WO2025005231A1 - Plant de tomate résistant aux virus du genre tobamovirus, cellules végétales de tomate et leur procédé de production - Google Patents

Plant de tomate résistant aux virus du genre tobamovirus, cellules végétales de tomate et leur procédé de production Download PDF

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WO2025005231A1
WO2025005231A1 PCT/JP2024/023481 JP2024023481W WO2025005231A1 WO 2025005231 A1 WO2025005231 A1 WO 2025005231A1 JP 2024023481 W JP2024023481 W JP 2024023481W WO 2025005231 A1 WO2025005231 A1 WO 2025005231A1
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gene
tomato
tobamovirus
mutation
seq
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泰規 新子
信光 佐々木
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Kikkoman Corp
Tokyo University of Agriculture and Technology NUC
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Kikkoman Corp
Tokyo University of Agriculture and Technology NUC
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • the present invention relates to tomato plants and tomato plant cells that are resistant to Tobamovirus viruses, and methods for producing the same.
  • tomatoes are the second most produced horticultural vegetable crop in the world after potatoes, with annual production exceeding 180 million tons, accounting for more than 10% of total vegetable production.
  • tomatoes during cultivation can become infected with various pathogens such as bacteria and viruses, often causing significant damage.
  • Tobamovirus viruses are a collective name for the Tobacco Mosaic Virus (TMV), which was the first virus discovered in the world. They are highly infectious, highly resistant to inactivation, and stable.
  • TMV Tobacco Mosaic Virus
  • Tomato brown rugose fruit virus (ToBRFV) is one of the emerging viruses that suddenly appeared in recent years and has spread rapidly around the world (Non-Patent Document 1). Currently, its occurrence is mainly noticeable in tomatoes and peppers.
  • ToBRFV was first discovered in Israel in the Middle East in 2014, but in the following years outbreaks have been found in Europe, including Jordan, Italy, Germany, and Turkey, as well as across the ocean in the United States, Canada, and China.
  • the source of the outbreak is likely to be around the Middle East, and because it is a highly contagious virus, it is speculated to be spreading rapidly around the world because trace amounts of the virus can adhere to or remain on seeds and materials, which can become sources of infection.
  • ToBRFV has not yet been discovered in Japan, and the Ministry of Agriculture, Forestry and Fisheries and the Plant Protection Station have designated it as a significant virus in the "Pest Risk Analysis for Quarantine Pests.” They are taking border control measures to prevent its entry and are issuing maximum warnings, but it is highly likely that it will invade the country in the near future.
  • ToBRFV tomato caused by ToBRFV
  • the symptoms of tomato caused by ToBRFV are similar to those of previous tobamoviruses, with mosaic, silky leaves, yellowing, necrotic stems, and discoloration, necrotic and ring-shaped leaves on the leaves, but the symptoms such as mosaic on the leaves are weaker, while the necrotic fruit symptoms are stronger than those of other tobamoviruses.
  • Tobamoviruses are primarily transmitted by contaminated seeds, mechanical contact (conventional agricultural work), or contaminated soil, water, or tools (Non-Patent Documents 2 and 3).
  • Tm-2 and Tm-2a also known as Tm-2 2
  • ToBRFV Tobamovirus resistance genes
  • Non-Patent Document 5 In light of these circumstances, attempts have been made to develop new resistance genes, but so far there have been few conclusively good resistance gene candidates, as there are often many resistance-related genes or loci, the effects are insufficient, or there are many conditions that make them difficult to use in breeding.
  • Non-Patent Document 6 Another example is the report in Non-Patent Document 6.
  • the description in this document indicates that resistance can be obtained for the first time in plants by introducing four gene mutations through gene editing, and there are a large number of related genes.
  • Non-Patent Document 7 published in 2021 reports that the BAM gene, a tomato receptor-like kinase, interacts with the TMV movement protein (MP). This suggests that tobamoviruses may use the BAM gene for infection or movement within plants after infection.
  • MP TMV movement protein
  • Tobamovirus viruses particularly ToBRFV
  • the inventors have discovered a new resistance gene that is effective in preventing tomato diseases caused by Tobamovirus viruses, and have developed a plant variety that carries this gene, thereby providing a method for controlling Tobamovirus viruses and a plant that is resistant to Tobamovirus viruses.
  • the present invention provides the following tomatoes (i.e., tomato plants), parts thereof, and processed products thereof.
  • a tomato having resistance to Tobamovirus viruses wherein at least one gene selected from the group consisting of receptor-like kinase RLK genes and genes homologous thereto has a mutation, and expression of the gene having the mutation is suppressed due to the mutation, or a protein encoded by the gene having the mutation is non-functional against Tobamovirus viruses.
  • the tomato according to [1], wherein the mutation is introduced into a gene in the genome by genome editing technology.
  • [4] The tomato according to any one of [1] to [3], wherein the receptor-like kinase RLK gene is an RLK1 gene (Solyc02g091840) whose cDNA sequence comprises the base sequence shown in SEQ ID NO:1, and the homologous gene of the receptor-like kinase RLK gene is a homologous gene of the RLK1 gene whose cDNA sequence comprises a base sequence having 85% or more sequence homology to the base sequence shown in SEQ ID NO:1.
  • [5] The tomato according to [4], which has a mutation in a region corresponding to the base sequence shown in SEQ ID NO:3 in the RLK1 gene or a gene homologous thereto.
  • [6] The tomato according to [5], wherein a region corresponding to the base sequence shown in SEQ ID NO: 3 is mutated to a base sequence shown in one type selected from SEQ ID NOs: 4 to 7.
  • [7] The tomato according to any one of [1] to [3], wherein the receptor-like kinase RLK gene is the RLK2 gene (Solyc03g043770) and its cDNA sequence comprises the base sequence shown in SEQ ID NO: 16, and the homologous gene of the receptor-like kinase RLK gene is a homologous gene of the RLK2 gene and its cDNA sequence comprises a base sequence having 85% or more sequence homology to the base sequence shown in SEQ ID NO: 16.
  • [8] The tomato according to [7], having a mutation in a region corresponding to the base sequence shown in SEQ ID NO: 18 in the RLK2 gene or a gene homologous thereto. [9] The tomato according to [8], wherein a region corresponding to the base sequence shown in SEQ ID NO: 18 is mutated to a base sequence shown in one type selected from SEQ ID NOs: 19 to 24. [10] The tomato according to any one of [1] to [9], wherein the Tobamovirus is at least one selected from the group consisting of Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (TMV), Tomato mosaic virus (ToMV), and Tomato mottle mosaic virus (ToMMV).
  • ToBRFV Tomato brown rugose fruit virus
  • TMV Tomato mosaic virus
  • ToMV Tomato mosaic virus
  • ToMMV Tomato mottle mosaic virus
  • [11] A part of a tomato according to any one of [1] to [10].
