Method for conferring disease resistance

Abstract

The present invention aims to provide a method for producing Solanum species with improved disease resistance, and disease-resistant Solanum species obtained by this method. [Solution] The above problem is solved by providing a method for producing disease-resistant plants, characterized by suppressing the activity of PAP2 in Solanum plants or suppressing the expression of the gene encoding the enzyme.

Description

[Technical Field]

[0001] The present invention relates to a method for producing a disease-resistant plant. The present invention also relates to a Solanaceae plant having improved disease resistance. [Background Art]

[0002] Potatoes are susceptible to multiple diseases including bacterial wilt, soft rot, late blight, common scab, mosaic disease, and the like. Infection with these diseases reduces yield, so imparting disease resistance to plants is an important issue from the perspective of improving productivity.

[0003] Regarding plants with improved disease resistance, for example, Non-Patent Document 1 describes that *Nicotiana benthamiana*, a species of the genus *Nicotiana*, acquires resistance to *Ralstonia solanacearum*, the causative bacterium of bacterial wilt, by silencing the DS1 gene encoding phosphatidic acid phosphatase 2. However, there have been no reports to date on *Solanum* plants (e.g., potatoes) with improved resistance to various diseases. [Prior Art Documents] [Non-Patent Documents]

[0004] [Non-Patent Document 1] Nakano et al., Suppression of DS1 Phosphatidic Acid Phosphatase Confirms Resistance to Ralstonia solanacearum in Nicotiana benthamiana, PLOSONE, 2013, Volume 8,Issue 9. [Summary of the Invention] [Problem to be Solved by the Invention]

[0005] In view of the above, the object of the present invention is to provide a method for producing Solanum species with improved disease resistance, and disease-resistant Solanum species obtained by this method. [Means for solving the problem]

[0006] The inventors, through diligent research to solve the above problems, have for the first time discovered that disease resistance in Solanum species (e.g., potato) can be improved by suppressing the expression of the gene encoding phosphatidic acid phosphatase 2 (hereinafter also referred to as "PAP2"), which is involved in phospholipid metabolism. Furthermore, the inventors have found that Solanum species obtained by this method are resistant not only to one disease but to multiple diseases (e.g., bacterial wilt, soft rot, late blight, and scab). These findings are astonishing results that could not have been predicted from conventional knowledge. This invention was completed based on these findings.

[0007] In other words, one aspect of the present invention relates to the following: [1] A method for producing disease-resistant plants, characterized by suppressing the activity of phosphatidic acid phosphatase 2 in plants of the genus Solanum, or suppressing the expression of the gene encoding the enzyme. [2] The Solanum species has two different types of first and second phosphatidic acid phosphatase 2, The method according to [1], wherein the activity of the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed, or the expression of the gene encoding the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed. [3] The method according to [2], wherein the activity of both the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed, or the expression of both the genes encoding the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed. [4] The method according to [2] or [3], characterized in that the gene encoding the first phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (a) to (e) below, and the gene encoding the second phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (f) to (j) below: (a) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NOs: 1, 3, or 5; (b) A gene encoding a protein having phosphatidic acid phosphatase II activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (c) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in Sequence ID No. 2, 4, or 6; (e) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (a) to (d) above, and that encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 7; (g) A gene encoding a protein having phosphatidic acid phosphatase II activity, consisting of an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence described in Sequence ID No. 7; (h) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence described in Sequence ID No. 7; (i) A gene consisting of the nucleotide sequence described in Sequence ID No. 8; (j) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (f) to (i) above, and that encodes a protein having phosphatidic acid phosphatase 2 activity. [5] The method according to any one of [1] to [4], wherein the disease resistance is resistance to multiple diseases. [6] The method according to any one of [1] to [5], wherein the disease resistance is resistance to at least two or more diseases from among bacterial wilt, soft rot, late blight, and scab. [7] The method according to any one of [1] to [6], characterized in that the expression of the gene encoding phosphatidic acid phosphatase 2 is suppressed by ZFN, TALEN, CRISPR / Cas family protein, PPR motif, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA, or homologous recombination. [8] The method according to any one of [1] to [7], wherein the Solanum plant is potato, tomato, eggplant, bell pepper, or chili pepper. A disease-resistant plant characterized by being obtained by any one of the methods described in [9], [1], to [8].

[10] A cultivated variety of the plant described in [9].

[11] Solanum plants having a mutation in the gene encoding phosphatidic acid phosphatase 2 and exhibiting improved disease resistance.

[0008] Furthermore, one aspect of the present invention relates to the following:

[12] The Solanum plant according to

[11] , characterized in that the activity of phosphatidic acid phosphatase 2 is suppressed, or the expression of the gene encoding the enzyme is suppressed.

[13] The Solanum species has two different types of first and second phosphatidic acid phosphatase 2, The Solanum plant according to

[11] or

[12] , wherein the activity of the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed, or the expression of the gene encoding the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed.

[14] The Solanum plant described in

[13] , wherein the activity of both the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed, or the expression of both the genes encoding the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed.

[15] The Solanum plant described in

[13] or

[14] , characterized in that the gene encoding the first phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (a) to (e) below, and the gene encoding the second phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (f) to (j) below: (a) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NOs: 1, 3, or 5; (b) A gene encoding a protein having phosphatidic acid phosphatase II activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (c) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in Sequence ID No. 2, 4, or 6; (e) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (a) to (d) above, and that encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 7; (g) a gene encoding a protein consisting of an amino acid sequence in which one or several amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence set forth in SEQ ID NO: 7, and having phosphatidic acid phosphatase 2 activity; (h) a gene encoding a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence set forth in SEQ ID NO: 7, and having phosphatidic acid phosphatase 2 activity; (i) a gene consisting of the nucleotide sequence set forth in SEQ ID NO: 8; (j) a gene that hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to any one of the genes (f) to (i) above, and encodes a protein having phosphatidic acid phosphatase 2 activity.

