JP2008212067A - Method for preparation of plant resistant to plant virus and use thereof - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a plant virus-resistant plant having high resistance to plant viruses. <P>SOLUTION: The method for the preparation of a plant virus-resistant plant comprises a step to increase the plant virus proliferation suppressing activity in a plant body by a polypeptide binding to the genom RNA of the plant virus to suppress the proliferation of the plant virus. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for producing a plant virus-resistant plant and its use. More specifically, the present invention relates to a method for producing a plant virus-resistant plant using a polypeptide having an activity of binding to a plant RNA genomic RNA and suppressing the growth of the plant virus and the use thereof.

  Diseases caused by plant viruses can seriously affect crop yield and quality. Until now, as countermeasures against viral diseases, prevention of spread of infection by early detection and removal of diseased strains, control of vector insects, cross-protection by attenuated viruses, and the like have been utilized. In addition, there are known loci in nature that confer resistance to specific viruses, and some of them have actually been introduced into crops. A technique for imparting virus resistance to plants by introducing a part of the virus genome into the plant genome (pathogen-derived resistance) is also expected as an effective countermeasure.

  An example of a plant virus that causes disease is Tobamovirus. Tobamovirus is a plus-strand RNA virus belonging to the alphavirus superfamily. Examples of tobamovirus members include tobacco mosaic virus and tomato mosaic virus (hereinafter also referred to as “ToMV”).

  The plus-strand RNA virus has a single-stranded RNA (polar RNA) functioning as mRNA in the virus particle as a genome. The majority of plant viruses, including tobamoviruses, and many animal viruses such as hepatitis C virus and poliovirus are positive-strand RNA viruses. When a plus-strand RNA virus enters a host cell, a protein involved in replication (replication protein) is translated from genomic RNA. The replication protein recognizes its own genomic RNA and forms an RNA replication complex on the cytoplasmic surface of the organelle membrane of the host cell. The RNA replication complex replicates genomic plus-strand RNA (offspring genomic RNA) via minus-strand RNA complementary to the plus strand (see Non-Patent Documents 1 and 2). In addition, the translocation protein and coat protein of the virus are translated through the subgenomic RNA generated during replication. When this translocation protein is expressed, the viral genomic RNA translocates to the adjacent cells through the plant plasmodesmata (see Non-Patent Document 3).

Methods for preventing diseases caused by tobamovirus include, for example, a method of expressing a protein that binds to a tobamovirus transfer protein in plants (for example, Patent Document 1), identifying host factors necessary for virus growth, A method of imparting virus resistance to plants by suppressing expression (for example, see Non-Patent Document 3) has been proposed.
International Publication No. 03/022039 Pamphlet (published on March 20, 2003) Buck, KW, Comparison of the replication of positive-stranded RNA viruses of plants and animals., Adv.Vrius Res., 1996, 47, 159-251 Schwartz, M., Chen, J., Janda, M., Sullivan, M., den Boon, J., and Ahlquist, P., A positive-strand RNA virus replicationcomplex parallels form and function of retrovirus capsids., Mol. Cell, 2002, 9, 505-514 Lucas, WJ, Plant viral movement proteins: Agents for cell-to-cell trafficking of viral genomes., Virology, 2006,344,169,184 Asano et al., Tobamovirus-resistant tobacco generated by RNA interference directed against host genes., FEBS Lett., 2005, 579 (20), 4479-4484

  However, there is currently little knowledge of host genes that can genetically confer virus resistance to plants. In consideration of the possibility that a virus that breaks resistance will appear in the future, it is required to secure a wide repertoire of target genes. That is, a host gene currently used for conferring virus resistance has a possibility of becoming invalid in the future due to the appearance of a virus that breaks down the resistance. For example, regarding the resistance imparted to plants by the methods of Patent Document 1 and Non-Patent Document 3, the possibility that a virus that breaks down the resistance will appear in the future cannot be denied.

  Pathogen-derived resistance has the advantage that virus resistance can be conferred regardless of plant species, but the risk of new viruses being generated by recombination with the introduced sequence has also been pointed out (for example, Goldbach et al., Resistance mechanisms to plant viruses: an overview. See Virus Res., 2003, 92, 207-212).

  Therefore, it is required to prepare as many genes that can confer virus resistance as much as possible and to use them in a complex manner.

  This invention is made | formed in view of the said problem, The objective is to provide the plant virus resistant plant which has high resistance with respect to a plant virus.

  As a result of intensive studies in view of the above problems, the present inventors have newly found a protein that binds to the genomic RNA of tobamovirus and suppresses the proliferation of tobamovirus. And it discovered that the proliferation of a tobamovirus could be suppressed by overexpressing the said protein, and came to completion of this invention. That is, the present invention includes the following inventions.

  The method for producing a plant virus-resistant plant according to the present invention includes a step of promoting plant virus growth inhibitory activity by a polypeptide that binds to plant virus genomic RNA and suppresses plant virus growth in the plant body. It is characterized by that.

In the method for producing a plant virus resistant plant according to the present invention, the polypeptide is more preferably a polypeptide described in the following (a) or (b):
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(B) An amino acid sequence represented by SEQ ID NO: 1 or 2, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and has plant virus growth inhibitory activity A polypeptide having

  In the method for producing a plant virus-resistant plant according to the present invention, the step is more preferably performed by introducing a polynucleotide encoding the polypeptide into the plant body.

In the method for producing a plant virus resistant plant according to the present invention, the polynucleotide is more preferably a polynucleotide described in the following (c), (d) or (e):
(C) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or 4;
(D) a polynucleotide comprising a base sequence in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 3 or 4;
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 4.

  In the method for producing a plant virus resistant plant according to the present invention, the plant virus is more preferably a tobamovirus.

  In order to solve the above-mentioned problems, the kit for producing a plant virus-resistant plant according to the present invention is characterized by comprising a polynucleotide encoding a polypeptide that binds to a plant virus genomic RNA.

  In order to solve the above-mentioned problems, the screening method for a plant virus resistant plant according to the present invention comprises an accumulation amount of a polypeptide that binds to a plant virus genomic RNA and a polypeptide that binds to the plant virus genomic RNA. It includes a step of measuring at least one of the expression levels of the polynucleotide encoding the peptide.

  In order to solve the above problems, a plant virus-resistant plant screening kit according to the present invention comprises an antibody that specifically binds to a polypeptide that binds to a plant virus genomic RNA, and a polypeptide that binds to a plant virus genomic RNA. It comprises at least one of probes that specifically bind to a polynucleotide encoding a peptide.

  In order to solve the above-mentioned problems, the plant virus-resistant plant according to the present invention is produced by the method for producing a plant virus-resistant plant according to the present invention described above. Further, the present invention is characterized in that the plant virus growth inhibitory activity by the polypeptide that binds to the plant virus genomic RNA is promoted.

  As described above, the method for producing a plant virus resistant plant according to the present invention has a plant virus growth inhibitory activity by a polypeptide that binds to a plant virus genomic RNA and suppresses the growth of the plant virus in the plant body. Since the step of promoting is included, the plant virus-resistant plant having high resistance to the plant virus can be provided.

  An embodiment of the present invention will be described as follows, but the present invention is not limited to this.

<1. Method for producing plant virus resistant plant>
The method for producing a plant virus-resistant plant according to the present invention includes a step of promoting plant virus growth inhibitory activity by a polypeptide that binds to plant virus genomic RNA and suppresses plant virus growth in the plant body. That's fine.

  By promoting the activity of a polypeptide that binds to the genomic RNA of a plant virus and inhibits the growth of the plant virus, the growth of the plant virus can be suppressed. Thereby, the plant with improved resistance to plant viruses can be produced. Thereby, the plant virus resistant plant to which the resistance with respect to various plant viruses was provided can be manufactured.

