Tobamovirus-resistant tomato plants

Abstract

Disclosed is a tomato plant or part thereof. The plant is a tomato plant or part thereof, which has a Tm-2 genotype having an amino acid sequence that renders the plant resistant to Tomato Brown Rugose Fruit Virus (ToBRFV). 2 The plant expresses a protein. Methods for producing the plant are also disclosed. Products produced from the plant are also described.

Description

[Technical Field]

[0001] Related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 108,476, filed on 2 November 2020, and this reference incorporates the entirety of its contents.

[0002] Statement regarding sequence listings This reference incorporates the 16,512-byte ASCII file named 89944SequenceListing.txt, created on November 1, 2021, which was submitted simultaneously with this application. [Background technology]

[0003] In some embodiments, the present invention relates to tomato plants resistant to the harmful effects of tomato brown rugos fruit virus (ToBRFV).

[0004] Several highly destructive plant viruses belong to the genus Tobamovirus, which includes tomato mosaic virus (ToMV), tobacco mosaic virus (TMV), and cucumber green mottle mosaic virus (CGMMV). Tomato resistance to Tobamovirus is due to the resistance (R) genes Tm-1, Tm-2, and Tm-2 2 These are conferred by, and all of them originate from wild-type tomatoes. Over the past few decades, resistance-breaking virus strains have emerged against Tm-1 and Tm-2, and Tm-2 2 Only Tm-2 remained as the key gene for controlling tobamovirus in tomatoes. 2encodes a member of the nucleotide-binding leucine-rich repeat (NB-LRR or NLR) family of plant immune receptors. Members of the NLR family recognize effectors encoded by specific pathogens, named avirulence (AVR) factors, either directly binding them or indirectly via mediator proteins. This recognition typically triggers an immune signaling cascade that confines the pathogen to the site of infection by inducing programmed cell death (PCD) in a process called the hypersensitive reaction (HR).

[0005] Like many plant NLRs, Tm-2 2 contains a coiled-coil (CC) at its N-terminus and a NB domain in the center, and a LRR domain at the C-terminus, which determines effector recognition specificity. To activate an antiviral immune response, Tm-2 2 associates with its AVR, the viral movement protein (MP). The MP is a virus-encoded protein that enables intercellular virus movement through plasmodesmata, channels that connect adjacent cells. Upon virus infection, Tm-2 2 binds directly to the tobamovirus MP. This binding triggers the self-association of the Tm-2 2 protein, which enables the generation of immune signals.

[0006] Tm-2 2 is an allele of the disrupted resistance gene Tm-2. Interestingly, both alleles have different pathogen recognition capabilities. For example, Tm-2 confers resistance to the N3 strain of ToMV but not to the B7 strain, while Tm-2 2 defends against B7 but not against N3. Tm-2 and Tm-2 2 differ by only four amino acids, two of which are located in the NB domain and two within the LRR domain. One of these LRR domain residues, Tyr-767, is essential for the recognition of the MP encoded by the ToMV B7 strain, and Tm-2 2This suggests that specific residues within the LRR domain determine its ability to recognize specific MPs.

[0007] The recent outbreak of a novel tobamovirus, named Tomato Brown Rugos Fruit Virus (ToBRFV), has virtually devastated the tomato industry in Israel and Jordan. ToBRFV is classified into Tm-1, Tm-2, and Tm-2 2 Overcomes all known tobermovirus resistance in tomatoes, including France, Italy, and Germany. 3 Recent reports of ToBRFV outbreaks in the Netherlands, China, Mexico, Turkey, and the United States indicate the emergence of an international pandemic. Sequence analysis shows that ToBRFV has 9–15% variation from other tobamoviruses such as TMV and ToMV, including 21 potential resistance-disrupting mutations, 12 of which are ToBRFV MP(MP) ToBRFV It is located in ).

[0008] Other relevant background technologies include Weber et al. (1993), Journal of virology, 67(11), 6432-6438 and Kobayashi, M., et al (2011), Journal of plant physiology, 168(10), 1142-1145. [Overview of the Initiative]

[0009] In one aspect of the present invention, Tm-2 has an amino acid sequence that makes plants resistant to tomato brown rugos fruit virus (ToBRFV). 2 A tomato plant or part thereof that expresses a protein is provided.

[0010] In one embodiment of the present invention, a method for propagating tomato plants by cuttings as described in this application is provided.

[0011] In one embodiment of the present invention, seeds of the plant described in this application are provided.

[0012] In one embodiment of the present invention, cells having the genome of the plant described in this application are provided.

[0013] In one aspect of the present invention, a culture containing a plurality of cells described in this application is provided.

[0014] In one aspect of the present invention, a method for breeding tomato plants is provided, which includes crossbreeding the plant described in this application with another tomato plant to breed tomato plants.

[0015] In one embodiment of the present invention, hybrid seeds prepared by the method described herein are provided.

[0016] In one aspect of the present invention, a hybrid plant or a portion thereof produced by cultivating the hybrid seeds described in this application is provided.

[0017] In one embodiment of the present invention, a method for cultivating plants is provided, which involves vegetatively propagating the plants described in this application and cultivating the plants.

[0018] In one embodiment of the present invention, a food product is provided which is a processed product containing the plant or a part thereof as described in this application.

[0019] According to embodiments of the present invention, the tomato plant is Tm-2 2 The mutation is homozygous, which makes the plants resistant to tomato brown goose fruit virus (ToBRFV).

[0020] According to embodiments of the present invention, the tomato plant is Tm-2 2 The mutation is heterozygous, which makes the plants resistant to tomato brown goose fruit virus (ToBRFV).

[0021] According to embodiments of the present invention, wild-type Tm-2 2 Compared to proteins, the mutations include amino acid modifications that enhance immune activation by the ToBRFV migration protein (MP).

[0022] According to embodiments of the present invention, wild-type Tm-2 2 Compared to proteins, amino acid modifications that enhance immune activation by the ToBRFV migration protein (MP) are found in Tm-2 2 It contains protein.

[0023] According to embodiments of the present invention, amino acid modification is performed on Tm-2 2 It is located in the leucine-rich repeat (LRR) domain of the protein.

[0024] According to embodiments of the present invention, wild-type Tm-2 2 Compared to proteins, Tm-2 2 Proteins have an amino acid modification at one of the following positions: 528, 604, or 652.

[0025] According to embodiments of the present invention, modification is substitution.

[0026] According to embodiments of the present invention, the modification at position 528 is F528S.

[0027] According to embodiments of the present invention, the modification at position 604 is S604N.

[0028] According to embodiments of the present invention, the modification at position 652 is I652M.

[0029] According to embodiments of the present invention, tomato plants are resistant to tomato mosaic virus (ToMV) and tobacco mosaic virus (TMV).

[0030] According to embodiments of the present invention, the plant part is selected from the group consisting of roots, stems, leaves, cotyledons, flowers, fruits, embryos, and pollen.

[0031] According to embodiments of the present invention, cross-pollination includes pollination.

[0032] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as those generally understood by those skilled in the art in which the invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing embodiments of the invention, but exemplary methods and / or materials are described below. In the event of any conflict in definitions, etc., this specification shall prevail. Furthermore, the materials, methods and examples are merely illustrative and not necessarily intended to be limiting.

[0033] Some embodiments of the present invention are described herein merely as examples, with reference to the accompanying drawings. While the following description will be specifically referenced in detail to the drawings, it is important to emphasize that the features described herein are illustrative and intended to provide an illustrative consideration of embodiments of the present invention. In this regard, the description accompanied by the drawings will make it clear to those skilled in the art how embodiments of the present invention can be carried out. [Brief explanation of the drawing]

[0034] [Figure 1] Figures 1A-1H: MPToBRFV overcomes Tm-22 resistance in tomatoes. (A) Comparison of an infectious clone of ToMV (top) and a recombinant ToMV (ToMVMP-ToBRFV) in which native MP is replaced with MPToBRFV (bottom). Dashed lines indicate the replacement region. (B-D) Representative leaves of tm-2 / tm-2 tomato plants (variety: Manimaker): (B) uninfected plant, (C) ToMV-infected plant, and (D) ToMV-MPToBRFV. (B-D) Representative leaves of tm-2 / tm-2 tomato plants (variety: Manimaker): (B) uninfected plant, (C) ToMV-infected plant, and (D) ToMVMP-ToBRFV-infected plant. (E~G) Representative leaves of Tm-22 / Tm-22 tomato plants (variety: Manimaker): (E) Uninfected plants, (F) ToMV-infected plants, and (G) ToMVMP-ToBRFV-infected plants. For each treatment, the third leaf from the tip was sampled. (H) Table summarizing the number of plants showing symptoms and infected plants for each treatment. Scale bar = 5cm. [Figure 2]Figures 2A-2C: MPToBRFV overcomes Tm-22 resistance in N. benthamiana. (A) Comparison of an infectious clone of TMV-GFP (top) and recombinant TMV-GFP (TMV-GFPMP-ToBRFV) in which native MP is replaced with MPToBRFV (bottom). Dashed lines indicate the substitution region. (B) N. benthamiana plants infected with TMV-GFP alone (left) or infected with p35S:Tm-22 expression (right). (C) N. benthamiana plants infected with TMV-GFPMP-ToBRFV alone (left) or infected with p35S:Tm-22 expression (right). [Figure 3] Figures 3A-3F: Overexpression of MPToBRFV does not induce Tm-22-mediated cell death in tomato and N. benthamiana. (A-C) Transient expression of MPTMV(A), MPToBRFV(B), and empty vectors in tomato (variety: Ikram) with the Tm-22 resistance gene. (D-E) Transient expression of MPTMV(D) and MPToBRFV(E) in N. benthamiana leaves with or without Tm-22. (F) Transient expression of Tm-22 in N. benthamiana leaves served as a negative control. [Figure 4] Figure 4: Library preparation and screening steps for Tm-22 mutant clones. The ORF of Tm-22 was amplified using two separate PCR reactions: high-fidelity PCR for the CC-NB region and error-prone PCR to induce mutations in the LRR region of the gene. Both PCR products were incorporated into a Golden Gate Level 0 cassette. The resulting colonies were pooled together, and the ORF was inserted into a Level 2 plant expression plasmid. After additional cycles of the pool, the resulting Level 2 plasmid was transformed into Agrobacterium, and the colonies were isolated to form a library. The mutant Tm-22 clones were screened by co-expression with MPToBRFV, and then necrotic lesions, which are indicators of HR, were detected. [Figure 5]Figures 5A-5C: Isolation of Tm-22 mutant clones whose product recognizes MPToBRFV using directional evolution. (A) Isolation of 10 different Tm-22 mutant clones whose expression initiates HR in response to MPToBRFV. (B) A table showing the number of clones, the intensity of HR in response to MPToBRFV (+ mild, ++ moderate, +++ severe), and the location of various mutations. Silent mutations are defined as synonymous mutations that do not change amino acid identity. (C) Schematic diagram of the Tm-22 protein with altered amino acids in at least two MPToBRFV-recognizing clones (I528, S604, and I652). [Figure 6] Figures 6A-6D: Demonstration of directional evolutionary Tm-22 mutations conferring MPToBRFV recognition. Transient expression of Tm-22 with the following mutations: F528S (A), S604N (B), and I652M (C). Tm-22 mutant clones were expressed alone (left) or co-expressed with MPToBRFV (right). (D) Co-expression of non-mutant Tm-22 with MPTMV or MPToBRFV served as positive and negative controls, respectively. [Figure 7] Figures 7A-7F: Transient expression of the Tm-22 variant confers resistance to resistance-disrupted TMV-GFP. Infection with a resistance-disrupted TMV-GFP vector containing MPToBRFV (TMV-GFPMP-ToBRFV) was used instead of native MP. The viral vector was expressed alone (A) or co-expressed with one of the following in N. benthamiana leaves: native Tm-22 (B), Tm-22 F528S (C), Tm-22 S604N (D), and Tm-22 I652M (E). Left panel: GFP fluorescence in infected leaves, center panel: GFP fluorescence in the entire plant, indicating systemic infection, right panel: viral symptoms. Images were obtained 6 days after infection. (F) Quantification of GFP fluorescence in the 6th leaf from the infection site. [Figure 8]Figure 8: Predicted structural changes in isolated Tm-22 mutants. This is a model based on the homology of Tm-22 and Tm-22 mutants using the ZAR1 structure (Wang et al., 2019). Small image: Structure of the entire Tm-22 protein. Large image: Alignment of the Tm-22 LRR domain of the following various Tm-22 variants. White: Non-mutant Tm-22, Green: Tm-22 F538S, Purple: Tm-22 S604N, Yellow: Tm-22 I652M. Red arrows indicate mutation sites within the structure. White arrows indicate loop regions where structural changes occur compared to wild-type Tm-22. [Modes for carrying out the invention]

