the virus spreads in the infected leaves, the viral genome forms particles by the coat protein and moves to the uninfected upper leaf through the vascular system.

[0004] Tobacco mosaic virus mainly uses solanaceous plants as hosts. Upon infection with the tobacco mosaic virus, the leaves of the developing plant develop shades, exhibit mosaic symptoms, and slow plant growth. For this reason, when tobacco mosaic virus infects crops, their quality and yield are reduced, causing serious agricultural damage.

[0005] In recent years, for the purpose of preventing such virus damage, attempts have been made to create virus-resistant plants using molecular biology. One of these is the use of attenuated viruses. This is because plants infected with a certain virus are less likely to be infected with the same or related viruses, or the "interference effect" between viruses, or when stress is applied to a part of a plant, that information is transmitted to the whole body. A phenomenon in which a new resistance to stress is transmitted systemically (systemic acquired resistance, SAR), and when a sequence stored in the plant invades from the outside, the RNA of an enemy invading the plant It utilizes "the resistance of the plant itself", such as specifically reducing synthesis (gene silencing). For example, TMV-M, which is an attenuated virus that shows only very faint symptoms at best when plants infect tobacco or other crops, has a strong interference with virulent tobacco mosaic virus. Has been reported (Holmes F, Phytopathology 24, 845-873 (1934)).

[0006] Attempts have also been made to produce virus-resistant plants by expressing the gene of a plant virus itself in a host plant. This also utilizes "the resistance of the plant itself". For example, it has been reported that a gene encoding a tobacco mosaic virus coat protein was successfully introduced into a tobacco plant and expressed to succeed in producing a tobacco plant resistant to tobacco mosaic virus (Powell). Abel, P et al, Science 232,738-743
(1986)).

[0007] However, the production of resistant plants using attenuated viruses requires safety because the attenuated virus used is a live virus that can be transmitted to the surroundings, although its toxicity is improved. There is a big problem in point. In addition, when creating a resistant plant into which a plant virus gene has been introduced, when a wild-type gene is used, there is a risk that other plant virus infections will be assisted and that a virus that cannot be infected will infect the plant. Or part of the viral RNA expressed in plants and infectious viral RNA
However, there is no denying the possibility that the virus will be rearranged and a virus not found in nature will appear. In addition, transgenic plants expressing a wild-type gene can be expected only to have resistance at the RNA level, and cannot be expected to suppress virus growth at the protein level. Therefore, it was necessary to suppress the propagation power by modifying the gene of the plant virus.

On the other hand, attempts have been made to utilize plant virus vectors in the production of useful substances in plants.
However, even if a viral vector is produced for such a purpose, the range of the host plant is limited, and a useful substance may not be produced sufficiently. Further, even if a virus vector is modified in order to expand the host range of a plant virus in a plant, intracellular replication can be performed, but transfer between cells cannot be performed in many cases. Further, at this time, even if the transfer protein of the viral vector is replaced with a transfer protein of a virus capable of systemically infecting a plant to be infected, a desired effect is often not obtained. From such a viewpoint, it has been desired to extend the host range by modifying the virus vector.

[0009]

DISCLOSURE OF THE INVENTION The present invention has been made in view of the above circumstances, and provides a modified replication enzyme for controlling the ability of a plant virus to move from cell to cell, and encodes the replication enzyme. A plant virus having a genome is provided. Furthermore, the present invention provides a transgenic plant having high virus resistance, a highly safe attenuated virus, and a virus vector having an improved host range for producing useful substances, of the replication enzyme and the plant virus. To provide applications.

[0010]

Means for Solving the Problems Tobacco mosaic virus mainly uses solanaceous plants as hosts, but strains using various plants as hosts have been found. In 1994, a new strain TMV-R was discovered from a monocotyledon, Rakkyo. TMV-U1, which is a common system, infects tobacco systemically, whereas TMV-R replicates intracellularly and can move between cells, but tobacco cannot move between tissues and spreads only in infected leaves.