  • [12] A part of the tomato according to [11], which is a fruit.
  • [13] A part of the tomato according to [11], which is a seed.
  • [14] A processed product of the tomato part according to any one of [11] to [13].
  • [15] The processed product according to [14], which is edible.
  • the present invention further provides the following tomato cells, as well as tomato plants and parts thereof having the same: [16] A tomato cell having resistance to Tobamovirus viruses, wherein at least one gene selected from the group consisting of receptor-like kinase RLK genes and genes homologous thereto has a mutation, and expression of the gene having the mutation is suppressed due to the mutation, or a protein encoded by the gene having the mutation is non-functional against Tobamovirus viruses. [17] The tomato cell according to [16], wherein the mutation is introduced into a gene in the genome by genome editing technology.
  • the tomato cell according to [19] which has a mutation in a region corresponding to the base sequence shown in SEQ ID NO:3 in the RLK1 gene or a gene homologous thereto.
  • ToBRFV Tomato brown rugose fruit virus
  • TMV Tomato mosaic virus
  • ToMV Tomato mosaic virus
  • ToMMV Tomato mottle mosaic virus
  • a tomato or a part thereof comprising the tomato cell according to any one of [16] to [25] and having resistance to Tobamovirus.
  • a part of the tomato according to [26] which is a fruit.
  • the processed product according to [29] which is edible.
  • the present invention further provides the following tomato production method and tomato plant obtained by said production method.
  • a method for producing a tomato that is resistant to a tobamovirus virus comprising the steps of: selecting at least one gene selected from the group consisting of receptor-like kinase RLK genes and genes homologous thereto; introducing into the selected gene in the genome of the tomato a mutation that suppresses expression of the selected gene, or a mutation that renders a protein encoded by the selected gene non-functional against Tobamovirus viruses; and selecting a tomato that is resistant to Tobamovirus viruses.
  • the method for producing a tobamovirus-resistant tomato according to [31] wherein the mutation is introduced into a gene in the genome by genome editing technology.
  • [34] The method for producing a tobamovirus-resistant tomato according to any one of [31] to [33], wherein the receptor-like kinase RLK gene is the RLK1 gene (Solyc02g091840) and its cDNA sequence comprises the base sequence shown in SEQ ID NO:1, and the homologous gene of the receptor-like kinase RLK gene is a homologous gene of the RLK1 gene and its cDNA sequence comprises a base sequence having 85% or more sequence homology to the base sequence shown in SEQ ID NO:1.
  • [37] The method for producing a tobamovirus-resistant tomato according to any one of [31] to [33], wherein the receptor-like kinase RLK gene is the RLK2 gene (Solyc03g043770) and its cDNA sequence comprises the base sequence shown in SEQ ID NO: 16, and the homologous gene of the receptor-like kinase RLK gene is a homologous gene of the RLK2 gene and its cDNA sequence comprises a base sequence having 85% or more sequence homology to the base sequence shown in SEQ ID NO: 16.
  • the receptor-like kinase RLK gene is the RLK2 gene (Solyc03g043770) and its cDNA sequence comprises the base sequence shown in SEQ ID NO: 16
  • the homologous gene of the receptor-like kinase RLK gene is a homologous gene of the RLK2 gene and its cDNA sequence comprises a base sequence having 85% or more sequence homology to the base sequence shown in SEQ ID NO: 16.
  • the method for producing a tobamovirus-resistant tomato according to any one of [31] to [39], wherein the tobamovirus is at least one selected from the group consisting of tomato brown rugose fruit virus (ToBRFV), tobacco mosaic virus (TMV), tomato mosaic virus (ToMV), and tomato mottle mosaic virus (ToMMV).
  • ToBRFV tomato brown rugose fruit virus
  • TMV tobacco mosaic virus
  • ToMV tomato mosaic virus
  • ToMMV tomato mottle mosaic virus
  • the present invention further provides the following method for producing a tomato breeding progeny and a tomato plant obtained by said method.
  • a method for producing a breeding progeny of a Tobamovirus virus-resistant tomato comprising a step of self-pollinating or cross-pollinating the Tobamovirus virus-resistant tomato or its progeny obtained by the method according to any one of [31] to [40].
  • the present invention provides a method for producing a tomato plant, tomato cell, and tomato plant resistant to tobamovirus viruses, which have the property of inhibiting infection with tobamovirus viruses, the property of suppressing the proliferation and movement of tobamovirus viruses after infection, and/or the property of suppressing the manifestation of symptoms of infection with tobamovirus viruses.
  • FIG. 1 shows the cDNA sequence of the tomato receptor-like kinase RLK1 gene (Solyc02g091840).
  • the underlined part is exon 1, the wavy underlined part is the guide RNA recognition portion in exon 1 (bases 790 to 809 of exon 1), and the boxed part is the PAM sequence.
  • FIG. 2 shows the cDNA sequence of the tomato receptor-like kinase RLK2 gene (Solyc03g043770).
  • the underlined part is exon 1, the wavy underlined part is the guide RNA recognition portion in exon 1 (bases 277 to 296 of exon 1), and the boxed part is the PAM sequence.
  • FIG. 1 shows the cDNA sequence of the tomato receptor-like kinase RLK1 gene (Solyc02g091840).
  • the underlined part is exon 1
  • the wavy underlined part is the guide RNA recognition portion in exon 1 (bases 277 to
  • FIG. 3 is an electrophoretic photograph showing the results of RT-PCR analysis of the ToBRFV inoculation test.
  • FIG. 4 is a diagram showing the mutation pattern in the tomato receptor-like kinase RLK1 gene. The wavy underlined portion in the wild-type sequence corresponds to the guide RNA recognition site in exon 1, while a - symbol indicates a base deletion, a box indicates a base insertion, and an underline indicates a base substitution.
  • FIG. 5 is a diagram showing the mutation pattern in the tomato receptor-like kinase RLK2 gene. The wavy underlined portion in the wild-type sequence corresponds to the guide RNA recognition site in exon 1, and - indicates a deletion of a base.
  • the present embodiment relates to a tomato that is resistant to a Tobamovirus virus.
  • the virus-resistant tomato is, for example, a tomato that has a property of inhibiting infection with a Tobamovirus virus, a property of suppressing proliferation and movement of the virus even if infected, and/or a property of suppressing the manifestation of an infection symptom of the virus.
  • the virus-resistant tomato is preferably a tomato that has a property of inhibiting infection with the virus or a property of suppressing proliferation and movement of the virus even if infected.
  • tobamovirus is a general term for viruses that have single-stranded RNA and are related to the tobacco mosaic virus (TMV), which was the first virus discovered in the world. Tobamovirus viruses are known to be highly infectious, highly resistant to inactivation, and are stable, causing leaf mosaics, silky leaves, yellowing symptoms, stem necrosis, and fruit discoloration, necrosis, ring spots, etc. in infected plants.
