CCA gene for viral resistance

Abstract

The present invention relates to a modified CCA gene encoding a CCA-adding enzyme, wherein the modified CCA gene confers resistance to positive-strand RNA viruses having TLS, and the modified CCA gene is selected from the group consisting of: a gene comprising a nucleotide sequence encoding a CCA-adding enzyme according to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; a gene comprising a promoter sequence comprising SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16; a gene comprising a nucleotide sequence encoding a CCA-adding enzyme having at least one amino acid deletion, substitution, or insertion when compared to SEQ ID NO:2 or SEQ ID NO:7; a gene comprising a nucleotide sequence encoding a CCA-adding enzyme having at least one amino acid deletion, substitution, or insertion when compared to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; a gene comprising a nucleotide sequence encoding a CCA-adding enzyme having at least 80% sequence identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11; and a gene comprising a promoter sequence having at least 80% sequence identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16. The invention further relates to plants and seeds containing the modified genes, methods for producing and identifying such plants, and uses of the genes.

Description

Technical Field

[0001] The present invention relates to a modified gene that confers resistance to positive-strand RNA viruses having TLS. The present invention further relates to a plant comprising the modified gene, a method for producing such a plant, and a method for the identification of the modified gene and the selection of such a plant. The present invention also relates to a marker for the identification of the modified gene in a plant and the use of such a marker.

Background Art

[0002] Viral diseases pose one of the major threats that growers must address in both protected and field crop cultivation. When a crop is infected, the spread of the virus can occur rapidly via vectors that are difficult to control, usually insects. Furthermore, cultivation methods often contribute to further spread of the virus by sap transmission via tools and workers.

[0003] Plant viruses are typically host-dependent for rapid replication and spread, thereby infecting the same host with disease. Different viruses employ different systems to achieve this goal. Certain groups of viruses belonging to the positive-strand RNA virus family appear to use a transfer RNA-like structure (TLS) in their 3'-terminal genome sequence as an essential component of these processes. These viral TLSs are capable of specific aminoacylations related to the order of the anticodon sequences present in the TLS structure, thereby mimicking the behavior of universally present transfer RNA (tRNA). However, the TLSs of these viral genomes typically lack a CCA tail, a tRNA property essential for the binding of the relevant amino acids. Instead, the viral genome often terminates at 3'-CC. This CC tail, however, can be adenylated using a tRNA nucleotidyltransferase, also known as the "CCA-adding enzyme," in the host plant into which the virus has entered. This adenylation and subsequent aminoacylation of the viral genome are recognized to play a crucial role in viral stabilization, translation, and replication, and are therefore thought to form essential steps in viral infection and spread. Several positive-strand RNA viruses belonging to the genera Tobamovirus, Thymovirus, or Bromovirus are examples of viruses that utilize this tRNA mimicry system. These viruses not only possess TLS at the 3' end of their genome, but also generally at the 3' end of their sgRNA transcript, and this transcript TLS can also function in interaction with the host's CCA-adding enzyme.

[0004] Many genes involved in plant virus resistance have been recognized. Viral resistance may be based on a variety of mechanisms, and many different stages of plant development and defense pathways may be involved. However, for many viruses, resistance genes have yet to be identified. In particular, for relatively new viruses, or viruses that are similar to other viruses but break known resistances, there is always the challenge of identifying new causes of resistance before viral damage becomes widespread. Newly identified resistance genes can also be added to the protection of crops against known viral diseases. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a modified gene that provides resistance to positive-strand RNA viruses having a 3' terminal transfer RNA-like structure (TLS). [Means for solving the problem]

[0006] The present invention provides a modified CCA gene encoding a CCA-adding enzyme, which confers resistance to positive-strand RNA viruses having TLS, and the modified CCA gene is selected from the group consisting of: - A gene containing a nucleotide sequence encoding a CCA-adding enzyme as indicated by SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; - A gene containing a promoter sequence including SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; - A gene containing a nucleotide sequence encoding a CCA-additive enzyme that has at least one amino acid deletion, substitution, or insertion when compared to SEQ ID NO: 2 or SEQ ID NO: 7; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme that has at least one amino acid deletion, substitution, or insertion when compared to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme having at least 80% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; and A gene containing a promoter sequence that has at least 80% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.

[0007] As used herein, a CCA gene is a gene that encodes a CCA-additive enzyme. As used herein, a CCA gene is a gene containing the wild-type CDS sequence represented by Sequence ID No. 1, or a homologous gene containing a sequence having at least 80% sequence identity to Sequence ID No. 1; or a gene encoding a CCA-additive enzyme containing Sequence ID No. 2; or a gene encoding a homologous CCA-additive enzyme containing a sequence having at least 80% sequence identity to Sequence ID No. 2. As used herein, a gene also includes its 5'-UTR sequence, promoter, and 3'-UTR sequence.

[0008] The promoter of the CCA gene contains sequence number 3, or a sequence having at least 80%, preferably 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 3. The homologous CCA gene contains a sequence having at least 80%, preferably 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 1. The homologous CCA-adding enzyme contains a sequence having at least 80%, preferably 85%, 90%, 93%, 95%, 96%, 97%, 98%, or 99% sequence identity with sequence number 2.

[0009] A CCA-adder having at least 80% sequence identity to SEQ ID NO: 8 preferably contains at least one of the following substitutions: N535D, R553S, or K579N. A CCA-adder having at least 80% sequence identity to SEQ ID NO: 9 preferably contains at least one of the following substitutions: K450E, R553S, or K579N. A CCA-adder having at least 80% sequence identity to SEQ ID NO: 10 preferably contains at least one of the following substitutions: K316N or A317V. A CCA-adder having at least 80% sequence identity to SEQ ID NO: 11 preferably contains at least one of the following substitutions: C211R. Alternatively, any of these substitutions are substitutions at the corresponding positions in homologous sequences.

[0010] As used herein, sequence identity is the percentage of nucleotides or amino acids that are identical between two sequences after proper alignment. Those skilled in the art know how to align sequences, for example, by using sequence alignment tools such as BLAST®, which can be used for both nucleotide and protein sequences. For the most important results, it is necessary to obtain the best possible alignment that gives the highest sequence identity score. The percentage of sequence identity is calculated by comparing the lengths of the shortest sequences in the evaluation.

[0011] CCA-adding enzymes play a crucial role in most organisms because they are active and essential for adding a CCA tail to the 3' end of universally present transfer RNA (tRNA). In almost all eukaryotes, this CCA tail, a prerequisite for tRNA aminoacylation, is not encoded by the tRNA gene and therefore must be added post-transcriptionally. Specialized CCA-adding enzymes recognize all tRNAs and synthesize the appropriate CCA tail in all of them. Most eukaryotic genomes contain only one copy of the CCA gene, which encodes an essential and highly conserved CCA-adding enzyme.

[0012] CCA-adding enzymes are also involved in other RNA-related processes. One of their tasks is, for example, tRNA quality control, where the enzymes are involved in tRNA repair and the degradation of unstable or deviant tRNAs. They tag RNAs identified as defective for any reason by adding a double CCA tail instead of a single one, thereby tagging these RNAs for degradation. CCA-adding enzymes are also involved in the processing of other non-coding RNAs, such as IncRNA.