[16] The Solanaceae plant according to any one of

[11] to

[15] , wherein the disease resistance is resistance to a plurality of diseases.

[17] The Solanaceae plant according to any one of

[11] to

[16] , wherein the disease resistance is resistance to at least two or more diseases selected from the group consisting of bacterial wilt, soft rot, late blight, and common scab.

[18] The Solanaceae plant according to any one of

[11] to

[17] , wherein suppression of expression of the gene encoding phosphatidic acid phosphatase 2 is performed by ZFN, TALEN, CRISPR / Cas family protein, PPR motif, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA, or homologous recombination.

[19] The Solanaceae plant according to any one of

[11] to

[18] , which is potato, tomato, eggplant, bell pepper, or capsicum.

[20] The Solanaceae plant according to any one of

[11] to

[19] , which is a cultivar. Effects of the Invention

[0009] According to the present invention, a Solanum plant with improved disease resistance can be provided. In one aspect of the present invention, a Solanum plant having resistance to multiple diseases can be provided, whereby agricultural crops can be produced efficiently. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] [Figure 1] Figure 1 is a diagram showing the target sites of CRISPR / Cas9 in StDS1-1 and StDS1-2. [Figure 2] Figure 2 is a diagram showing the sequence of target site 2 (which may be denoted as "circled number 1" in the figure or a table) of a StDS1-1 single knockout (TF3). In Figure 2, the underlined highlighted portion indicates target site 2. Details of the mutation and the number of clones of the sequence in sequence analysis (the number in parentheses) are shown at the right end of the sequence. [Figure 3] Figure 3 is a diagram showing the sequence of target site 1 (which may be denoted as "circled number 1" in the figure or a table) of a StDS1-2b single knockout (V4_2). In Figure 3, the underlined highlighted portion indicates target site 1. Details of the mutation and the number of clones of the sequence in sequence analysis (the number in parentheses) are shown at the right end of the sequence. Since the originally frameshift-containing StDS1-2a allele and the full-length protein-encoding StDS1-2b allele contain no polymorphism within the amplified region and cannot be distinguished from each other, they are denoted as "StDS1-2a or StDS1-2b". [Figure 4] Figure 4 is a diagram showing the sequence of StDS1-2 target site 1 of a double knockout (NS1_R3). In Figure 4, the underlined highlighted portion indicates target site 1. Details of the mutation and the number of clones of the sequence in sequence analysis (the number in parentheses) are shown at the right end of the sequence. [Figure 5]Figure 5 shows the amplicon sequence analysis of StDS1-2 target site 1 in the double knockout organism (NS1_R3). In Figure 5, the underlined highlighted area indicates target site 1. The right end of the sequence shows the details of the mutation and the relative abundance and number of occurrences of the sequence in the sequence analysis (numbers in parentheses). [Figure 6] Figure 6 shows the bacterial wilt resistance test. [Figure 7] Figure 7 shows the method for testing bacterial wilt resistance through xylem inoculation. [Figure 8] Figure 8 shows the results of the bacterial wilt resistance test using xylem inoculation. From left to right in Figure 8, the data are shown for the wild type (WT), a single DS1-1 knockout line (TF3, labeled "T3" in the figure), a single DS1-2 knockout line (V4_2, labeled "V2" in the figure), a double knockout line (NS1_R3, labeled "NS" in the figure), and a bacterial wilt-resistant variety (Nagasaki Kogane) (NK). [Figure 9] Figure 9 shows the results of the bacterial wilt resistance test using vascular inoculation. [Figure 10] Figure 10 shows the results of the soft rot resistance test. [Figure 11] Figure 11 shows the results of the soft rot resistance test. [Modes for carrying out the invention]

[0011] One embodiment of the present invention will be described in detail below. All academic and patent documents cited herein are incorporated herein by reference. Unless otherwise specified herein, "A to B" representing a numerical range means "A or greater (including A and greater than A) to B or less (including B and less than B)."

[0012] In this specification, the term “gene” is interchangeable with “polynucleotide,” “nucleic acid,” or “nucleic acid molecule,” and refers to a polymer of nucleotides. Here, a gene may exist in the form of DNA (e.g., cDNA or genomic DNA) or RNA (e.g., mRNA). DNA or RNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be a coding strand (sense strand) or a non-coding strand (antisense strand). Genes may also be chemically synthesized, and their codon usage may be modified to improve the expression of the protein they encode. Substitutions are also possible between codons that code for the same amino acid. The term “protein” is interchangeable with “peptide” or “polypeptide.” In this specification, bases and amino acids are represented using the single-letter or three-letter notation as defined by IUPAC and IUB, as appropriate.

[0013] (1. Method for creating disease-resistant plants) One embodiment of the present invention provides a method for producing disease-resistant plants (hereinafter also referred to as "this production method") characterized by suppressing the activity of PAP2 in Solanum plants or suppressing the expression of the gene encoding the enzyme.

[0014] In this specification, "plants of the genus Solanum" refers to plants belonging to a genus within the family Solanaceae, such as potatoes, tomatoes, eggplants, bell peppers, and chili peppers.