  • TMV tobacco mosaic virus
  • the Tobamovirus genus virus targeted for infection and control is not particularly limited as long as it is a Tobamovirus genus virus that infects tomatoes, and specific examples include Tomato brown rugose fruit virus (ToBRFV), Tomato mosaic virus (TMV), Tomato mosaic virus (ToMV), and Tomato mottle mosaic virus (ToMMV).
  • Tobamovirus genus viruses may be abbreviated to "tobamovirus.”
  • tomato refers to Solanum lycopersicum, and refers to the entire plant, not just the fruit.
  • the tobamovirus-resistant tomato of this embodiment has a mutation in at least one gene selected from the group consisting of the receptor-like kinase RLK gene and its homologous genes.
  • RLK gene is a tomato gene that codes for a "Receptor-Like Kinase.”
  • BAM1 Barley Any Meristem 1
  • CLAVATA1-related receptor-like kinase protein that is necessary for shoot and floral meristem functions involved in leaf and gamete formation.
  • BAM2 which is highly homologous to BAM1 in Arabidopsis, has also been confirmed, and recent research has revealed the existence of highly homologous homologs in tomato.
  • Such homologs include the tomato "RLK1 gene” (Solyc02g091840, on chromosome 2) and "RLK2 gene” (Solyc03g043770, on chromosome 3).
  • RLK gene refers collectively to the "RLK1 gene” (Solyc02g091840) or the “RLK2 gene” (Solyc03g043770), or even both, and the individual genes are referred to as the "RLK1 gene” or "RLK2 gene.”
  • homologous genes of the RLK gene may include both “homologous genes of the RLK1 gene” and “homologous genes of the RLK2 gene” described below.
  • the "RLK1 gene” has a cDNA sequence that includes the base sequence shown in SEQ ID NO:1 or consists of the base sequence shown in SEQ ID NO:1.
  • a "homologous gene of the RLK1 gene” preferably has a cDNA sequence that includes a base sequence having sequence homology to the base sequence shown in SEQ ID NO: 1, or is composed of a base sequence having sequence homology to the base sequence shown in SEQ ID NO: 1.
  • sequence homology means sequence identity. There is no particular limit to the homology with the base sequence of SEQ ID NO: 1, but it is preferably 85% or more and less than 100%. The lower limit of the homology may be any value as long as it is 85% or more, such as 87% or more, 90% or more, 93% or more, 95% or more, 97% or more, 99% or more, or 99.5% or more.
  • the "RLK2 gene” has a cDNA sequence that includes the base sequence shown in SEQ ID NO: 16 or consists of the base sequence shown in SEQ ID NO: 16.
  • the "homologous gene of the RLK2 gene” preferably has a cDNA sequence that includes a base sequence having sequence homology to the base sequence shown in SEQ ID NO: 16, or is composed of a base sequence having sequence homology to the base sequence shown in SEQ ID NO: 16.
  • sequence homology means sequence identity. There is no particular limit to the homology with the base sequence of SEQ ID NO: 16, but it is preferably 85% or more and less than 100%. The lower limit of the homology may be any value as long as it is 85% or more, such as 87% or more, 90% or more, 93% or more, 95% or more, 97% or more, 99% or more, or 99.5% or more.
  • the homology (i.e., identity) between a specific base sequence and the cDNA sequence of a homologous gene can be determined by known methods.
  • the homology of base sequences can be determined using a known homology search program such as BLAST.
  • the tomato has a mutation in at least one gene selected from the group consisting of RLK gene or its homologous genes (hereinafter, the gene having the mutation is also referred to as a "virus resistance gene” or a "tobamovirus resistance gene”).
  • the mutation suppresses the expression of the gene having the mutation, or makes a protein encoded by the mutated gene or a mutually related gene non-functional against a tobamovirus.
  • a protein non-functional against the virus refers to a protein that cannot be used by the virus when the virus infects, grows, and moves in a plant, or that reduces the infection and growth of the virus.
  • the tobamovirus resistance gene may be mutated so as not to encode a protein.
  • tobamovirus when tobamovirus infects tomatoes, it interacts with a specific RLK among the multiple RLK isoforms present in tomatoes. At this time, if a gene encoding the specific isoform used by tobamovirus (i.e., an RLK functional against tobamovirus) mutates and the specific RLK protein used by tobamovirus is no longer produced, or if the RLK protein produced is non-functional against tobamovirus, it is believed that translation of the protein encoded on the viral genome and necessary for infection and proliferation does not proceed. Alternatively, it is believed that tobamovirus proteins that require interaction with RLK are no longer able to function, inhibiting tobamovirus infection and proliferation, resulting in tomato acquiring resistance to tobamovirus.
  • the tomato itself can utilize other homologs, or the tomato itself can utilize a non-functional RLK protein for tobamoviruses, so it is thought that it is possible to confer resistance to tobamoviruses without affecting the growth of the host tomato.
  • tomatoes having a tobamovirus resistance gene acquire tobamovirus resistance. For example, if the amount of tobamovirus accumulated in the plant body is equal to or less than that of a non-tobamovirus inoculated strain even after 18 days or more have passed since inoculation with tobamovirus, and/or if no tobamovirus infection symptoms are visually observed, the plant can be judged to have "tobamovirus resistance". Specifically, the tobamovirus resistance of a plant can be judged by infecting the plant with tobamovirus in a conventional manner and confirming the accumulation of tobamovirus in the plant body by a known method such as ELISA or PCR.
  • the tobamovirus resistance of a tomato can also be judged by confirming the presence or absence of tobamovirus infection symptoms (mosaic leaves, silky leaves, yellowing symptoms, necrotic symptoms of stems, and further discoloration, necrotic and ring-shaped symptoms of fruits, etc.) in a plant infected with tobamovirus.
  • tobamovirus infection symptoms mosaic leaves, silky leaves, yellowing symptoms, necrotic symptoms of stems, and further discoloration, necrotic and ring-shaped symptoms of fruits, etc.
  • tobamoviruses can infect plants not only in the form of virus particles but also with the genomic RNA alone, and as described in Non-Patent Document 6, it is also possible to test ToBRFV resistance by using a synthetic infectious RNA (e.g., SEQ ID NO: 45) equivalent to the ToBRFV genomic RNA instead of the virus.
  • a synthetic infectious RNA e.g., SEQ ID NO: 45
  • the genetic mutation may be present in at least one gene selected from the group consisting of the RLK1 gene and its homologous genes, and the RLK2 gene and its homologous genes.
  • the tobamovirus-resistant tomato in this embodiment has a mutation in the RLK1 gene, it may have mutations in all of the genes encoding RLK1 proteins functional against tobamoviruses.
  • the gene encoding the RLK1 protein functional against tobamoviruses is mutated, such a tobamovirus-resistant tomato may have another normal RLK1 gene.