  In the method for producing a plant virus resistant plant according to the present invention, the plant virus to be resistant to plants is not limited as long as it is a virus whose genome is RNA, for example, plus strand RNA Although a virus etc. are mentioned, if the BTR polypeptide etc. mentioned later are used among them, the resistance with respect to a tobamovirus can be provided suitably.

  Therefore, in the present embodiment, a method for producing a tobamovirus-resistant plant imparted with resistance against tobamovirus will be described. However, within the spirit of the present invention, plant virus resistance imparted with resistance against various plant viruses. Sex plants can be produced.

  Here, the tobamovirus refers to a virus belonging to the genus Tobamovirus. Examples of viruses belonging to the genus Tobamovirus include Cucumber green mottle mosaic virus (CGMMV), Frangipani mosaic virus (FrMV), Kyuri green mottle mosaic virus (KGMMV), Obuda pepper virus (ObPV), Odontoglossum ringspot virus (ORSV), Paprika mild mottle virus (PaMMV), Pepper mild mottle virus (PMMV), Ribgrass mosaic virus (RMV), Sammons's Opuntia virus (SOV), Sunn-hemp mosaic virus (SHMV), Tobacco mild green mosaic virus (TMGMV), Tobacco mosaic Examples include viruses (TMV), Tomato mosaic virus (ToMV), Turnip vein-cleaning virus (TVCV), Ullucus mild mottle virus (UMMV), Yucari mosaic virus (YoMV), and the like.

  In addition, a polypeptide having an activity of binding to tobamovirus genomic RNA and suppressing tobamovirus growth is hereinafter referred to as “BTR polypeptide”, and a polynucleotide encoding the polypeptide is referred to as “btr polynucleotide”. . Moreover, the activity that binds to the genomic RNA of tobamovirus and suppresses the proliferation of tobamovirus is referred to as “BTR activity”. “BTR” or “btr” is an abbreviation of “Binding to Tobamovirus RNA”. Further, in this specification, “to promote the tobamovirus growth inhibitory activity by the polypeptide that binds to the genome RNA of tobamovirus” is intended to promote the tobamovirus growth inhibitory activity by the BTR polypeptide more than the wild type strain. It does not limit the degree of advancement.

[1-1. BTR polypeptide and btr polynucleotide]
In the present invention, the polypeptide (BTR polypeptide) that binds to the tobamovirus genomic RNA is not limited as long as it binds to the tobamovirus genomic RNA and exhibits the activity of suppressing the proliferation of tobamovirus.

Examples of the BTR polypeptide include the polypeptides described in (a) below.
(A) A polypeptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2.

  Polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 2 are registered in GenBank with accession number NM_120525 and accession number NM_180433, respectively. In addition, the amino acid sequences shown in SEQ ID NOs: 1 and 2 are two types generated from the base sequence of the gene shown by Locus tag At5g04430 among the genomic DNAs of Arabidopsis thaliana registered with GenBank under the accession number NC_003076. Is a splicing variant.

  As will be described in detail in the Examples below, the polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 2 each have three KH domains. The KH domain is known as the RNA binding domain. However, conventionally, the function of the polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 1 and 2 has been completely unknown. The present inventors have found that Arabidopsis thaliana has a protein that binds to the genomic RNA of ToMV and suppresses the growth of ToMV, as will be described in later examples. And as a result of identifying this, it discovered that the said protein has an amino acid sequence shown to sequence number 1 and 2. The present invention is an invention that cannot be easily conceived even by those skilled in the art based on this completely new knowledge.

  In animals, PCBP2 (αCP2), which is a kind of poly-C binding protein and has a KH domain, has been reported to bind to the 5 ′ end region of poliovirus RNA and increase the translation efficiency of the RNA (Blyn , LB, Swiderek, KM, Richards, O., Stahl, DC, Semler, BL and Ehrenfeld, E., Proc. Natl. Acad. Sci. USA, 1996, 93, 11115-11120, Blyn, LB, Towner, JS Semler, BL and Ehrenfeld, E., J. Virol., 1997, 71, 6243-6246). That is, as a polypeptide having a KH domain, conventionally, a polypeptide that enhances the translation efficiency of viral genomic RNA has been known. In this case, even a person skilled in the art cannot easily conceive activation of the polypeptide consisting of the amino acid sequences of SEQ ID NOs: 1 and 2. This is because increasing the activity of a polypeptide comprising an amino acid sequence having a KH domain in plants in which diseases caused by various viruses cause problems may even cause the virus that causes the disease to grow.

In one embodiment, the BTR polypeptide is a variant of the BTR polypeptide. Examples of such mutants include the polypeptides described in the following (b).
(B) In the amino acid sequence shown in SEQ ID NO: 1 or 2, the amino acid sequence consists of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted or added, and has a tobamovirus growth inhibitory activity. Having a polypeptide.

  Such variants include deletions, insertions, inversions, repeats and type substitutions (eg, replacement of a hydrophilic residue with another residue, but usually leaving a strongly hydrophilic residue strongly hydrophobic). Variants that contain no substitution on the group are included.

  It is well known in the art that some amino acids in the amino acid sequence that make up a polypeptide can be easily modified without significantly affecting the structure or function of the polypeptide. Furthermore, it is also well known that there are mutants in natural proteins that do not change artificially, but do not significantly alter the structure or function of the protein.

  One skilled in the art can readily mutate one or several amino acids in the amino acid sequence of a polypeptide using well-known techniques. For example, according to a known point mutation introduction method, an arbitrary base of a polynucleotide encoding a polypeptide can be mutated. In addition, a deletion mutant or an addition mutant can be prepared by designing a primer corresponding to an arbitrary site of a polynucleotide encoding a polypeptide. Furthermore, if the method for measuring the amount of BTR polypeptide accumulation or btr polynucleotide expression described in the present specification is used, it can be easily determined whether the prepared mutant suppresses the growth of the desired tobamovirus. Can be determined.

  Typical conservative substitutions are substitutions from one amino acid to another in the aliphatic amino acids Ala, Val, Leu and Ile; exchange of hydroxyl residues Ser and Thr, acidic residue Asp And Glu exchange, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg, and substitution between aromatic residues Phe, Tyr.

  The BTR polypeptides described in (a) and (b) above bind to the 5 'terminal region of tobamovirus genomic RNA. More specifically, the BTR polypeptides described in the above (a) and (b) bind to RNA at positions 1 to 277 at the 5 'end of tobamovirus genomic RNA. However, the region on the genomic RNA of tobamovirus to which the polypeptide having BTR activity binds is not limited to this. In other words, any substance that binds to the genomic RNA of tobamovirus and suppresses the proliferation of tobamovirus may be used. For example, in Tobamovirus, in addition to the 5 ′ end region of the viral genomic RNA, the 3 ′ end untranslated region (positions 6280-6384) also plays an essential role in its own growth (Takamatsu, N., Watanabe, Y., Meshi, T. and Okada, Y., J. Virol., 1990, 64, 3686-3693, Takamatsu, N., Watanabe, Y., Iwasaki, T., Meshi, T. and Okada, Y., J. Virol., 1991, 65, 1619-1622). Therefore, even a BTR polypeptide that binds to the 3'-terminal untranslated region and inhibits its function can efficiently suppress virus growth.

  In addition, the BTR polypeptides (a) and (b) above specifically bind to tobamovirus. In the present invention, the BTR polypeptide is not particularly limited as long as it binds to the tobamovirus and suppresses the proliferation of the tobamovirus. However, like the BTR polypeptides (a) and (b) above, It is preferably one that specifically binds and suppresses proliferation. A BTR polypeptide that specifically binds to tomovirus and suppresses proliferation does not inhibit the growth of the plant itself by the BTR polypeptide. That is, it is possible to provide a tobamovirus resistant plant having improved resistance to tobamovirus without inhibiting the growth of the plant itself. In fact, in the examples described later, the present inventors have produced plants with improved resistance to tobamoviruses without inhibiting plant growth.