In order to elucidate the cause of the difference between the two virus strains, the present inventors first prepared a chimeric virus encoding a chimeric protein derived from both virus strains for each protein, and produced those chimeric viruses in tobacco. Behavior was studied. As a result, it was found that the ability of the virus to move between tissues was determined depending on the replication enzyme.

On the basis of the results, the present inventors have prepared chimeric viruses for the replication enzyme in order to clarify the site that determines the host range of the virus. Since it is thought that there are three domains in the replication enzyme, these three domains were targeted for chimeric virus production.
As a result, we found an interesting chimeric virus UR-hel by chance.

UR-hel is prepared by genetic recombination such that only the RNA helicase domain of the replication enzyme is a sequence derived from TMV-R, and all others are sequences derived from TMV-U1. Therefore, the nucleotide sequence encoding the UR-hel translocation protein is the same as that of TMV-U1.

Surprisingly, however, it has been found that UR-hel cannot transfer between cells. In addition, in the synthesis of the UR-hel transfer protein, the quantitative and peak patterns were almost the same as those of TMV-U1, indicating that the replication enzyme functions in the cells.

From these results, it was found that the UR-hel replication enzyme plays a sufficient role of replication, but completely lacks the ability to translocate between cells, which was previously considered to be dependent on the translocation protein. did. In other words, the present inventors have elucidated for the first time that not only a transfer protein but also a replication enzyme originally considered to be involved in the intracellular replication of the virus is directly involved in the movement of tobacco mosaic virus between cells. Was successful.

The present inventors further studied to apply the obtained knowledge to the production of virus-resistant plants. As a result, by modifying the replication enzyme, the ability of the virus to move from cell to cell can be lost and attenuated, thereby avoiding the risk of virus transmission, which has been a problem with conventional attenuated viruses. I found sex. Furthermore, the present inventors express the modified replication enzyme in a plant, thereby infecting a plant with a virus that cannot be infected originally, which has conventionally been a problem in a virus-resistant plant expressing a wild-type virus gene. In addition, this modified replication enzyme inhibits the growth of infectious virus not only at the RNA level but also at the protein level by competing with the wild-type replication enzyme produced by virus infection. We also found the potential. The present inventors have further attempted to extend the host range by modifying a plant virus gene for the purpose of, for example, producing a useful substance in a plant body. It has been found that the problem of inability to solve this problem can be solved by modifying the replication enzyme.

That is, the present invention relates to a modified plant virus replication enzyme, a plant virus having the replication enzyme, and use thereof, and more particularly, to (1) a replication enzyme, a coat protein, and a transfer protein. A replication enzyme derived from a plant virus having a genome encoding, and having a mutation introduced into its amino acid, wherein the introduction of the mutation causes the plant virus to alter the ability of the plant virus to move between cells of the plant. Enzymes, (2) retaining a genome encoding a replicating enzyme, a coat protein, and a transfer protein, and having the ability to replicate in plant cells, the ability to transfer between plant cells, and the ability to transfer between plant tissues A replication enzyme derived from a plant virus having translocation ability and having a mutation introduced into its amino acid, wherein the introduction of the mutation is caused by the plant virus Replication enzymes to lose the migration ability,
(3) the replication enzyme according to (1) or (2), wherein a mutation is introduced into at least the amino acid of the RNA helicase domain; (4) a replication-derived single-stranded RNA virus derived from (1) to (3) The replication enzyme according to any of the above,
(5) a replication enzyme according to any one of (1) to (3), which is derived from a virus belonging to the Sindbis-like virus superfamily; (6) a replication enzyme derived from a virus belonging to the genus Tobamovirus, (1) to (3) The replication enzyme according to any one of (1) to (3), which is derived from tobacco mosaic virus;
(8) a nucleic acid molecule encoding the replication enzyme according to any one of (1) to (7), and (9) a nucleic acid molecule encoding the replication enzyme according to any one of (1) to (7), (10) a nucleic acid molecule encoding the replication enzyme according to any one of (1) to (7) or a plant cell carrying the vector according to (9), (11)
(12) A plant comprising the plant cell according to (10).
A plant that is a progeny or a clone of the plant according to (11), (13) a propagation material of the plant according to (11) or (12), (14) a replication enzyme, a coat protein,
And a plant virus having a genome encoding a transfer protein, wherein the genome encoding the replication enzyme is modified to encode the replication enzyme according to (1),
It does not lose the ability of the plant to replicate in cells,
A plant virus having an altered ability to transfer between cells of a plant,
(15) Retaining a genome encoding a replicating enzyme, a coat protein, and a transfer protein, and replicating ability in plant cells, transfer ability between plant cells, and transfer ability between plant tissues. In a plant virus having, the genome encoding the replication enzyme is modified to encode the replication enzyme according to (2), whereby the replication ability in the plant cell is not lost. (16) The plant virus according to (14) or (15), wherein the plant virus is a plus-strand single-stranded RNA virus, and (17) the plant virus is a Sindbis-like virus superfamily. (18) The plant virus according to (14) or (15), wherein the plant virus is a virus belonging to the genus Tobamovirus. (14) The plant virus according to (15), (19) the plant virus according to (14) or (15), wherein the plant virus is a tobacco mosaic virus, and (20) (14) to (1).
9) A plant infected with the plant virus according to any one of 9).