[0015] In this specification, "phosphatidic acid phosphatase 2 (PAP2)" means a protein that has the function of dephosphorylating phosphatidic acid (PA) and forming diacylglycerol (DAG).

[0016] In one embodiment of the present invention, a plant of the genus Solanum may have two different types of PAP2. For convenience, these will be referred to as "first PAP2" and "second PAP2" in this specification.

[0017] In one embodiment of the present invention, the present production method may be a method for suppressing the activity of the first PAP2 and / or the second PAP2 of a Solanum plant, or for suppressing the expression of the genes encoding the first PAP2 and / or the second PAP2.

[0018] Furthermore, in one embodiment of the present invention, the present production method may be a method for suppressing the activity of both the first PAP2 and the second PAP2 of a Solanum plant, or for suppressing the expression of both the genes encoding the first PAP2 and the second PAP2.

[0019] In one embodiment of the present invention, PAP2 is not particularly limited as long as it is a protein having the above-described activity. The gene encoding the first PAP2 is, for example, any gene selected from the group consisting of (a) to (e) below. (a) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NOs: 1, 3, or 5; (b) A gene encoding a protein having PAP2 activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (c) A gene encoding a protein having PAP2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in Sequence ID No. 2, 4, or 6; (e) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (a) to (d) above, and that encodes a protein having PAP2 activity.

[0020] Furthermore, the gene encoding the second PAP2 is, for example, one of the genes selected from the group consisting of (f) to (j) below. (f) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 7; (g) A gene encoding a protein having PAP2 activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence described in Sequence ID No. 7; (h) A gene encoding a protein having PAP2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence described in Sequence ID No. 7; (i) A gene consisting of the nucleotide sequence described in Sequence ID No. 8; (j) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (f) to (i) above, and that encodes a protein having PAP2 activity.

[0021] In the gene described in (a) above, sequence numbers 1, 3, or 5 represent PAP2 encoded by the potato-derived DS1-1 gene. In other words, the genes encoding the protein consisting of the amino acid sequences shown in sequence numbers 1, 3, and 5 are polymorphisms of the DS1-1 gene. For convenience, in this specification, the genes encoding the protein consisting of the amino acid sequences shown in sequence numbers 1, 3, and 5 may be referred to as "StDS1-1a," "StDS1-1b," and "StDS1-1c," respectively.

[0022] In the gene described in (f) above, sequence number 7 is PAP2 encoded by the potato-derived DS1-2 gene. For convenience, in this specification, the gene encoding the protein consisting of the amino acid sequence shown in sequence number 7 may be referred to as "StDS1-2b".

[0023] The genes in (b) and (g) above are functionally equivalent mutants, derivatives, variants, alleles, homologs, orthologues, partial peptides, or fusion proteins with other proteins / peptides, etc., of proteins having the amino acid sequences shown in SEQ ID NOs: 1, 3, 5, and 7, respectively, and the specific sequences are not limited as long as they encode proteins having PAP2 activity. Here, the number of amino acids that may be deleted, substituted, or added is not limited as long as the above function is not lost, but refers to a number that can be deleted, substituted, or added by known introduction methods such as site-directed mutagenesis. The number of such amino acids is usually within 30 amino acids, preferably within 20 amino acids, more preferably within 10 amino acids, even more preferably within 7 amino acids, and particularly preferably within 5 amino acids (e.g., 5, 4, 3, 2, or 1 amino acid). Furthermore, in this specification, "mutation" mainly refers to mutations artificially introduced by site-directed mutagenesis, etc., but may also refer to similar mutations that exist in nature.

[0024] It is preferable that the mutated amino acid residue is mutated to another amino acid whose amino acid side chain properties are conserved. For example, amino acid side chain properties include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (all in parentheses represent the single letter notation of the amino acid). It is already known that polypeptides having amino acid sequences modified by the deletion, addition, and / or substitution of one or more amino acid residues with other amino acids maintain their biological activity. Furthermore, it is more preferable to mutate the target amino acid residue to an amino acid residue that shares as many common properties as possible.

[0025] In this specification, "functionally equivalent" means that the target protein has the same (identical and / or similar) biological and biochemical functions as the protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1, 3, 5, and 7. In this specification, examples of the biological and biochemical functions of the protein consisting of the amino acid sequence described in any of SEQ ID NOs: 1, 3, 5, and 7 include, for example, the function of dephosphorylating phosphatidic acid (PA) to form diacylglycerol (DAG). Biological functions may also include the specificity of the expression site and the expression level. Whether a mutated protein is functionally equivalent can be determined by obtaining a transformant that expresses the gene encoding the protein and examining whether this transformant can form diacylglycerol using phosphatidic acid as a substrate.

[0026] The genes described in (c) and (h) above are intended to be functionally equivalent variants, derivatives, variants, alleles, homologs, orthologues, partial peptides, or fusion proteins with other proteins or peptides of proteins having the amino acid sequences shown in SEQ ID NOs. 1, 3, or 5, and SEQ ID NOs. 7, respectively, and are not limited to specific sequences as long as they encode proteins with PAP2 activity.