  • it may be a tomato in which all endogenous genes encoding RLK1 proteins functional against tobamoviruses have been deleted, destroyed, or otherwise rendered nonfunctional, and an exogenous RLK1 gene has been introduced instead.
  • the tobamovirus-resistant tomato has a mutation in the RLK2 gene. That is, so long as the gene encoding any protein functional against the tobamovirus of the present invention is mutated, it may have other normal genes. It may also be one in which all endogenous genes encoding any RLK2 protein functional against the tobamovirus have been deleted, destroyed, or otherwise rendered nonfunctional, and an exogenous homologous gene has been introduced instead.
  • the tobamovirus-resistant tomato of this embodiment has a mutation in a gene in its genome. There are no particular limitations on the genetic mutation, so long as it confers virus resistance.
  • mutations in the RLK gene include the following (a) to (d).
  • (c) a consecutive or non-consecutive deletion of 3n bases (n 1 to 5); and
  • a frameshift mutation is a mutation in which the reading frame of a codon is shifted due to the deletion or insertion of bases, resulting in the coding of a different amino acid sequence. By changing the encoded amino acid sequence, the mutated gene becomes a tobamovirus resistance gene.
  • a nonsense mutation is a mutation that changes a codon that normally codes for an amino acid into a stop codon, resulting in a tobamovirus resistance gene.
  • the insertion or substitution of 1 to 10 bases changes the reading frame of the amino acid sequence encoded by the base sequence downstream of the mutated region.
  • the change in reading frame alters the originally encoded amino acid sequence, resulting in a change in the protein structure, etc., resulting in a tobamovirus resistance gene.
  • this mutation is preferably a mutation in a base other than the third base of the codon.
  • the number of bases inserted or substituted is not particularly limited as long as a tobamovirus resistance gene is obtained, but can be, for example, 1 to 5, 1 to 3, or 1 to 2.
  • the mutation in the tobamovirus resistance gene is preferably at least one selected from the group consisting of (a) to (d) above. Note that the mutations (a) to (d) above are not alternative; for example, mutations (a) and (b) may occur as a result of mutations (c) and (d).
  • the mutation when tomato has a mutation in the RLK1 gene, the mutation is preferably present in exon 1 (SEQ ID NO: 2) of the RLK1 gene, and more preferably present in a region including bases 790 to 809 of exon 1, i.e., a region including TCTCTAGAGTACCTTGCAGT shown in SEQ ID NO: 3.
  • the mutation is preferably present in a region in the homologous gene corresponding to the base sequence shown in SEQ ID NO: 2, and more preferably present in a region in the region corresponding to the base sequence shown in SEQ ID NO: 3.
  • the mutation may also be present in a portion of exon 1 other than the region.
  • the mutation is preferably a 7-base deletion, a 5-base deletion, a 1-base insertion, or a base substitution.
  • Such mutations include a 7-base deletion at positions 793-799 of exon 1, a 5-base deletion at positions 793-797, an insertion of a 1-base between positions 795 and 796, and a substitution of a 806-808 base.
  • the mutations include a 5-base deletion at positions 793-797 (mutation C1), a 7-base deletion at positions 793-799 (mutation C2), an insertion of a 1-base (adenine) between positions 795 and 796 (mutation C3), and a 5-base deletion at positions 793-797 and a substitution of a 806-808 base (substitution of CAG to ACG) (mutation C4) (see Figure 4).
  • the base sequences of the mutated regions are shown in SEQ ID NOs:4-7.
  • the mutation when tomato has a mutation in the RLK2 gene, the mutation is preferably present in exon 1 (SEQ ID NO: 17) of the RLK2 gene, and more preferably in a region including bases 790 to 809 of exon 1, i.e., a region including GAGGTGTCACGTGTGACCGGTATCGTCACGTGACTTCTCTC as shown in SEQ ID NO: 18.
  • the mutation is preferably present in a region in the gene corresponding to the base sequence shown in SEQ ID NO: 17, and more preferably in a region in the gene corresponding to the base sequence shown in SEQ ID NO: 18. Note that when a mutation is present in the region shown in SEQ ID NO: 18 or a corresponding region, the mutation may also be present in a portion of exon 1 other than the region.
  • the mutation is preferably a 12-base deletion, a 6-base deletion, a 5-base deletion, a 3-base deletion, or a 1-base deletion.
  • Specific examples of such mutations include a 12-base deletion at positions 269-280 of exon 1 (mutation P1), a 12-base deletion at positions 272-283 (mutation P2), a 6-base deletion at positions 279-285 (mutation P3), a 5-base deletion at positions 279-284 (mutation P4), a 3-base deletion at positions 279-281 (mutation P5), and a 1-base deletion at position 279 (mutation P6) (see Figure 5).
  • the base sequences of the mutated regions are shown in SEQ ID NOs:19-24.
  • the present embodiment relates to a mutant RLK1 gene itself having a mutated exon 1 as shown in any one of SEQ ID NOs:8 to 11, which is a tobamovirus resistance gene, and a mutant RLK2 gene itself having a mutated exon 1 as shown in any one of SEQ ID NOs:25 to 30.
  • the present embodiment also relates to the use of the mutant RLK1 gene and/or mutant RLK2 gene for imparting tobamovirus resistance to tomatoes.
  • the mutations in tomatoes are not limited to the above-mentioned regions, and mutations may exist in other regions of the RLK1 gene and/or the RLK2 gene, or in other genes, as long as tobamovirus resistance is not impaired.
  • the mutation in the tomato gene is preferably introduced into a gene in the genome by genome editing technology such as the CRISPR system described below.
  • the mutated gene in the genome may be homozygous, present in both alleles, or heterozygous, present in only one allele; however, homozygous is preferable. This is because the homozygous type, in which the two alleles are characterized by the same mutated sequence, is thought to more strongly reflect the properties brought about by the mutated gene.
  • the tobamovirus-resistant tomato in this embodiment may be a multiple-resistant tomato that exhibits resistance to other viruses and bacteria, so long as it exhibits resistance to Tobamovirus viruses.
  • the present embodiment relates to a part of a tomato plant having tobamovirus resistance.
  • the part includes a part taken from the tomato plant and its progeny or clones having the above characteristics, or a derivative obtained from the plant or part.
  • Parts include organs such as fruits, shoots, stems, roots, shoots, anthers, as well as plant tissues and cells.
  • Such parts may be in any form, including suspension cultures, protoplasts, embryos, callus tissue, leaf pieces, gametophytes, sporophytes, pollen, and microspores.
  • Derivatives of tomatoes include seeds.
  • the tobamovirus-resistant tomato portion of this embodiment may be a scion, rootstock, etc., used for grafting.
  • this embodiment also relates to plant cells (including callus) etc. that can regenerate the above-mentioned virus-resistant tomato, and the tobamovirus-resistant tomato of this embodiment also includes plants obtained from such plant cells.