[0018]

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a modified plant virus replication enzyme. The present invention is based on the fact first discovered by the present inventors that the replication enzyme of a plant virus is involved not only in the replication of the virus in plant cells but also in the transfer of the virus between cells. The modification of the replication enzyme in the present invention is performed from two viewpoints. One of them is a modification to eliminate the cell-to-cell movement ability of a plant virus that originally has a cell-to-cell movement ability. It is a modification to give.

In other words, for the purpose of eliminating the ability of a plant virus to move from cell to cell, the replication enzyme of the present invention, by its modification, can inhibit plant virus having a genome encoding the replication enzyme from intercellularity of the plant. It has the ability to lose the transfer ability. For the purpose of imparting the ability to transfer cells from one plant cell to another, the replication enzyme of the present invention, by its modification, is capable of transferring a plant virus carrying a genome encoding the replication enzyme between cells of a plant. Is given. The "replication enzyme" in the present invention is intended to include the case where the original replication ability has been lost as long as it has the above-mentioned ability.

These replication enzymes are preferably modified in at least a part of the amino acids of the RNA helicase domain. In this case, amino acids in regions other than the RNA helicase domain may be modified as long as the modified replication enzyme has the above ability. Regarding the RNA helicase domain, see the literature (Fernandez et al., (1995) Nucleic. Aci.
ds.Res.23, 1327-1332).

The amino acid modification in the replication enzyme of the present invention may be any of amino acid substitution, deletion and insertion mutations or a combination of these mutations, as long as the replication enzyme has the above ability. . The introduction of such a mutation can be carried out using a gene recombination technique or a mutation introduction technique (Molecular Cloning (Sambrook et al., 1989)) known to those skilled in the art. Whether or not the mutation-introduced replication enzyme has the above ability can be determined by infecting a host plant with a virus having a genome encoding the mutation-introduced replication enzyme, and determining the presence or absence of cell-to-cell movement. . For the purpose of eliminating the ability of plant virus to transfer from cell to cell,
The plant virus derived from the replication enzyme of the present invention includes a replication enzyme, a coat protein, and a genome encoding a movement protein, and has a replication ability in plant cells,
The plant virus is not particularly limited as long as it is a plant virus having a transfer ability between cells of a plant and a transfer ability between tissues of a plant.
For the purpose of imparting cell-to-cell movement ability to a plant virus, the replication-enzyme of the present invention is derived from a plant virus that retains a genome encoding a replication enzyme, a coat protein, and a movement protein, There is no particular limitation as long as it is a plant virus that has an ability to replicate in cells but does not have the ability to transfer between cells of a plant.