[0013] Due to the crucial role of CCA-adding enzymes, mutations in the CCA gene, particularly those in highly conserved regions of the gene sequence, are expected to have a strong adverse effect on plant growth and development. Therefore, even though many viruses are known to possess a 3' terminal transfer RNA-like structure (TLS) that utilizes the host plant's CCA-adding enzyme to infect the same host plant, the CCA gene has not been considered a likely target in approaches to acquiring viral resistance due to its essential function.

[0014] However, this invention proposes a modification of the CCA gene that confers virus resistance in plants.

[0015] Modifications to the CCA gene that confer resistance to positive-strand RNA viruses possessing TLS are selected from the following group: - Modification of the promoter sequence of the CCA gene; - Modification of the genome sequence of the CCA gene; - Modification of the coding sequence (CDS) of the CCA gene; - Modification of the regulatory sequence of the CCA gene; and - Modification of the conserved domain of the CCA gene, or any combination thereof.

[0016] Modifications to the CCA gene that result in resistance will alter the expression of the gene. Alternatively, or as a result, the modification may affect the activity and / or function of the encoded protein, or the protein may not be encoded. Modifications to the CCA gene of the present invention include modifications that result in amino acid changes, modifications that result in early stop codons, modifications that result in truncated proteins, or modifications that result in frameshifts. The modifications may alter, reduce, or render the encoded protein non-functional.

[0017] The altered expression of the CCA gene according to the present invention includes reduced expression, no expression, or silencing. The modification of the CCA gene according to the present invention includes the deletion, substitution, or insertion of at least one nucleotide in the nucleotide sequence of SEQ ID NO: 1 or its homologous sequence, or at least one amino acid encoded in SEQ ID NO: 2 or its homologous sequence. The modification includes modifications that affect a conserved domain such as the active site or catalytic domain of the encoded protein which is a CCA-additive enzyme.

[0018] In one embodiment, a modification constituting resistance to positive-strand RNA viruses having TLS includes a deletion of the promoter sequence of the CCA gene. Promoters of the CCA gene suitable for modification to constituting resistance include, in order of priority, sequences having at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity with sequence number 3, provided that the promoter sequence includes sequence number 4. A deletion of the promoter sequence of the CCA gene results in a change in the expression of the gene, thereby constituting resistance, including the deletion of a regulatory sequence, particularly the deletion of a TATA box, or a deletion including sequence number 4 (Table 1).

[0019] In a preferred embodiment, the deletion containing SEQ ID NO: 4 is the same as the deletion containing SEQ ID NO: 18, or the deletion containing SEQ ID NO: 19.

[0020] In one embodiment, a modification that results in resistance to positive-strand RNA viruses having TLS includes a SNP in the CDS of the CCA gene resulting in an amino acid substitution. Optionally, the SNP results in an amino acid substitution in a conserved domain, such as the active site or catalytic domain of the CCA adductor. The conserved domain of the CCA adductor includes the PolyA_pol_head_ domain (domain ID IPR002646, accessible online in the InterPro database), which includes positions 82 to 241 of SEQ ID NO: 2, or the corresponding position in a homologous sequence having at least 80% sequence identity to SEQ ID NO: 2. The conserved domain also includes a polyA_pol_C-terminal region-like domain (domain ID SSF81891, accessible online in the Superfamily Database). This domain includes three active sites located at amino acids 244 to 583 of the CCA adductor containing SEQ ID NO: 2, or the corresponding position in a homologous CCA adductor sequence having at least 80% sequence identity to SEQ ID NO: 2.

[0021] Surprisingly, the species Solanum lycopersicum deviates from general rules by containing two CCA genes in its genome, and these genes were found to be highly homologous, sharing 95% sequence identity. The first CCA gene, identified herein as SlCCA1, is represented by Sequence ID 1. The second CCA gene of S. lycopersicum, identified herein as SlCCA2, has an 11 bp deletion compared to the SlCCA1 gene (this deletion results in a frameshift, thereby being an early stop codon). The SlCCA2 gene of S. lycopersicum is represented by Sequence ID 5. The deletion in the SlCCA2 gene compared to the SlCCA1 gene is located in exon 9 of the gene and leads to an early stop codon in exon 10 of SlCCA2. The deletion is specifically an 11 bp deletion corresponding to positions 1062 to 1072 in Sequence ID 1. The deletions include, in particular, those involving sequence number 6 (Table 1).

[0022] [Table 1]

[0023] According to the research, despite the premature stop codon in the SlCCA2 gene of S. lycopersicum, this gene is still expressed. RNAseq reads spanning regions with deletions were found, such as reads containing sequences covering positions 1055 to 1065 of SEQ ID NO: 5. Therefore, the SlCCA2 gene of S. lycopersicum is predicted to result in a truncated protein. The truncated protein deviates from the protein encoded by SlCCA1 after position 350 and ends after position 366 within a domain such as the polyA_pol_C terminal region. As a result, only the first of the three active sites in this domain still exists in the CCA-adding enzyme encoded by SlCCA2. Therefore, this domain is predicted to have its function altered, reduced, or not functioning at all. The CCA-adding enzyme encoded by wild-type SlCCA2 contains SEQ ID NO: 7 and has 90% sequence identity to SEQ ID NO: 2.

[0024] Further research on the wild-type SlCCA2 gene of S. lycopersicum showed that it contains several polymorphisms compared to wild-type SlCCA1, as can be inferred from the sequence alignment of SEQ ID NO: 1 and SEQ ID NO: 5. One of these polymorphisms, C at position 631 of SEQ ID NO: 1 versus T of SEQ ID NO: 5, results in the amino acid variant R211C of the protein encoded by wild-type SlCCA2. This position was determined to be within an essential and highly conserved site of the PolyA_pol_head_domain involved in nucleotide binding of the enzyme. Notably, a S. lycopersicum line containing a mutation that reverts this amino acid substitution of SlCCA2 from C to R, i.e., the T631C mutation resulting in the C211R substitution shown in SEQ ID NO: 11, was found to exhibit a field tolerant ToBRFV phenotype (see also Table 3).

[0025] In one embodiment, a modification that results in resistance to positive-strand RNA viruses having TLS includes a T-to-C SNP (T631C) at position 631 of SEQ ID NO: 5 or at a corresponding position in its homologous sequence, which results in a C211R amino acid substitution at a corresponding position in SEQ ID NO: 7 or its homologous sequence. This embodiment relates in particular to a genome containing two CCA genes, thereby both CCA genes having R at position 211 of the encoded protein after modification. This embodiment results in resistance including at least field tolerance. A plant containing this modification is preferably a S. lycopersicum plant containing a modification of the SlCCA2 gene, preferably the modification represented by SEQ ID NO: 11, where the presence of SEQ ID NO: 11 results in ToBRFV resistance, particularly ToBRFV field tolerance.