  As used herein, the term “polypeptide” is used interchangeably with “peptide” or “protein”. In addition, “fragment” of a polypeptide is intended to be a partial fragment of the polypeptide. Also, as used herein, the term “BTR polypeptide” refers to those produced by expression of btr polynucleotides in plants, those isolated from natural sources, or obtained by chemical synthesis. Also included. Note that the term “isolated” polypeptide or protein is intended to be a polypeptide or protein removed from its natural environment. For example, recombinantly produced polypeptides and proteins expressed in host cells can be isolated in the same manner as natural or recombinant polypeptides and proteins that have been substantially purified by any suitable technique. It is thought that there is.

  BTR polypeptides also include natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). Including products produced by recombinant technology. Depending on the host used in the recombinant production procedure, it can be glycosylated or non-glycosylated. Further, the BTR polypeptide may in some cases contain an initial modified methionine residue as a result of a host-mediated process.

  The BTR polypeptide may be any polypeptide in which amino acids are peptide-bonded, but is not limited thereto, and may be a complex polypeptide including a structure other than the polypeptide. As used herein, examples of the “structure other than the polypeptide” include sugar chains and isoprenoid groups, but are not particularly limited.

  The BTR polypeptide may also contain an additional polypeptide. Examples of the additional polypeptide include epitope-tagged polypeptides such as His, Myc, and Flag.

  As used herein with respect to a polypeptide or protein, the term “variant” intends a polypeptide having BTR activity (ie, a polypeptide that binds to tobamovirus genomic RNA and inhibits growth), eg, A “variant of a polypeptide consisting of the amino acid sequence shown in SEQ ID NO:” is a polypeptide that suppresses the growth of tobamovirus, and one or several amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 1. It is intended to be a polypeptide consisting of an amino acid sequence inserted, substituted, or added.

In one embodiment, the BTR polypeptide is a polynucleotide encoding a polypeptide having BTR activity, and is encoded by the polynucleotide described in the following (c), (d), or (e): Can be mentioned.
(C) A polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or 4.
(D) A polynucleotide comprising a base sequence in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 3 or 4.
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 4.

  Polynucleotides having the base sequences shown in SEQ ID NOs: 3 and 4 are registered in GenBank with accession numbers NM_120525 and accession numbers NM_180433, respectively. Polynucleotides having the amino acid sequences shown in SEQ ID NOs: 1 and 2, respectively. A polynucleotide encoding a peptide.

  The above “stringent conditions” means that hybridization occurs only when at least 90% identity, preferably at least 95% identity, most preferably 97% identity exists between sequences. Means that.

  The hybridization can be performed by a known method such as the method described in Sambrook et al., Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, 1989. Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize), and a more homologous polynucleotide can be obtained. As hybridization conditions, conventionally known conditions can be preferably used, and are not particularly limited. For example, 42 ° C., 6 × SSPE, 50% formamide, 1% SDS, 100 μg / ml salmon sperm DNA, 5 × Denhardt's solution (however, 1 × SSPE; 0.18M sodium chloride, 10 mM sodium phosphate, pH 7.7, 1 mM EDTA, 5 × Denhardt's solution; 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% Polyvinylpyrrolidone).

  As used herein, the term “polynucleotide” is used interchangeably with “gene”, “nucleic acid” or “nucleic acid molecule”, and is intended to be a polymer of nucleotides. Further, as used herein, the term “base sequence” is shown as a sequence of deoxyribonucleotides (abbreviated as A, G, C, and T).

[1-2. Step of promoting the tobamovirus growth inhibitory activity of the BTR polypeptide]
As described above, the method for producing a tobamovirus-resistant plant according to the present embodiment may include a step of promoting the tobamovirus growth inhibitory activity by the BTR polypeptide.

  Such a step is carried out by introducing a polynucleotide encoding the above-mentioned polypeptide in the plant, a method for improving the expression level of the btr polynucleotide present in the plant in advance, or purified BTR. Examples thereof include a method for introducing a polypeptide. In particular, the above step is preferably performed by introducing a polynucleotide encoding a BTR polypeptide into the plant body. If btr polynucleotide is introduced, the expression of btr polynucleotide in the plant body can be maintained for a long period of time, and a plant having tobamovirus resistance can also be obtained by obtaining its progeny.

  When a btr polynucleotide is introduced into a plant, the plant part or tissue into which the polynucleotide is introduced is the whole plant, plant organ (eg, leaf, petal, stem, root, seed, etc.), plant tissue (eg, epidermis, Phloem, soft tissue, xylem, vascular bundle, palisade tissue, spongy tissue, etc.) or plant cultured cells, or various forms of plant cells (eg suspension cultured cells), protoplasts, leaf sections, callus, etc. Either of these may be used. In addition, the type of plant used for introduction of the btr polynucleotide is not particularly limited, and may be any plant belonging to the monocotyledonous plant class or the dicotyledonous plant class.

  For introducing btr polynucleotide into a plant, a conventionally known method such as introduction of a vector provided with the polynucleotide can be suitably used.

  Here, a method for introducing a polynucleotide into a plant that can be used in the method for producing a plant virus-resistant plant according to the present invention is exemplified, but the present invention is not limited thereto.

  For introduction of polynucleotides into plants, transformation methods known to those skilled in the art (for example, Agrobacterium method, gene gun, PEG method, electroporation method, etc.) are used. It is divided roughly into the method of introducing into plant cells. When the Agrobacterium method is used, the constructed plant expression vector is introduced into an appropriate Agrobacterium (for example, Agrobacterium tumefaciens), and this strain is introduced into the leaf disk method (Hirofumi Uchimiya). The plant genetic manipulation manual (1990) pages 27-31, Kodansha Scientific, Tokyo) can be used to infect sterile cultured leaf pieces to obtain transformed plants. The method of Nagel et al. (Micribiol. Lett., 1990, 67, 325) can also be used. In this method, for example, an expression vector is first introduced into Agrobacterium, and then transformed Agrobacterium is plant cell or plant tissue by the method described in Plant Molecular Biology Manual (SBGelvin et al., Academic Press Publishers). It is a method to introduce into. Here, “plant tissue” includes callus obtained by culturing plant cells. When transforming using the Agrobacterium method, pBI binary vectors (for example, pBIG, pBIN19, pBI101, pBI121, pBI221, and pPZP202) can be used.

  In addition, electroporation and gene gun methods are known as methods for directly introducing foreign polynucleotides such as btr polynucleotides into plant cells or plant tissues. When using a gene gun, a plant body, a plant organ, and a plant tissue itself may be used as they are, or may be used after preparing a section, or a protoplast may be prepared and used. The sample thus prepared can be processed using a gene introduction apparatus (for example, PDS-1000 (BIO-RAD)). The treatment conditions vary depending on the plant or sample, but are usually performed at a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

  A cell or plant tissue into which a foreign polynucleotide has been introduced is first selected for drug resistance such as hygromycin resistance, and then regenerated into a plant body by a conventional method. Regeneration of a plant body from a transformed cell can be performed by methods known to those skilled in the art depending on the type of plant cell. The selection marker is not limited to hygromycin resistance, and examples thereof include drug resistance to bleomycin, kanamycin, gentamicin, chloramphenicol and the like.

  When plant cultured cells are used as the host, the recombinant vector can be obtained by, for example, microinjection, electroporation (electroporation), polyethylene glycol, gene gun (particle gun), protoplast fusion, calcium phosphate, etc. It is introduced into a cultured cell and transformed. Callus, shoots, hairy roots, etc. obtained as a result of transformation can be used for cell culture, tissue culture or organ culture as they are, and can be used at a suitable concentration using a conventionally known plant tissue culture method. Of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.).