Among plant viruses, the ability to transfer from cell to cell may vary depending on the strain, even if the viruses are the same species. The plant virus from which the replication enzyme of the present invention is derived is generally a plus-strand single-stranded RNA virus. Examples of the positive-sense single-stranded RNA virus applicable to the present invention include viruses belonging to the Sindbis-like virus superfamily, luteo-like virus superfamily, and picorna-like virus superfamily. Viruses belonging to the Sindbis-like virus superfamily are preferable. . Viruses belonging to the Sindbis-like virus superfamily include:
Viruses belonging to the genus Potexvirus, genus Cucumovirus, genus bromovirus and the like can be exemplified, and among them, particularly preferred viruses are viruses belonging to the genus Tobamovirus. Viruses belonging to the genus Tobamovirus include, for example, tobacco mosaic virus, tomato mosaic virus, and obda pepper virus, with tobacco mosaic virus being particularly preferred.

The present invention also provides a nucleic acid molecule encoding the replication enzyme of the present invention. The nucleic acid molecule encoding the replication enzyme of the present invention includes genomic RNA, cDNA, chemically synthesized RNA, and chemically synthesized DNA. The nucleic acid molecule encoding the replication enzyme of the present invention can be used, for example, for producing a virus-resistant transgenic plant. The vector used for the transformation of plant cells for producing a transgenic plant is not particularly limited as long as it can express the inserted gene in the cells. A specific vector is pBI121 which is a binary vector.
(Toyobo) and pCaMVCN and pNCN (Pharmacia) which are vectors for electroporation. As a promoter in a vector, for example, a promoter for performing constant gene expression in plant cells (for example, cauliflower mosaic virus)
It is possible to use a promoter that is inducibly activated by an external stimulus.

The "plant cells" into which the vector is introduced include:
Various forms of plant cells, such as suspension culture cells, protoplasts, leaf sections, callus, and the like, are included. The type of plant cell into which the vector is introduced varies depending on the origin of the replication enzyme of the present invention expressed from the vector. For example, if the replication enzyme is a virus belonging to the genus Tobamovirus,
Hosts are mainly solanaceous plants, and if the virus belongs to the genus Bromovirus, the host is mainly grasses and legumes. If the virus belongs to the genus Cucumovirus, various species including mainly cucurbitaceae and legumes are used. Plants are the host. For the relationship between the virus and its host, see, for example, the literature (Virology, Shoichi Hatanaka, Asakura Shoten, Dictionary of Plant Virology, Kiyora Kora, Asakura Shoten, Crop Virology Dictionary, Tsuneo Tsuchizaki, National Rural Education Association, etc. )It is described in.

Various methods known to those skilled in the art, such as a polyethylene glycol method, an electroporation method, an Agrobacterium-mediated method, and a particle gun method, can be used for introducing a vector into a plant cell.

Regeneration of a plant from a transformed plant cell comprises:
It can be performed by a method known to those skilled in the art depending on the type of plant cell. Regeneration of a plant from a plant tissue or cell generally includes a method of inducing adventitious buds from a plant tissue or cell to regenerate, and a method of inducing and regenerating an adventitious embryo. For example, in tobacco, through the formation of shoots (adventitious shoots) from tissue pieces such as leaves or dedifferentiated tissues such as callus,
Regenerate plants. On the other hand, plant species, such as carrots, which are difficult to induce adventitious buds, regenerate plants from dedifferentiated cells such as callus and suspension cultured cells through the development of embryos (adventive embryos). For the method of plant regeneration, see the literature (Katsuji Osawa, Basic Knowledge of Plant Biotech, pp. 72-78) and p7
See the references cited in 2-78.

Once a transformed plant in which the DNA of the present invention has been introduced into the genome is obtained, progeny can be obtained from the plant by sexual or asexual reproduction. In addition, it is also possible to obtain a propagation material (eg, seed, fruit, cutting, tuber, tuber, root, strain, callus, protoplast, etc.) from the plant, its progeny or clone, and mass-produce the plant based on them. It is possible. In the present invention, plant cells into which DNA encoding the replication enzyme of the present invention has been introduced, plants containing the cells, progeny and clones of the plant, and propagation material of the plant, progeny thereof, and clones included. The plant thus produced can have improved virus resistance as compared to a wild-type plant.