  • the part of the tomato that has virus resistance is preferably the fruit, which is useful for eating raw or processing. It is also preferable that the part is the seed, since this is useful for producing progeny.
  • the present embodiment relates to a processed product of tomatoes or parts thereof.
  • the processed product is not particularly limited, and may be for edible, industrial, or medical use, and is particularly preferably for edible use.
  • examples of edible processed tomato products include canned tomatoes, tomato paste, ketchup, tomato sauce, tomato soup, dried tomatoes, tomato juice, tomato powder, and tomato concentrate.
  • Nutritional supplements made from tomatoes are also examples of processed products.
  • Tomato Cells Having Resistance to Tobamoviruses In one aspect, the present embodiment relates to tomato cells having resistance to tobamoviruses.
  • the tomato cells of this embodiment have a mutation in at least one gene selected from the group consisting of the receptor-like kinase RLK gene and its homologous genes. These genes and their mutations are as described above in relation to tobamovirus-resistant tomatoes.
  • the tobamovirus resistance of tomato cells can be confirmed by the methods described above.
  • the presence or absence of virus resistance can be confirmed by infecting plant cells with a tobamovirus using standard methods and detecting the accumulation of the virus in the cells using known methods such as ELISA and PCR.
  • the tobamovirus-resistant tomato cells of this embodiment may be isolated from the plant body or parts of the above-mentioned tobamovirus-resistant tomato and its progeny or clone, or may be plant cells into which a genetic mutation has been introduced, obtained by the method for producing a tobamovirus-resistant tomato described below. Furthermore, there are no particular limitations on the form of the tobamovirus-resistant tomato cells, and they may be suspension cultures or protoplasts.
  • the present embodiment relates to a tomato plant or a part thereof that has the above-mentioned tomato cell and has tobamovirus resistance.
  • the tomato plant or part thereof includes a plant or a part such as a tissue or organ regenerated from a tomato plant cell into which a genetic mutation has been introduced.
  • the part of the plant regenerated from the tomato plant cell is also a part that has the above-mentioned tomato cell. Details of the part are as described above in relation to the virus-resistant tomato.
  • the tomato part is preferably the fruit, which is useful for eating raw or processing. It is also preferable that the tomato part be the seed, since this is useful for producing progeny.
  • the present embodiment relates to a processed product of tomatoes or parts thereof.
  • the processed product is not particularly limited, and examples include processed products for edible, industrial, and medical purposes, and is particularly preferably an edible processed product.
  • the present embodiment relates to a method for producing a virus-resistant tomato of the present invention, specifically, the method includes the following steps: Selecting at least one gene from the group consisting of receptor-like ligase RLK genes and their homologous genes; introducing into a selected gene in the genome of a tomato a mutation that suppresses expression of the selected gene or that renders a protein encoded by the selected gene non-functional against a Tobamovirus that causes yellow leaf curl symptoms in tomato; A process for selecting tomatoes having resistance to Tobamovirus.
  • a target gene to be mutated is selected from the group consisting of the RLK gene and its homologous genes.
  • the selected gene may be one kind or a combination of two or more different kinds of genes. These genes are as described above in relation to the virus-resistant tomato. Next, a method for producing a virus-resistant tomato will be specifically described.
  • a mutation is introduced into the selected gene.
  • Methods for introducing a mutation into a gene in a genome can be roughly classified into the following two methods.
  • Direct genome editing This method involves directly editing the genome of a plant that has a functional RLK gene against tobamovirus, thereby introducing mutations into targeted locations and producing a plant that has a tobamovirus resistance gene.
  • Mutant gene introduction This method combines the following procedures (A) and (B).
  • a tobamovirus resistance gene is prepared and introduced into a plant using an appropriate promoter.
  • B Among the endogenous genes possessed by the plant that correspond to the tobamovirus resistance gene prepared in (A) above, a gene functional against tobamovirus is made non-functional against tobamovirus. Each method will be explained below.
  • Direct genome editing can be performed using known genome editing techniques using site-specific nucleases such as CRISPR and TALEN.
  • site-specific nucleases such as CRISPR and TALEN.
  • a double-strand break is introduced using a restriction enzyme capable of cleaving a specific site in the genome, various mutations are introduced due to repair errors when the break is repaired.
  • a mutation is introduced into the target gene (in this embodiment, a gene encoding RLK functional against tobamovirus).
  • the CRISPR system is preferably used, and the CRISPR/Cas9 system is particularly preferred, since it allows mutations to be introduced with particularly high specificity and efficiency.
  • a guide RNA sgRNA
  • sgRNA guide RNA
  • the Cas9 protein cuts the double strand.
  • NHEJ non-homologous end joining
  • the Cas protein and sgRNA can be delivered to plants via vectors encoding them using methods known to those skilled in the art, such as the Agrobacterium method, standard transfection methods, electroporation, particle bombardment, etc.
  • a binary vector incorporating the Cas gene and sgRNA can be constructed, used to transform Agrobacterium, and then used to transform a plant, thereby delivering the Cas protein and sgRNA to the plant (see Friedrich Fauser et al., "The Plant Journal," 2014, 79: 348-359, and Osawa Ryo and Ezura Hiroshi, "Understanding New Plant Breeding Techniques - NBT (New plant breeding techniques),” Kokusai Bunkensha, 2013, etc.).
  • the form of the plant transformed with Agrobacterium is not particularly limited as long as it is capable of regenerating the plant body, and examples include suspension culture cells, protoplasts, leaf slices, callus, etc. After removing the Agrobacterium, the plant is cultured in a medium containing a drug appropriate to the vector used, and the slices into which the target gene has been incorporated can be selectively cultured using drug resistance as an indicator.
  • Guide RNA can be designed to allow highly efficient introduction of mutations into the target site.
  • cleavage generally occurs three bases before a three-base sequence called the PAM sequence (NGG when using the most common S. pyogenes-derived Cas9). Since the PAM sequence must be present immediately after the target sequence, guide RNA can be designed with the upstream of the PAM sequence as the target sequence.
  • guide RNA In the design of guide RNA, it is preferable to consider the GC content because the higher the GC content of the base sequence, the higher the cleavage efficiency. In addition, it can be designed to minimize non-specific cleavage due to off-target effects.
  • FIG. 1 showing the cDNA sequence (SEQ ID NO: 1) of the RLK1 gene present on chromosome 2 of tomato
  • the squared portion present in exon 1 is used as the PAM sequence
  • the guide RNA can be designed targeting the usual 20 bases upstream from this 3 bases (wavy underlined portion in FIG. 1, SEQ ID NO: 3).
  • FIG. 2 showing the cDNA sequence (SEQ ID NO: 16) of the RLK2 gene present on tomato chromosome 3, the boxed portion present in exon 1 (single underlined portion in FIG. 2, SEQ ID NO: 17) is used as the PAM sequence, and a guide RNA can be designed targeting usually 20 bases (wavy underlined portion in FIG. 2, bases 20 to 39 of SEQ ID NO: 18) three bases upstream from this PAM sequence.