[0027] Amino acid sequence identity means that the entire amino acid sequence (or the region necessary for functional expression) has sequence identity of at least 80%, more preferably 90%, and particularly preferably 95% or more (e.g., 95%, 96%, 97%, 98%, 99% or more). Amino acid sequence identity is achieved using the BLASTN (nucleic acid level) or BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). This can be determined. The program is based on the BLAST algorithm by Karlin and Altschul (Proc. Natl. Acad. Sci. USA, 87:2264-2268, 1990, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993). The base sequence is analyzed by BLASTN. In this case, the parameters should be, for example, score=100 and wordlength=12. When analyzing amino acid sequences using BLASTX, the parameters should be, for example, score=50 and wordlength=3. Furthermore, when analyzing amino acid sequences using the Gapped BLAST program, the procedure can be carried out as described by Altschul et al. (Nucleic Acids Res. 25: 3389-3402, 1997). When using BLAST and the Gapped BLAST program, the default parameters of each program should be used. Specific methods for these analysis techniques are publicly known. Additions or deletions (e.g., gaps) may be permitted to optimally align the comparison base sequences or amino acid sequences.

[0028] In this specification, "identity" refers to the proportion of identical amino acid residues. The properties of amino acids are as described above.

[0029] For the genes in (d) and (i) above, SEQ ID NOs. 2, 4, or 6, and SEQ ID NOs. 8 represent the nucleotide sequences (Open Reading Frames: ORFs) of the genes encoding polypeptides consisting of the amino acid sequences shown in SEQ ID NOs. 1, 3, or 5, and SEQ ID NOs. 7, respectively.

[0030] The gene described in (e) above is intended to hybridize under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes described in (a) to (d) above. Similarly, the gene described in (j) above is intended to hybridize under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes described in (f) to (i) above. Here, stringent conditions refer to conditions in which a so-called base sequence-specific double-stranded polynucleotide is formed, and a non-specific double-stranded polynucleotide is not formed. In other words, it can be described as conditions in which highly homologous nucleic acids, for example, hybridize at a temperature range of 15°C, preferably 10°C, and even more preferably 5°C lower than the melting temperature (Tm value) of a perfectly matched hybrid. For example, one example is hybridization at 68°C for 20 hours in a general hybridization buffer. More specifically, the following conditions can be mentioned: hybridization for 16 to 24 hours in a buffer consisting of 0.25 M Na2HPO4, pH 7.2, 7% SDS, 1 mM EDTA, and 1 × Denhardt's solution at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C; followed by two washes of 15 minutes each in a buffer consisting of 20 mM Na2HPO4, pH 7.2, 1% SDS, and 1 mM EDTA at a temperature of 60 to 68°C, preferably 65°C, and more preferably 68°C. Those skilled in the art can easily obtain such genes by referring to Molecular Cloning (Sambrook, J. et al., Molecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)), etc.

[0031] In this specification, "suppressing gene expression" means that the protein encoded by the gene is not produced or its production is reduced within the target plant, and this includes loss of gene function (so-called knockout), gene disruption, etc. It may also mean inhibiting the transcription process from DNA to mRNA of the gene, or inhibiting the translation process from mRNA to protein of the gene. There are no particular restrictions on the degree of inhibition, as long as the plant whose gene expression has been suppressed exhibits resistance to disease.

[0032] The method used to suppress the expression of the gene encoding PAP2 is not particularly limited and any method used in this field may be used. For example, ZFNs, TALENs, CRISPR / Cas family proteins, PPR (Pentatrico Peptide Repeat) Methods include UV irradiation, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA, and homologous recombination. The Cas family proteins in CRISPR / Cas family proteins are not particularly limited, but examples include Cas9, Cpf1 (also known as Cas12a), C2C1 (also known as Cas12b), Cas12f, C2C2 (also known as Cas13a), CasX, CasY, Cas1, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, and Cas10. Cas family proteins in which the function has been appropriately modified are also included in the above Cas family proteins. Each method is described, for example, in the following publications: *Genome engineering for crop improvement and future agriculture. Gao C. Cell. 2021 Mar 18;184(6):1621-1635. doi: 10.1016 / j.cell.2021.01.005. Epub 2021 Feb 12. PMID: 33581057 Free article Review.* and *Plant genome editing: ever more precise and wide reaching. Sukegawa S, Saika H, ​​Toki S. Plant J. 2021 Jun;106(5):1208-1218. doi: 10.1111 / tpj.15233. Epub 2021 Apr 24. PMID: 33730414 Free article Review.* , ZFN (Kim YG et al., Proc. Natl. Acad. Sci. USA 93:1156-1160, 1996), T ALEN (Christian et al., Genetics, 186:757-761, 2010), Cas family tan Proteins (e.g., CRISPR / Cas9 (Gilbert et al., Cell, 154:442-451, 2013) ), Cas12f(Hino, T., Omura, SN, Nakagawa, R., Togashi, T., Takeda, SN, Hiramoto, T., et al. (2023). An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis. Cell 186, 4920-4935.e4923. and Ishibashi K, Sukegawa S, Endo M, Hara N, Nureki O, Saika H and Toki S (2024) Systemic delivery of engineered compact AsCas12f by a positive-strand RNA virus vector enables highly efficient targeted mutagenesis in plants. Front. Plant Sci. 15:1454554.)), PPR motif (International Publication No. 2014 / 175284), ion beam irradiation (A. Tanaka et a1., Int. J. Radiat. Bio1. 72: 121-127, 1997), ultraviolet irradiation (Yasuo Ukai, Plant Breeding, University of Tokyo Press (2003)), s iRNA (Elbashir SM et al., Nature. 2001 May 24;411(6836):494-8), miRNA (Caudy AA et al, Genes & Devel 16: 2491-96, 2002), shRNA (Bernstein E, Caudy AA et al, Nature; 409(6818): 363-6, 2001), antisense RNA (Ching et al., Proc. Natl. Acad. Sci. USA 86:10006-10010 (1989)). From the viewpoint of simple and practical use, genome editing is preferred. Furthermore, it is preferable to use null isolates for gene expression suppression.