The present invention also provides a plant virus having a genome encoding the above-mentioned replication enzyme of the present invention. One embodiment of the plant virus of the present invention does not lose the ability of the plant to replicate in cells but loses the ability of the plant to migrate between cells due to the modification of the replication enzyme. In another embodiment of the plant virus of the present invention, the ability to transfer between plant cells, which is not originally possessed, is imparted by modifying a replication enzyme.
The origin of these plant viruses is the same as the above-mentioned plant viruses from which the replication enzyme of the present invention is derived.

A plant virus that has lost the ability to transfer between cells can be used, for example, as an attenuated virus. That is, by infecting a host plant with the plant virus of the present invention, virus resistance of the plant can be improved. On the other hand, a plant virus having the ability to transfer between cells can be used, for example, for producing useful substances in plants. By inoculating a host plant with the plant virus of the present invention, the virus can be transferred from cell to cell, and thus can be infected systemically, and useful substances can be effectively produced in the plant. The useful substance is not particularly limited as long as it is encoded by a nucleic acid molecule. For example, it is conceivable to express an antigen (epitope) in a plant as a fusion protein with a coat protein. See the literature (virology, Asakura Shoten) for the construction of plant virus vectors.

The method of infecting a plant with a virus includes a method of causing a virus to physically invade a plant and infecting the plant, and a method of transmitting the virus through an insect or fungus. For viruses that physically infect (such as TMV and BMV), the viral genome is already cD
Some are reverse transcribed into NA and integrated into plasmids such as E. coli and yeast (Ahlquist et al., (1984) Pr.
oc.Natl.Acad.Sci.USA.81,7066-7070, Ishikawa, M.et a
l., (1997) J. Virol. 71,7781-7790). Since the T7 promoter exists upstream of the cDNA in the plasmid, transcription is performed in vitro using T7 RNA polymerase to synthesize viral RNA during infection. The RNA solution is sprayed with a carborundum (abrasive) in advance, spotted on the leaf whose surface has been damaged, and rubbed with a cotton swab or a glass stick to establish infection. If the virus has a coat protein, purified virus particles formed in plants infected with these viruses, or a slightly crushed leaf of the infected plant, can be similarly applied to the leaves of the plant to be infected. By rubbing, it can be infected. On the other hand, a virus that is transmitted by insect transmission can be transmitted as described above. However, if the infection is not mediated by insects (for example, the infected leaves are ground and inoculated to the non-infected leaves), the insects can be infected. Genes to be transmitted through the virus may drop out. For this reason, it is preferable to establish the infection by binding the infected leaves and virus-free insects for several days to one week to transmit the virus to the insects, and then similarly closing the infected insects and non-infected plants. The present invention includes a plant thus infected with the plant virus of the present invention.

[0031]

EXAMPLES Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples. [Example 1] Preparation of UR-hel and intracellular replication ability The full-length TMV-U1 and TMV-R were reverse-transcribed, and a plasmid having UR-hel cDNA was prepared from the plasmid incorporated downstream of the T7 promoter. The arrangement of UR-hel is based on TMV-U1, N
The restriction fragment of si I (base 2354) -Bam HI (base 3332)
It is derived from VR. This fragment contains 12 TMVs
There is an amino acid different from -U1. Similarly, from U1Δc-GFP and UR-hel that have already been created, UR-helΔC-G
Created FP.

FIG. 1 shows a schematic diagram of the UR-hel genome structure. UR-hel is based on TMV-U1 and TMV-R Nsi I-B
The am HI restriction fragment was recombined. In this part
There are 12 different amino acids.

The ability of UR-hel to replicate intracellularly was determined by measuring the accumulation of the most synthesized coat protein 16 hours after infection with protoplasts (cultured cells excluding cell walls).
It was confirmed by detection on S-PAGE. As a result, UR-h
el accumulates almost the same amount of coat protein as TMV-U1, indicating that it has sufficient intracellular replication ability.