  Whether or not a polynucleotide has been introduced into a plant can be confirmed by PCR, Southern hybridization, Northern hybridization, or the like. For example, DNA is prepared from a transformed plant, PCR is performed by designing a DNA-specific primer. PCR can be performed under the same conditions as those used for preparing the plasmid. Thereafter, the amplified product is subjected to agarose gel electrophoresis, polyacrylamide gel electrophoresis, capillary electrophoresis, etc., stained with ethidium bromide, SYBR Green solution, etc., and the amplified product is detected as a single band, It can be confirmed that it has been transformed. Further, PCR can be performed using a primer previously labeled with a fluorescent dye or the like to detect an amplification product. Furthermore, it is possible to employ a method in which the amplification product is bound to a solid phase such as a microplate and the amplification product is confirmed by fluorescence or enzyme reaction.

  Once a plant in which a foreign polynucleotide such as a btr polynucleotide is integrated into the genome is obtained, offspring can be obtained by sexual or asexual reproduction of the plant. Further, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc. can be obtained from the plant or its progeny, or clones thereof, and the plant can be mass-produced based on them. it can. Therefore, the present invention also includes a plant into which the polynucleotide according to the present invention is introduced so that it can be expressed, or a progeny of the plant having the same properties as the plant, or a tissue derived therefrom.

  The foreign polynucleotide to be introduced into the plant may include a sequence such as an untranslated region (UTR) sequence or a vector sequence (including an expression vector sequence).

  In addition, the source for obtaining the foreign polynucleotide to be introduced into the plant is not particularly limited, but is preferably a biological material. As used herein, the term “biological material” intends a biological sample (a tissue sample or cell sample obtained from an organism). In the examples described below, btr polynucleotides are obtained using Arabidopsis thaliana, but are not limited thereto.

  In addition, as a method for promoting the tobamovirus growth inhibitory activity of the BTR polypeptide, as described above, the expression level of the btr polynucleotide present in the plant body in advance may be improved. For example, a BTR polynucleotide endogenous to a plant and / or its regulatory gene may be mutated to enhance the BTR activity of the BTR polypeptide.

That is, the plant obtained by the method for producing a tobamovirus resistant plant according to the present embodiment may be a plant variant. Such a plant mutant can be obtained, for example, by selecting a plant in which the BTR activity of the BTR polypeptide is enhanced from naturally occurring mutants. The plant mutant can also be obtained by a method (chemical mutagenesis method) in which seeds are treated with a known compound that is a chemical mutagen. The chemical mutagens include sodium azide (NaN ₃ ), ethyl methanesulfonate (EMS), azidoglycerol (AG, 3-azido-1,2-propanediol), methylnitrosourea (MNU), and Examples thereof include maleic hydrazide (MH). Further, UV irradiation may be used. By such mutation, for example, when the function as a repressor is reduced by introducing a mutation of one or more nucleotides in a region encoding a repressor of a btr polynucleotide, the expression of the btr polynucleotide is improved. , Can enhance the BTR activity of the BTR polypeptide.

  As a method for promoting the tobamovirus growth inhibitory activity of the BTR polypeptide, as described above, a BTR polypeptide produced outside the plant body may be introduced externally.

  The method for externally introducing the BTR polypeptide is not particularly limited, and for example, it may be sprayed directly or administered to the veins.

  Here, a method for producing a BTR polypeptide used for external introduction will be described. The method for producing a BTR polypeptide is not particularly limited, and for example, a vector containing a btr polynucleotide may be used. The vector is preferably used in a cell-free protein synthesis system. When using a cell-free protein synthesis system, various commercially available kits can be used.

  Cell-free protein synthesis systems include systems using wheat germ extract, systems using rabbit reticulocyte extract, systems using Escherichia coli S30 extract, and cell component extracts obtained from plant devolatilized protoplasts. . Generally, eukaryotic cells are selected for translation of eukaryotic genes, i.e., systems using wheat germ extract or rabbit reticulocyte extract. In view of the origin of the gene (prokaryotes / eukaryotes) and the intended use of the protein after synthesis, it may be selected from the above synthesis system.

  After synthesizing the BTR polypeptide using the above vector, it may be purified from a cell or tissue extract containing the BTR polypeptide by a conventionally known method. BTR polypeptide can be purified by preparing a cell extract from cells or tissues by a conventionally known method (for example, a method in which cells or tissues are disrupted and then centrifuged to recover a soluble fraction), and then the cell extract Known methods (eg, ammonium sulfate precipitation or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography). The step of purifying by a) is preferred, but not limited thereto.

  A BTR polypeptide may also be obtained by chemical synthesis. Those skilled in the art will readily understand that a BTR polypeptide can be chemically synthesized by applying well-known chemical synthesis techniques based on the amino acid sequences of the BTR polypeptides described herein.

  Thus, the BTR polypeptide production method may use a known and conventional technique based on at least the amino acid sequence of the BTR polypeptide or the base sequence of the btr polynucleotide.

  A method for selecting a plant having enhanced tobamovirus growth inhibitory activity from the plant produced by the above method is not particularly limited, and a conventionally known method can be used. For example, the expression level of a polynucleotide encoding a BTR polypeptide may be measured by Northern blotting or the like, and immunity such as ELISA or Western blotting using an antibody that specifically recognizes the BTR polypeptide. The accumulated amount of the polypeptide may be measured by a biological method. The antibody specifically recognizing the BTR polypeptide will be described in detail in the description of the plant virus resistant plant screening kit according to the present invention.

  The tobamovirus resistant plant having improved tobamovirus resistance can be obtained by the method for producing a tobamovirus resistant plant according to the present embodiment described above. Since the tobamovirus resistant plant thus obtained has improved resistance to tobamovirus, a plant in which plant diseases caused by the virus are prevented can be obtained.

<II. Plant Virus-Resistant Plant Production Kit According to the Present Invention>
The present invention also provides a kit for producing a plant virus resistant plant. The plant virus resistant plant production kit according to the present invention only needs to have a plant virus growth inhibitory activity in the plant body, and encodes a polypeptide that binds to a plant virus genomic RNA. It is preferable that the polynucleotide comprises For example, a tobamovirus-resistant plant production kit is preferably provided with a btr polynucleotide.

  The production kit according to the present invention includes reagents necessary for carrying out the above-described method for producing a plant, in addition to a polynucleotide encoding a polypeptide that binds to a plant virus genomic RNA, such as a btr polynucleotide. Etc. are preferably provided. Examples of such a reagent include a reagent for performing the above-described Agrobacterium method and the like.

<III. Screening Method and Screening Kit for Plant Virus-Resistant Plant>
[3-1. Plant virus-resistant plant screening method according to the present invention]
The plant virus-resistant plant screening method according to the present invention comprises an accumulation amount of a polypeptide that binds to a plant virus genomic RNA and an expression level of a polynucleotide that encodes the polypeptide that binds to the plant virus genomic RNA. Of these, a step of measuring at least one amount may be included. For example, in the case of a tobamovirus-resistant plant screening method, a step of measuring at least one of the accumulated amount of BTR polypeptide and the expression amount of btr polynucleotide in the plant body (hereinafter also referred to as “measurement step”). ). Plants screened by the screening method for plant virus resistant plants according to the present invention may be natural, transformants, or mutants.