[0033] In this specification, "suppressing activity" means that the activity of the target enzyme is eliminated or reduced within the target plant. There are no particular restrictions on the degree of suppression; the goal is simply for the plant, whose gene expression has been suppressed, to exhibit resistance to disease.

[0034] The method for inhibiting PAP2 activity is not particularly limited, and any method used in this field can be used. For example, in addition to the method described above, methods using enzyme inhibitors and aptamers can be used.

[0035] Examples of plant materials targeted for suppressing the expression of the gene encoding PAP2, or for suppressing the activity of PAP2, include, but are not limited to, plant tissues such as roots, stems, leaves, seeds, embryos, ovules, ovaries, shoot apices, anthers, and pollen, as well as their sections, cells, callus, and plant cells such as proplasts obtained by enzymatic treatment to remove the cell wall.

[0036] In one embodiment of the present invention, a suitable vector capable of causing modification (destruction) of the above-mentioned genes (a) to (j) can be constructed, introduced into plant cells, and a transformed plant can be regenerated from these cells to produce the plant of the present invention. The transformed plant cells described above can be regenerated into a complete plant by methods known to those skilled in the art. For example, callus-like transformed cells can be transferred to a culture medium with varying hormone types and concentrations and cultured to form a somatic embryo and obtain a complete plant.

[0037] In addition, when plant tissue, such as leaf discs, is used as the plant material for transformation, after infection with Agrobacterium, stems and leaves can be formed by culturing them on a sterilized redifferentiation solid medium, which has been treated with inorganic salts, vitamins, carbon sources (such as sugars as energy sources), plant growth regulators (plant hormones such as auxin and cytokinin), and selected agents such as kanamycin, under appropriate light and temperature conditions. Next, adventitious roots can be induced by culturing the stems and leaves on a medium (rooting medium) from which the plant growth regulators have been removed from the above solid medium, thereby regenerating into a complete plant. Common media such as LS medium and MS medium can be used. In the above-mentioned transformed organism, confirmation of whether or not gene expression is suppressed is performed by determining the presence or absence of any of the genes (a) to (j) above, or whether or not the expression of said gene is suppressed.

[0038] Since the genes (a) to (j) above encode PAP2, it is possible to easily determine whether a plant can have improved disease resistance by determining whether these genes are present in the plant, whether the expression of these genes is suppressed, or whether the activity of PAP2 is suppressed.

[0039] While conventionally known methods can be used for specific determination methods, examples include: (i) obtaining a DNA sample from the target plant and examining whether the gene is present or absent, or whether a mutation has been introduced into the gene and its expression is suppressed; (ii) examining the presence or amount of mRNA, which is the transcript of the above gene; and (iii) examining the presence, amount or activity of protein, which is the transcript of the above gene.

[0040] Methods for investigating the above-mentioned DNA, RNA, or proteins can be conventionally known methods and are not particularly limited. Examples include probe-based methods, PCR methods, RT-PCR methods, various immunoassay methods using antibodies, microarray-based methods, and enzyme activity measurement methods.

[0041] In one embodiment of the present invention, disease resistance may be resistance to multiple diseases. In another embodiment of the present invention, disease resistance may be resistance to at least two or more diseases from among bacterial wilt, soft rot, late blight, and scab.

[0042] (2. Solanum species with improved disease resistance) In one embodiment of the present invention, a Solanum plant (hereinafter also referred to as "this Solanum plant") having a mutation in the gene encoding PAP2 and exhibiting improved disease resistance is provided.

[0043] In this specification, "having a mutation in the gene encoding PAP2" means that the base sequence of the gene encoding PAP2 has been altered to the extent that the expression of the gene encoding PAP2 is suppressed. The method for introducing a mutation into the gene encoding PAP2 is not particularly limited, but examples include the above-mentioned ZFN, TALEN, CRISPR / Cas family proteins, ion beam irradiation, ultraviolet irradiation, etc.

[0044] In relation to this Solanum species, the terms "Solanum species," "disease resistance," and "improved disease resistance" are as described in section (1. Method for producing disease-resistant plants) above.

[0045] This Solanum genus includes any of the following: the entire plant body, plant organs (e.g., roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, etc.), plant cells, callus, etc. It also includes protoplasts, bud primordia, multiple buds, and hairy roots. In one embodiment of the present invention, preferably the entire plant body, and more preferably a cultivated variety.

[0046] Furthermore, once a transgenic plant is obtained in which the genes (a) to (j) above within the chromosome are disrupted or their expression is suppressed, it is possible to obtain offspring from the plant through sexual or asexual reproduction. It is also possible to obtain reproductive materials (e.g., seeds, fruits, cuttings, tubers, rhizomes, plants, callus, protoplasts, etc.) from the plant, its offspring, or clones, and mass-produce the plant based on these materials. In addition, this Solanum genus includes progeny such as the "T0 generation," which is the redifferentiated current generation after transgenic treatment, and the "T1 generation," which is the self-pollinated seed of the T0 generation plant, as well as hybrid plants and their progeny obtained by crossing them with one parent.

[0047] In one embodiment of the present invention, the Solanum plant may be a Solanum plant obtained by the present manufacturing method. [Examples]

[0048] The present invention will be described in more detail below using examples, but this will not limit the scope of the invention. References made throughout this specification are incorporated entirely by reference.

