Method for imparting disease resistance

Abstract

The objective of this invention is to provide a method for producing a plant of the genus Solanacea having improved disease resistance, and also to provide a plant of the genus Solanacea having disease resistance obtained with the method. A solution to the above is a method for producing a disease-resistant plant, characterized by suppressing the activity of PAP2 of a plant of the genus Solanacea or suppressing the expression of a gene encoding that enzyme.

Description

Method for imparting disease resistance

[0001] The present invention relates to a method for producing a disease-resistant plant. The present invention also relates to a Solanum plant with improved disease resistance.

[0002] Potato is susceptible to multiple diseases including bacterial wilt, soft rot, late blight, common scab, mosaic disease and the like. Infection with these diseases leads to reduced yield, and therefore, 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 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., potato) with improved resistance to various diseases.

[0004] Nakano et al., Suppression of DS1 Phosphatidic Acid Phosphatase Confirms Resistance to Ralstonia solanacearum in Nicotiana benthamiana, PLOSONE, 2013, Volume 8,Issue 9.

[0005] In view of the above, an object of the present invention is to provide a method for producing a Solanum plant with improved disease resistance, and a disease-resistant Solanum plant obtained by said method.

[0006] As a result of diligent research to solve the above problems, the inventors have discovered for the first time 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 Solanum plants or suppressing the expression of the gene encoding the enzyme. [2] The method according to [1], wherein the Solanum plant has two different types of first and second phosphatidic acid phosphatase 2, and 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 first phosphatidic acid phosphatase 2 and the gene encoding 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 NO: 1, 3, or 5; (b) A gene encoding a protein having phosphatidic acid phosphatase 2 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 shown in SEQ ID NO: 1, 3, or 5; (c) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in SEQ 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 encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene that encodes a protein having an amino acid sequence described in Sequence ID No. 7; (g) A gene that encodes a protein having phosphatidic acid phosphatase II activity, having 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 that encodes a protein having phosphatidic acid phosphatase II activity, having an amino acid sequence having 80% or more identity with the amino acid sequence described in Sequence ID No. 7; (i) A gene having a base 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 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 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 suppression of the 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. [8] The method according to any one of [1] to [7], wherein the Solanum plant is potato, tomato, eggplant, bell pepper, or chili pepper. [9] A disease-resistant plant characterized by being obtained by the method according to any one of [1] to [8].

[10] The plant according to [9], which is a cultivar.

[11] A Solanum plant having a mutation in the gene encoding phosphatidic acid phosphatase 2 and having 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 plant according to

[11] or

[12] , wherein the Solanum plant has two different types of first and second phosphatidic acid phosphatase 2, and 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 according to

[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] A Solanum plant according to

[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 NO: 1, 3, or 5; (b) A gene encoding a protein having phosphatidic acid phosphatase 2 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 shown in SEQ ID NO: 1, 3, or 5; (c) A gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in SEQ 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 encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene that encodes a protein having an amino acid sequence described in Sequence ID No. 7; (g) A gene that encodes a protein having phosphatidic acid phosphatase II activity, having 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 that encodes a protein having phosphatidic acid phosphatase II activity, having an amino acid sequence having 80% or more identity with the amino acid sequence described in Sequence ID No. 7; (i) A gene having a base 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.

[16] A Solanum plant according to any one of

[11] to

[15] , wherein the disease resistance is resistance to multiple diseases.

[17] A Solanum plant according to any one of

[11] to

[16] , wherein the disease resistance is resistance to at least two diseases from among bacterial wilt, soft rot, late blight, and scab.

[18] A Solanum plant according to any one of

[11] to

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

[19] A Solanum plant according to any one of

[11] to

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

[20] A Solanum plant according to any one of

[11] to

[19] , which is a cultivar.

[0009] According to the present invention, it is possible to provide Solanum plants with improved disease resistance. In one aspect of the present invention, it is possible to provide Solanum plants that have resistance to multiple diseases, thereby enabling efficient production of agricultural products.