[Example 2] Deficiency of UR-hel in cell-to-cell transferability The virus-to-cell transferability of Nicotiana having the N gene
Confirmed using tabacum cv. Xanthi-nc. The N gene is a gene that is resistant to tobacco mosaic virus. When infected with tobacco mosaic virus, it produces local necrotic spots called local regions (hereinafter abbreviated as necrotic spots).
This is a phenomenon in which cells that have not been infected with the virus, surrounding the infected cell, commit suicide first, thereby confining the spread of the virus and thereby preventing systemic infection. It has been empirically found that the size of the necrotic spot is proportional to the ability to move between cells. That is, the smaller the necrotic spot, the lower the ability of the used tobacco mosaic virus to move between cells.

Using Xanthi-nc, a tobacco having this N gene, the ability of UR-hel to move between cells was analyzed. As a result, no necrotic spots were unexpectedly generated (FIG. 2A).
UR-hel replicated intracellularly, so this surprising result is due to UR-hel not migrating or very weak, UR-hel migrating from cell to cell but not necrotic , Either thought.

Then, whether or not it was the cause was analyzed next. Four days after infection, the infected leaves were ground and subjected to Western blot with an antibody against the coat protein, and the presence of the virus was confirmed by detection of the protein.

In the Western blot, first, proteins were extracted from leaves. That is, after adding and suspending and centrifuging a phosphate buffer four times as much as the leaf, the supernatant was mixed with an equal amount of SDS-PAGE buffer and boiled for 5 minutes.
This was subjected to SDS-PAGE (12.5% acrylamide), and then the band of interest was detected using an antibody against the coat protein.

As a result, when TMV-U1 was infected,
While the coat protein was sufficiently detected, UR-hel
Was not detected at all (FIG. 3). This ruled out the possible cause, and found that UR-hel was unable to transfer from cell to cell or was very weak.

[Example 3] The reason that UR-hel cannot transfer from cell to cell is not due to the amount of transfer protein synthesized. The sequence of UR-hel transfer protein is the same as that of TMV-U1. However, in the UR-hel, the replication enzyme has been recombined. The replication enzyme synthesizes mRNA for the transfer protein, but if the UR-hel replication enzyme synthesizes no or very small amounts of the mRNA for the transfer protein, even if the sequence of the transfer protein is OK, No transfer between cells is possible.

In order to confirm this point, intracellular replication was analyzed in more detail using radioisotopes (FIG. 4). Infect TMV-U1 and UR-hel with protoplasts,
Up to 24 hours post-infection, the synthesis of translocation and coat proteins was detected by 35S methionine / cysteine.

For the preparation of protoplasts, BY2 tobacco cultured cells were used. At the time of virus infection to protoplasts, RNA transcribed from a plasmid using T7 RNA polymerase was used. After infection with protoplasts, 3, 6, 9,
At 12, 12, and 24 hours, 0.1 MBq of 35S methionine / cysteine, a radioisotope, was added, and sampling was performed 30 minutes later. The protoplasts were centrifuged, the supernatant was removed, mixed with SDS-PAGE buffer and boiled for 5 minutes.
After that, the extracted protein was subjected to SDS-PAGE, and Fuji I
35S was detected by mage Analyazer.

As a result, the synthesis of the UR-hel transfer protein was almost the same as that of TMV-U1 in both quantity and peak pattern (FIG. 4).

[Example 4] Transgenic tobacco expressing the transfer protein, no necrotic spots were generated in 2005 From the above results, the UR-hel transfer protein was identical to TMV-U1 in both sequence and synthesis. Turned out to be.
However, as shown in Example 2, UR-hel has no or very weak ability to move between cells. This fact suggests that tobacco mosaic virus proteins
This overturns the common belief that each protein corresponds one to one for each step.