[0049] (1. Gene cloning of DS1-1 and DS1-2) To identify the putative DS1 orthologue gene in potato, a homology search was performed using the potato SpudDB sequence database with the full-length coding region sequence of the tobacco benthamiana DS1 gene as the query. As a result, two orthologue candidates were found from Solanum tuberosum Group Phureja clone DM1-3 516R44. The full-length coding region sequences of these genes are shown below.

[0050] [Table 1]

[0051] Total RNA was extracted from the stems and leaves of the potato variety Sayaka using RNAiso Plus (Takara Bio). The obtained total RNA was treated with DNase I (Takara Bio) to degrade the genomic DNA, and then the RNA was re-purified using RNeasy Mini Kit (Qiagen). 2.5 μg of the re-purified RNA was used for reverse transcription using PrimeScript™ RT Master Mix (Takara Bio), and then the full-length coding region derived from Sayaka was amplified using PrimeSTAR Max DNA Polymerase (Takara Bio). The primers used are as follows.

[0052] [Table 2]

[0053] PCR products were cloned into plasmid vectors using the Zero Blunt™ TOPO™ PCR Cloning Kit (Invitrogen). Plasmid DNA was extracted from multiple colonies obtained by transforming E. coli (DH5α) and the nucleotide sequences were determined. As a result, three different sequences (StDS1-1a to 1c) differing by a few nucleotides were obtained for StPAP2-1 (StDS1-1a corresponds to the amino acid sequence described in SEQ ID NO: 1 and the nucleotide sequence described in SEQ ID NO: 2; StDS1-1b corresponds to the amino acid sequence described in SEQ ID NO: 3 and the nucleotide sequence described in SEQ ID NO: 4; StDS1-1c corresponds to the amino acid sequence described in SEQ ID NO: 5 and the nucleotide sequence described in SEQ ID NO: 6). Furthermore, while four distinct sequences were obtained for StPAP2-2, three of them exhibited frameshift mutations due to interallelic DNA polymorphism. Therefore, only one sequence (StDS1-2b) could encode the full-length protein (corresponding to the amino acid sequence described in SEQ ID NO: 7 and the nucleotide sequence described in SEQ ID NO: 8).

[0054] (2. Construction of genome editing constructs for DS1-1 and DS1-2) Using CRISPR direct (http: / / crispr.dbcls.jp / ), StDS1-1 Two target sites were set within the second exon (Figure 1). For StDS1-1, the target was designed in a region where no polymorphism was observed between alleles. For StDS1-2, one target site was set in the second and fourth exons, respectively (Figure 1). See Table 3. This shows the target sites for each gene.

[0055] [Table 3]

[0056] For the creation of the CRISPR / Cas9 construct for genome editing, the pMgP237-2A-GFP vector (Hashimoto, R., Ueta, R., Abe, C., Osakabe, Y. and Osakabe, K. (2018) Front. Plant Sci. 9: 916) was used. Constructs were created to simultaneously express gRNAs targeting both target sites in StDS1-1 and StDS1-2, respectively, according to the method. The resulting constructs were introduced into Agrobacterium tumefaciens GV3101 (pMP90) strain by electroporation. Transformed Agrobacterium were selected on LB agar medium containing 50 mg / L kanamycin, a final concentration of 100 mg / L rifampicin, and a final concentration of 25 mg / L gentamicin.

[0057] (3. Creation of genome-edited plants using StDS1-1 and StDS1-2) From wild-type (Sayaka) plants cultivated under sterile conditions for approximately one month after subculturing in a plant box, stem sections 5-7 mm in length and without lateral buds were prepared. 1.5 ml of Agrobacterium culture solution containing the genome editing construct was transferred to tubes, and the cells were collected by centrifugation. The cells were resuspended in 1.5 ml of MS liquid medium to prepare the infection solution. The infection solution was added to a petri dish containing the stem sections, and the dish was shaken for several seconds to induce infection. After transferring the stem sections onto sterile filter paper to remove excess bacterial suspension, they were transferred to 3C5ZR agar medium (co-culture medium) supplemented with a final concentration of 20 mg / L acetosyringone and co-cultured in a chamber at 20°C for 3 days. The stem sections after co-culture were transferred to 3C5ZR medium (selective medium 1) supplemented with a final concentration of 250 mg / L carbenicillin and 50 mg / L kanamycin. Subsequently, every two weeks, the stem sections were transferred to selective medium 2, which was selective medium 1 from which indoleacetic acid had been removed. Adventitious buds that regenerated within two months of Agrobacterium infection were transferred to root selection medium. The root selection medium was changed every two weeks, and root selection was performed a total of three times to identify candidate transformants. The various culture media were prepared as follows.

[0058] [Table 4]

[0059] The above was dissolved in approximately 900 mL of deionized water, the pH was adjusted to 5.9 using 1N KOH, and then the volume was made up to 1 L. 8 g of Agar (SIGMA, for plant culture medium) was added, and the mixture was heated at 121°C. It was autoclaved for 15 minutes.

[0060] The R3VD used in the 3C5ZR medium described above was prepared as follows.

[0061] [Table 5]

[0062] [Table 6]

[0063] The above was dissolved in approximately 900 mL of deionized water, the pH was adjusted to 5.9 using 1N KOH, and then the volume was made up to 1 L. 8 g of Agar (SIGMA, for plant culture medium) was added, and the mixture was heated at 121°C. After autoclaving for 15 minutes, carbenicillin at a final concentration of 250 mg / L and kanamycin at a final concentration of 50 mg / L were added.