Hereinafter, as an embodiment of the screening method for plant virus-resistant plants according to the present invention, a screening method for tobamovirus-resistant plants will be described below, but the present invention is not limited thereto and is within the spirit of the present invention. The screening method for a tobamovirus resistant plant according to the present embodiment can provide a screening method capable of screening a plant having resistance to various plant viruses. The BTR polypeptide extracted from the target plant (a crude extract, a partially purified product, or a purified product) is reacted with an antibody against the BTR polypeptide. And a step of measuring the amount of BTR polypeptide accumulated in the plant body. More specifically, a step of measuring the amount of BTR polypeptide accumulated in the plant body may be performed using Western blotting or ELISA. According to the said process, the plant body with the high accumulation amount of BTR polypeptide in a plant body can be easily detected (screening) by comparison with a wild strain. In many cases, such a plant body having a high accumulation amount of BTR polypeptide has a tobamovirus growth inhibitory activity in plants. Therefore, a plant with improved resistance to tobamovirus can be easily screened by this step. As the antibody used in the above step, an antibody described later can be used.

  In addition, as the measurement step, for example, a probe having at least a part of the base sequence of a polynucleotide consisting of a base sequence of a btr polynucleotide or a complementary sequence thereof is used as a target plant-derived genomic DNA, mRNA or A step of measuring the expression level of the btr polynucleotide can be exemplified by hybridizing to cDNA for mRNA and detecting the polynucleotide hybridized to the probe. According to such a process, the plant body in which the expression level of the btr polynucleotide is improved can be easily detected (screened) by comparison with the wild type. In many cases, the plant body in which the expression level of the btr polynucleotide is improved has an improved tobamovirus growth inhibitory activity in the plant. Therefore, a plant with enhanced tobamovirus growth inhibitory activity can be easily screened by this step.

  The process illustrated as the said measurement process may be used independently, and may be used in combination of multiple. When used alone, a tobamovirus resistant plant can be screened rapidly. On the other hand, the accuracy of screening can be increased by using a plurality of combinations.

  The probe used in the tobamovirus-resistant plant screening method according to the present embodiment can also be used as a PCR primer or a hybridization probe for obtaining a btr polynucleotide or a fragment thereof. The probe can also be used for the following applications. (1) isolation of btr polynucleotides or allelic or splicing variants thereof in a cDNA library; (2) in situ hybridization to metaphase chromosome spreads to provide the exact chromosomal location of the btr polynucleotide. (See, eg, “FISH”) (see Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York, 1988); and (3) detecting mRNA expression of btr polynucleotides in specific tissues Northern blot analysis for. In addition, those skilled in the art have attributed to the hybridization generated between the probe used in the tobamovirus-resistant plant screening method according to the present embodiment and the target gene (polynucleotide) in any of the above applications. It will be easily understood that the probe is used to hybridize with a gene of interest (polynucleotide).

  The probe used in the tobamovirus-resistant plant screening method according to the present embodiment is at least 7 nt (nucleotide), 10 nt, 12 nt, preferably about 15 nt, and more preferably at least about 20 nt, still more preferably at least about Fragments (probes) with a length of 30 nt, and even more preferably at least about 40 nt are contemplated, but those skilled in the art can appropriately set the preferred length depending on the application described above. By “a fragment having a length of at least 20 nt”, for example, a fragment (probe) comprising 20 or more consecutive base sequences from the base sequence shown in SEQ ID NO: 3 or a complementary sequence thereof is intended. By referring to the present specification, the base sequence shown in SEQ ID NO: 3 is provided, so that those skilled in the art can easily prepare a probe based on SEQ ID NO: 3. For example, restriction endonuclease cleavage or ultrasonic shearing can be readily used to create fragments of various sizes. Alternatively, such fragments can be made synthetically. Appropriate fragments are synthesized, such as by Applied Biosystems Incorporated (ABI, 850 Lincoln Center Dr., Foster City, CA 94404) type 392 synthesizer. The probe includes not only double-stranded DNA but also single-stranded DNA and RNA such as a sense strand and an antisense strand constituting the DNA.

  Thus, by using the probe used in the tobamovirus-resistant plant screening method according to the present embodiment as a hybridization probe for detecting a btr polynucleotide or a PCR primer for amplifying the polynucleotide. It is possible to easily detect (screen) a plant or tissue in which the expression level of the btr polynucleotide is improved.

[3-2. Plant virus-resistant plant screening kit according to the present invention]
The present invention also provides a screening kit for plant virus resistant plants. The plant virus resistant plant screening kit according to the present invention is specific to an antibody that specifically binds to a polypeptide that binds to a plant virus genomic RNA and a polynucleotide that encodes a polypeptide that binds to the plant virus genomic RNA. It suffices to have at least one of the probes that bind to. For example, in the case of a tobamovirus resistant plant screening kit, it is sufficient to have at least one of an antibody that specifically binds to a BTR polypeptide and a probe that specifically binds to a btr polynucleotide.

Hereinafter, as an embodiment of a plant virus resistant plant screening kit according to the present invention, a tobamovirus resistant plant screening kit will be described below, but the present invention is not limited thereto, and is within the spirit of the present invention. A screening kit capable of screening plants having resistance to various plant viruses can be provided. A screening kit for a tobamovirus resistant plant according to the present embodiment is specific to a BTR polypeptide. In addition to the antibody that binds to the Btr and the probe that specifically binds to the btr polynucleotide, it is preferable to include reagents necessary for carrying out the above-described screening method for tobamovirus-resistant plants. For example, a buffer solution used when mixing the antibody and / or the probe and a plant tissue to be screened may be provided, and a reagent or a buffer solution for stably holding the antibody and / or the probe may be provided. You may prepare.

(An antibody that specifically binds to a BTR polypeptide)
Here, an antibody that specifically binds to a BTR polypeptide will be described. An antibody that specifically binds to a BTR polypeptide is not only included in the screening kit according to the present embodiment, but can be used in various ways. For example, when selecting a plant with enhanced tobamovirus growth inhibitory activity from the plant produced by the method for producing a tobamovirus resistant plant according to one embodiment of the present invention described above, or in the produced tobamovirus resistant plant, It can also be used to confirm peptide accumulation or btr polynucleotide expression.

  The antibody that specifically binds to the BTR polypeptide is not limited as long as it can specifically bind to the BTR polypeptide. For example, a polyclonal antibody against BTR polypeptide may be used, but a monoclonal antibody against BTR polypeptide is preferable. Monoclonal antibodies have advantages such as uniform properties, easy supply, and semipermanent storage as hybridomas.

As used herein, the term “antibody” means an immunoglobulin (IgA, IgD, IgE, IgG, IgM and their Fab fragments, F (ab ′) ₂ fragments, Fc fragments), eg Examples include, but are not limited to, polyclonal antibodies, monoclonal antibodies, single chain antibodies, and anti-idiotype antibodies.

  As a method for producing an antibody specific for a BTR polypeptide, various known methods can be suitably used with the BTR polypeptide obtained by the above-described BTR polypeptide production method as an antigen.

  For example, for monoclonal antibodies, the hybridoma method (see Kohler, G. and Milstein, C., Nature, 1975 256, 495-497), the trioma method, the human B-cell hybridoma method (Kozbor, Immunology Today, 1983, 4, 72) and the EBV-hybridoma method (see Monoclonal Antibodies and Cancer Therapy, Alan R Liss, Inc., 1985, 77-96) and the like.

  Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention.

  In addition, all of the academic literatures and patent literatures described in this specification are incorporated herein by reference.

[Example 1: Identification of BTR1]
The 5 ′ and 3 ′ terminal regions of the genomic RNA of the positive-strand RNA virus play an important role in the translation and replication of viral RNA. In this Example, the present inventors attempted to identify plant cell-derived factors that bind to these terminal regions in order to identify host factors involved in the control of tobamovirus growth.

  More specifically, a protein that binds to RNA containing a 5'-terminal or 3'-terminal region of ToMV genomic RNA was obtained from an Arabidopsis cell extract and identified.