The present inventors have proved that UR-hel has no loss of cell-to-cell movement ability, but does not have a problem with the amount of transfer protein synthesized, and has no problem with the recombination enzyme replication. To do so, experiments were performed using Xanthi-nc transgenic plants, 2005. 2005 is TMV-U1
Has the property of assisting tobacco mosaic virus that has lost the function of the translocation protein. For example, U1ΔMP did not produce necrotic spots in Xanthi-nc (FIG. 2A), but did infect 2005 (FIG. 2B). As described above, in 2005, even a virus having a problem with a transfer protein is transferred between cells.

Then, UR-hel was infected in 2005 and the formation of necrotic spots was confirmed (FIG. 2B). UR-hel did not produce necrotic spots in 2005, ie, even after recruiting translocation protein, whereas U1ΔMP did.

From the results, it was found that the fact that UR-hel cannot move between cells (very weak) was not caused by the transfer protein. This proved that the replication enzyme was directly involved in cell-to-cell movement.

[Example 5] Detailed observation of UR-hel by GFP It was determined whether UR-hel could not migrate between cells at all, or it migrated a little, but the necrotic spots were simply not visible. Was analyzed using. FIG. 1B is a schematic diagram of the virus used.

As described above, the coat protein is not involved in intracellular replication or cell-to-cell movement, and it has already been found that without this, the virus can spread to infected leaves. Therefore, GFP was expressed instead of the coat protein, and the behavior of the virus was observed.

FIG. 5 shows the results. When necrotic spots are formed, it becomes difficult to observe the GFP signal. Therefore, in this experiment, a tobacco without the N gene, Samsun, was used. In macroscopic observation (FIG. 5A), TMV-U1 spread on a circle of about 1 mm on the fourth day after infection, like necrotic spots. In contrast, UR-hel did not show GFP expression.

However, observation at the cell level with a microscope (FIG. 5B) revealed that the UR-hel glowed in one cell. That is, it was confirmed that UR-hel was unable to move between cells at all.

[0051]

Industrial Applicability According to the present invention, a replication enzyme whose amino acid is modified in order to modify the ability of a plant virus to move between cells, and a plant having a genome encoding the replication enzyme and having an improved ability to move between cells Virus provided. This has made it possible to produce transgenic plants with high virus resistance and highly safe attenuated viruses. It is considered that if the present invention is applied to crops, it is possible to improve the virus resistance of useful crops and to prevent a decrease in quality and a decrease in yield due to a virus disease. In addition, the present invention makes it possible to expand the host range of plant viruses, and it is thought that the use of such plant viruses enables effective production of useful substances in plants.

[Brief description of the drawings]

FIG. 1 shows a schematic diagram of virus genome construction. ORF
The red line in indicates the difference in amino acid position between TMV-U1 and TMV-R. The gray part in the chimeric virus indicates a sequence obtained from the TMV-R genome. A: TMV-U1 and TMV-R parent virus, and the obtained chimeric virus. B: virus in which CP was replaced with GFP.

FIG. 2 is a photograph showing a state of a local lesion on the fourth day after infection in a plant having an N gene. All the left half leaves were inoculated with TMV-U1. A: Xanthi inoculated with UR-hel and U1ΔMP in the right half of the leaf
-nc leaves. No local lesions appeared in the right half of any lobe. B: 2005 leaves in which the right half of the leaves were inoculated with UR-hel and U1ΔMP. The 2005 strain expressed the TMV-U1 MP gene. Leaves inoculated with U1ΔMP showed localized lesions, but not in leaves inoculated with UR-hel.

FIG. 3 is a photograph showing the result of detection of a tobacco mosaic virus coat protein by Western blot. Protein was extracted from the inoculated leaves. The first lane shows the location of the coat protein obtained from purified tobacco mosaic virus virions (0.2 μg). Total soluble proteins were extracted on day 4 after infection.

FIG. 4 is a photograph showing a comparison of transprotein and coat protein synthesis in protoplasts infected with TMV-U1 and UR-hel. UR-hel synthesized translocation and coat proteins to the same level as TMV-U1.
“Hpi” in the figure indicates the time since infection.