[0035] In some embodiments, the present invention relates to tomato plants resistant to the harmful effects of tomato brown rugos fruit virus (ToBRFV).

[0036] Before describing in detail at least one embodiment of the present invention, it should be understood that the uses of the present invention are not necessarily limited by the details illustrated by the following description or examples. The present invention can be carried out or implemented in other embodiments or in various ways. It should also be understood that the expressions and terms used herein are for illustrative purposes only and should not be considered limiting.

[0037] ToBRFV is a recently emerged, devastating tobamovirus that is causing significant damage to tomato crops worldwide. The main cause of the ToBRFV outbreak is Tm-2, which had been resistant to tobamovirus for over 50 years. 2 The objective is to overcome all genetic resistances in tomatoes, including resistance genes. Here, the inventors have introduced ToBRFV to Tm-2 2 The cause of resistance breakdown is MP ToBRFV It was established that this was a loss of recognition (Figures 1A-1H and 2A-2C). Tm-2 2 Genes are MP ToBRFVA directional evolutionary method was used to modify the recognition of . Based on the onset of necrosis in the leaves of N. benthamiana (Figures 1B-1F), the inventors of MP ToBRFV Tm-2 for identifying mutations that confer recognition 2 A screening system has been established. Tm-2 2 A variant library was constructed (Figure 4), and then the MP was screened. ToBRFV Eight individual Tm-2s that recognize each other 2 Clones were obtained (Figures 5A-5B). These clones were then used in MP ToBRFV Recognition (Figures 6A-6D) and TMV-GFP expression MP ToBRFV This allowed for the identification of three single-nucleotide mutations that confer resistance to the resistance-disrupting clone (Figures 7A-7F). All three mutations were Tm-2 2 Located on the convex side of the LRR, this region was suggested to play a role in MP recognition specificity (Figure 8).

[0038] In summary, these results pave the way for the development of novel tomato varieties resistant to the devastating effects of ToBRFV.

[0039] Therefore, according to the first aspect of the present invention, Tm-2 has an amino acid sequence that makes plants resistant to tomato brown rugos fruit virus (ToBRFV). 2 A tomato plant or a portion thereof that expresses a protein is provided.

[0040] As used in this application, the term "plant" includes the whole plant, grafted plants, ancestral and descendant plants and their parts (seeds, shoots, stems, roots, rootstocks, twigs, and plant cells, tissues, and organs). A plant may be in any form, including suspension cultures, embryos, meristem regions, callus tissue, leaves, gametophytes, sporophytes, pollen, and microspores.

[0041] The tomato plants may have a cultivated genetic background or a wild-type tomato genetic background.

[0042] In this application, the term "tomato" refers to the species Solanum lycopersicum (synonyms include Lycopersicon lycopersicum or Lycopersicon esculentum), or to plants, lineages, or groups previously known under the genus name Lycopersicon. The genus Lycopersicon includes, but is not limited to, L. cerasiforrne, L. cheesmanii, L. chilense, L. chmielewskii, L. esculentum (now S. pennellii), L. hirsutum, L. parviborum, L. pennellii, L. peruvianum, L. pimpinellifolium, or S. lycopersicoides. The newly proposed scientific name for L. esculentum is S. pennellii. Similarly, the names of wild species may have been changed. L. pennellii can become S. pennellii, L. hirsutum can become S. habrochaites, L. peruvianum can be divided into S. 'N peruvianum' and S. 'Callejon de Hueyles', or S. peruvianum and S. corneliomuelleri, L. parviflorum can become S. neorickii, L. chmielewskii can become S. chmielewskii, L. chilense can become S. chilense, L. cheesmaniae can become S. cheesmaniae or S. galapagense, and L. pimpinellifolium can become S. pimpinellifolium.

[0043] Generally, cultivated tomatoes refer to tomatoes that are suitable for consumption and meet the requirements for commercial cultivation, such as those usually classified as Solanum lycopersicum. In addition to the whole tomato plant and edible parts such as the fruit, the present invention includes plant parts or derivatives suitable for propagation. Examples of parts suitable for propagation include organ tissues such as leaves, stems, roots, and shoots, as well as protoplasts, somatic cell embryos, anthers, petioles, and cultured cells. Examples of derivatives suitable for propagation include seeds. The plants in the present invention can be cultivated and propagated from plant parts in the conventional manner and by tissue culture techniques.

[0044] The present invention aims to utilize any tomato variety, such as those for home use, fresh market tomatoes, and processing tomatoes.

[0045] Variety selection varies depending on market demand, regional adaptability, disease resistance, and the end use of the product. Exemplary classifications of fresh market tomatoes include, but are not limited to, Beef (fruit weight approximately 220–400g), Standard (fruit weight approximately 160–220g), and Cluster (uniform fruit weight approximately 120–180g). Such varieties are available from major seed companies, such as Grodena, Macarena, Estatio, Zouk, Climbo, and Climstar, all of which are also available from Syngenta. Other varieties are either exclusive to this company or available from other vendors, and include, but are not limited to, Cherry-micro (up to 5g), round cherry, mini round cherry (7.5-15g), and mini plum elongated cherry (10-25g). Examples of such varieties include Creativo (Clause), Batico (Nirit Seeds), and Shiren (Hazera Genetics). Round and elongated Cocktail (25-40g): Romanita, Cherry, and Cocktail, with red, yellow, orange, pink, zebra, and chocolate backgrounds. Examples include, but are not limited to, Summer Sun (Hazera Genetics), Black Pearl (Burpee), and Tyty (Tomodori). Examples of intermediate markers for finite-growth and indefinite-growth Roma, weighing 120-200g, include, but are not limited to, Lancelot (Vilomorin) and Parsifal (Vilomorin).Examples of pink tomatoes, categorized as Beef (220-400g), Standard (160-220g), and Cluster (120-180g), include Momotaro type, Cor di bue tomato (150-350g), Pinton (250-300g), and field-grown tomatoes - finite or semi-finite growth (180-400g).

[0046] Examples of tomato varieties used for processing include, but are not limited to, Roma, SUN6366, AB2, Heinz 9780, Heinz 9557, Halley 3155, and Hyper 303.

[0047] There are two main modes of tomato growth: finite growth and infinite growth. Finite growth produces "bush" tomatoes, which have been bred for compactness. When the terminal fruit matures, the growth of the entire plant stops, all the remaining fruits mature almost simultaneously, and then the plant dies. Infinite growth produces tomatoes that can grow up to 10 feet tall (so-called "vining" tomatoes) and grow until they die (e.g., due to frost). Their fruits mature sequentially. In a typical plant, all growth arises from the repetition of modular sympodial units, each unit producing an inflorescence with three leaves and multiple flowers. Most field cultivars of tomatoes, including M82, are finite growth types, with their shoots producing an average of six sympodial units, each unit having a single inflorescence where the number of leaves gradually decreases before the early termination of growth. In general, finite growth tomatoes are suitable for open-field production. Semi-finite growth and infinite growth "cultivation" varieties are suitable for open-field or staked cultivation within protective netting, as well as greenhouse cultivation.

[0048] According to one embodiment of the present invention, the tomato plant is a finite-growth type tomato.

[0049] According to one embodiment of the present invention, the tomato plant is an infinitely growing tomato.

[0050] According to one embodiment of the present invention, the tomato plant is a semi-finite growth type tomato.

[0051] According to one embodiment, the tomato is selected from the group consisting of single-fruit-per-cluster tomatoes, branched tomatoes, and cherry tomatoes.

[0052] Tomato plants according to this embodiment of the present invention are modified Tm-2 under the same genetic background. 2 Compared to control tomato plants that do not express the protein, these plants show increased resistance to tomato brown rugos fruit virus (ToBRFV).

[0053] "Same genetic background" means that at least 95%, 96%, 97%, 98%, 99%, or 99.9% of the genome is common between the plant and the unmodified plant.

[0054] The term "increased resistance" as used in this application refers to modified Tm-2 2 This represents an increase in viral resistance of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or even 95% compared to tomato plants with the same genetic background that do not express the protein, as evidenced by delayed or gradual symptom onset, or a decrease in viral RNA accumulation as analyzed by methods known in the industry (see examples below).

[0055] According to certain embodiments, increased resistance was demonstrated for periods of at least 10, 20, 30 days, or longer.

[0056] "Tm-2 2 The term refers to the receptor that confers tobamovirus resistance to tomato plants. Wild-type Tm-2 2 An example amino acid sequence is shown in SEQ ID NO: 25. (Wild-type Tm-2) 2 An exemplary nucleic acid sequence encoding this is shown in Sequence ID No. 26.