[0026] ToBRFV was first described by Luria et al ((2017): A new Israeli tobamovirus isolate infects tomato plants harboring Tm-22 resistance genes. PLoS ONE 12(1):e0170429. Doi:10.1371 / journal.pone.0170429). At the time of its publication, Tomato Brown Rugose Fruit Virus was still abbreviated as TBRFV, but over time, the commonly used abbreviation for this virus became ToBRFV, and is therefore currently used in this application.

[0027] Further research identified several SlCCA gene polymorphisms that confer ToBRFV resistance. Specific modifications were found in the CCA gene of wild tomato species, particularly Solanum pimpinellifolium, and when these modifications were transferred to ToBRFV-sensitive S. lycopersicum plants, the plants exhibited resistance to ToBRFV. SNPs that result in resistance-inducing amino acid changes include the A-T SNP at position 948 of SEQ ID NO: 1 or 5 (A948T), the C-T SNP at position 950 of SEQ ID NO: 1 or 5 (C950T), the A-G SNP at position 1348 of SEQ ID NO: 1 (A1348G), the A-G SNP at position 1603 of SEQ ID NO: 1 (A1603G), the A-T SNP at position 1659 of SEQ ID NO: 1 (A1659T), the G-T SNP at position 1737 of SEQ ID NO: 1 (G1737T), or any SNP at the corresponding position in the homologous sequence of SEQ ID NO: 1. Each of these nucleotide changes results in a K316N substitution, A317V substitution, K450E substitution, N535D substitution, R553S substitution, or K579N substitution in SEQ ID NO: 2, or an amino acid substitution at the corresponding position in the homologous sequence of SEQ ID NO: 2.

[0028] As used herein, an X000Y mutation, SNP, or substitution means that the wild-type sequence has a nucleotide or amino acid X at position 000, which is changed to a nucleotide or amino acid Y in the modified sequence.

[0029] Sequence ID 8 contains the N535D, R553S, and K579N mutations. Sequence ID 9 contains the K450E, R553S, and K579N mutations. Sequence ID 10 contains the K316N and A317V mutations.

[0030] Furthermore, other polymorphisms correlated with ToBRFV resistance in S. lycopersicum were found in the promoter of the CCA gene. The resistant CCA1 gene had a deletion containing SEQ ID NO: 4 in its promoter sequence compared to the wild-type SEQ ID NO: 3. Other polymorphisms contained nucleotide substitutions within the promoter sequence, as shown, for example, in the promoter sequence alignment in Figure 3. Notably, the wild-type CCA2 gene of S. lycopersicum, containing SEQ ID NO: 17, also has a deletion containing SEQ ID NO: 4 compared to SEQ ID NO: 3. The deletion of SEQ ID NO: 4 appears to be a deletion in the TATA box of the promoter region of the CCA gene.

[0031] In one embodiment, the promoter of the modified CCA gene of the present invention includes SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16. All of these promoter sequences have a deletion including SEQ ID NO: 4 when compared to the wild-type promoter sequence including SEQ ID NO: 3 (Figure 3b).

[0032] As used herein, resistance to positive-strand RNA viruses with TLS, particularly resistance to tobamovirus, and more specifically to ToBRFV, includes resistance to viruses and / or field resistance. Viral resistance can be expressed at various levels, thereby involving various mechanisms. When a plant is truly resistant to a virus, viral infection and / or replication in the host plant are limited by resistance mechanisms. When bioassays are performed on young plants, resistant plants do not show susceptibility symptoms.

[0033] When used herein, if the plant is resistant to the virus, viral replication and proliferation can occur in the plant and can be measured, for example, through a qPCR experiment. Although some mild symptoms may be observed in bioassays, the impact of the virus's presence on the plant's fitness is significantly reduced compared to the impact on susceptible plants.

[0034] A specific form of resistance, as used herein, is field resistance. When a plant is field-resistant, the host plant is unable to limit the replication and proliferation of the virus, and in bioassays performed on young plants under controlled conditions, the plant exhibits symptoms. However, when such plants are grown in the field under normal cultivation practices, the host can reduce the impact of the virus's presence on the plant's health, resulting in no symptoms or only limited symptoms. Furthermore, crop yields are not significantly reduced and are comparable to those of virus-free crops.

[0035] In one embodiment, a modification that results in resistance to positive-strand RNA viruses having TLS includes a combination of two or more such modifications in a single CCA gene, the combination of which may be a modification in the coding sequence, a modification in the promoter sequence, or a modification in both the promoter and coding sequences. The modification may also be a combination of at least one modification in each of two CCA genes if two CCA genes are present in the plant genome, where the modifications in both CCA genes may be different or the same. The modification may be, in particular, a combination of at least one modification in the gene indicated by SEQ ID NO: 1 and at least one modification in the gene indicated by SEQ ID NO: 5; or a combination of at least one modification in the gene indicated by SEQ ID NO: 1 and at least one modification in the promoter indicated by SEQ ID NO: 3; or a combination of at least one modification in the gene indicated by SEQ ID NO: 5 and at least one modification in the promoter indicated by SEQ ID NO: 17; or a modification of homologous sequences of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and SEQ ID NO: 17.

[0036] Positive-strand RNA viruses having TLS include viruses of the Tobamovirus, Thymovirus, or Bromovirus genera. Preferably, positive-strand RNA viruses having TLS are viruses of the Tobamovirus genus, particularly viruses of the ToBRFV, TMV, ToMV, or CGMMV species. The modification of the CCA gene of the present invention preferably results in ToBRFV resistance, and optionally in combination with resistance to other viruses, particularly other tobamoviruses.

[0037] The present invention relates to plants containing the modified CCA gene of the present invention. Plants containing the modified CCA gene are preferably plants of the Solanaceae family and include species of Solanum lycopersicum, Capsicum annuum, Solanum melongena, Capsicum frutescens, Solanum tuberosum, Petunia spp, or Nicotiana tabacum. The plants of the present invention are preferably cultivated plants that have agricultural value and are particularly agriculturally elite, rather than being wild.

[0038] In one embodiment, plants containing the modified CCA gene of the present invention are resistant to positive-strand RNA viruses having TLS, particularly viruses of the genus Tobamovirus, Thymovirus, or Bromovirus, preferably viruses of the genus Tobamovirus. The positive-strand RNA virus is most preferably the tomato brown rugos fruit virus (ToBRFV), tobacco mosaic virus (TMV), or tomato mosaic virus (ToMV).

[0039] In a preferred embodiment, the plant of the present invention is a Solanum lycopersicum species containing a modified CCA gene, which is resistant to tobamovirus, particularly tomato brown rugos fruit virus (ToBRFV). Modifications to the CCA gene in the S. lycopersicum plant of the present invention include modifications selected from the group comprising: A to T SNP at position 948 of SEQ ID NO: 1 and / or SEQ ID NO: 5; C to T SNP at position 950 of SEQ ID NO: 1 and / or SEQ ID NO: 5; A to G SNP at position 1348 of SEQ ID NO: 1; A to G SNP at position 1603 of SEQ ID NO: 1; A to T SNP at position 1659 of SEQ ID NO: 1; G to T SNP at position 1737 of SEQ ID NO: 1; T to C SNP at position 631 of SEQ ID NO: 5; a deletion of the promoter of the CCA gene, particularly a deletion including SEQ ID NO: 4 from a promoter sequence including SEQ ID NO: 3; or a corresponding modification in homologous sequences of SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5. Certain modifications in the CCA gene can result in resistance to one or more categories of resistance.