FIG. 5 is a photograph in which the reaction of the virus with GFP was observed on the fourth day after infection. A: GFP in Samsun leaves inoculated with U1ΔC-GFP (left half) and UR-helΔC-GFP (right half). The bar is 10mm. B: Image of each half of the leaf under a fluorescence microscope. UR
In the right half inoculated with helicase ΔC-GFP, GFP was observed in only one cell. Bars are 200 μm.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) // (C12N 15/09 (C12N 7/00 C12R 1:94) C12R 1:94) (C12N 5/10 C12N 15/00 A C12R 1:94) 5/00 C (C12N 7/00 C12R 1:94) C12R 1:94) (72) Inventor Keitaro Hirashima 6-23-51 Re, Mizuguchi, Takatsu-ku, Kawasaki City, Kanagawa Prefecture Bion Takatsu 204 F term (reference) 2B030 AB03 AD05 CA14 CA17 4B024 AA08 BA07 CA04 DA01 EA01 GA11 GA17 GA21 HA01 HA15 4B050 CC01 CC04 DD20 LL10 4B065 AA88X AA95X AA95Y AB01 AC14 BA03 BA10 CA27 CA53

Claims (20)

[Claims] 1. A replication enzyme derived from a plant virus having a genome encoding a replication enzyme, a coat protein, and a movement protein, wherein a mutation is introduced into the amino acid thereof, and the introduction of the mutation is carried out by the plant virus. A replication enzyme that alters the ability of plants to move between cells. 2. A method for retaining a genome encoding a replication enzyme, a coat protein, and a transfer protein, and having the ability to replicate in a plant cell, the ability to transfer between cells of a plant, and the ability to transfer between tissues of a plant. A replication enzyme derived from a plant virus having translocation ability and having a mutation introduced into its amino acid, wherein the introduction of the mutation causes the plant virus to lose the translocation ability between plant cells. 3. The replication enzyme according to claim 1, wherein a mutation is introduced into at least amino acids of the RNA helicase domain. 4. The replication enzyme according to claim 1, which is derived from a positive-sense single-stranded RNA virus. 5. The replication enzyme according to claim 1, which is derived from a virus belonging to the Sindbis-like virus superfamily. 6. The replication enzyme according to claim 1, which is derived from a virus belonging to the genus Tobamovirus. 7. The replication enzyme according to claim 1, which is derived from tobacco mosaic virus. 8. A nucleic acid molecule encoding the replication enzyme according to claim 1. 9. A vector into which a nucleic acid molecule encoding the replication enzyme according to claim 1 has been inserted. 10. A plant cell carrying a nucleic acid molecule encoding the replication enzyme according to any one of claims 1 to 7 or a vector according to claim 9. 11. A plant comprising the plant cell according to claim 10. 12. A plant which is a progeny or a clone of the plant according to claim 11. 13. A propagation material for a plant according to claim 11 or 12. 14. A plant virus having a genome encoding a replication enzyme, a coat protein, and a movement protein, wherein the genome encoding the replication enzyme is provided.
A plant virus modified so as to encode the replication enzyme described in 1 above, thereby not losing the ability of the plant to replicate in cells, but having an altered ability to transfer between cells of the plant. 15. A plant which retains a genome encoding a replication enzyme, a coat protein, and a transfer protein, and has the ability to replicate in plant cells, the ability to transfer between plant cells,
And a plant virus having the ability to transfer between tissues of a plant, wherein the genome encoding the replication enzyme is modified to encode the replication enzyme according to claim 2, whereby the ability of the plant to replicate in cells. A plant virus that does not lose its capacity but loses the ability to transfer between cells of the plant. 16. The plant virus according to claim 14, wherein the plant virus is a positive-sense single-stranded RNA virus. 17. The plant virus according to claim 1, wherein the plant virus is a virus belonging to the Sindbis-like virus superfamily.
16. The plant virus according to 4 or 15. 18. The plant virus according to claim 14, wherein the plant virus is a virus belonging to the genus Tobamovirus. 19. The plant virus according to claim 14, wherein the plant virus is a tobacco mosaic virus. 20. A plant infected with the plant virus according to any one of claims 14 to 19.