  • the tobamovirus resistance gene of this embodiment has a variety of mutations.
  • the mutation introduced does not necessarily have to be limited to the target site.
  • the mutation may exist in a region other than the target site, for example, in a region adjacent to the target site.
  • the mutation introduced into the RLK1 gene was limited to the target site (see Figure 4), but the mutation introduced into the RLK2 gene was also present in a region adjacent to the target site (see Figure 5).
  • this embodiment also relates to the guide RNA and the vector containing the guide RNA used to create the tobamovirus-resistant tomato.
  • the sequence of the guide RNA is as described above.
  • This embodiment also relates to a kit containing the guide RNA.
  • the kit may contain a site-specific nuclease, etc., necessary for performing genome editing using the CRISPR system, and can be used to create a tobamovirus-resistant tomato.
  • Mutant gene introduction is a method that combines the following procedures (A) and (B).
  • A) A tobamovirus resistance gene is constructed and introduced into a plant using an appropriate promoter.
  • B) Among the endogenous genes contained in a plant and corresponding to the tobamovirus resistance gene prepared in (A) above, a gene functional against a tobamovirus is made non-functional against a tobamovirus.
  • the order of carrying out the above steps (A) and (B) is not particularly limited as long as the plant does not die, and (B) may be carried out first. Note that the method of carrying out only (B) at a specific site is the above-mentioned (1) direct genome editing.
  • step (A) a mutant gene encoding a RLK protein that is non-functional against tobamovirus is prepared and introduced into a plant using an appropriate promoter.
  • the mutant gene can be prepared using a method known to those skilled in the art. For example, a base sequence having the desired mutation can be synthesized and amplified by PCR or the like. The mutation introduced here is as described above in relation to tobamovirus-resistant tomatoes.
  • the mutant gene thus created can also be introduced into a plant using methods known to those skilled in the art. Conveniently, this can be done using a vector carrying the mutant gene, for example, the polyethylene glycol method, electroporation method, the Agrobacterium method, the particle gun method, etc.
  • the mutant gene introduced here is a tobamovirus resistance gene obtained by mutating the RLK gene (or a homologous gene) derived from tomato, but it may also be a tobamovirus resistance gene from another type of plant.
  • the form of the plant into which the vector is introduced is not particularly limited as long as it is capable of regenerating the plant body, and examples include suspension culture cells, protoplasts, leaf slices, callus, etc.
  • step (B) endogenous RLK genes (or homologous genes) possessed by the plant that are functional against tobamoviruses are changed to non-functional against tobamoviruses.
  • known methods for introducing mutations into plants can be used. For example, mutagen treatment such as ion beams or EMS can be used. It can also be carried out using genome editing techniques such as the above-mentioned CRISPR and TALEN. It is desirable to make all endogenous RLK genes that are functional against tobamoviruses non-functional against tobamoviruses.
  • a plant body is regenerated from a part of the plant (such as a leaf piece or a plant cell) that has the tobamovirus resistance gene.
  • Regeneration of the plant body can be carried out by a method known to those skilled in the art depending on the type of plant. For example, in the case of tomato, regeneration can be carried out by referring to Sun H.J. et al., "Plant Cell Physiol.," 2006, 47: 426, etc.
  • tomatoes resistant to tobamovirus are selected from the regenerated plants.
  • the selection can be performed by the above-mentioned method for confirming tobamovirus resistance.
  • a plant resistant to tobamovirus can be selected by infecting a plant with tobamovirus in a conventional manner and confirming the accumulation of tobamovirus in the plant body by a known method such as ELISA or PCR.
  • Tomatoes resistant to tobamovirus can also be selected by confirming the presence or absence of tobamovirus infection symptoms (mosaic leaves, silky leaves, yellowing symptoms, necrotic symptoms of stems, and further discoloration, necrotic symptoms and ring spots of fruits, etc.) in the plants infected with tobamovirus.
  • RNA corresponding to the genomic RNA of ToBRFV e.g., SEQ ID NO: 45
  • SEQ ID NO: 45 a synthetic infectious RNA corresponding to the genomic RNA of ToBRFV
  • the present embodiment relates to a tomato produced by the method described above.
  • the tomato is similar to the tobamovirus-resistant tomato described above.
  • the tobamovirus-resistant tomato plant of this embodiment includes these progeny and clones.
  • the present embodiment relates to a method for producing a breeding progeny of a tobamovirus-resistant tomato, comprising a step of self-pollinating or cross-pollinating a tobamovirus-resistant tomato (first generation) obtained by the above-mentioned production method or a progeny thereof.
  • Self-pollination or cross-pollination of a plant can be carried out by a method known in the art, and may be carried out naturally or artificially.
  • the progeny thus obtained can be further self-pollinated or cross-pollinated to produce further progeny.
  • the tomatoes crossed with the primary or progeny in cross-pollination may be tomatoes with the same mutation in the same gene, tomatoes with different mutations in the same gene, or tomatoes with mutations in different genes.
  • Example 1 Preparation of recombinant Agrobacterium A for introducing a mutation into the RLK1 gene
  • a site recognized by a guide RNA was arbitrarily designed within exon 1 of the RLK1 gene (Solyc02g091840) (SEQ ID NO: 1), which is believed to be present on chromosome 2 of tomato.
  • a double-stranded DNA corresponding to the set 20-base length site (SEQ ID NO: 3: TCTCTAGAGTACCTTGCAGT) was synthesized and inserted into the restriction enzyme BbsI site in the vector pUC19_AtU6oligo (obtained from the National Institute of Agrobiological Sciences, currently the National Agriculture and Food Research Organization) to construct a recombinant binary vector.
  • the cDNA sequence of the RLK1 gene present on chromosome 2 of wild-type tomato is shown in FIG. 1 and SEQ ID NO: 1.
  • the cassette portion containing the guide RNA sequence region was excised from each of the constructed recombinant vectors and inserted into the restriction enzyme I-SceI site in the binary vector pZD_OsU3gYSA_HolgerCas9_NPTII to obtain a recombinant binary vector.
  • These binary vectors were used to transform Agrobacterium LBA4404 (Takara Bio Inc.) by a conventional method to obtain recombinant Agrobacterium A.
  • the cassette portion containing the guide RNA sequence region was excised from each of the constructed recombinant vectors and inserted into the restriction enzyme I-SceI site in the binary vector pZD_OsU3gYSA_HolgerCas9_NPTII to obtain a recombinant binary vector.
  • These binary vectors were used to transform Agrobacterium LBA4404 (Takara Bio) using standard methods to obtain recombinant Agrobacterium B.