[0040] Table 4 outlines SNP modifications in the CCA gene that result in amino acid substitutions in the protein it encodes, which is a CCA-adding enzyme and forms part of the present invention. Modifications are shown from sensitive (before the indicated position) to tolerant (after the indicated position).

[0041] In one embodiment, the S. lycopersicum plant of the present invention comprises two modified CCA genes. In one embodiment, the S. lycopersicum plant of the present invention comprises a CCA1 gene comprising SEQ ID NOs. 8 and SEQ ID NOs. 12, and a CCA2 gene comprising SEQ ID NOs. 10 and SEQ ID NOs. 14; or, the plant comprises a CCA1 gene comprising SEQ ID NOs. 8 and SEQ ID NOs. 12, and a CCA2 gene comprising SEQ ID NOs. 7 and SEQ ID NOs. 15; or, the plant comprises a CCA1 gene comprising SEQ ID NOs. 9 and SEQ ID NOs. 13, and a CCA2 gene comprising SEQ ID NOs. 11 and SEQ ID NOs. 16.

[0042] ToBRFV resistance is determined by comparison with a control variety known to be susceptible to ToBRFV. Examples of ToBRFV-sensitive tomato varieties that can be used as controls are Endeavour F1 and Ramyle F1. As a resistance control, plants deposited as NCIMB43511 or NCIMB43512 containing the modified CCA gene of the present invention can be used. NCIMB43511 contains the CCA1 gene encoding SEQ ID NO: 8 and the CCA2 gene encoding SEQ ID NO: 10. NCIMB43512 contains the CCA1 gene encoding SEQ ID NO: 8 and the CCA2 gene encoding SEQ ID NO: 7. The promoter of the CCA1 gene in NCIMB43511 and NCIMB43512 contains SEQ ID NO: 12. The promoter of the CCA2 gene in NCIMB43511 contains SEQ ID NO: 14. The promoter of the CCA2 gene in NCIMB43512 contains SEQ ID NO: 15.

[0043] To determine resistance, seeds of the strain to be tested are sown in standard seedling trays, and seedlings are inoculated four weeks after sowing. The inoculation material is prepared by grinding ToBRFV-infected tomato leaves in 0.01 M phosphate buffer (pH 7.0) mixed with Celite. Next, the seedlings are dusted with carborundum powder before gently rubbing the leaves with the inoculation material. Resistance is appropriately scored on a scale of 0-5; a description of the scoring scale is provided in Table 2. Observation of symptoms in young tomato plants in bioassays is preferably performed 14-21 days after inoculation (dai).

[0044] As used herein, Solanum lycopersicum plants resistant to ToBRFV due to the presence of a modified CCA gene will have a score of 3 or less than 3, preferably less than 2.5, when the bioassay described above is performed and scoring according to Table 2 is used. In one embodiment, the plants are resistant to ToBRFV and have a score of 2 or less than 2, preferably less than 1. In other embodiments, the plants have field tolerance (FT) to ToBRFV, with a score of 3 or less than 3, preferably less than 2.5, in the bioassay and a score of 2 or less than 2 under field conditions. As is the standard for any bioassay, to obtain a reliable assessment, a representative number of plants, e.g., 10 plants of a particular strain, should be scored and the average score obtained. The susceptibility control of this test should have a score higher than 3, preferably higher than 3.5, if the test is performed properly.

[0045] The plants of the present invention include homozygous or heterozygous modified CCA genes; that is, the modified CCA genes may be present on both chromosomes of a chromosome pair in the plant genome, or on only one chromosome of a chromosome pair. If two modified CCA genes are present in a particular species, e.g., Solanum lycopersicum, they may be in a mutually exclusive phase, i.e., two modified CCA genes on the same chromosome, or in a mutually exclusive phase, i.e., one modified CCA gene on each complementary chromosome. The plants of the present invention include inbred lines, hybrids, open-pollinated varieties, doubling haploids, or plants of segregated populations.

[0046] In one embodiment, the plant of the present invention is a Solanum lycopersicum plant whose representative seeds contain a modified CCA gene found in the genome of a S. lycopersicum plant deposited under deposit number NCIMB43511 or NCIMB43512.

[0047] In one embodiment, the plant of the present invention is a Solanum lycopersicum plant deposited as NCIMB43511 or NCIMB43512, or a progeny containing one or more or all polymorphisms of the CCA gene present in said deposit.

[0048] The virus resistance, particularly ToBRFV resistance, in the plants of the present invention is inherited in an intermediate manner. As used herein, intermediate means that a higher level of resistance is found when the modified CCA gene of the present invention is present homozygously. However, the presence of heterozygosity of the modified CCA gene of the present invention still confers a certain level of ToBRFV resistance. ToBRFV resistance in both homozygous and heterozygous plants makes the plants more suitable for cultivation under conditions in which ToBRFV is present. The improvement in heterozygosity levels can also be expressed when the heterozygous plant has two different modified CCA genes, thereby each modified CCA gene originating from a different parent. Therefore, both heterozygous and homozygous plants are considered to have improved agricultural properties. Furthermore, heterozygous plants can be used to develop homozygous plants by cross and selection, and therefore heterozygous plants also form part of the present invention.

[0049] The present invention further relates to seeds containing the modified CCA gene of the present invention. These seeds can grow into the plant of the present invention. The present invention also relates to the use of the seeds for the production of the plant of the present invention by growing the seeds into a plant. The present invention also relates to a plant part of the plant of the present invention, comprising the fruit of the plant of the present invention or the seeds of the plant of the present invention, wherein the plant part contains the modified CCA gene in its genome.

[0050] The present invention further relates to a method for seed production, comprising growing a plant from the seeds of the present invention, causing the plant to produce fruit having seeds, harvesting the fruit, and extracting the seeds therefrom. Seed production can be appropriately carried out by self-pollination or by crossing with another plant, which is also the plant of the present invention, by optional selection. Seeds thus produced have the ability to grow into plants resistant to positive-strand RNA viruses having TLS, particularly viruses of the genus Tobamovirus, more specifically ToBRFV.

[0051] The present invention further relates to hybrid seeds and a method for producing said hybrid seeds, comprising crossing a first parent plant with a second parent plant and harvesting the resulting hybrid seeds. Herein, the first parent plant and / or the second parent plant are plants of the present invention containing the modified CCA gene of the present invention. The hybrid plants obtained by growing these hybrid seeds are also plants of the present invention that contain the CCA gene of the present invention and are resistant to positive-strand RNA viruses having TLS, particularly viruses of the genus Tobamovirus, more specifically ToBRFV.