[0057] In one embodiment, the plant is made resistant to Tomato Brown Rugos Fruit Virus (ToBRFV) by Tm-2 2 The mutation is homozygous. In other embodiments, the plant is Tm-2 2 The mutation is heterozygous.

[0058] The tomato plant of this embodiment of the present invention has a mutation called Tm-2 2 The gene may be characterized by being present in both alleles, thereby enhancing ToBRFV resistance. In such cases, Tm-2 2 This can be homozygous or heterozygous. According to this embodiment, the homozygous type is Tm-2 2 This is a condition in which both alleles at a gene locus are characterized by identical nucleotide sequences. A heterozygous state is defined as Tm-2 2 This refers to a state where genes are different at a specific gene locus.

[0059] As used in this application, the term "allele" refers to one or more alternative forms of a gene locus, all of which are associated with a trait or characteristic. In diploid cells or organisms, the two alleles of a given gene occupy the corresponding gene locus on a pair of homologous chromosomes.

[0060] As used in this application, the term "gene" refers to a heritable factor that determines the biological characteristics of an organism (i.e., a tomato plant), and "allele" refers to the individual genes of a gene pair present in a (diploid) tomato plant.

[0061] In plants, a gene is called "homozygous" if it has the same allele, and "heterozygous" if it has two different alleles. Uppercase letters indicate dominant genes, and lowercase letters indicate recessive genes. Thus, "X,X" represents a homozygous dominant genotype for gene or trait X, "X,x" and "x,X" represent heterozygous genotypes, and "x,x" represents a homozygous recessive genotype. As is generally known, only homozygous recessive genotypes usually provide the corresponding recessive phenotype (i.e., plants exhibiting trait "x"). On the other hand, heterozygous and homozygous dominant genotypes usually provide the corresponding dominant phenotype (i.e., plants exhibiting trait "X"), unless other genes and / or factors such as multiple alleles, suppressors, and codominance also play a role in phenotypic determination.

[0062] In one embodiment, the genome of a tomato plant results in enhanced resistance to ToBRFV (wild-type Tm-2 2 (compared to) mutated Tm-2 2 It contains nucleic acid sequences that code for proteins.

[0063] The mutation may be any insertion, deletion, or substitution.

[0064] Mutations, also referred to as an increase or alteration of functional mutations, are responsible for acquiring resistance to ToBRFV.

[0065] Tm-2 of this embodiment of the present invention 2 The protein is wild-type Tm-2 2 Compared to proteins, they may contain single mutations, two mutations, three mutations, or more.

[0066] Preferably, the mutation occurs via the ToBRFV transfer protein (MP) in Tm-2 2 It enables the activation of proteins.

[0067] Tm-2 2 Receptor activation typically enables the generation of immune signals, such as Tm-2 2 It leads to the self-assembly of proteins.

[0068] In one embodiment, the mutation is wild-type Tm-2 2 Compared to proteins, Tm-2 2 It enhances the binding of the receptor to the ToBRFV transfer protein (MP).

[0069] Preferably, Tm-2 2 The amino acid modification within the leucine-rich repeat (LRR) region of the receptor occurs between amino acids 388 and 861.

[0070] Therefore, for example, the present invention relates to Tm-2, which is at least 90% identical to the amino acid sequence of SEQ ID NO: 25. 2 It expresses the receptor and wild-type Tm-2 2 We also plan to develop plants that express proteins that have an amino acid modification at one of the following positions: 528, 604, and / or 652.

[0071] The "percent identicality" of two amino acid sequences can be determined using the algorithm from Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as described in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such algorithms are incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. To obtain amino acid sequences homologous to the target protein molecule, a BLAST protein search can be performed using the XBLAST program with a score of 50 and a word length of 3. If a gap exists between two sequences, Gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997 can be used. When using the BLAST program and the Gapped BLAST program, the default parameters of each program (e.g., XBLAST and NBLAST) can be used. To obtain nucleotide sequences homologous to the nucleic acid molecules described in this application, a BLAST nucleotide search can be performed with the parameter set of the NBLAST nucleotide program, e.g., score=100, word length=12. To obtain amino acid sequences homologous to the protein molecules described in this application, a BLAST protein search can be performed with the parameters of the XBLAST program, e.g., score=50, word length=3. To obtain gapped alignments for comparison purposes, Gapped BLAST as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402 can be used. Alternatively, you can use PSI BLAST or PHI BLAST to perform iterative searches to detect distance-dependent relationships between molecules (Id.).When using the BLAST, Gapped BLAST, PSI Blast, and PHI Blast programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used (e.g., see the National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi(dot)nlm(dot)nih(dot)gov). Other specific and non-restrictive examples of mathematical algorithms for sequence comparison include the algorithm described by Myers and Miller, 1988, CABIOS 4:11 17. Such algorithms are incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When using the ALIGN program for amino acid sequence comparison, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. Percent identity between two sequences can be determined using techniques similar to those described above, i.e., any software for protein sequence alignment, with or without gap tolerance. In calculating percentage identity, typically only exact matches are counted.

[0072] In one embodiment, the modification at position 528 is the substitution of F528S.

[0073] In one embodiment, the modification at position 604 is S604N substitution.

[0074] In one embodiment, the modification at position 652 is an I652M substitution.

[0075] Further targeted mutations include P579Q, S604I, S651I, M704T, L842S, N522D, S723F, L560S, N522D, H737R, I652V, H817R, and P736L. Other mutations are summarized in Figure 5B.

[0076] In one embodiment, the modification is Tm-2 against tomato mosaic virus (ToMV) and / or other viruses of the Tobamovirus genus such as tobacco mosaic virus (TMV). 2 It does not affect resistance.

[0077] In other embodiments, the modification is Tm-2 against other viruses of the Tobamovirus genus, such as tomato mosaic virus (ToMV) and / or tobacco mosaic virus (TMV). 2 This reduces resistance to ToBRFV. In this embodiment, only one allele has a mutation that enhances resistance to ToBRFV, while the other allele does not contain these mutations, and this allele is assumed to confer resistance to ToMV and TMV.

[0078] The inventors intend to use both chemical mutation and recombination techniques to produce the tomato plants of the present invention.

[0079] Therefore, the tomato plant of the present invention can be produced by exposing the tomato plant body or a part thereof to a chemical mutagenic agent. Examples of chemical mutagenic agents, but not limited to these, include alkylating agents such as nitrite, ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), and diethyl sulfate (DES), and base analogs such as 5-bromodoxyuridine (5BU). In such cases, the plant becomes Tm-2 2 It is non-genetically modified by factors that induce mutations.

[0080] Following the initial exposure, the process typically continues with further steps of self-pollination, selection, hybridization, and self-pollination or a combination thereof, leading to Tm-2 2 Any step may be repeated two or more times, as long as gain-of-function (i.e., acquired resistance) exists in the gene. Selection may be phenotypic or involve marker-assisted breeding, as further described below in this application.

[0081] According to another specific embodiment, the non-genetically modified plants of the present invention are the result of spontaneously occurring genetic events that occur through polyphasic hybridization / self-pollination.

[0082] The following methods are all Tm-2, as long as the acquisition of the function is achieved (the function is acquired). 2 Any part of the gene can be targeted. In a particular embodiment, the action factor is the Tm-2 2 This targets the leucine-rich repeat (LRR) domain.

[0083] If necessary, further self-pollination steps are taken to achieve homozygous mutations.

[0084] In the context of this application, "target sequence" means Tm-2 2 This refers to DNA-coding products or RNA transcripts.

[0085] Genome editing using genetically modified endonucleases. This approach refers to reverse genetics that uses artificially genetically modified nucleases to cut the genome at one or more desired locations to create specific double strands, which are then repaired by cellular endogenous processes such as homology-directed repair (HDR) or non-homologous end-joining (NHEJ). NHEJ directly joins the DNA ends after a double-strand break, while HDR uses homologous sequences as templates to reconstruct the DNA sequences lost at the break point. In order to introduce specific nucleotide modifications into genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases. This is because most restriction enzymes recognize only a few base pairs of DNA as their target, and therefore the probability of finding the recognized base pair combination at numerous locations in the genome is extremely high, resulting in multiple breaks not limited to the desired location. To overcome this challenge and create site-specific single- or double-stranded cleavage sites, several distinct classes of nucleases have been discovered and modified through bioengineering. Specific examples include meganucleases, zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and the CRISPR / Cas system.

[0086] Meganucleases are generally grouped into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of the conserved LAGLIDADG motif. The four families of meganucleases are broadly separated from each other in terms of DNA recognition sequence specificity and catalytic activity, as a result of conserved structural elements. Meganucleases are commonly found in microbial species and have the unique characteristic of having extremely long recognition sequences (over 14 bp). Because of this characteristic, they naturally exhibit extremely high specificity for cleavage at desired locations. This can be utilized in genome editing to create site-specific double-strand break sites. Those skilled in the art can use these naturally occurring meganucleases, although the number of such naturally occurring meganucleases is limited. To overcome this challenge, mutagenesis and high-throughput screening methods have been used to create meganuclease variants that recognize specific sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize novel sequences. Alternatively, to design sequence-specific meganucleases, the amino acids that interact with DNA in the meganuclease may be modified (see, for example, U.S. Patent No. 8,021,867). Meganucleases can be designed, for example, using the methods described in Certo, MT et al. Nature Methods (2012) 9:073-975, U.S. Patents Nos. 8,304,222, 8,021,867, 8,119,381, 8,124,369, 8,129,134, 8,133,697, 8,143,015, 8,143,016, 8,148,098, or No. 8,163,514, the contents of which are incorporated herein by reference in their entirety.Alternatively, meganucleases with site-specific cleavage properties can be obtained using commercially available technologies, such as Precision Biosciences' Directed Nuclease Editor™ genome editing technology.

[0087] ZFNs and TALENs—two distinct classes of genetically engineered nucleases, zinc finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs)—have both been proven effective in producing targeted double-strand breaks (Christian et al., 2010; Kim et al., 1996; Li et al., 2011; Mahfouz et al., 2011; Miller et al., 2010).

[0088] Essentially, ZFN and TALEN restriction endonuclease technologies utilize nonspecific DNA-cleaving enzymes linked to specific DNA-binding domains (a series of zinc finger domains or TALE repeat sequences, respectively). Typically, restriction enzymes are selected in which the DNA recognition site and the cleavage site are separated from each other. By separating the cleavage site and then linking it to the DNA-binding domain, an endonuclease with extremely high specificity for the desired sequence can be obtained. Fokl is an exemplary restriction enzyme with such properties. Furthermore, Fokl has the advantage of requiring dimerization to possess nuclease activity, which means that specificity is significantly increased because each nuclease partner recognizes a unique DNA sequence. To enhance this effect, Fokl nucleases are genetically engineered to function only as heterodimers, thereby increasing catalytic activity. Heterodimeric functional nucleases increase the specificity of double-strand break sites by avoiding the possibility of unwanted homodimeric activity.