  • Tomato transformation was performed using the known fixed variety Manymaker (hereinafter abbreviated as "MM”) or our own fixed variety S.
  • Tomato transformation using Agrobacterium was performed according to the method described in a general textbook (e.g., "Protocols for plant transformation” edited by Yutaka Tabei, Kagaku Dojinsha, 2012). Specifically, cotyledon pieces obtained by germinating tomato seeds in a sterile medium, or sterilized cotyledon pieces or primary leaf pieces of seedlings sown normally, were prepared.
  • a culture solution was prepared by culturing the recombinant Agrobacterium A obtained in Example 1 or the recombinant Agrobacterium B obtained in Example 2 until the turbidity reached 0.5 to 2.0. Tomato leaf pieces were immersed in the culture solution for about 10 minutes to be infected with Agrobacterium A or B.
  • Tomato leaf pieces were transferred onto Murashige and Skoog medium (sometimes abbreviated as MS basal medium; MS basal medium supplemented with 3% sucrose, 1.5 mg/L zeatin, and 1% agar) supplemented with carbenicillin (100-500 mg/ml) and kanamycin (20-100 mg/ml) and subjected to selective culture under illumination at 25°C (16 hours light/8 hours darkness). Callus formation from the leaf pieces was promoted by replacing the medium and subculturing every 10 days to 2 weeks from the start of culture. Adventitious buds were induced by subsequent repeated subculture.
  • MS basal medium sometimes abbreviated as MS basal medium; MS basal medium supplemented with 3% sucrose, 1.5 mg/L zeatin, and 1% agar
  • carbenicillin 100-500 mg/ml
  • kanamycin 20-100 mg/ml
  • rooting medium MS basic medium with 1.5% sucrose, 1% agar, 50-250 mg/ml carbenicillin, 20-100 mg/ml kanamycin, and sometimes naphthalene acetic acid (NAA) added
  • NAA naphthalene acetic acid
  • T0 transgenic current generation
  • Example 4 Selection of gene-edited lines To confirm whether or not genetic recombination and editing sites (base deletion, insertion, or substitution) were present in the target gene of the transgenic generation, T0 tomato leaves were crushed and genomic DNA was extracted using a NucleoSpin Plant II kit (Takara Bio Inc.) or the like according to the method attached to the kit.
  • primer 1 (5'-TTAACACGTCTGCGTAACCTC-3', SEQ ID NO: 37) and primer 2 (5'-CCGGTGAAGGTATTGTAGTATCC-3', SEQ ID NO: 38) were used for the region in the RLK1 gene (Solyc02g091840), and primer 3 (5'-CTCCACTTCACCACCGCAAA-3', SEQ ID NO: 39) and primer 4 (5'-TACCGGAGATGGGTAAGCTG-3', SEQ ID NO: 40) were used for the region in the RLK2 gene (Solyc03g043770).
  • PCR was performed using a T100 thermal cycler (BIORAD) and KOD One (Toyobo) according to the attached manual to amplify DNA.
  • the amplified DNA was purified using NucleoSpin Gel and PCR Clean-up (Takara Bio) according to the kit's instructions, and the base sequence of the amplified site was sequenced using the Sanger method with the forward primer (primer 1 or primer 3 mentioned above) of the primers used in PCR for the gene in question to confirm the presence or absence of editing.
  • the edited part differs depending on the allele, i.e., it may be a heterozygote. In that case, when DNA amplification products of the same region are sequenced, different base signals will be detected overlapping, making it impossible to decipher.
  • the amplified fragment obtained by PCR of the relevant region was cloned into a TA vector using the TArget Clone-Plus (Toyobo) kit, and then sequencing was performed using the Sanger method (when cloning, the amplified DNA is cloned molecule by molecule, so the sequence can be confirmed for each clone.
  • sequencing multiple clones if they all have the same editing pattern sequence, it can be inferred that the allele is homozygous, and if there are two patterns, the allele is heterozygous).
  • Example 5 Generation of transgenic progeny Furthermore, these edited lines were self-pollinated, and seeds of the progeny (the generation next to the TO generation was designated T1, and the subsequent generation was designated T2) were collected. Genomic DNA was extracted from the sown seedlings using the method of [Example 4], the base sequence near the edited site was amplified by PCR, and the editing pattern was confirmed by sequencing.
  • Example 6 Preparation of ToBRFV inoculum (infectious RNA) Tomato brown rugose fruit virus (ToBRFV) was used as a Tobamovirus virus. Due to quarantine issues, it is very difficult to bring the ToBRFV virus or virus-infected leaves into Japan. Therefore, following the report in Non-Patent Document 6, an infectious RNA (SEQ ID NO: 45) was synthesized in vitro by linking the genome sequence (6388 bases long, with the 6th to 9th bases deleted) of the Jordan strain of ToBRFV (Genbank: KT383474) under the T7 promoter (17 bases long). Note that ToBRFV is an RNA virus, and its genome sequence is an RNA sequence. SEQ ID NO: 45 shows the DNA sequence in which U (uracil) of RNA is converted to T (thymine) of DNA.
  • PCR was performed using primer 5 (5'-AGCTTGCATGCCTGCAGGTCT-3', SEQ ID NO:41), primer 6 (5'-TGGGCCCCTCCGGGGTTCCGGGGAATTCGAATC-3', SEQ ID NO:42), and KOD One (Toyobo Co., Ltd.) under the conditions attached to KOD One to amplify a full-length viral fragment beginning with the T7 promoter sequence.
  • the primers were removed from the PCR amplified fragment using NucleoSpin (registered trademark) Gel and PCR Clean-up (Takara Bio Inc.) according to the kit's instructions.
  • RNA was transcribed and synthesized at 37°C for 4 hours using T7 polymerase (HiScribe T7 Quick High Yield RNA Synthesis Kit; New England Biolabs) according to the method attached to the reagent.
  • T7 polymerase HiScribe T7 Quick High Yield RNA Synthesis Kit; New England Biolabs
  • Example 6 Inoculation of ToBRFV One-tenth to one-half of the 20 ⁇ L of RNA synthesized in Example 6 was inoculated into the true leaves (compound leaves) at approximately the middle position of test tomato seedlings (3- to 5-leaf stage) by mechanical inoculation (rubbing inoculation) using the carborundum method.
  • Example 7 Extraction of total RNA
  • leaves inoculated with RNA (infectious RNA instead of the virus) inoculated leaves
  • upper leaves not inoculated upper leaves
  • about 100 mg were frozen with liquid nitrogen, ground, and dissolved in 1 mL of TRIsol Reagent (Ambion) heated to 50 ° C.
  • the leaves were transferred to a microtube and heated in a thermostatic bath at 50 ° C for 10 minutes to extract RNA. Then, 1/5 volume of chloroform was added, stirred, and centrifuged at 15,000 rpm for 15 minutes.