[0010] Figure 1 shows the target sites of CRISPR / Cas9 in StDS1-1 and StDS1-2. Figure 2 shows the sequence of target site 2 (sometimes indicated as "circled number 2" in the figure or table) of the StDS1-1 single knockout (TF3). In Figure 2, the underlined highlighted portion indicates target site 2. The right end of the sequence shows the details of the mutation and the number of clones of the sequence in the sequence analysis (number in parentheses). Figure 3 shows the sequence of target site 1 (sometimes indicated as "circled number 1" in the figure or table) of the StDS1-2b single knockout (V4_2). In Figure 3, the underlined highlighted portion indicates target site 1. The right end of the sequence shows the details of the mutation and the number of clones of the sequence in the sequence analysis (number in parentheses). The StDS1-2a allele, which originally contains a frameshift, and the StDS1-2b allele, which codes for the full-length protein, do not contain polymorphisms within the amplified region and cannot be distinguished from each other, so they are labeled as "StDS1-2a or StDS1-2b". Figure 4 shows the sequence of StDS1-2 target site 1 in the double knockout (NS1_R3). In Figure 4, the underlined highlighted portion indicates target site 1. The right end of the sequence shows the details of the mutation and the number of clones of the sequence in the sequence analysis (number in parentheses). Figure 5 shows the amplicon sequence analysis of StDS1-2 target site 1 in the double knockout (NS1_R3). In Figure 5, the underlined highlighted portion indicates target site 1. The right end of the sequence shows the details of the mutation and the abundance ratio and number of the sequence in the sequence analysis (number in parentheses). Figure 6 shows the bacterial wilt resistance test. Figure 7 shows the bacterial wilt resistance vascular inoculation test method. Figure 8 shows the results of the bacterial wilt resistance vascular inoculation test method. In Figure 8, from left to right, the data are shown for the wild type (WT), the single DS1-1 knockout line (TF3, labeled "T3" in the figure), the single DS1-2 knockout line (V4_2, labeled "V2" in the figure), the double knockout line (NS1_R3, labeled "NS" in the figure), and the bacterial wilt resistant variety (Nagasaki Kogane) (NK). Figure 9 shows the results of the bacterial wilt resistance vascular inoculation test method. Figure 10 shows the results for soft rot resistance.Figure 11 shows the results of the soft rot resistance test.

[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) and 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, the notation of bases and amino acids shall be in the single-letter or three-letter notation as defined by IUPAC and IUB, as appropriate.

[0013] (1. Method for producing 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 gene 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 NO: 1, 3, or 5; (b) A gene encoding a protein having PAP2 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 shown in SEQ ID NO: 1, 3, or 5; (c) A gene encoding a protein having PAP2 activity, consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 3, or 5; (d) A gene consisting of the nucleotide sequence shown in SEQ ID NO: 2, 4, or 6; (e) A gene encoding a protein having PAP2 activity, which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to any of the genes in (a) to (d).

[0020] Furthermore, the second PAP2 encoding gene 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, 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 PAP2 activity, consisting of an amino acid sequence having 80% or more identity with 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 encoding a protein having PAP2 activity, which hybridizes under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to any of the genes in (f) to (i) above.

[0021] In the gene described in (a) above, sequence numbers 1, 3, or 5 are 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 ID 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 ID 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 or peptides, etc., of the protein 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 a protein 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 (for example, 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 group-containing side chains (H, F, Y, W) (all in parentheses are single-letter abbreviations of amino acids). 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 that the target amino acid residue be mutated to an amino acid residue that has 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 their specific sequences are not limited 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 can be determined using the BLASTN (nucleic acid level) or BLASTX (amino acid level) programs (Altschul et al. J. Mol. Biol., 215: 403-410, 1990). These programs are 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). When analyzing nucleotide sequences using BLASTN, 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 techniques for these analysis methods are publicly known. Additions or deletions (e.g., gaps) may be permitted to optimally align the nucleotide or amino acid sequences being compared.

[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, respectively, represent the nucleotide sequence (Open Reading Frame: ORF) of the gene encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 1, 3, or 5, and SEQ ID NOs: 7.