[0089] Therefore, for example, to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, with each member of the pair designed to bind to an adjacent sequence at the target site. During transient expression in cells, the nuclease binds to its target site, and the FokI domain heterodimerizes to create a double-strand break site. Repair of these double-strand break sites by the non-homologous end joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Because each repair performed by NHEJ is unique, the use of a single nuclease pair can generate allele lines with various different deletions at the target site. The deletions are usually in the range of a few to several hundred base pairs, but by using two sets of nuclease pairs simultaneously, it has also been successful to create larger deletions in cultured cells (Carlson et al., 2012, Lee et al., 2010). Furthermore, when a DNA fragment homologous to the target region is introduced along with a nuclease pair, the double-strand break site can be repaired by homologous recombination repair, resulting in specific modifications (Li et al., 2011, Miller et al., 2010, Urnov et al., 2005).

[0090] Although the nuclease moieties of both ZFNs and TALENs have similar properties, the difference between these genetically engineered nucleases lies in their DNA-recognizing peptides. ZFNs rely on Cys2-His2 zinc fingers, while TALENs rely on TALE. Both of these DNA-recognizing peptide domains have protein combinations that are characteristic of those found in nature. Cys2-His2 zinc fingers are found in nucleic acid interacting proteins in a wide variety of combinations, usually in a repeating form separated by 3 bp. TALE, on the other hand, is found in repeats with a 1:1 recognition ratio between amino acids and the nucleotide pair that is recognized. Since both zinc fingers and TALE are found in repeating patterns, various combinations can be tried to create a wide range of sequence specificities. Approaches for constructing site-specific zinc finger endonucleases include, for example, modular assembly (continuously linking zinc fingers associated with triplet sequences to cover the desired sequence), OPEN (low-stringency selection of peptide domains versus triplet nucleotides, followed by high-stringency selection of peptide combinations versus the final target in a bacterial system), and bacterial one-hybrid screening of zinc finger libraries. ZFNs can also be designed and are commercially available, for example, from Sangamo Biosciences® (Richmond, California).

[0091] Methods for designing and obtaining TALENs are described, for example, in Reyon et al. Nature Biotechnology 2012 May;30(5):460-5, Miller et al. Nat Biotechnol. (2011) 29:143-148, Cermak et al. Nucleic Acids Research (2011) 39 (12): e82, and Zhang et al. Nature Biotechnology (2011) 29 (2):149-53. A recently developed web-based program called Mojo Hand was introduced by the Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (accessible at www.talendesign.org). Designed TALENs are also commercially available from Sangamo Biosciences® (Richmond, California).

[0092] Another factor that can downregulate AGL6 is RNA-guided endonuclease technology, such as the CRISPR system (which is illustrated in great detail in the Examples section below).

[0093] As used in this application, the term “CRISPR system” is also known as Clustered Regularly Interspaced Short Palindromic Repeats and refers to the collective set of transcripts and other elements that are involved in or direct the activity of CRISPR-related genes. CRISPR-related genes include sequences encoding the Cas9 gene (e.g., CRISPR-related endonuclease 9), tracr (trans-activated CRISPR) sequences (e.g., tracrRNA or the active portion of tracrRNA), tracr-mate sequences (including “direct repeats” and tracrRNA processing moiety direct sequences), or guide sequences (also called “spacers”), the guide sequences including crRNA sequences (i.e., endogenous bacterial RNA that confers target specificity but requires tracrRNA to bind to Cas) or sgRNA sequences (i.e., single guide RNA).

[0094] In some embodiments, one or more elements of the CRISPR system are derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of the CRISPR system (e.g., Cas) are derived from specific organisms containing an endogenous CRISPR system, such as Streptococcus pyogenes, Neisseria meningitides, Streptococcus thermophilus, or Treponema denticola.

[0095] Generally, the CRISPR system is characterized by elements that promote the formation of the CRISPR complex at target sequence sites (also called protospacers in relation to the endogenous CRISPR system).

[0096] In the formation of the CRISPR complex, the “target sequence,” in this invention AGL6, refers to a sequence designed to have complementarity with a guide sequence (i.e., a guide RNA, e.g., sgRNA or crRNA), and hybridization of the target sequence and the guide sequence promotes the formation of the CRISPR complex. Complete complementarity is not necessarily required, however sufficient complementarity is necessary to induce hybridization and promote the formation of the CRISPR complex. Thus, according to some embodiments, the overall homology to the target sequence can be 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. The target sequence may include any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is located in the nucleus or cytoplasm of a cell.

[0097] Therefore, the CRISPR system comprises two distinct components: a guide RNA (gRNA) that hybridizes to a target sequence, and a nuclease (e.g., type II Cas9 protein), where the gRNA targets the target sequence and the nuclease (e.g., Cas9 protein) cleaves it. The guide RNA may include a combination of endogenous bacterial crRNA and tracrRNA; that is, the gRNA combines the target specificity of the crRNA with the scaffolding properties of the tracrRNA (required for Cas9 binding). Alternatively, the guide RNA may be a single guide RNA capable of directly binding to Cas.

[0098] Typically, in relation to the endogenous CRISPR system, the formation of a CRISPR complex (including a guide sequence that hybridizes with the target sequence and forms a complex with one or more Cas proteins) results in a cleavage of one or both strands within or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). While we do not wish to impose logical limitations, a tracr sequence containing or consisting of all or part of the wild-type tracr sequence (e.g., approximately 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence) may also form part of a CRISPR complex. This complex formation occurs, for example, through hybridization with all or part of a tracr mate sequence that is expressibly linked to the guide sequence along at least part of the tracr sequence.

[0099] In some embodiments, the tracr sequence has sufficient complementarity to hybridize with the tracr mate sequence and participate in the formation of the CRISPR complex. As with the target sequence, complete complementarity is not required, but sufficient homology is necessary for it to be functional. In some embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity when optimally aligned along the chain length of the tracr mate sequence.

[0100] CRISPR / Cas can be introduced into cells using one or more vectors that drive the expression of one or more elements of the CRISPR system, resulting in the formation of a CRISPR complex at one or more target sites. For example, the Cas enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence can be expressibly linked to separate regulatory elements on separate vectors. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined within a first vector, and any components of the CRISPR system not included in the first vector can be provided by one or more other vectors. Multiple elements of the CRISPR system combined within a single vector can be arranged in any suitable orientation. For example, one element can be positioned 5' (upstream) or 3' (downstream) of a second element. The coding sequence of the first element may be co-stranded with or reverse-stranded with the coding sequence of the second element, and may be arranged in the same or opposite directions. A single promoter may drive the expression of a transcript encoding a CRISPR enzyme with one or more sequences selected from guide sequences embedded within one or more intron sequences (e.g., each contained within a different intron, two or more contained within at least one intron, or all contained within a single intron), tracr mate sequences (optionally expressibly ligated to the guide sequences), and tracr sequences.

[0101] "Hit and run" or "in-out" is a recombination procedure involving two steps. The first step involves using an insertion vector containing a double-positive / negative selection marker cassette to introduce the desired sequence modification. The insertion vector contains a single continuous region homologous to the target locus and is modified to retain the target mutation. This targeted construct is linearized using restriction enzymes at one site within the homologous region, transformed into cells, and subjected to positive selection to isolate homologous recombinants. These homologous recombinants contain local duplications separated by the intervening vector sequence containing the selection cassette. The second step involves negative selection of target clones to identify cells that have lost the selection cassette due to intrachromosomal recombination between the duplication sequences. The local recombination event removes the duplication, and depending on the site of recombination, the allele either retains the introduced mutation or reverts to the wild type. The final result is the introduction of the desired modification, without retaining any exogenous sequences.

[0102] The "double substitution" or "tag and exchange" strategy involves a two-step selection procedure similar to the hit-and-run approach, but requires the use of two different targeting constructs. In the first step, a standard targeting vector with 3' and 5' homology arms is used to insert a double positive / negative selectable cassette near the site where the mutation is to be introduced. After transformation and positive selection, the targeted clone is identified with homology. The target clone is then transformed with a second targeting vector containing the homology region with the desired mutation, and negative selection is applied to remove the selection cassette and introduce the mutation. The final allele contains the desired mutation while eliminating unwanted exogenous sequences.

[0103] Site-directed recombinases—Cre recombinase from the P1 bacteriophage and Flp recombinase from the yeast Saccharomyces cerevisiae—are site-directed DNA recombinases that recognize unique 34-base pair DNA sequences (called "Lox" and "FRT," respectively). Sequences adjacent to either the Lox or FRT site can be easily removed by site-directed recombination during expression of Cre recombinase or Flp recombinase, respectively. For example, the Lox sequence consists of an asymmetric 8-base pair spacer region flanked by 13-base pair reverse repeats at both ends. Cre binds to the 13-base pair reverse repeats, catalyzes strand breaks, and religates within the spacer region, thereby recombining the 34-base pair lox DNA sequence. The twisted DNA break sites created by Cre within the spacer region are separated by 6 base pairs to create overlapping regions that act as homology sensors, ensuring that only recombination sites with identical overlapping regions are recombined.

[0104] Essentially, site-directed recombinase systems provide a means for removing the selected cassette after homologous recombination. This system also allows for the creation of conditionally altered alleles that can be inactivated or activated in a transient or tissue-specific manner. Notably, Cre recombinase and Flp recombinase leave behind a 34-base pair Lox "scar" or FRT "scar." While the residual Lox or FRT site typically remains within the intron or 3'UTR of the modified locus, recent evidence suggests that these sites generally do not significantly interfere with gene function.

[0105] Therefore, Cre / Lox and Flp / FRT recombinations involve the introduction of a targeted vector having 3' and 5' homology arms containing the target mutation, two Lox or FRT sequences, and a selectable cassette typically positioned between the two Lox or FRT sequences. Positive selection is applied to identify homologous recombinants containing the target mutation. Transient expression of Cre or Flp, combined with negative selection, results in the excision of the selectable cassette, leading to cells without the cassette. The final targeted allele contains an exogenous Lox or FRT scar.

[0106] As described above, the tomato plant of the present invention is Tm-2 2 These may be created by other technologies, including but not limited to gene genome editing.

[0107] Therefore, for example, Tm-2 2 Gene knock-in or gene knock-out constructs containing sequences homologous to the gene can be created and used to modify the enzyme-coding gene by inserting auxiliary sequences into its coding sequence.