  • RNA analysis of ToBRFV Using 5-10 ⁇ L of the RNA sample obtained in Example 7, first strand cDNA was synthesized using SuperScript VILO cDNA Synthesis Kit (Invitrogen) according to the conditions of the kit's instructions. Furthermore, PCR was performed using 2 ⁇ L of this cDNA sample, primer 7 (5'-GAGACTTACGTCGCCGATTC-3', SEQ ID NO: 43) and primer 8 (5'-GTACCACGTGTGTTTGCAGAC-3', SEQ ID NO: 44) with GoTaq Green Master Mix (Promega). PCR was carried out under the conditions of 95° C. for 2 minutes, 30 cycles of "95° C. for 30 seconds, 53° C. for 30 seconds, and 72° C. for 30 seconds", and 72° C. for 5 minutes. After PCR, 1/10 of the PCR product was subjected to 1.5% agarose gel electrophoresis to confirm the presence or absence of ToBRFV.
  • SuperScript VILO cDNA Synthesis Kit Invitrogen
  • FIG. 3 An electrophoretic photograph showing the results of the RT-PCR analysis of the ToBRFV inoculation test is shown in Figure 3.
  • M represents the ladder marker
  • WT represents the wild type cultivar (Many Maker)
  • R1 represents a mutant with a frameshift mutation in the RLK1 gene
  • R2 represents a mutant with a frameshift mutation in the RLK2 gene
  • PC represents the positive control.
  • ToBRFV was detected in both the inoculated leaves and upper leaves of the control cultivar Many Maker, but in the RLK1 and RLK2 mutants, ToBRFV was detected in the inoculated leaves but not in the upper leaves. This result indicated that the movement of the virus within the plant body was restricted or inhibited in the RLK1 and RLK2 mutants, and the mutants were resistant.
  • Figure 4 shows the mutations C1 to C4 found in the RLK1 gene together with the wild-type sequence
  • Figure 5 shows the mutations P1 to P6 found in the RLK2 gene together with the wild-type sequence.
  • the present invention provides a method for producing tomatoes, tomato cells, and tomatoes resistant to tobamovirus viruses, which have the property of inhibiting infection with tobamovirus viruses, the property of suppressing the proliferation and migration of tobamovirus viruses after infection, and/or the property of suppressing the manifestation of symptoms of infection with tobamovirus viruses.
  • the present invention can solve problems mainly in the agricultural field, such as reduced tomato yields due to infection with tobamovirus viruses.
  • SEQ ID NO: 1 cDNA sequence of RLK1 gene, bases 1 to 2818 are exon 1, bases 790 to 809 are target sequence.
  • SEQ ID NO: 2 Exon 1 of RLK1 gene, bases 790 to 809 are target sequence.
  • SEQ ID NO: 3 Wild-type target sequence in exon 1 of RLK1 gene.
  • SEQ ID NO: 4 Mutation region C1 of RLK1 gene.
  • SEQ ID NO: 5 Mutation region C2 of the RLK1 gene
  • SEQ ID NO: 6 Mutation region C3 of the RLK1 gene
  • SEQ ID NO: 7 Mutation region C4 of the RLK1 gene
  • SEQ ID NO: 8 Exon 1 of the RLK1 gene containing the mutated region C1
  • SEQ ID NO: 9 Exon 1 of the RLK1 gene containing the mutated region C2
  • SEQ ID NO: 10 Exon 1 of the RLK1 gene containing the mutated region C3
  • SEQ ID NO: 11 Exon 1 of the RLK1 gene containing the mutated region C4
  • 12 cDNA sequence of mutant RLK1 gene having mutated region C1
  • SEQ ID NO: 13 cDNA sequence of mutant RLK1 gene having mutated region C2
  • 14 cDNA sequence of mutant RLK1 gene having mutated region C3
  • 15 cDNA sequence of mutant RLK1 gene having mutated region C4 SEQ ID NO:

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Abstract

Le problème à résoudre par la présente invention est de fournir : une tomate résistant au virus du genre Tobamovirus qui présente une propriété d'inhibition d'une infection par des virus appartenant au genre Tobamovirus, une propriété de suppression de la prolifération des virus appartenant au genre Tobamovirus après avoir été infectée, et/ou une propriété de suppression de l'apparition de symptômes provoqués par une infection par les virus appartenant au genre Tobamovirus ; des cellules de tomate ; et leur procédé de production. Le problème est résolu par la fourniture d'une tomate qui présente une mutation d'au moins un gène choisi dans le groupe constitué par des gènes de kinase de type récepteur (RLK) et des gènes homologues de ces derniers, et dans laquelle, grâce à ladite mutation, l'expression du gène muté est supprimée ou une protéine codée par le gène muté n'est pas fonctionnelle par rapport à des virus appartenant au genre Tobamovirus et qui est ainsi résistante aux virus appartenant au genre Tobamovirus.
PCT/JP2024/023481 2023-06-29 2024-06-28 Plant de tomate résistant aux virus du genre tobamovirus, cellules végétales de tomate et leur procédé de production Ceased WO2025005231A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020111149A1 (fr) * 2018-11-28 2020-06-04 キッコーマン株式会社 Plante solanacée résistante au virus du genre begomovirus causant les symptômes d'enroulement des feuilles jaunes de la tomate, cellule végétale solanacée et procédé de production d'une plante solanacée
WO2021131628A1 (fr) * 2019-12-24 2021-07-01 キッコーマン株式会社 Plante solanacée et cellule de plante solanacée ayant une résistance au virus de la maladie bronzée de la tomate, et procédé de production d'une plante solanacée
WO2022186197A1 (fr) * 2021-03-04 2022-09-09 キッコーマン株式会社 Plante solanacée résistante à un virus du genre begomovirus provoquant des symptômes de feuilles jaunes en cuillère chez les tomates, cellule de plante solanacée et procédé pour la production d'une plante solanacée

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020111149A1 (fr) * 2018-11-28 2020-06-04 キッコーマン株式会社 Plante solanacée résistante au virus du genre begomovirus causant les symptômes d'enroulement des feuilles jaunes de la tomate, cellule végétale solanacée et procédé de production d'une plante solanacée
WO2021131628A1 (fr) * 2019-12-24 2021-07-01 キッコーマン株式会社 Plante solanacée et cellule de plante solanacée ayant une résistance au virus de la maladie bronzée de la tomate, et procédé de production d'une plante solanacée
WO2022186197A1 (fr) * 2021-03-04 2022-09-09 キッコーマン株式会社 Plante solanacée résistante à un virus du genre begomovirus provoquant des symptômes de feuilles jaunes en cuillère chez les tomates, cellule de plante solanacée et procédé pour la production d'une plante solanacée

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN121385043A (zh) * 2025-12-25 2026-01-23 酒泉市农业技术推广服务中心 一种结合传感网节点的番茄褐色皱果病毒监测方法

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