(Synthesis of strepttag-added RNA probe)
In this example, in order to obtain a protein that binds to the genomic RNA of ToMV, an RNA probe (hereinafter referred to as “strept tag”) in which RNA containing the 5′-terminal or 3′-terminal region of the genomic RNA of ToMV is bound to a strepttag. An “additional RNA probe” was used.

  The strepttag means a 46-base streptomycin-binding RNA motif and is described in Bachler, M., Schroeder, R. and von Ahsen, U., RNA, 1999, 5, 1509-1516. It was synthesized by the method.

  Strepttag-added RNA probes were prepared as follows.

  First, a DNA fragment having a SacI recognition site, a strep tag, a SphI recognition site, a NotI recognition site, an MluI recognition site, and an XbaI recognition site in this order was synthesized.

  Next, the DNA fragment was inserted into the SacI and XbaI recognition sites of pBluescript II SK (+) (manufactured by TOYOBO, GenBank accession number X52328) to prepare a vector (pStM).

  Next, among ToMV genomic RNAs (6384 bases, GenBank accession number X02144), DNAs having base sequences corresponding to RNAs at positions 1 to 510 and 6166 to 6384 are respectively represented by SphI of pStM, A plasmid was prepared by inserting into the MluI recognition site. For comparison, the same operation was performed using DNA having a base sequence corresponding to RNA in the region of positions 764 to 1004. The plasmids derived from DNAs having base sequences corresponding to the RNA at positions 1 to 510, 764 to 1004, and 6166 to 6384 are referred to as pL5-5, pLR1, and pL3-2, respectively. “DNA having a base sequence corresponding to RNA” is intended to mean DNA having the same base sequence as the base sequence except that U (uracil) in the base sequence of the RNA is replaced with T (thymine). To do.

  Next, pL5-5 was linearized with EcoRI. Moreover, pLR1 and pL3-2 were linearized with MluI.

  Next, transcription of DNA obtained by linearizing each plasmid was performed using T3 RNA polymerase. As a result, a transcript containing the region of positions 1 to 277, 764 to 1004, or 6166 to 6384 of the ToMV genome and having a strept tag added to the 5 ′ end side was obtained.

  Here, the structure of the strepttag-added RNA probe obtained in this example will be described with reference to FIG. FIG. 1 is a diagram schematically showing the structure of a strepttag-added RNA probe obtained in this example. The upper part shows the structure of a ToMV genomic RNA, and the lower part shows the structure of a strepttag-added RNA probe. Yes.

  The “Probe RNA” shown at the 3 ′ end of the strep-tagged RNA probe of FIG. 1 has a region at positions 1 to 277 indicated as “L5” in FIG. 1, a region at positions 764 to 1004 indicated as “LR”, And an RNA fragment consisting of the region from positions 6166 to 6384 designated as “L3”. That is, a strepttag-added RNA probe in which an RNA fragment in any region is bound to the 3 'end can be obtained by the above-described method.

  Hereinafter, a Strept-tagged RNA probe having a base sequence of positions 1 to 277 of ToMV genomic RNA is denoted as “L5”, and a Strept-tagged RNA probe having a base sequence of positions 764 to 1004 is denoted as “LR”. A strepttag-added RNA probe having a nucleotide sequence at positions 6166-6384 is denoted as “L3”.

(Arabidopsis cell extract)
As a material for the cell extract, Arabidopsis thaliana ecotype Col-0 strain (hereinafter simply referred to as “Col-0”) was used.

The cell extract of Col-0 was prepared as follows. First, Col-0 MM2d cultured cells were devacuated and then disrupted in disruption buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl ₂ , 2 mM DTT, Complete mini). The extract obtained by crushing was centrifuged at 30,000 × g for 15 minutes, and the supernatant (S30) from which biological membrane components were removed was used as a Col-0 cell extract in subsequent experiments. MM2d cultured cells were obtained by the method described in Menges, M. and Murray, JAH, Plant J., 2002, 30, 203-212. Further, devolatilization was performed by the method described in Sonobe, S., J. Plant Res., 1996, 109, 437-448.

(Mixing of cell extract of Col-0 and RNA probe)
Next, the cell extract of Col-0 and each of the L5, LR, and L3 RNA probes were mixed under the following conditions to form an RNA-protein complex.

  First, the RNA probe was added to 400 μl of a cell extract of Col-0 adjusted to a protein concentration of 15 mg / ml. The amount of RNA probe added was 50 μg for L5 and 20 μg for L3 and LR. After adding the RNA probe, the mixture was allowed to stand on ice for 20 minutes. Thereafter, 8 μl of heparin (100 mg / ml) was added, and the mixture was further allowed to stand on ice for 30 minutes.

(Affinity purification)
The RNA-protein complex obtained from each of the above RNA probes was affinity purified using sepharose beads conjugated with streptomycin.

Affinity purification was carried out according to the method described in Bachler, M., Schroeder, R. and von Ahsen, U., RNA, 1999, 5, 1509-1516. Specifically, 0.8 ml of the Sepharose beads were packed in a column, equilibrated with 7 ml column buffer (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl ₂ ), and then a sample was added. . Washing was performed 10 times with 2 ml of column buffer, and elution was performed with the above column buffer further containing 3 ml of 10 μM streptomycin. The eluted sample was concentrated by acetone precipitation, and 1/15 of the total amount was separated by 8-12% SDS-PAGE and detected by silver staining. All operations were performed at 4 ° C.

  As samples, RNA-protein complexes obtained from the RNA probes of L5, LR, and L3 were respectively subjected to phenol / chloroform extraction and those not subjected to phenol / chloroform extraction. The result is shown in FIG. FIG. 2 is a diagram showing the results of subjecting the RNA-protein complex obtained in this example to SDS-PAGE. “+” And “−” of “P / C” shown in FIG. 2 indicate a sample subjected to the phenol / chloroform extraction and a sample not subjected to the phenol / chloroform extraction, respectively.

  As shown by the black triangles in FIG. 2, in the RNA-protein complex obtained from L5, a protein band disappeared by phenol / chloroform extraction was confirmed.

(LC-MS / MS analysis)
The protein in the band indicated by the black triangle in FIG. 2 was analyzed by LC-MS / MS. As the protein, a sample cut from an SDS-PAGE gel and treated with trypsin was used. The analysis by LC-MS / MS used a contract analysis service of Apro Science Co., Ltd.

  As a result, the amino acid sequence of the protein subjected to LC-MS / MS analysis matched the amino acid sequence of Locus tag At5g04430 registered in GenBank. Hereinafter, this protein is referred to as “BTR1”. The amino acid sequence shown in Locus tag At5g04430 is one of the amino acid sequences of Arabidopsis thaliana.

  Here, the structure of the BTR 1 obtained in this example will be described with reference to FIG. FIG. 3 is a diagram showing two types of splicing variants generated from the base sequence of Locus tag At5g04430 and the results of LC-MS / MS analysis of this example. The proteins having amino acid sequences obtained by the two types of splicing variants are denoted as “BTR1S” and “BTR1L”, respectively. That is, the polypeptide encoded by the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 and the polypeptide encoded by the polynucleotide consisting of the base sequence shown in SEQ ID NO: 3 is BTR1S. The polypeptide encoded by the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 2 and the polypeptide encoded by the polynucleotide consisting of the base sequence shown in SEQ ID NO: 4 is BTR1S. Further, I, II, and III in FIG. 3 indicate KH domains. The underlined amino acids in FIG. 3 indicate the amino acid sequences of the peptide fragments detected by the LC-MS / MS analysis described above.

  As shown in FIG. 3, the amino acid sequences of BTR1S and BTR1L are identical except for the presence or absence of 21 amino acid residues present between KH domains II and III.