[0052] The present invention relates to a method for producing plants resistant to positive-strand RNA viruses having TLS, particularly viruses of the genus Tobamovirus, more specifically ToBRFV, the method comprising introducing a resistance-inducing modification to the CCA gene. The method comprises introducing a deletion, substitution, or insertion in the coding sequence and / or promoter sequence of the CCA gene. The introduction of such a modification can be carried out by a mutagenesis approach using a compound such as ethyl methanesulfonate (EMS) or by physical means such as UV irradiation, fast neutron irradiation, or other irradiation techniques.

[0053] The introduction of modifications can also be carried out using more specific targeted approaches, including homologous recombination, oligonucleotide-based mutagenesis, zinc finger nucleases (ZFNs), activator-like effector nucleases (TALENs), or targeted genome editing using clustered regular scattered short palindromic repeat (CRISPR) systems.

[0054] The introduction of the modified CCA gene of the present invention can also be carried out by gene transfer from a plant containing the modified CCA gene, for example, from a plant deposited as NCIMB43511 or NCIMB43512, or from its progeny, or from another plant resistant to a positive-strand RNA virus having TLS, particularly a virus of the genus Tobamovirus, more specifically ToBRFV, in which the modified CCA gene has been identified. Breeding methods such as crossing and selection, backcrossing, recombinant selection, or other breeding methods that result in the transfer of a gene sequence from a resistant plant to a susceptible plant can be used. The resistant plant may be of the same species, a different species, and / or a wild species. Difficulties in interspecies crossing can be overcome by techniques known in the art, such as embryo rescue, or by applying cisgenesis. The progeny of a deposit may be sexual or vegetative offspring of that deposit, and may be F1, F2, or subsequent generations, as long as the progeny of the deposit still contain the modified CCA gene of that deposit. Plants produced by such methods are also part of the present invention.

[0055] In one embodiment, the modified CCA gene is transferred from a plant of the species S. pimpinellifolium to a plant of the species S. lycopersicum. In another embodiment, the modified CCA gene is transferred from a S. lycopersicum plant containing the modified CCA gene to a S. lycopersicum plant lacking the modified CCA gene, or to a S. lycopersicum plant containing different modifications in arbitrarily different CCAs.

[0056] Transgenic techniques used to introduce sequences between sexually incompatible plants can also be used to produce the plants of the present invention by introducing a modified CCA gene from one species to another. Suitable techniques include common plant transformation techniques known to those skilled in the art, such as the use of Agrobacterium-mediated transformation.

[0057] The present invention also, a) Crossing a plant containing the modified CCA gene of the present invention with another plant; b) Optionally, perform one or more rounds of self-pollination and / or crossbreeding the plants resulting from step a) to obtain a population of further generations; c) Select plants resistant to positive-strand RNA viruses, particularly ToBRFV, that have TLS, including a modified CCA gene as defined herein, from a population resulting from the cross in step a) or from a population of further generations in step b). This relates to a method for producing plants resistant to positive-strand RNA viruses with TLS, particularly ToBRFV.

[0058] The present invention also, a) Crossing a first parent plant of the present invention containing the modified CCA gene of the present invention with a second parent plant which is another plant that does not contain the modified CCA gene of the present invention, or which is another plant that contains a different modification to the CCA gene; b) Backcross the plants resulting from step a) with the second parent plant for at least three generations; c) Select a plant from a backcross population of three generations or more that contains at least the modified CCA gene of the first parent plant from step a), This relates to a method for producing plants resistant to positive-strand RNA viruses with TLS, particularly ToBRFV.

[0059] The present invention further, a) Crossing a plant containing the modified CCA gene of the present invention with a second plant containing other desired traits for producing F1 offspring; b) Optionally, select plants in the F1 generation that possess virus resistance and other desirable traits; c) Crossing an arbitrarily selected F1 progeny with one of the parents for at least three generations to produce a backcross progeny; d) Selecting backcross progeny that include virus resistance and other desired traits; and e) optionally repeat steps c) and d) one or more times in succession to produce a selected fourth or more backcross progeny containing virus resistance and other desired traits. This invention provides a method for introducing other desired traits into plants resistant to positive-strand RNA viruses with TLS, particularly ToBRFV.

[0060] Optionally, a self-pollination step is performed after the crossing or backcrossing step. Alternatively, selection of plants with virus resistance and other desired traits can be performed after any crossing or self-pollination step of the method. Other desired traits can be selected from, but are not limited to, the following groups: resistance to bacterial, fungal, or viral diseases; insect or pest resistance; improved germination; improved plant size; improved plant type; improved shelf life; tolerance to water and heat stress; and male sterility. The present invention includes plants produced by this method and fruits obtained therefrom.

[0061] The present invention further relates to a method for producing plants resistant to positive-strand RNA viruses, particularly tobamovirus, and more specifically ToBRFV, that have TLS, comprising the modified CCA gene of the present invention, by using tissue culture or vegetative propagation.

[0062] The present invention relates to a method for identifying plants resistant to positive-strand RNA viruses having TLS, particularly ToBRFV, which include the modified CCA gene of the present invention, the identification comprising determining the presence of a modification in the CCA gene of Sequence ID No. 1 or its homologous sequence, and analyzing whether the plant containing the modification is resistant to positive-strand RNA viruses having TLS, particularly tobamovirus, and more specifically ToBRFV. Determining the presence of a modification in the CCA gene includes identification by appropriately using markers designed to identify any of the modifications described herein, particularly the SNP modifications shown in Table 4, such that their sequence contains the particular modification.

[0063] The present invention further relates to a method for selecting plants resistant to positive-strand RNA viruses having TLS, particularly ToBRFV, the method comprising identifying the modified CCA gene of the present invention in the plants, and subsequently selecting the plants as plants resistant to positive-strand RNA viruses having TLS, particularly tobamovirus, more specifically ToBRFV. Optionally, viral resistance can be confirmed by performing a bioassay as described in Example 1. Selected plants obtained by such a method are also part of the present invention.

[0064] The present invention also relates to a reproductive material suitable for producing the plant of the present invention, wherein the reproductive material is suitable for sexual reproduction and is selected in particular from microspores, pollen, ovaries, ovules, embryo sacs, or egg cells, or is suitable for vegetative reproduction and is selected in particular from cuttings, roots, stem cells, or protoplasm, or is suitable for tissue culture of regenerative cells and is particularly leaves, pollen, embryos, cotyledons, hypocotyls, meristem cells, roots, root tips, anthers, flowers, seeds, and stems, wherein the plant produced from the reproductive material contains the modified CCA gene of the present invention that provides resistance to positive-strand RNA viruses having TLS, in particular tobamovirus, more specifically ToBRFV. The plant of the present invention can be used as a source of reproductive material. Tissue cultures containing regenerative cells also form part of the present invention.

[0065] The present invention further relates to the cells of the plant of the present invention. Such cells may be either isolated forms or parts or portions of a complete plant, but such cells still constitute the cells of the present invention because they contain the modified CCA gene of the present invention. Each cell of the plant of the present invention possesses the modified CCA gene of the present invention. The cells of the present invention may also be regenerative cells that can be regenerated into new plants of the present invention.