[0108] These constructs preferably include positive and negative selection markers and may therefore be used for the selection of homologous recombination events. Those skilled in the art can easily design knock-in / knockout constructs containing both positive and negative selection genes to efficiently select transformed plant cells that have undergone homologous recombination events using the construct. Such cells can then be grown into complete plants. Standard methods known in the art can be used to carry out the knock-in / knockout techniques.Such methods are described, for example, in U.S. Patents No. 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384, 5,175,383, and 4,736,866, as well as in Burke and Olson, Methods in Enzymology, 194:251-270, 1991, Capecchi, Science 244:1288-1292, 1989, and Davies et al., Nucleic Acids Research, 20 (11) 2693-2698. 1992, Dickinson et al., Human Molecular Genetics, 2(8):1299-1302, 1993, Duff and Lincoln, "Insertion of a pathogenic mutation into a yeast artificial chromosome containing the human APP gene and experission in ES cells", Research Advances in Alzheimer's Disease and Related Disorders, 1995, Huxley et al., Genomics, 9:742-750 1991, Jakobovits et al., Nature, 362:255-261 1993, Lamb et al., Nature Genetics, 5: 22-29, 1993, Pearson and Choi, Proc. Natl. Acad. Sci. USA, 1993, 90:10578-82, Rothstein, Methods in Enzymology, This is available in 194:281-301, 1991, Schedl et al., Nature, 362:258-261, 1993, Strauss et al., Science, 259:1904-1907, 1993, and international publications 94 / 23049, 93 / 14200, 94 / 06908, and 94 / 28123.

[0109] According to a particular embodiment, the plant is a transgenic plant (e.g., for the genome editing factors described above in this application).

[0110] In certain embodiments, the plant may be a transgenic plant, and the transgene may not be related to (i.e., not the cause of) the ToBRFV resistance described herein. For example, the transgene may function to improve abiotic stress tolerance, insecticide tolerance, or abiotic stress tolerance.

[0111] According to a particular embodiment of the present invention, tomato plants are created by introducing a nucleic acid construct, the nucleic acid construct having a mutation that results in enhanced resistance to ToBRFV (Tm-2). 2 It includes a nucleic acid sequence encoding a polynucleotide factor that upregulates the expression of [the polynucleotide factor], and a cis-element capable of inducing the expression of the polynucleotide factor in plants.

[0112] Constructs useful in the method according to the present invention can be constructed using recombinant DNA technology well known to those skilled in the art. Gene constructs are commercially available, suitable for transformation into plants, and can be inserted into vectors suitable for the expression of the target gene in transformed cells. The gene construct may also be an expression vector in which a nucleic acid sequence is expressibly ligated to one or more regulatory sequences, enabling expression in plant cells.

[0113] The polynucleotides in this embodiment of the present invention are, for example, Tm-2 having the F528S mutation, the S604N mutation and / or the I652M mutation. 2 This may also be coded. An example Tm-2 having the above mutation. 2The polypeptide sequence is typically at least 90% homologous, at least 91% homologous, at least 92% homologous, at least 93% homologous, at least 94% homologous, at least 95% homologous, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, or 100% homologous to the sequence of SEQ ID NO: 25. The nucleic acid sequence of an exemplary polynucleotide encoding such a protein is at least 90% homologous, at least 91% homologous, at least 92% homologous, at least 93% homologous, at least 94% homologous, at least 95% homologous, at least 96% homologous, at least 97% homologous, at least 98% homologous, at least 99% homologous, or 100% homologous to the nucleic acid sequence of SEQ ID NO: 26.

[0114] In certain embodiments of the present invention, the regulatory element is a plant-expressible promoter.

[0115] As used in this application, the term "plant-expressible" means a promoter sequence, including any additional regulators attached thereto, that is capable of inducing, conferring, activating or enhancing expression in at least melon cells, tissues, or organs.

[0116] The promoter may be a controllable promoter, a constitutive promoter, or an organization-related promoter.

[0117] As used in this application, the term “adjustable promoter” means any promoter whose activity is affected by specific environmental or genomic conditions.

[0118] As used in this application, the term “constitutive promoter” means a promoter that induces the majority of time RNA production in many or all tissues of a plant transformant.

[0119] As used in this application, the term “tissue-related promoter” means a promoter that induces higher levels of RNA synthesis in specific types of cells and tissues (e.g., fruit-related promoter).

[0120] Exemplary promoters that can be used for the expression of functionally linked nucleic acid sequences (i.e., transgenes) include the cauliflower mosaic virus (CaMV) promoter and the tobacco mosaic virus (TMV) promoter.

[0121] Other promoters that can be used in the context of the present invention include those described in U.S. Patent No. 20060168699 and Hector G. Numez-Palenius et al. [Critical Reviews in Biotechnology, Volume 28, Issue 1 March 2008, pages 13-55], which are incorporated herein by reference.

[0122] Plant cells can be stably or transiently transformed using the nucleic acid constructs of the present invention. In stable transformation, the nucleic acid molecules of the present invention are incorporated into the plant genome, resulting in the expression of stable inheritable traits. In transient transformation, the nucleic acid molecules are expressed by the transformed cells but are not incorporated into the genome, resulting in the expression of transient traits.

[0123] Various methods exist for introducing foreign genes into both monocots and dicots (Potrykus, I., Annu. Rev. Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225, Shimamoto et al., Nature (1989) 338:274-276).

[0124] The following two main approaches are fundamental to inducing the stable integration of exogenous DNA into plant genomic DNA:

[0125] (i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38:467-486, Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25, Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, CJ, Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

[0126] (ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J., and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 52-68. This method also includes a method of directly uptake of DNA into protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6:1072-1074. DNA uptake induced by short-term electric shocks in plant cells: Zhang et al. Plant Cell Rep. (1988) 7:379-384, Fromm et al. Nature (1986) 319:791-793. DNA injection into plant cells or tissues using a microparticle gun, Klein et al. Bio / Technology (1988) 6:559-563, McCabe et al. Bio / Technology (1988) 6:923-926, Sanford, Physiol. Plant. (1990) 79:206-209. Method using a micropipette system: Neuhaus et al., Theor. Appl. Genet. (1987) 75:30-36, Neuhaus and Spangenberg, Physiol. Plant. (1990) 79:213-217.Transformation of cultured cells, embryos, or callus tissue with glass fiber or silicon carbide whiskers, U.S. Patent No. 5,464,765, or direct incubation of DNA with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, GP and Mantell, SH and Daniels, W. Longman, London, (1985) p. 197-209, and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

[0127] The Agrobacterium system involves the use of plasmid vectors containing defined DNA segments that are integrated into plant genomic DNA. The method of plant tissue seeding varies depending on the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disk procedure, which can be carried out using any extratissue graft that provides a good genetic resource for initiating overall plant differentiation. (Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9). A supplementary approach utilizes the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is particularly well-suited for the creation of transgenic dicotyledonous plants.

[0128] Various methods exist for the direct introduction of DNA into plant cells. Electroporation involves briefly exposing protoplasts to a strong electric field. Microinjection mechanically injects DNA into cells using extremely small micropipettes. Microparticle guns adsorb DNA onto micro-exjectors such as magnesium sulfate crystals or tungsten particles, and then physically accelerate these micro-exjectors toward cells or plant tissue.

[0129] Following stable transformation, the plants are propagated. The most common method of plant propagation is by seed. However, seed propagation has the disadvantage of producing non-uniform crops due to heterozygosity. This is because plants produce seeds based on genetic dispersion governed by Mendel's laws. Essentially, each seed is genetically distinct and grows with its own unique properties. Therefore, it is preferable that transformed plants be produced so that the regenerated plants have the same properties and characteristics as the parent transgenic plant. Thus, it is preferable that transformed plants be regenerated by micropropagation, which provides rapid and consistent propagation of transformed plants.

[0130] Micropropagation is the process of growing a new generation of plants from a single tissue excised from a selected parent plant or cultivar. This process allows for the mass propagation of plants with desirable tissue expressing fusion proteins. The newly produced plants are genetically identical to the original plant and possess all the characteristics of the original plant. Micropropagation enables the mass production of high-quality plant material in a short period of time and provides rapid propagation of selected cultivars while preserving the characteristics of the original transgenic or transformed plant. The advantages of cloning plants are the speed of plant propagation as well as the quality and uniformity of the plants produced.

[0131] Micropropagation is a multi-stage procedure that requires changes in culture medium or cultivation conditions between stages. Therefore, the micropropagation process consists of four basic stages: Stage 1: Initial tissue culture, Stage 2: Proliferation of cultured tissue, Stage 3: Differentiation and plant formation, and Stage 4: Greenhouse cultivation and environmental acclimatization. In Stage 1, initial tissue culture, the cultured tissue is established and its absence of contaminants is ensured. In Stage 2, the initial cultured tissue is doubled until a sufficient number of tissue samples are produced to achieve the production target. In Stage 3, the tissues propagated in Stage 2 are divided and grown into individual seedlings. In Stage 4, the transformed seedlings are moved to a greenhouse to strengthen them and gradually increase the light tolerance of the plants, enabling cultivation in a natural environment.

[0132] Currently, stable transformations are preferred, but this invention also envisions transient transformations of leaf cells, meristematic cells, or the entire plant.

[0133] Transient transformation can be carried out by either the direct DNA transfer method described above or by viral infection using a modified plant virus.

[0134] Viruses that have been shown to be useful for transforming plant hosts include CaMV, TMV, and BV. Plant transformation using plant viruses is described in U.S. Patent No. 4,855,237 (BGV), European Patent Publication No. 67,553 (TMV), Japanese Patent Publication No. 63-14693 (TMV), European Patent Publication No. 194,809 (BV), European Patent Publication No. 278,667 (BV), and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudoviral particles for use in the expression of foreign DNA in various hosts, including plants, are described in International Publication No. 87 / 06261.

[0135] Regardless of the method used to produce tomato plants in some embodiments of the present invention, once the plants or any propagating material are available, they are selected for their ToBRFV resistance traits.

[0136] Therefore, according to one aspect of the present invention, a method for selecting tomato plants resistant to ToBRFV, Tm-2 2 This invention provides a method for detecting gain-of-function mutations in genes from the genome of tomato plants, where the presence of the mutation indicates that the tomato plant is resistant.

[0137] Numerous methods for analyzing mutations are known in the art, including, for example, single-base extension (SBE), allele-specific primer extension sequencing (ASPE), DNA sequencing, RNA sequencing, microarray-based analysis, universal PCR, melting curve SNP method, allele-specific extension, hybridization, mass spectrometry, ligation, extension-ligation, flap endonuclease-mediated assay, restriction fragment length polymorphism (RFLP), electrophoresis, sequence alignment, allele-specific oligonucleotide hybridization (ASO), and randomly amplified polymorphic DNA (RAPD).