[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. 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 double-stranded polynucleotide specific to the base sequence is formed, and a non-specific double-stranded polynucleotide is not formed. In other words, it can be said that these are 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 described: 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 for suppressing the expression of the gene encoding PAP2 is not particularly limited, and any method used in this field can be used. Examples include ZFN, TALEN, CRISPR / Cas family proteins, PPR (Pentatrico Peptide Repeat) motifs, 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, Cas10, etc. The above-mentioned Cas family proteins also include those whose functions have been appropriately modified. Each method is described, for example, in the following literature: a review on plant genome editing (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.), and ZFN (Kim YG et al., Proc. Natl. Acad. Sci. USA 93:1156-1160, 1996), TALEN (Christian et al., Genetics, 186:757-761, 2010), Cas family 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 Science, University of Tokyo Press (2003)), siRNA (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. USA86:10006-10010 (1989). From the viewpoint of simplicity and practical use, genome editing is preferred. Furthermore, suppression of gene expression is preferably performed using a null isolate.

[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) is constructed, introduced into plant cells, and a transformed plant is 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 somatic embryos, thereby obtaining a complete plant.

[0037] In addition, when plant tissue, such as leaf discs, are 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, to which 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 have been added, 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. Examples of media that can be used include common media such as LS medium and MS medium. In the above-mentioned transformants, 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] The methods used to examine the above-mentioned DNA, RNA, or proteins are conventionally known methods and are not particularly limited, but 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 a plurality of diseases. Further, in one embodiment of the present invention, the disease resistance may be resistance to at least two or more diseases selected from the group consisting of bacterial wilt, soft rot, late blight and scab.

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

[0043] As used herein, the phrase "having a mutation in a gene encoding PAP2" means that the nucleotide sequence of the gene encoding PAP2 is altered to such an 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, and examples thereof include the aforementioned ZFN, TALEN, CRISPR / Cas family proteins, ion beam irradiation, and ultraviolet irradiation.

[0044] In the present Solanum plant, the descriptions in the above section (1. Method for producing a disease-resistant plant) are incorporated herein by reference for the definitions of "Solanum plant", "disease resistance", and "improved disease resistance".

[0045] The present Solanum plant includes any of the whole plant, plant organs (e.g., roots, stems, leaves, petals, seeds, fruits, etc.), plant tissues (e.g., epidermis, phloem, parenchyma, xylem, vascular bundles, etc.), plant cells, calli, and the like. It also includes protoplasts, shoot primordia, multiple shoots, and hairy roots. In one embodiment of the present invention, it is preferably the whole plant, 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.

[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]

[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]

[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 their 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) was capable of encoding 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. Genome editing construct construction for DS1-1 and DS1-2) Using CRISPR direct (http: / / crispr.dbcls.jp / ), two target sites were set in the second exon of StDS1-1 (Figure 1). For StDS1-1, the target sites were designed in regions where no polymorphisms were observed between alleles. For StDS1-2, one target site each was set in the second and fourth exons (Figure 1). Table 3 shows the target sites for each gene.

[0055]

[0056] For the preparation 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. Following the method described in Hashimoto et al., 2018, constructs were created that simultaneously expressed gRNAs targeting both target sites for StDS1-1 and StDS1-2, respectively. The obtained constructs were introduced into the Agrobacterium tumefaciens GV3101 (pMP90) strain by electroporation. Transformed Agrobacterium were selected on LB agar 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 of StDS1-1 and StDS1-2) From wild-type (Sayaka) individuals cultivated under sterile conditions for about 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 solution, they were transferred to 3C5ZR agar medium (coexistence medium) supplemented with a final concentration of 20 mg / L acetosyringone, and coexistence culture was carried out in a chamber at 20°C for 3 days. After co-culture, stem sections 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 rooting selection medium. The rooting selection medium was changed every two weeks, and rooting selection was performed a total of three times to select candidate transformants. The various media were prepared as follows.

[0058]

[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 media) was added, and the mixture was autoclaved at 121°C for 15 minutes.