[0066] The present invention further relates to plant tissue of the plant of the present invention, comprising the modified CCA gene of the present invention. The tissue may be undifferentiated or already differentiated. Undifferentiated tissue may be, for example, a shoot apex, anther, petal, or pollen, and can be used for micropropagation to obtain new plant bodies that will grow into the new plant of the present invention. The tissue may also be grown from the cells of the present invention.

[0067] Furthermore, the present invention relates to the progeny of the plant, cell, tissue, or seed of the present invention. This progeny comprises the modified CCA gene of the present invention. Such progeny may itself be a plant, cell, tissue, or seed. The progeny may, in particular, be the progeny of the plant of the present invention deposited under NCIMB number 43511 or NCIMB 43512. As used herein, progeny includes the first and all further progeny from a cross of the plant of the present invention with itself or with other plants. Offspring determined to be progeny contain the modified CCA gene of the present invention. Offspring may be obtained by self-pollination and / or further cross of the deposit. The progeny also includes material obtained by vegetative propagation or other forms of propagation.

[0068] The present invention also relates to markers for identifying modified CCA genes in plants. These markers include any of the modifications in the CCA gene as described herein, thereby enabling the identification of such modifications. The markers of the present invention include, in particular, SNP modifications, i.e., polymorphisms, as shown in Table 4, and are suitable markers for identification by such modifications. The use of such markers for the identification of modified CCA genes is also part of the present invention. [Brief explanation of the drawing]

[0069] [Figure 1] Figure 1 shows the CDS sequences of Sequence ID No. 1 (wild-type CCA1 gene of Solanum lycopersicum) and Sequence ID No. 5 (wild-type CCA2 gene of Solanum lycopersicum). [Figure 2-1]Figure 2 shows the protein sequences of SEQ ID NO: 2 (wild-type CCA adductor enzyme encoded by SEQ ID NO: 1), SEQ ID NO: 7 (wild-type CCA adductor enzyme encoded by SEQ ID NO: 5), and SEQ ID NOs: 8-11 (CCA adductor enzymes with modifications leading to resistance). CCA1_NCIMB43511 and CCA1_NCIMB43512 are the same as SEQ ID NO: 8. CCA1_TO1 is the same as SEQ ID NO: 9. CCA1_Ramyle F1 and CCA1_Sl3_00 are the same as SEQ ID NO: 2. CCA2_NCIMB43512, CCA2_Sl3_00, CCA2_Ramyle F1, and CCA2_Endeavour F1 are the same as SEQ ID NO: 7. TO1 is the same as SEQ ID NO: 11. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 2-4] Same as above [Figure 3-1] Figure 3a shows the promoter sequences of SEQ ID NO: 3 (promoter of the wild-type CCA1 gene in Solanum lycopersicum), SEQ ID NO: 17 (promoter of the wild-type CCA2 gene in Solanum lycopersicum), and SEQ ID NOs: 12-16. CCA1_NCIMB43511_43512 is the same sequence as SEQ ID NO: 12. Figure 3b shows an alternative alignment with a stretch preceding nucleotide 917, which includes deletions in all of these sequences when compared to SEQ ID NO: 3. [Figure 3-2] Same as above [Figure 3-3] Same as above [Figure 4] Figure 4 shows the domains of the CCA-adding enzyme. [Modes for carrying out the invention]

[0070] The present invention is further illustrated in the following examples, which are for illustrative purposes only. These examples are not intended to limit the invention in any way. The examples and this application refer to the figures.

[0071] Deposit Seeds of tomato Solanum lycopersicum containing the modified CCA gene of the present invention were deposited on November 7, 2019, with accession numbers NCIMB43511 and NCIMB43512 at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen AB21 9YA, UK. [Examples]

[0072] Example 1 Bioassay for ToBRFV resistance in Solanum lycopersicum S. lycopersicum lines with modifications in one or both CCA genes were observed in the ToBRFV bioassay. Three S. pimpinellifolium sources were included as resistance controls. Endeavor F1 and Ramir F1 were used as susceptibility controls.

[0073] Seeds of the test varieties were sown in standard seedling trays, and 10 seedlings per variety were inoculated four weeks after sowing. Inoculation material was prepared by grinding Tomato leaves infected with ToBRFV in 0.01 M phosphate buffer (pH 7.0) mixed with Celite. Before gently rubbing the leaves with the inoculation material, the plants were coated with carborundum powder. Symptom scoring was performed 19 days after inoculation according to Table 2.

[0074] [Table 2]

[0075] The bioassay results are shown in Table 3. The average score of the 10 inoculated seedlings is shown. TO313 is a hybrid between a line with the CCA genotype NCIMB43511 and a line with the CCA genotype NCIMB43512. “CCA genotype” means that the line has both CCA1 and CCA2 sequences, as in the referenced deposit.

[0076] [Table 3]

[0077] Example 2 Identification of CCA gene modifications that lead to ToBRFV resistance. Various Solanum lycopersicum populations isolated for ToBRFV resistance were finely mapped to a small region on chromosome 11 containing only four potential genes likely to contribute to ToBRFV resistance. Whole-genome sequences were available in-house for the background of the resistant and susceptible lines used to develop these populations. Therefore, a SNP calling approach was performed on that region. This meant that unique polymorphisms within the region were identified by comparing the sequences with each other.

[0078] Among the genes in the region of interest were two CCA genes called CCA1 and CCA2. CCA1 is a complete CCA gene, and various polymorphisms were observed between susceptible and resistant materials, but all of them led to proteins containing the essential domain and active site of the CCA-adder. However, although the CCA2 gene also contained various polymorphisms, in all lines containing susceptible material, the CCA2 gene had an early stop codon, producing a cleaved protein that lacked all the essential active sites of the CCA-adder. The encoded protein was truncated within a domain such as the poly(A_pol_C-terminal region), and as a result, only the first of the three active sites of this domain is present in the CCA2 gene of S. lycopersicum (Figure 4).

[0079] Different resistance strains were observed to possess different polymorphisms. Many of these polymorphisms resulted in non-conservative amino acid changes, which are shown in Table 4.

[0080] [Table 4]

[0081] Furthermore, the presence of ToBRFV resistance was found to correlate with a deletion in the promoter of the CCA1 gene. In all cases where a promoter deletion was present, the deletion contained at least the sequence ATATTTATTT (SEQ ID NO: 4; Table 1), but the deletion could also contain several nucleotides or more, e.g., 1 to 10 nucleotides or more, in addition to a simple deletion of SEQ ID NO: 4. In some cases, for example, the deletion contained the sequence represented by SEQ ID NO: 18 or the sequence represented by SEQ ID NO: 19, both of which were found to contain SEQ ID NO: 4.

[0082] Since the promoter deletion was located in a TATA-rich region, it was considered to be a deletion of the promoter's TATA box.

[0083] Through analysis of the correlation between phenotype and genotype segregation, it was determined that modifications to the CCA gene, specifically modifications to the CCA1 gene and / or the CCA2 gene, which may involve promoter modifications and / or coding sequence modifications, are responsible for ToBRFV resistance in resistant Solanum lycopersicum plants.