[0138] Therefore, the present invention relates to Tm-2 2 These are intended to be oligonucleotides (e.g., primers) that can be used to distinguish between mutant and non-mutant forms of a gene.

[0139] Therefore, once a plant is identified that retains a gain-of-function gene modification, it is considered to be ToBRFV resistant. This plant material can be used as breeding material in the development of tomato varieties with agriculturally desirable traits.

[0140] According to one embodiment, the plant of the present invention is a hybrid variety, namely, both Tm-2 2 This is a variety that arises from the crossbreeding (i.e., mating) of two heterogeneous plants that are homozygous for gain-of-function mutations in their genes. The hybrid can also be an F1 hybrid.

[0141] When used in this application, "F1 hybrid" refers to the first generation offspring of a cross between two non-isogenic plants.

[0142] The development of tomato hybrids according to this invention requires the development of stable parent lines. The breeding program combines desirable traits derived from two or more germplasm resources or gene pools to develop superior breeding varieties. The development of desirable self-pollination or parent lines is carried out by successive self-pollination and / or backcrossing and selection of the best breeding lines, sometimes utilizing molecular markers to expedite the selection process.

[0143] Once multiple parent lines that produce the best hybrid performance, for example, both being Tm-2 2 Once a gene carrying the above-described gain-of-function mutation is identified, it becomes possible to produce hybrid seeds indefinitely, as long as the parent's homozygous state is maintained. According to one embodiment, the tomato plant of the present invention is (heterozygous or homozygous, for example, Tm-2 2 It is a stable parent plant lineage that retains gain-of-function mutations in its genes.

[0144] As defined herein, the term “stable parent line” refers to a self-pollinating, open-pollinated line of the desired plant that is stable throughout the self-pollination and planting cycle. According to certain embodiments, 95% of the genome is homozygous in the parent line of the present invention.

[0145] A common procedure in plant breeding is to develop new varieties through single trait transformation using backcrossing.

[0146] As used in this application, the term "single trait conversion" refers to the incorporation of a new single gene into a parent line, where, in addition to the introduced single gene, substantially all of the desired morphological and physiological characteristics of the parent line are restored.

[0147] In this application, the term "backcross" refers to the repeated cross between a hybrid progeny and one of the parent tomato plants. The parent tomato plant that contributes the gene for the desired trait is called the non-repeating parent or donor parent. This technical term refers to the fact that a non-repeating parent is used once in the backcross protocol and is therefore not repeated. The plant of the parent Solanaceae plant (i.e., eggplant, tomato, and pepper, e.g., tomato) into which the gene derived from the non-repeating parent is transferred is known as the repeating parent because it is used in several rounds of the backcross protocol.

[0148] In a typical backcross protocol, a plant derived from the original variety of interest (recurrent parent) is crossed with a plant selected from a second variety (non-recurrent parent) that carries the single gene to be introduced. The offspring from this cross are then crossed again with the recurrent parent. This process is repeated until a tomato plant is obtained in which, in addition to the introduced single gene from the non-recurrent parent, substantially all of the desired morphological and physiological characteristics of the recurrent parent line are restored.

[0149] Therefore, near-isogenic lines (NILs) are matched with the trait or genomic region being matched (in this case, for example, Tm-2). 2 It can be produced by numerous backcrosses to create an array consisting of individuals that are nearly identical in gene composition, except for genes (gain-of-function gene modifications).

[0150] The characteristics of the parent lines can be improved or introduced by using the backcross method in conjunction with the present invention. In this method, marker-assisted breeding (selection) as described above can be used.

[0151] According to certain embodiments, the plant or plant seed is an inbred line.

[0152] According to certain embodiments, the plant is a hybrid plant, or the seeds are hybrid seeds.

[0153] The present invention also relates to progeny of the tomato, eggplant, and pepper plants of the present invention. Such progeny can be produced by sexual or vegetative reproduction of the plants or their progeny of the present invention. The regenerated progeny develop fruits independently of fertilization, in the same or similar manner as the parent. In addition, the progeny may be modified in one or more other characteristics. Such further modifications can be carried out, for example, by mutagenesis or transformation using transgenes.

[0154] As used in this application, the word “offspring” means the offspring or first generation and all subsequent descendants obtained from a cross with the plant of the present invention that exhibits fertilization-independent fruit formation. The offspring of the present invention are the offspring resulting from any cross with the plant of the present invention that retains (in a homozygous) variant traits leading to fertilization-independent fruit formation.

[0155] "Progeny" also includes plants that possess the traits of the present invention, obtained from other plants of the present invention by vegetative propagation or growth.

[0156] Embodiments of the present invention, as described herein, further relate to hybrid seeds and methods for producing hybrid seeds. The production method includes crossing a first parent plant with a second parent plant and harvesting the resulting hybrid seeds. In this case, since the trait is recessive, both parent plants must be homozygous for the trait of fertilization-independent fruit formation in order for all hybrid seeds to retain the trait of the present invention. They do not necessarily need to be uniform for other traits.

[0157] The embodiments described herein also relate to the germplasm of a plant. The germplasm is composed of all the heritable features of the organism and, in the present invention, includes at least the conditional fertilization-independent fruit formation of the present invention.

[0158] The embodiments described herein also relate to plant cells exhibiting a conditional fertilization-independent fruit formation trait. Each such plant cell contains the genetic information (i.e., Tm-2) that leads to ToBRFV resistance. 2 It possesses a gain-of-function mutation. The cells may be individual cells, or they may be parts of a plant, such as a plant or a fruit.

[0159] This technology involves genomic (DNA) information (i.e., Tm-2) that leads to ToBRFV resistance. 2 Further details about the consumed products, including mutations.

[0160] The fruits of any of the plants described in this application may be selected or certified at ripening or after harvest for fruit color, Brix, pH, sugar, organic acid, and defect levels (e.g., insect damage, mold). For example, tomatoes are typically transported to a large processing facility where they are collected, then typically washed with chlorinated water, rinsed with tap water, and further selected to remove any exhibiting defects (e.g., improper ripening, disease damage, mold, etc.). Tomatoes may be stored or immediately sent to consumers (fresh market tomatoes), especially those with improved shelf life as described above. Tomatoes for processing may be processed into a variety of products.

[0161] For the production of juice or pulp, tomatoes can be dehydrated in an oven, crushed, and softened (broken down and destroyed) with liquid to obtain a mass that can be pumped up. As will be obvious to those skilled in the art, these operations are known in themselves and are common in the field of tomato processing, and any modifications to the method can be made in this regard without departing from the scope.

[0162] Methods for processing tomatoes and / or producing tomato-based compositions are well known in the art; see U.S. Patent No. 6,924,420 for a general description. Specific methods for preparing, for example, paste (U.S. Patent No. 7,074,451), sterilized paste (U.S. Patent No. 4,206,239), puree (U.S. Patent No. 4,556,576), sauce (U.S. Patent No. 7,122,217), solidified sauce (U.S. Patent No. 4,038,424), barbecue sauce (U.S. Patent No. 6,869,634), salsa (U.S. Patent No. 5,914,146), ketchup (U.S. Patent No. 6,689,279), tomato fiber compositions (U.S. Patent No. 7,166,315), and dehydrated tomato products (U.S. Patent No. 5,035,909) have also been reported. Methods for modifying the texture and consistency of tomato paste, pulp, and puree have also been reported; see, for example, U.S. Patent No. 6,720,019.

[0163] Tomato products for food processing, including tomatoes or their edible parts (e.g., the fruit or its edible parts), are also provided.

[0164] Tomato paste produced according to this technology is also provided.

[0165] Examples of such food products, but not limited to, include canned whole tomatoes, tomato paste, ketchup, tomato sauce, tomato soup, dehydrated tomatoes, tomato juice, tomato powder, diced tomatoes, crushed tomatoes, chopped tomatoes, and tomato concentrates.

[0166] According to some embodiments, the product (e.g., paste, dried fruit, juice, etc.) causes Tm-2 (which causes ToBRFV resistance). 2 Contains tomato DNA (which retains a gain-of-function mutation).

[0167] The terms "comprises," "comprising," "includes," "including," and "having," along with their cognates, all mean "including but not limited to."

[0168] The term "consisting of" means "including and limited to."

[0169] The term "consisting essentially of" means that a composition, method, or structure may include additional components, steps, and / or parts, provided that such additional components, steps, and / or parts do not materially alter the fundamental and novel features of the composition, method, or structure described in the claims.

[0170] As used in this application, the singular forms "a," "an," and "the" include multiple references unless the context otherwise explicitly states. For example, the term "a compound" or "at least one compound" may include multiple compounds, such as mixtures thereof.

[0171] Throughout this application, various embodiments of the invention may be described in scope form. It should be understood that descriptions in scope form are merely for convenience and brevity and should not be construed as immutable limitations on the scope of the invention. Accordingly, a scope description should be deemed to include all possible subranges specifically disclosed, as well as the individual numbers within that range. For example, a scope description such as 1 to 6 should be deemed to include the specifically disclosed subranges, e.g., 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, as well as the individual numbers within that range, e.g., 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0172] Whereever a numerical range is indicated in this application, it means that it encompasses all cited digits (fractions or integers) within the indicated range. The phrases “range / range” between a first specified digit and a second specified digit, and “range / range” from the first specified digit to the second specified digit are used synonymously in this application and mean that they encompass the first and second specified digits and all fractional and integer digits between them.

[0173] As used in this application, the term “method” means a way, means, technique and procedure for accomplishing a given task, including, but not limited to, ways, means, techniques and procedures that are publicly known or readily developed from ways, means, techniques and procedures known to technicians in the fields of chemistry, pharmacology, biology, biochemistry and medicine.

[0174] When referring to a specific sequence listing, it should be understood that such references also include sequences that substantially correspond to the complementary strand, including minor sequence variations. Sequence variations are the result of, for example, sequencing errors, cloning errors, or other changes that result in base substitutions, base deletions, or base additions, but the frequency of such variations is less than 1 per 50 nucleotides, or less than 1 per 100 nucleotides, or less than 1 per 200 nucleotides, or less than 1 per 500 nucleotides, or less than 1 per 1000 nucleotides, or less than 1 per 5000 nucleotides, or less than 1 per 10000 nucleotides.

[0175] Even if certain features of the present invention are described in relation to separate embodiments for clarity, it should be understood that these features can be combined and provided as a single embodiment. Conversely, various features of the present invention described in relation to one embodiment for brevity may be provided individually, in preferred partial combinations, or as other embodiments of the present invention described herein that are deemed preferable. Certain features described in relation to various embodiments are not considered essential requirements of those embodiments unless the embodiments would be impossible to implement without those elements.