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

[0061]

[0062]

[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 autoclaved at 121°C for 15 minutes. After that, 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 of StDS1-1 and StDS1-2) A small amount of leaves from the candidate transformants obtained in the previous section was 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 incubated at 95°C for 10 minutes using a thermal cycler. 50 μL of sterile water was added to the reaction solution, vortexed, and spun down to obtain the genome extract. Next, the extracted genomic DNA was used as a template to PCR amplify the fragment containing the target site. KOD FX Neo (TOYOBO) and the following primers were used to amplify the fragment 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]

[0066] For the StDS1-1 single knockout candidate, a deletion of several bases was observed at target site 2, and the wild-type sequence was not found in any of the StDS1-1a, 1b, or 1c alleles (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] (5. 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 a StDS1-1 knockout plant (TF3) as the background. The same construct used to create 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 identified. PCR fragments containing the target site of StDS1-2 were cloned from stem sections of the obtained line (redifferentiation) and their base sequences were 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), leading to the conclusion 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 was tested using in vitro resistance testing (Habe 2018 American Journal of Potato Research (2018) 95:311-316) and stem injection inoculation 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]

[0070] Sterile cultured plants were transplanted into test tubes using vermiculite as a growing medium and cultured in MS liquid medium. In in vitro resistance testing, 1 × 10⁻⁶ plants were cultured. ２Inoculation was performed by dropping a bacterial suspension adjusted to cfu / ml onto the growing medium in test tubes containing plant cultures. The degree of disease development 20 days after inoculation was evaluated according to the following criteria. A disease 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 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 existing bacterial wilt-resistant varieties (Nagasaki Kogane) (Figure 6 and Table 9).

[0071]

[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 x 10⁻⁶ doses were administered. ４ Inoculation 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 knockout line of DS1-1 (TF3, labeled "T3" in the figure) and the single knockout line of DS1-2 (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 additional lines of StDS1-1 and StDS1-2 double mutants 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 lines of StDS1-1 and StDS1-2 double mutants (denoted as D7, D41, and E12) were obtained. Bacterial wilt resistance testing was performed using the same method. As a result, similar to NS1_R3 mentioned above, they 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 ８ Inoculation was performed by dropwise adding 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 single knockout line of DS1-1 (TF3) showed delayed progression of decay (resistance) (Figure 10). Furthermore, soft rot resistance was quantitatively examined using the single knockout line of DS1-1 (TF3), the single knockout line of DS1-2 (V4_2, labeled "V4" in the figure), and the double knockout line (NS1_R3, labeled "NS" in the figure) (Figure 11). 1 × 10 ８ A 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, decay was suppressed (resistance was shown) in the single-deficient lines (TF3, V4) and double-knockout lines (NS) of DS1-1, with the double-knockout lines (NS) showing the strongest resistance to soft rot.

[0075] (8. Late blight resistance test) The late blight resistance test is performed using tubers and acclimatized plants (Yoshioka et al. 1999 Plant Cell Physiol 40:993-8). Phytophora insectans Race 0 is used as the test fungus. 1 × 10 ４ The fungal solution, adjusted to zoospores / ml, is inoculated onto the 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 8 Tubers or acclimatized plants are grown in soil containing vermiculite and potting mix 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.

[0077] The present invention is extremely useful in the field of agricultural production because it enables the production of Solanum species with improved disease resistance. This application is based on Japanese Patent Application No. 2025-029699 (filed on February 27, 2025) and Japanese Patent Application No. 2026-028413 (filed on February 25, 2026), the contents of which are fully incorporated herein.

Claims

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

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

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 NO: 1, 3, or 5; (b) a gene encoding a protein having phosphatidic acid phosphatase 2 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 shown in SEQ ID NO: 1, 3, or 5; (c) a gene encoding a protein having phosphatidic acid phosphatase 2 activity, consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, 3, or 5; (d) a gene consisting of the nucleotide sequence shown in SEQ 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 encodes a protein having phosphatidic acid phosphatase II activity; (f) A gene that encodes a protein having an amino acid sequence described in Sequence ID No. 7; (g) A gene that encodes a protein having phosphatidic acid phosphatase II activity, having 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 that encodes a protein having phosphatidic acid phosphatase II activity, having an amino acid sequence having 80% or more identity with the amino acid sequence described in Sequence ID No. 7; (i) A gene having a base 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 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. The plant according to claim 9, which is a cultivated variety.

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

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