[0064] (4. Confirmation of genome editing in StDS1-1 and StDS1-2) A small amount of leaves from the candidate transformants obtained in the previous step were taken and transferred to a PCR tube. 50 μL of lysis buffer (100 mM Tris-HCl (pH 9.5), 1 M KCl, 10 mM EDTA) was added to this tube, and the mixture was incubated at 95°C for 10 minutes using a thermal cycler. 50 μL of sterile water was added to the reaction solution, and the mixture was vortexed and spun down to obtain the genome extract. Next, the extracted genomic DNA was used as a template for PCR amplification of fragments containing the target site. KOD FX Neo (TOYOBO) and the following primers were used for amplification of the fragments containing the target site. 10 μL of the reaction product was subjected to a heteroduplex mobility assay using MultiNA (Shimadzu Corporation). For samples in which bands of different sizes from the wild type, presumably derived from the edited genome, were observed, cloning to plasmid vectors and sequencing analysis were performed using the CloneJET PCR Cloning Kit.

[0065] [Table 7]

[0066] In the StDS1-1 single knockout candidate, several base deletions were observed at target site 2, and no wild-type sequence was found in any of StDS1-1a, 1b, or 1c (Figure 2). No mutations were found at target site 1 in any of the alleles. This StDS1-1 single knockout was named (TF3). For the StDS1-2 single knockout candidate, a deletion of 1 to 10 bases was observed at target site 1. Of the four alleles, the StDS1-2b allele, the only one capable of encoding a full-length protein, showed a deletion of 10 bases, and the wild-type sequence was not found (Figure 3). This StDS1-2b single knockout was named (V4_2).

[0067] <Creation and analysis of double mutants of StDS1-1 and StDS1-2> To create double mutants of StDS1-1 and StDS1-2, genome editing of StDS1-2 was performed using StDS1-1 knockout plants (TF3) as a background. The same construct used to produce the single knockout of StDS1-2 was used for genome editing of StDS1-2. PCR and heteroduplex mobility assays were performed on 169 shoot lines obtained using the same method as above, and one candidate double mutant line was found. PCR fragments containing the target site of StDS1-2 were cloned from stem sections of the obtained line (NS1_R3), and the base sequence was analyzed. Similar to the single knockout, a deletion was confirmed at target site 1, and no wild-type sequence was found (Figure 4). Furthermore, amplicon sequencing revealed that wild-type StDS1-2a or StDS1-2b sequences were detected with extremely low probability (Figure 5), so it was determined that a double mutant of StDS1-1 and StDS1-2 was obtained. This double mutant was named (NS1_R3).

[0068] (5. Resistance test for bacterial wilt) Resistance to bacterial wilt is tested using in vitro resistance testing (Habe 2018 American Journal). The inoculation was performed using the method described in Potato Research (2018) 95:311-316 and the stem injection method using acclimatized plants (Date 2006 Okayama Prefectural Agricultural Experiment Station Research Report 24 29-41). Ralstonia solanacearum MAFF 327001 and Ralstonia solanacearum MAFF 327040 were used as test organisms (Table 8).

[0069] [Table 8]

[0070] Sterile cultured plants were transplanted into test tubes using vermiculite as a growing medium and cultured in MS liquid medium. In vitro resistance testing was performed at 1 × 10⁻⁶ 2 Inoculation was performed by dropping a bacterial suspension adjusted to cfu / ml onto the growing medium in test tubes containing plant cultures. The severity of the disease 20 days after inoculation was evaluated according to the following criteria. A disease severity index of "0" was defined as no wilting at all in the stem, "1" as wilting in 1-25% of the stem, "2" as 26-50%, "3" as 51-75%, and "4" as 76-100%, with the average value for each group being used as the disease severity index. As a result, the single knockout line of DS1-1 (TF3) and the single knockout line of DS1-2 (V4_2) showed moderate resistance, while the double knockout line (NS1_R3) showed resistance comparable to that of an existing bacterial wilt-resistant variety (Nagasaki Kogane) (Figure 6 and Table 9).

[0071] [Table 9]

[0072] In the stem injection inoculation method using acclimatized plants, sterile cultured plants were transplanted into culture pots with vermiculite as the growing medium and subjected to a two-week acclimatization period. A micropipette tip was inserted into the base of the petiole, and 1 × 10⁻⁶ doses were administered. 4Inoculation was performed by dropping a bacterial suspension adjusted to cfu / ml into the tip (Figure 7). Similar to the in vitro resistance test, the disease incidence index 20 days after inoculation was evaluated. As a result, even in tests using stem injection inoculation with acclimatized plants, the single DS1-1 knockout line (TF3, labeled "T3" in the figure) and the single DS1-2 knockout line (V4_2, labeled "V2" in the figure) showed greater resistance than the wild-type Sayaka (WT). Furthermore, the double knockout line (NS1_R3, labeled "NS" in the figure) showed resistance comparable to the existing bacterial wilt-resistant variety (Nagasaki Kogane) (NK) (Figure 8).

[0073] (6. Creation of double mutant lines of StDS1-1 and StDS1-2 and bacterial wilt resistance testing) Using the same method as before, StDS1-2 genome editing was performed on the same StDS1-2 target sequence (Table 3) with StDS1-1 knockout plants (TF3) as the background, and three new double mutant lines of StDS1-1 and StDS1-2 (denoted as D7, D41, and E12) were obtained. Resistance to bacterial wilt was tested using the same method. As a result, similar to NS1_R3 mentioned above, the mutants showed resistance comparable to the existing bacterial wilt-resistant variety (Nagasaki Kogane) (NK) (Figure 9).