[0176] Experimental support for the various embodiments and aspects of the present invention described above and claimed in the claims described below is provided by the following examples. [Examples]

[0177] As described above, the various embodiments and aspects of the present invention described in this application and claimed in the claims are experimentally supported by the following examples.

[0178] Generally, the nomenclature used in this application and the experimental procedures used in the present invention include molecular techniques, biochemical techniques, microbiological techniques, and recombinant DNA techniques. Such techniques are fully described in the literature. For example, see: "Molecular Cloning: A Laboratory Manual" Sambrook et al., (1989), "Current Protocols in Molecular Biology" Volumes I-III Ausubel, RM, ed. (1994), Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989), Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988), Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998), methodologies as shown in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057, "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994), "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, NY (1994), Third Edition, "Current Protocols in Immunology" Volumes I-III Coligan, JE, ed.See Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994), Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980). Available immunoassays are extensively described in patents and scientific literature, e.g., U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, and 3,879,262. Detailed Statement, Specification No. 3,901,654, Specification No. 3,935,074, Specification No. 3,984,533, Specification No. 3,996,345, Specification No. 4,034,074, Specification No. 4,098,876, Specification No. 4,879,219, Specification No. 5,011,771 and Specification No. 5,281,521, "Oligonucleotide "Synthesis" Gait, MJ, ed. (1984), "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985), "Transcription and Translation" Hames, BD, and Higgins SJ, Eds. (1984), "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986), "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol.1-317, Academic Press, "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual", CSHL Press (1996). All of these are incorporated herein by reference as fully described herein. Other general references are provided hereby; the procedures described therein are considered well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

[0179] Materials and methods Plant materials: Nicotiana benthamiana plants and Solanum lycopersicum L. cv. Ikram (Syngenta) plants were cultivated under long light conditions at 25°C. Well-developed leaves from 5-week-old plants were used for agroinfiltration.

[0180] Agro-infiltration and N. benthamiana infection for transient expression: A. tumefaciens EHA105 strain containing a binary vector was cultured overnight at 28°C, and the cultured cells were resuspended in MES buffer (10 mM MgCl2, 10 mM MES, 150 μM acetosilinogon, pH 5.6) to an optical density of OD600 = 0.5. p35S:MP vector and p35s:Tm-2 2 Bacterial cells containing the vector were expressed individually or in a 1:1 mixture and then infiltrated into the abaxial surface of the fourth or fifth leaf of N. benthamiana or Solanum lycopersicum.

[0181] Construction of a directional evolution library: All golden gate vectors were provided by Addgene. Tm-2 2 The (AF536201) gene was cloned into the level 0 CDS1 golden gate vector pICH41308 (Weber et al., 2011). The internal BsaI region was removed in a one-step cloning process using primers 60 and 63, and 62 and 61 (Table 1) (sequence adaptation). The gene was then separated into two regions that share a 4-nucleotide sequence adjacent to the BpiI region and function as a fusion site; that is, the sequence encoding the CC-NBS region (1,409 bp) located at amino acids 1-470 was amplified with PCRBIO HiFi polymerase (Cat#PB10.41-10) (primers 60 and 99, Table 1). The sequence encoding the LRR (1,405-2,586 bp) served as a template for random mutation as described in Xu et al. (1999). In summary, the PCR reaction performed with Taq Ready Mix (HyLabs Cat.#EZ-3007) was mixed with 40 μM Mn 2+ The first reaction involved amplifying 100 ng of template DNA by adding [component name]. 2 μl of the product served as the template for the second PCR, with 40 μM dITP added to the reaction (primers 98 and 61 used in both reactions). Tm-2 2 Two portions of the gene were then inserted into the pICH41308 level 0 vector by Golden Gate assembly, and DH5α cells were transformed. The colonies were then pooled together, plasmid DNA was extracted, and cloned into the Golden Gate level 2 binary vector pICH486988. The colonies were pooled again, and the extracted DNA was transformed into A. tumefaciens. Individual A. tumefaciens clones were isolated, cataloged, and frozen at -80°C for Tm-2 2 A mutant library was formed.

[0182] [Table 1]

[0183] Site-directed mutation: Using a 30 bp complementary oligonucleotide containing the target mutation, PCRBIO HiFi polymerase (Cat.#PB10.41-10) is used to mutate Tm-2 in the level 0 vector. 2 Site-directed mutations were performed on the gene.

[0184] ToMV MP-ToBRFV Construction: To replace the ToMV MP gene in the pTLW3 vector (Masayuki Ishikawa), which contains the entire ToMV genome under the T7 promoter, with that of ToBRFV, the ToBRFV MP ORF was amplified using primers that overlapped with the sequence adjacent to the ToMV MP (primers 161 and 162, Table 1). The regions between the internal KpnI site and the MP, and between the MP and the internal XmaI site, were amplified using primers that overlapped with the ToBRFV MP (primers 158 and 160, and 163 and 159, respectively, Table 1). The three PCR products then served as templates for fusion PCR (primers 158 and 159). The products were digested with KpnI and XmaI and ligated into a similarly digested pTLW3 vector.

[0185] In vitro transcription and infection of tomato plants: 2 μl of ToMV pTLW3 and ToMV(MP-TB) were linearized with the restriction enzyme SmaI and washed with a gel extraction kit (Zymo Research, ZR-D4002). In vitro transcription was performed according to the instructions for the mMESSAGE mMACHINE® T7 kit (Invitrogen, Thermo Fisher Scientific, AM1344). The transcript was diluted fourfold, and then 5 μl of the transcript was used for mechanical inoculation into N. benthamiana plants that had been sprinkled with silicon carbide powder before inoculation. Leaves of systemically infected N. benthamiana were used as inoculation material in 2-week-old tomato plant cultivars Manimaker (LA2706, LA3310, University of California, Davis Conservation Center). Samples were collected 3 weeks after infection.

[0186] TMV-GFP MP-ToBRFVConstruction: The binary vector pJL24 (TMV-GFP) contains the entire TMV genome and the gene of GFP following the MP gene (Lindbow, 2007). Cloning of MP into this vector was carried out as follows. The MP ORF was amplified by PCR from infected plants (primers 39 and 40, Table 1), and at this time, a sequence overlapping with the pJL24 vector at the 5' end and reaching the internal PacI site on the 3' side was added. The second PCR amplified the sequence between the internal AgeI site approximately 1,660 bp upstream from the original MP of TMV-GFP and added a sequence overlapping with MP (primers 37 and 38, Table 1). Two PCR products with a molar ratio of 1:1 functioned as templates for fusion PCR (primers 37 and 40, Table 1), resulting in an amplification product of 2,500 bp with the restriction enzyme sites AgeI and PacI adjacent to each other, which was cloned in place of the original sequence. Next, colonies were tested for MP using specific primers (50), and DNA sequencing was performed using primers located upstream of MP (41). ToBRFV To test whether MP is a resistance-breaking factor, the MP sequence of the ToMV infectious clone (Hamamoto et al., 1997) was replaced with MP ToBRFV (ToMV ToBRFV ) (Figure 1A). In ToMV-sensitive tomato plants (tm-2 / tm-2) (Figure 1B), both ToMV and ToMV ToBRFV were infectious and caused systemic diseases (Figures 1C, 1D, and 1H). As expected, the Tm-2

[0187] Results MP ToBRFV is a resistance-breaking factor 2 homozygous tomato plants (Figure 1E) were immune to ToMV (Figures 1F and 1H). However, when these plants were inoculated with ToMV MP ToBRFV , systemic diseases were clear and viral RNA was detected from leaves throughout the plant (Figures 1G and 1H). This result indicates that the Tm-2 of tomato ToBRFV (ToMV MP-ToBRFV ), systemic diseases were clear and viral RNA was detected from leaves throughout the plant (Figures 1G and 1H). This result indicates that the Tm-2 of tomato MP-ToBRFV both were infectious and caused systemic diseases (Figures 1C, 1D, and 1H). As expected, the Tm-2 2 homozygous tomato plants (Figure 1E) were immune to ToMV (Figures 1F and 1H). However, when these plants were inoculated with ToMV MP-ToBRFV , systemic diseases were clear and viral RNA was detected from leaves throughout the plant (Figures 1G and 1H). This result indicates that the Tm-2 of tomato 2To overcome resistance, MP ToBRFV It was established that this alone is sufficient.

[0188] MP ToBRFV Tm-2 2 To reinforce its role as a resistance disruption factor, parallel experiments were conducted in N. benthamiana. Here, the original MP of the TMV-GFP vector (Lindbow, 2007) was used in MP ToBRFV (TMV-GFP) replaced MP-ToBRFV (Figure 2A). These viruses are expressed either alone or under the Tm-2 promoter in the leaves of Nicotiana benthamiana. 2 (p35S:Tm-2 2 It was expressed along with ). As expected, Tm-2 2 This conferred resistance to TMV-GFP (Figure 2B). In contrast, Tm-2 2 TMV-GFP MP-ToBRFV It did not confer resistance to Tm-2 (Figure 2C). In summary, these experiments showed that ToBRFV does not confer resistance to Tm-2 2 The reason for overcoming resistance is MP ToBRFV It was established that there was a lack of awareness.

[0189] MP ToBRFV Tm-2 2 It does not cause mediated hypersensitivity reactions (HR). Tm-2 2 The recognition of Tm-2 and the recognition of tobamovirus MP are separate processes and depend on specific elements within both proteins. 2 Specific mutations within MP ToBRFV We thought that this could confer the ability to recognize such modifications. For the identification of such modifications, Tm-2 2 A robust screening system is needed to identify the activation of the immune response. Farnham and Baulcombe (2006) have already used the appearance of HR necrotic lesions as an indicator of pathogen recognition and immune response. Tm-2 2 MP TMVSince it is possible to induce HR in the presence of Tm-2 (Zhang et al., 2013, Chen et al., 2017, Wang et al., 2020), the inventors of this invention have found that Tm-2 2 MP when co-expressed with ToBRFV We considered confirming that the expression of does not cause cell death. To test this hypothesis, MP ToBRFV and MP TMV Each of the code sequences was cloned under the control of the 35S promoter (p35S:MP ToBRFV and p35S:MP TM Next, using agroin filtration, tomato cv. Ikram (Tm-2 2 The clone was transiently expressed in the lobules of / tm-2) (Figures 3A-3C). p35S:MP TMV Cell death was observed in lobules permeated with (Figure 3A). On the other hand, p35S:MP ToBRFV The lobules treated with Tm-2 showed only slight necrosis, similar to the control treatment (Figure 3C) (Figure 3B). 2 To create an efficient screening system for variants, the same system was tested with N. benthamiana. Here too, p35S:MP TMV and p35S:Tm-2 2 Transient co-expression of p35S:MP caused tissue necrosis consistent with the establishment of HR (Figure 3D), but p35S:MP ToBRFV and p35S:Tm-2 2 Co-expression of did not induce cell death, similar to the negative control (Figure 3F) (Figure 3E). These results suggest that MP ToBRFV Tm-2 with recognition 2 This demonstrates that HR necrosis can be used as a platform for screening to identify novel variants.