[0084] Example 3 Modification of the CCA gene to obtain resistance to positive-strand RNA viruses with TLS Modifications are introduced into the seeds of target plants that require resistance to positive-strand RNA viruses possessing TLS, such as tobamoviruses like ToBRFV, ToMV, or TMV. Modifications are introduced by mutagenesis, such as EMS treatment, radiation, or specific targeted approaches such as CRISPR. When non-targeted approaches such as EMS are used, they are combined with identification techniques such as TILLING. Thus, modifications to the CCA gene can be generated and identified using both mutagenesis and targeted modification methods. Those skilled in the art are familiar with these methods for introducing modifications into the genome of target plants.

[0085] Next, the modified seeds germinate, the plants grow, and they are crossbred or self-pollinated to produce M2 seeds. Subsequently, plant screening is performed to identify CCA gene modifications based on comparison with the wild-type sequence of one or more CCA genes of that species. For example, in the case of Solanum lycopersicum, comparison can be made with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 17. Those skilled in the art are familiar with tilling (McCallum et. al. (2000) Nature Biotechnology, 18:455-457) for identifying mutations in specific genes, and techniques for identifying nucleotide changes, such as DNA sequencing, in particular.

[0086] Plants possessing the modified CCA gene are homozygous or made homozygous by self-pollination, crossbreeding, or the use of doubling haploid techniques well known to those skilled in the art. Plants identified and selected based on the modification of the CCA gene can then be tested for resistance to positive-strand RNA viruses with TLS, such as tobamoviruses like ToBRFV, ToMV, or TMV. Plants produced, identified, and selected in this manner are confirmed to possess viral resistance as a result of one or more modifications to the CCA gene. This disclosure provides, for example, the following embodiments. [Section 1] A modified CCA gene encoding a CCA-adding enzyme, wherein the modified CCA gene provides resistance to positive-strand RNA viruses with TLS, and the modified CCA gene: - A gene containing a nucleotide sequence encoding a CCA-adding enzyme, as shown in SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; - A gene containing a promoter sequence, including SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; - A gene containing a nucleotide sequence encoding a CCA-additive enzyme that has at least one amino acid deletion, substitution, or insertion when compared to SEQ ID NO: 2 or SEQ ID NO: 7; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme that, when compared to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11, has at least one amino acid deletion, substitution, or insertion; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme having at least 80% sequence identity with SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11; and - A gene containing a promoter sequence having at least 80% sequence identity with SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16, A modified CCA gene selected from a group consisting of the following. [Section 2] A modified CCA gene as described in item 1, wherein at least one amino acid deletion, substitution, or insertion is present in a conserved domain or active site of the encoded CCA-adding enzyme. [Section 3] A modified CCA gene as described in item 1, comprising a modification of the promoter sequence, wherein the promoter sequence comprises SEQ ID NO: 3, and in particular comprises a modification of the regulatory sequence of the promoter sequence, wherein the modification particularly comprises a deletion. [Section 4] A modified CCA gene as described in any one of items 1 to 3, comprising a combination of two or more modifications in a single CCA gene, particularly a combination of modifications in the promoter sequence and modifications in the coding sequence. [Section 5] A modified CCA gene according to any one of items 1 to 4, wherein the CCA adductor having at least 80% sequence identity to SEQ ID NO: 8 contains at least one of the following substitutions: N535D, R553S, or K579N; the CCA adductor having at least 80% sequence identity to SEQ ID NO: 9 contains at least one of the following substitutions: K450E, R553S, or K579N; the CCA adductor having at least 80% sequence identity to SEQ ID NO: 10 contains at least one of the following substitutions: K316N or A317V; the CCA adductor having at least 80% sequence identity to SEQ ID NO: 11 contains at least one of the following substitutions: C211R; or the modified CCA gene according to any one of items 1 to 4, wherein the CCA adductor having at least 80% sequence identity to SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11 contains one of these substitutions at the corresponding position in the homologous sequence. [Section 6] A plant containing a modified CCA gene as described in any one of items 1 to 5. [Section 7] A plant according to item 6, resistant to a positive-strand RNA virus having TLS, preferably tobamovirus, most preferably ToBRFV. [Section 8] The plant described in item 6 or 7 is a plant of the Solanaceae family, preferably a plant of the Solanum lycopersicum species. [Section 9] A Solanum lycopersicum plant, as described in item 8, containing two modified CCA genes. [Section 10] Solanum lycopersicum plants described in section 8 or 9, in which the modified CCA gene is present in the genome of a Solanum lycopersicum plant whose representative seeds are deposited with NCIMB under deposit number 43511 or NCIMB43512. [Section 11] A seed wherein the plant grown from the seed contains the modified CCA gene described in any one of items 1 to 5. [Section 12] A marker for identifying a modified CCA gene, wherein the marker is: - SNPs A through T at position 948 of sequence number 1, - From position 950 of sequence number 1, T SNP, - SNPs A through G at position 1348 of sequence number 1, - SNPs A through G at position 1603 of sequence number 1, - SNPs A through T at position 1659 of sequence number 1, - From position 1737 of sequence number 1, G to T SNP, - T to C SNP at position 631 of SEQ ID NO: 5, and -Deletion of sequence number 3, including sequence number 4. Detect modifications selected from the group consisting of, or The marker detects a modification at the corresponding position of a homologous sequence that has at least 80% sequence identity with sequence number 1, sequence number 3, or sequence number 5. marker. [Section 13] Use of the markers described in Section 12 for the identification of ToBRFV resistance in Solanum lycopersicum plants and / or for the selection of ToBRFV-resistant Solanum lycopersicum plants. [Section 14] A method for producing a ToBRFV-resistant Solanum lycopersicum plant, comprising introducing a modification to the CCA gene, wherein the modified CCA gene is one of those described in any one of sections 1 to 5. [Section 15] A method for selecting ToBRFV-resistant Solanum lycopersicum plants, comprising identifying the presence of a modification in the CCA gene, optionally testing the plants for ToBRFV resistance, and selecting the plants containing the said modification as ToBRFV-resistant plants. [Section 16] The method described in item 15, wherein identification is performed by using the markers described in item 12. [Section 17] A method for producing plants resistant to positive-strand RNA viruses, including TLS: a) Crossing a first parent plant containing the modified CCA gene described in any one of items 6 to 10 with a second parent plant; b) Optionally, perform one or more rounds of self-pollination and / or cross-pollination of the plants resulting from the cross in step a) to obtain a population of further generations; c) Select plants containing the modified CCA gene from the plants resulting from the cross in step a) or from a population of further generations in step b), where the selected plants are resistant to positive-strand RNA viruses including TLS. Methods that include... [Section 18] A method for producing Solanum lycopersicum plants resistant to ToBRFV: a) Crossing a first parent plant containing the modified CCA gene described in any one of items 8 to 10 with a second parent plant; b) Optionally, perform one or more rounds of self-pollination and / or cross-pollination of the plants resulting from the cross in step a) to obtain a population of further generations; c) Select plants containing the modified CCA gene from the plants resulting from the cross in step a) or from a population of further generations in step b), where the selected plants are resistant to ToBRFV. Methods that include... [Section 19] The method according to section 17 or 18, wherein the second parent plant also contains a modified CCA gene. [Section 20] The method according to any one of sections 17 to 19, wherein the selection of plants including modifications of the CCA gene is carried out by using the markers described in section 12. [Section 21] The method according to section 18 or 19, wherein plants resistant to ToBRFV are phenotypically selected, particularly by using a ToBRFV resistance bioassay. [Section 22] The method described in any one of sections 6 to 10, wherein the plant is a plant grown from a seed deposited under NCIMB accession number 43511 or NCIMB 43512, or a progeny of such plant, as described in any one of sections 17 to 21. [Section 23] A method for producing hybrid seeds, comprising crossing a first parent plant with a second parent plant and harvesting the resulting hybrid seeds, wherein the first parent plant and / or the second parent plant contain a modified CCA gene as described in any one of claims 1 to 5, and the presence of the modified CCA gene results in ToBRFV resistance in plants grown from the hybrid seeds, according to any one of claims 1 to 5.