[0074] (7. Soft rot resistance test) The soft rot resistance test was conducted using tubers. The test fungus used was Pectobacterium carotovorum E7105R. 1 × 10⁶ samples were applied to the cut surface of the potato tubers. 8 Inoculation was performed by dropping a bacterial suspension adjusted to cfu / ml. After inoculation, the cells were cultured at 25°C in a humid chamber, and disease symptoms were observed 4 and 7 days after inoculation. As a result, the DS1-1 single-deficient strain (TF3) showed delayed progression of putrefaction (resistance) (Figure 10). Furthermore, resistance to soft rot was quantitatively investigated using a single knockout line of DS1-1 (TF3), a single knockout line of DS1-2 (V4_2, labeled "V4" in the figure), and a double knockout line (NS1_R3, labeled "NS" in the figure) (Figure 11). 1 × 10 8A bacterial suspension adjusted to cfu / ml was injected into microtiter plates. Tubers approximately 2 cm in diameter were cut in half lengthwise with a razor and weighed. Inoculation was performed by inserting the cut surface into the microtiter plate so that it was immersed in the bacterial suspension. Two days after inoculation, the cut surface was washed away, and the tuber tissue decomposed by decay was removed before weighing. The decay rate was calculated by subtracting the post-inoculation weight from the pre-inoculation weight. As a result, single-deficient lines (TF3, V4) and double-knockout lines (NS) of DS1-1 showed suppressed decay (resistance), with the double-knockout line (NS) showing the strongest resistance to soft rot.

[0075] (8. Disease resistance testing) Disease resistance tests are conducted using tubers and acclimatized plants (Yoshioka et al. 1999 Plant Cell Physiol 40:993-8). The test fungus used is Phytophthora infestans. Use Race0. 1×10 4 The fungal solution, adjusted to zoospores / ml, is inoculated onto leaves and tubers by dropping or spraying. This allows for the demonstration of disease resistance in single-deficient lines of DS1-1, single-deficient lines of DS1-2, and double-mutant lines of DS1-1 and DS1-2.

[0076] (9. Scab resistance test) Scab resistance tests are conducted using tubers and acclimatized plants (Clarke et al. 2019 Phytopathology 109:1544-54). Bacteria of the genus Streptomyces, which cause scab, are used as test organisms. Approximately 1 × 10⁻⁶ 8 Tubers or acclimatized plants are grown in soil containing vermiculite and culture medium adjusted to CFU / g. By evaluating the disease symptoms on the tuber surface after growth, scab resistance can be demonstrated in single-deficient lines of DS1-1, single-deficient lines of DS1-2, and double mutant lines of DS1-1 and DS1-2. [Industrial applicability]

[0077] This invention is extremely useful in the field of agricultural production because it enables the creation of Solanum plants with improved disease resistance.

Claims

1. A method for producing disease-resistant plants, characterized by suppressing the activity of phosphatidic acid phosphatase 2 in plants of the genus Solanum, or suppressing the expression of the gene encoding the enzyme.

2. The aforementioned Solanum species has two different types of first and second phosphatidic acid phosphatase 2, The method according to claim 1, wherein the activity of the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed, or the expression of the gene encoding the first phosphatidic acid phosphatase 2 and / or the second phosphatidic acid phosphatase 2 is suppressed.

3. The method according to claim 2, wherein the activity of both the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed, or the expression of both the genes encoding the first phosphatidic acid phosphatase 2 and the second phosphatidic acid phosphatase 2 is suppressed.

4. The method according to claim 2, characterized in that the gene encoding the first phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (a) to (e) below, and the gene encoding the second phosphatidic acid phosphatase 2 is one of the genes selected from the group consisting of (f) to (j) below: (a) A gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NOs: 1, 3, or 5; (b) A gene encoding a protein having phosphatidic acid phosphatase II activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence shown in Sequence ID No. 1, 3 or 5; (c) A gene encoding a protein having phosphatidic acid phosphatase II activity, comprising an amino acid sequence having 80% or more identity with the amino acid sequence shown in Sequence ID No. 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in SEQ ID NOs: 2, 4, or 6; (e) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (a) to (d) above, and that encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene encoding a protein consisting of the amino acid sequence described in Sequence ID No. 7; (g) A gene encoding a protein having phosphatidic acid phosphatase II activity, comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence described in Sequence ID No. 7; (h) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence that is 80% or more identical to the amino acid sequence described in Sequence ID No. 7; (i) A gene consisting of the nucleotide sequence described in Sequence ID No. 8; (j) A gene that hybridizes under stringent conditions with a polynucleotide having a base sequence complementary to any of the genes in (f) to (i) above, and encodes a protein having phosphatidic acid phosphatase II activity.

5. The method according to claim 1, wherein the disease resistance is resistance to multiple diseases.

6. The method according to claim 1, wherein the disease resistance is resistance to at least two or more diseases among bacterial wilt, soft rot, late blight, and scab.

7. The method according to claim 1, characterized in that the suppression of the expression of the gene encoding phosphatidic acid phosphatase 2 is performed by ZFN, TALEN, CRISPR / Cas family proteins, PPR motifs, ion beam irradiation, ultraviolet irradiation, siRNA, miRNA, shRNA, antisense RNA, or homologous recombination.

8. The method according to claim 1, wherein the Solanum plant is a potato, tomato, eggplant, bell pepper, or chili pepper.

9. A disease-resistant plant characterized by being obtained by the method described in any one of claims 1 to 8.

10. A cultivated variety of the plant according to claim 9.

11. Solanum plants with a mutation in the gene encoding phosphatidic acid phosphatase 2 and improved disease resistance.

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