  As shown in the underlined portion of FIG. 3, as a result of LC-MS / MS analysis, 11 peptide fragments that matched a part of the amino acid sequence of BTR1S were detected. As shown in FIG. 3, one of the peptide fragments had an amino acid sequence straddling the 21 amino acid residues specific to BTR1L. And the area | region which has the said 21 amino acid residue was not detected. From this, it can be said that BTR1 obtained in this example is BTR1S.

[Example 2: Evaluation of binding ability of BTR1S to tobamovirus]
In this example, the binding property of BTR1S expressed in E. coli to the genomic RNA of ToMV was evaluated by gel shift assay. As the base sequence of ToMV genomic RNA, the sequence registered in GenBank with accession number X02144 was used as in Example 1.

(Preparation of transcript having 300 bases of ToMV 5 ′ terminal region and transcript having 218 bases of ToMV 3 ′ terminal region)
A transcript having 300 bases of the ToMV 5 ′ terminal region and a transcript having 218 bases of the ToMV 3 ′ terminal region were prepared by the following method.

  Downstream of the T3 RNA polymerase promoter, DNA having a base sequence corresponding to 300 bases of the 5 'end region of ToMV or DNA having a base sequence corresponding to 218 bases of the ToMV 3' end region was ligated. Furthermore, a MluI recognition site was linked to each 3 'end to synthesize cDNA. Next, the cDNA was cloned using pCR-Blant II-TOPO (Invitrogen). Hereinafter, a transcript having 300 bases of ToMV 5 ′ terminal region is referred to as “ToMV5 ′”, and a transcript having 218 bases of ToMV 3 ′ terminal region is referred to as “ToMV3 ′”.

The cDNA obtained by cloning was linearized with MluI, and then transcribed using Ampliscribe T3 Transcription kit (Epicentre) in the presence of ³² P CTP.

For comparison with gel shift assay, a transcript having 318 bases of the 5 ′ end region of the firefly luciferase gene was linearized with pSP-luc + vector (manufactured by Promega) with Cfr10I, and SP6 RNA was present in the presence of ³² P CTP. It was obtained by transcription using a polymerase. Hereinafter, a transcript having 318 bases of the 5 ′ end region of the firefly luciferase gene is referred to as “luc5 ′”.

(Synthesis of BTR1S by E. coli)
BTR1S fused with a histidine tag at the N-terminus was expressed in E. coli (BL21 (DE3), Novagen) using the pDEST-His vector (Tsunoda et al., 2005).

  Next, purification was performed using His Select Nickel affinity gel (manufactured by Sigma) to obtain BTR1S.

(Gel shift assay)
The final concentration of BTR1S expressed in the above-mentioned Escherichia coli in 20 μl of a reaction solution (50 mM Tris-HCl (pH 7.5), 100 mM NaCl, 3 mM MgCl ₂ , 2 mM DTT, 100 mM imidazole, 5% Glycerol, 2 mg / ml heparin) It added so that it might become 0, 200, 400, 600 nM. Next, after standing at 30 ° C. for 10 minutes, 1 μl of ³² P-labeled ToMV5 ′, ToMV3 ′ or luc5 ′ (0.1 pmol / μl) and 2.5 μl of Glycerol-BPB solution (50% Glycerol,. 05% bromophenol blue).

Next, it was electrophoresed on a 4% TBE gel at 100 V for 1.5 to 2 hours. ³² P-RNA was detected with BAS-2500 (Fujifilm).

In addition, ³² P-labeled ToMV5 ′ and unlabeled ToMV5 ′, ToMV3 ′, or luc5 ′ were mixed in the reaction solution to conduct a competition experiment. In the competition experiment, the final concentration of BTR1S was 600 nM, the mixed amount of ³² P-labeled ToMV5 ′ was 5 nM, and unlabeled ToMV5 ′, ToMV3 ′ or luc5 ′ was 1, 3, and 10 pmol. The amount of these unlabeled transcripts is 10 times, 30 times, and 100 times the number of moles of mixed ³² P-labeled ToMV 5 ′. Next, electrophoresis was performed on a 4% TBE gel at 100 V for 1.5 to 2 hours. ³² P-RNA was detected with BAS-2500 (Fujifilm).

These results are shown in FIG. FIG. 4 is a diagram showing the results of gel shift assay in this example, (a) shows the results of examining the binding to BTR1S using ³² P-labeled ToMV5 ′, ToMV3 ′ or luc5 ′; (B) is a figure which shows the result of having examined the binding property to BTR1S using < ³² > P labeled ToMV5 'and unlabeled ToMV5', ToMV3 'or luc5'. In FIG. 4, the region marked with “F” is a rough region where a band indicating RNA not bound to protein appears, and the region marked with “C” is a complex in which RNA and protein are bound. This indicates the approximate area where a band indicating 現 appears.

As shown in FIG. 4A, a band was confirmed in the “C” region only with ³² P-labeled ToMV5 ′. Further, as shown in FIG. 4B, it was confirmed that only unlabeled ToMV5 ′ functions competitively with ³² P-labeled ToMV5 ′ and binds to BRT1S. From this, it was confirmed that BTR1S specifically binds to the 5 ′ terminal region of ToMV RNA.

[Example 3: Effect of BTR1 on virus propagation]
Involvement of BTR1 in virus growth in plants was confirmed using a BTR1 knockout strain and a strain overexpressing BTR1S.

(Preparation of BTR1 knockout strain)
A BTR1 knockout strain of Arabidopsis thaliana was obtained from the T-DNA tag line of Arabidopsis Biological Resource Center (ABRC) (SALK — 007924). Hereinafter, this BTR1 knockout strain is simply referred to as “SALK — 007924”. In SALK — 007924, T-DNA is inserted at the location of Locus tag At5g04430. Thereby, BTR1 is knocked out.

  The strain obtained from ABRC is a mixed state of homo and hetero individuals. Therefore, in order to identify homozygous individuals, primer LP (5′-ACGCTTAACCACCCAAATATC-3 ′: SEQ ID NO: 5), primer RP (5′-ATCCAGCATCTACTACTTGAC-3 ′: SEQ ID NO: 6), primer LB (5′-CGTGGACCGCTGTCGCCAACT- It confirmed by providing SALK_007924 to genomic PCR using 3 ': sequence number 7). Primers LP, RP, and LB are designed as follows. That is, based on the Arabidopsis genome sequence information, primers that are homologous to a site about 400 bp apart (total of about 800 bp) around the T-DNA insertion site are prepared. Primers within the DNA insertion region were designed.

  Arabidopsis genomic DNA was obtained according to the following method. A DNA extraction buffer (200 mM Tris-HCl (pH 7.5), 250 mM NaCl, 25 mM EDTA, 0.5% (wt / vol) SDS) was added to 1-2 rosette leaves about 3 weeks after sowing, and Tissue Lyser. The leaves were crushed using (Qiagen). Total DNA was recovered from the supernatant obtained by centrifugation at 15,000 × g and room temperature for 5 minutes by isopropanol precipitation.

(Preparation of BTR1 overexpression strain)
A BTR1 overexpression strain of Arabidopsis thaliana was prepared as follows. As plant material, Col-0 which is a wild strain of Arabidopsis thaliana was used.

  First, using XbaI and SacI recognition sites, a plasmid was constructed in which the polynucleotide consisting of SEQ ID NO: 3 or SEQ ID NO: 4 was inserted downstream of the 35S promoter of pBI121 (GenBank accession number AF485833). Next, Arabidopsis thaliana was transformed by introducing the plasmid into Arabidopsis thaliana using Agrobacterium tumefacience. Transformation was performed by the Floral dip method (see Clough, S. J. and Bent, A. F., Plant J., 1998, 16, 735-743). Among the BTR1 overexpression strains of Arabidopsis thus obtained, the BTR1S overexpression strain is hereinafter referred to as “35S: BTR1S”, and the BTR1L overexpression strain is hereinafter referred to as “35S: BTR1L”.