Claims

1. A cultivated Solanum lycopersicum plant resistant to ToBRFV, wherein the plant contains a modified CCA1 gene encoding a CCA-adding enzyme, and the modified CCA1 gene is - The CCA1 gene, which includes the nucleotide sequence encoding the CCA-adding enzyme shown in Sequence ID No. 8, and - The CCA1 gene containing the nucleotide sequence encoding the CCA-adding enzyme shown in Sequence ID No. 9 A plant selected from a group consisting of the following.

2. The plant according to claim 1, wherein the modified CCA1 gene matches the CCA1 gene contained in the genome of a Solanum lycopersicum plant deposited with NCIMB in a representative seed form under deposit number NCIMB43511 or NCIMB43512.

3. Seeds of a cultivated Solanum lycopersicum plant, wherein the plant grown from the seeds contains the modified CCA1 gene described in Claim 1 or 2.

4. A modified CCA1 gene encoding a CCA-adding enzyme, wherein the modified CCA1 gene provides resistance to positive-strand RNA viruses with TLS, and the modified CCA1 gene: - A gene containing the nucleotide sequence encoding the CCA-adding enzyme shown in Sequence ID No. 8; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme that has a deletion, substitution, or insertion of one amino acid compared to SEQ ID NO: 8; - A gene containing a nucleotide sequence encoding a CCA-adding enzyme that has a deletion, substitution, or insertion of one amino acid and includes an N535D substitution, compared to SEQ ID NO: 8; and - A gene containing a nucleotide sequence that has at least 95% sequence identity with SEQ ID NO: 8 and encodes a CCA-adding enzyme that includes the N535D substitution. A modified CCA1 gene selected from the group consisting of the following.

5. The modified CCA1 gene according to claim 4, wherein the deletion, substitution, or insertion of the one amino acid is located in a conserved domain or active site of the encoded CCA-adding enzyme.

6. The modified CCA1 gene according to claim 4, further comprising modification of the promoter sequence, wherein the promoter sequence comprises sequence number 3.

7. The modified CCA1 gene according to claim 6, wherein the modification includes modification of a regulatory sequence of the promoter sequence.

8. The modified CCA1 gene according to claim 6 or 7, wherein the modification includes a deletion.

9. A modified CCA1 gene according to any one of claims 6 to 8, comprising a promoter sequence including sequence number 12, 13, 14, 15, or 16.

10. A modified CCA1 gene according to any one of claims 6 to 9, comprising a combination of two modifications in a single CCA1 gene.

11. The use of genetic markers for the identification of ToBRFV resistance in Solanum lycopersicum plants and / or the selection of ToBRFV-resistant Solanum lycopersicum plants, wherein the genetic marker is: - SNPs from A at position 1603 of sequence number 1, - From A at position 1659 of sequence number 1, T SNP, - From G at position 1737 of sequence number 1 to T SNP, - SNPs starting from A at position 1348 of sequence number 1, It detects modifications selected from the group consisting of and A combination of gene markers that detects the G SNP from position 1603 of SEQ ID NO: 1, the T SNP from position 1659 of SEQ ID NO: 1, and the T SNP from position 1737 of SEQ ID NO: 1, or a combination of gene markers that detects the G SNP from position 1348 of SEQ ID NO: 1, the T SNP from position 1659 of SEQ ID NO: 1, and the T SNP from position 1737 of SEQ ID NO: 1, detects a modified CCA gene that provides resistance to positive-strand RNA viruses with TLS. Use of genetic markers.

12. A method for producing a ToBRFV-resistant Solanum lycopersicum plant, comprising introducing a modification to the CCA1 gene, wherein the modified CCA1 gene is one of those described in any one of claims 4 to 10.

13. A method for selecting a ToBRFV-resistant Solanum lycopersicum plant, comprising: identifying the presence of a modification in the CCA1 gene by using the gene marker described in claim 11; optionally testing the plant for ToBRFV resistance; and selecting the plant containing the modification as a ToBRFV-resistant plant.

14. A method for producing plants resistant to positive-strand RNA viruses, including TLS, is as follows: a) Crossing a first parent plant containing the modified CCA1 gene described in any one of claims 4 to 10 with a second parent plant; b) Optionally, perform one or more rounds of self-pollination and / or cross-pollination of the plants resulting from the cross in step a) to obtain a population of further generations; c) Select a plant containing the modified CCA1 gene from the plants resulting from the cross in step a) or from a population of further generations in step b), wherein the selected plant is resistant to positive-strand RNA viruses including TLS. Methods that include...

15. A method for producing Solanum lycopersicum plants resistant to ToBRFV, wherein: a) Crossing a first parent plant containing the modified CCA1 gene described in any one of claims 4 to 10 with a second parent plant; b) Optionally, perform one or more rounds of self-pollination and / or cross-pollination of the plants resulting from the cross in step a) to obtain a population of further generations; c) Select plants containing the modified CCA1 gene from the plants resulting from the cross in step a) or from a population of further generations in step b), wherein the selected plants are resistant to ToBRFV. Methods that include...

16. The method according to claim 14 or 15, wherein the second parent plant also contains the modified CCA1 gene.

17. The method according to any one of claims 14 to 16, wherein the selection of a plant containing the modified CCA1 gene is performed by using the gene marker described in claim 11.

18. The method according to claim 15 or 16, wherein plants phenotypically resistant to ToBRFV are selected.

19. The method according to claim 18, wherein phenotypic selection is performed by using a ToBRFV resistance bioassay.

20. The method according to any one of claims 14 to 19, wherein a plant containing the modified CCA1 gene described in any one of claims 4 to 10 is a plant grown from a seed deposited pursuant to NCIMB accession number NCIMB 43511 or NCIMB 43512, or a progeny of such a plant.

21. A method for producing hybrid seeds, comprising crossing a first parent plant with a second parent plant and harvesting the resulting hybrid seeds, wherein the first parent plant and / or the second parent plant are cultivated Solanum lycopersicum plants containing the modified CCA1 gene described in any one of claims 4 to 10, and the presence of the modified CCA1 gene results in ToBRFV resistance in plants grown from the hybrid seeds.

Images