[0190] Tm-2 using Golden Gate Cloning 2 Building a variant library Next, using the Golden Gate cloning method (Weber et al, 2011), which enables modular cloning of various components, we performed Tm-2 2A mutant library was created (Figure 4). Tm-2 2 Random mutations in the LRR region were detected by error-prone PCR. The mutated LRR portion was identified as Tm-2 2 The CC-NB portion was assembled into a level 0 plasmid. The resulting clones were pooled together and subcloned into a level 2 plant expression cassette containing a 35S promoter and OCS terminator, yielding multiple expression clones. These expression clones were pooled again and transformed into Agrobacterium tumefaciens to produce Tm-2. 2 A mutant library was created. Next, each Agrobacterium isolate was converted to p35S:MP ToBRFV It spread along with it. The appearance of necrotic lesions was due to MP ToBRFV Recognition and Tm-2 2 This demonstrated the success of revitalizing the HR brokerage sector.

[0191] MP ToBRFV Tm-2 recognizes 2 Isolation of mutants From a total of 1000 screened colonies, MP ToBRFV 13 types of Tm-2 that trigger HR in response to 2 The mutant alleles were isolated and sequenced (Figure 5A). Interestingly, these clones were MP TMV It does not generate HR in response to MP TMV and MP ToBRFVThis suggested a trade-off in recognition (Figure 5A). HR intensity varied among the various clones (Figure 5B), ranging from mild to moderate to severe. The mean mutation rate among the eight isolated clones was 2.4 mutations per clone, with three clones having one mutation (colonies 22, 67, and 905), four clones having two mutations (colonies 14, 24, 51, 58, and 67), two clones having three mutations (colonies 18 and 547), one clone having four mutations (colony 872), and one clone having five mutations (colony 184) (Figure 5B). In addition, six clones had only non-synonymous mutations (colonies 14, 22, 58, 67, 547, and 905), and seven had a combination of synonymous and non-synonymous mutations (colonies 18, 24, 51, 65, 184, 311, and 872). Surprisingly, the three nonsynonymous mutations were each independently repeated in two or more colonies (Figure 3C). These mutations were at amino acids F528, S604, and I652. The repetition of these mutations in more than one colony suggests that these mutations are related to MP ToBRFV This indicates a tendency to play a part in perception.

[0192] To test the function of mutations in F528, S604, and I652, three Tm-2 mutations were performed using site-directed mutations in these amino acids. 2 Mutant clone, i.e., Tm-2 2 F528S, Tm-2 2 S604N and Tm-2 2 We created I652M (Figures 6A-6D). Single expression of the mutant clone had no effect in the leaves. However, these MPs ToBRFV Co-expression with led to the appearance of necrotic lesions (Figures 6A-6C). This result was observed in non-mutant Tm-2. 2 Genetic MP TMV Its expression was similar to that of (Figure 6D). Non-mutant Tm-2 2 MP ToBRFV The expression of Tm-2 had no effect on the leaves. (Note Figure 6D.) These results suggest that Tm-2 2 The mutations F528S, S604N, and I652M are MP ToBRFVThis suggests that it can provide recognition and may be used to protect plants from ToBRFV.

[0193] Tm-2 2 The expression of mutants provides protection against resistance-destroying viruses. The objective of the following experiment is to study mutant Tm-2 2 The objective was to determine whether the variant could provide protection against infection-resistant viruses. To that end, various Tm-2 2 Variant resistance destruction TMV-GFP MP-ToBRFV The TMV-GFP gene was co-expressed, and the progression of the viral infection was monitored using GFP fluorescence. MP-ToBRFV Single expression (Figure 7A) or non-mutant Tm-2 2 (Figure 7B) shows that systemic infection and viral symptoms occurred in young leaves. In contrast, TMV-GFP MP-ToBRFV and mutated Tm-2 2 Co-expression with the variant resulted in necrosis of infected leaves, inhibition of systemic infection, and absence of symptoms (Figure 7C-7E). Quantification of GFP fluorescence in systemic leaves was determined to be Tm-2 2 We demonstrated that the mutant significantly inhibited viral infection (Figure 7F). These results indicate that Tm-2 2 The expression of the variant may halt the systemic spread of resistance-destroying tobermovirus, thus providing a potential cause of ToBRFV resistance.

[0194] Tm-2 confirmed by various mutations 2 Structural changes Tm-2 2 Enhanced functionality, MP ToBRFV In response to Tm-2 2 This is thought to be caused by modifications to the protein structure that improve binding or enhance the transmission of immune signals. Tm-2 2To explore the structural changes caused by the mutations, we performed 3D homology modeling based on the recently published Arabidopsis thaliana NLR ZAR1 (Wang et al., 2019) (Figure 8). According to this model, the spatial location of all three mutations is on the convex side of the LRR, suggesting that this region plays a role in MP recognition specificity. F528 and S604 are both located on the α-helix, and the S604M mutation results in a disruption of this structure. I652 is located in one of the two loops extending from the convex side of the NLR. All three mutations result in changes in the location of these loop structures, which may play a major role in MP recognition.

[0195] While the present invention has been described with regard to its specific embodiments, it is evident that numerous alternatives, modifications, and variations will be apparent to those skilled in the art. Therefore, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0196] All publications, patents, and patent applications referenced herein are intended to be incorporated in their entirety by reference to the same extent as if each individual publication, patent, or patent application were specifically indicated separately as being incorporated by reference herein. Furthermore, any reference or identification of any reference in this application should not be considered an endorsement that such reference is available as prior art of the present invention. Section headings, to the extent that they are used, should not necessarily be considered limitations. In addition, the basic application of this application is also incorporated by reference herein.

[0197] References Broadbent L (1976). Epidemiology and control of tomato mosaic virus. Annu Rev Phytopathol, 14: 75-96. Cambron-Crisantos, J. M., Rodriguez-Mendoza, J., Valencia-Luna, J. B., Rangel, S. A., de Jesus Garcia-Avila, C., & Lopez-Buenfil, J. A. (2018). First report of Tomato brown rugose fruit virus (ToBRFV) in Michoacan, Mexico. Mexican Journal of Phytopathology, 37(1), 185-192. Chen, T., Liu, D., Niu, X., Wang, J., Qian, L., Han, L., & Liu, Y. (2017). Antiviral resistance protein Tm-22 functions on the plasma membrane. Plant physiology, 173(4), 2399-2410. Dahan-Meir, T., Filler-Hayut, S., Melamed-Bessudo, C., Bocobza, S., Czosnek, H., Aharoni, A., & Levy, A. A. (2018). Efficient in planta gene targeting in tomato using geminiviral replicons and the CRISPR / Cas9 system. The Plant Journal, 95(1), 5-16. Farnham G, Baulcombe DC (2006). Artificial evolution extends the spectrum of viruses that are targeted by a disease-resistance gene from potato. 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[0198] Sequence ID 1: Single-stranded DNA oligonucleotide Sequence ID 2: Single-stranded DNA oligonucleotide Sequence ID 3: Single-stranded DNA oligonucleotide Sequence ID 4: Single-stranded DNA oligonucleotide Sequence ID 5: Single-stranded DNA oligonucleotide Sequence ID 6: Single-stranded DNA oligonucleotide Sequence ID 7: Single-stranded DNA oligonucleotide Sequence ID 8: Single-stranded DNA oligonucleotide Sequence ID 9: Single-stranded DNA oligonucleotide Sequence ID 10: Single-stranded DNA oligonucleotide Sequence ID 11: Single-stranded DNA oligonucleotide Sequence ID 12: Single-stranded DNA oligonucleotide Sequence ID 13: Single-stranded DNA oligonucleotide Sequence ID 14: Single-stranded DNA oligonucleotide Sequence ID 15: Single-stranded DNA oligonucleotide Sequence ID 16: Single-stranded DNA oligonucleotide Sequence ID 17: Single-stranded DNA oligonucleotide Sequence ID 18: Single-stranded DNA oligonucleotide Sequence ID 19: Single-stranded DNA oligonucleotide Sequence ID 20: Single-stranded DNA oligonucleotide Sequence ID 21: Single-stranded DNA oligonucleotide Sequence ID 22: Single-stranded DNA oligonucleotide Sequence ID 23: Single-stranded DNA oligonucleotide Sequence ID No. 24: Single-stranded DNA oligonucleotide Sequence ID 25: Wild-type Tm-2 2 Example amino acid sequence Sequence ID 26: Wild-type Tm-2 2 Exemplary nucleic acid sequences encoding

Claims

1. Modified Tm-22 protein having at least 95% homology to the amino acid sequence of the wild-type Tm-22 protein shown in Sequence ID No. 25, and comprising at least one amino acid modification to the wild-type Tm-22 protein. ２ A tomato plant expressing a protein, wherein at least one of the amino acid modifications is F528S, S604N, and / or I652M, and the modified Tm-22 protein makes the tomato plant resistant to tomato brown goose fruit virus (ToBRFV).

2. The tomato plant according to claim 1, wherein the modified Tm-22 protein has at least 97% homology to the amino acid sequence of the wild-type Tm-22 protein shown in Sequence ID No.

25.

3. The tomato plant according to claim 1, wherein the modified Tm-22 protein has an amino acid sequence obtained by adding at least one amino acid modification to the amino acid sequence of the wild-type Tm-22 protein shown in SEQ ID NO: 25, and the at least one amino acid modification is F528S, S604N, and / or I652M.

4. A tomato plant cutting according to any one of Claims 1 to 3.

5. The part of a tomato plant according to any one of claims 1 to 3.

6. A part of a tomato plant according to claim 5, selected from the group consisting of seeds, roots, stems, leaves, cotyledons, flowers, fruits, embryos, and pollen.

7. A cell having the genome of a plant as described in any one of Claims 1 to 3.

8. A culture comprising the cells according to multiple claims 7.

9. A method for breeding tomato plants, comprising crossbreeding the plant described in claim 1 with another tomato plant to breed a tomato plant.

10. The method according to claim 9, wherein the aforementioned mating includes pollination.

11. A method for cultivating plants, comprising vegetatively propagating the plant described in any one of claims 1 to 3 and cultivating the plant.

12. A processed food product comprising a plant or a part thereof as described in any one of Claims 1 to 3.

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