(Study on the relationship between BTR1 and virus growth)
For the measurement of virus growth, T2 generation plants obtained by kanamycin selection were used for each transformed plant.

  ToMV (0.1 mg / ml) or CMV RNA (0.1 mg / ml) was inoculated into rosette leaves of each transformed plant 4-5 weeks after sowing. In SALK_007924 inoculated with ToMV, inoculated leaves were collected 7 days after the inoculation. In 35S: BTR1S and 35S: BTR1L inoculated with ToMV, inoculated leaves were collected 4 days after the inoculation. In each transformed plant inoculated with cucumber mosaic virus (hereinafter referred to as “CMV”), inoculated leaves were collected on the second day after inoculation.

  The collected inoculated leaves were prepared as follows and subjected to SDS-PAGE. First, add 5 times the wet weight of the leaf gel sample buffer (50 mM Tris-HCl (pH 6.8), 2% SDS, 5% β-mercaptoethanol, 10% glycerol, 0.02% BPB) After crushing, a sample heated at 100 ° C. for 3 minutes was used as an SDS-PAGE sample.

  Accumulation of ToMV coat protein (hereinafter referred to as “CP”) in SALK_007924 was detected by Coomassie brilliant blue staining, and accumulation of ToMV CP in BTR1 overexpressing strains (35S: BTR1S and 35S: BTR1L), Accumulation of CP of CMV in SALK_007924 and BTR1 overexpressing strains was detected by Western blotting.

  Moreover, in the inoculated leaves collected from 35S: BTR1S and 35S: BTR1L, the expression of BTR1S and BTR1L was detected by Western blotting, respectively. A BTR1-specific antibody was produced using BTR1S fused with the above histidine tag at the N-terminus as an antigen using a contract service of Shibayagi Co., Ltd.

  The result is shown in FIG. FIG. 5 is a diagram showing the results of studies on the relationship between BTR1 and virus growth, (a) shows the results of detecting virus CP contained in the leaves of Col-0 and SALK_007924, and (b) shows Col. -0 and 35S: shows the results of detecting the viral CP contained in the leaves of BTR1S and the results of detecting the expression of BTR1S in the respective leaves, (c) is contained in the leaves of Col-0 and 35S: BTR1L The result of detecting ToMV CP and the result of detecting the expression of BTR1L in each leaf are shown.

  As shown in FIG. 5 (a), ToMV proliferation in SALK_007924 was accelerated compared to wild-type Col-0. Further, as shown in FIG. 5 (b), it was confirmed that 35S: BTR1S decreased ToMV proliferation. In addition, such a decrease in virus growth was not confirmed for CMV. Moreover, as shown in FIG.5 (c), it has confirmed that the effect which suppresses the proliferation of ToMV was acquired also by 35S: BTR1L.

(Effects of BTR1 expression on plant formation)
The effect of BTR1 expression on the formation of Arabidopsis plants was confirmed.

  First, ToMV (0.1 mg / ml) was seed | inoculated to each of Col-0, SALK_007924, 35S: BTR1S, and 35S: BTR1L. Next, it was grown for 4 weeks under long day conditions of 23 ° C. and 16 hours long. And the plant body after breeding was observed. The result is shown in FIG. FIG. 6 is a diagram showing the results of observation of Arabidopsis thaliana after seeding and growing ToMV.

  As shown in FIG. 6, overexpression of BTR1S and BTR1L had no noticeable effect on plant growth.

  From the above results, it was shown that BTR1 specifically binds to the 5 'terminal region of ToMV RNA and specifically inhibits the growth of ToMV.

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the claims. That is, embodiments obtained by combining technical means appropriately changed within the scope of the claims are also included in the technical scope of the present invention.

  As described above, according to the present invention, since a plant body imparted with plant virus resistance can be produced, plant viruses can be controlled in various crops. Therefore, the present invention can be applied not only in the field of breeding but also in industrial fields such as agriculture and horticulture using plants infected with plant viruses.

It is the figure which showed typically the structure of the strept tag addition RNA probe produced in the Example. In an Example, it is a figure which shows the result of having used the RNA-protein complex for SDS-PAGE. It is a figure which shows the result of LC-MS / MS analysis of two types of splicing variants produced from the base sequence of Locus tag At5g04430, and a present Example. In an Example, it is a figure which shows the result of having performed the gel shift assay, (a) shows the result of having examined the binding property to BTR1S using ³² P-labeled ToMV5 ′, ToMV3 ′ or luc5 ′, (b ) Shows the results of examining the binding to BTR1S using ³² P-labeled ToMV5 ′ and unlabeled ToMV5 ′, ToMV3 ′ or luc5 ′. In an Example, it is a figure which shows the result examined about the relationship between BTR1 and a virus proliferation, (a) shows the result of having detected CP of the virus contained in the leaf of Col-0 and SALK_007924, (b) is Col. -0 and 35S: The results of detecting the CP of the virus contained in the leaves of BTR1S and the results of detecting the expression of BTR1S in the respective leaves are shown, (c) is contained in the leaves of Col-0 and 35S: BTR1L The result of detecting ToMV CP and the result of detecting the expression of BTR1L in each leaf are shown. In an Example, it is a figure which shows the result of having observed the Arabidopsis thaliana after towing and growing ToMV.

Claims (10)

  A method for producing a plant virus-resistant plant, comprising a step of promoting the growth inhibitory activity of a plant virus by a polypeptide that binds to the genomic RNA of the plant virus and inhibits the growth of the plant virus in the plant body . The said polypeptide is a polypeptide as described in the following (a) or (b), The manufacturing method of the plant virus resistant plant of Claim 1 characterized by the above-mentioned:
(A) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 or 2;
(B) An amino acid sequence represented by SEQ ID NO: 1 or 2, consisting of an amino acid sequence in which one or several amino acids are deleted, inserted, substituted, or added, and has plant virus growth inhibitory activity A polypeptide having   2. The method for producing a plant virus-resistant plant according to claim 1, wherein the step is performed by introducing a polynucleotide encoding the polypeptide into the plant body. The said polynucleotide is a polynucleotide as described in the following (c), (d) or (e), The manufacturing method of the plant virus resistant plant of Claim 3 characterized by the above-mentioned:
(C) a polynucleotide comprising the base sequence represented by SEQ ID NO: 3 or 4;
(D) a polynucleotide comprising a base sequence in which one or several bases have been deleted, inserted, substituted or added in the base sequence shown in SEQ ID NO: 3 or 4;
(E) a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a base sequence complementary to the base sequence represented by SEQ ID NO: 3 or 4.   The method for producing a plant virus-resistant plant according to claim 1, wherein the plant virus is a tobamovirus.   A plant virus-resistant plant production kit comprising a polynucleotide encoding a polypeptide that binds to a plant virus genomic RNA.   A step of measuring at least one of an accumulated amount of a polypeptide that binds to a plant virus genomic RNA and an expression level of a polynucleotide encoding a polypeptide that binds to a plant virus genomic RNA in a plant body. A screening method for plant virus-resistant plants characterized by the above.   It comprises at least one of an antibody that specifically binds to a polypeptide that binds to a plant virus genomic RNA and a probe that specifically binds to a polynucleotide encoding a polypeptide that binds to a plant virus genomic RNA. A plant virus-resistant plant screening kit.   A plant virus-resistant plant produced by the method for producing a plant virus-resistant plant according to any one of claims 1 to 5.   A plant virus resistant plant characterized in that the growth inhibitory activity of a plant virus by a polypeptide that binds to the genomic RNA of the plant virus is promoted.