Potato cyst nematode disease control agent

Abstract

The present invention aims to make potato cyst nematode disease resistance genes available, and to provide a method for producing resistant varieties possessing such genes, as well as a simple method for determining resistance. [Solution] A control agent for potato cyst nematode disease in target plants, comprising a nucleic acid containing a base sequence encoding a polypeptide consisting of a specific amino acid sequence, and a transformant that expresses the agent and exhibits resistance to potato cyst nematode disease.

Description

[Technical Field]

[0001] The present invention relates to a control agent for potato cyst nematode disease, a transformant exhibiting resistance to potato cyst nematode disease, and a method for producing potato cyst nematode-resistant plants. [Background technology]

[0002] The potato cyst nematode is a nematode belonging to the order Stemonematoda, and it infests plants in the Chenopodiaceae and Solanaceae families, among others. In particular, it is one of the most important pests and diseases of potatoes, causing poor growth, reduced yield, and even death.

[0003] Methods used to control these nematodes include chemical control, cultural control methods such as crop rotation, and physical control methods such as flooding and soil reduction. Of these, the most cost-effective, environmentally friendly method is the use of resistant varieties.

[0004] The search for resistance genes against these nematodes has been extensively conducted, and the surrounding sequences of the region where the resistance gene H1 is believed to exist have been examined in detail (Non-Patent Literature 1).

[0005] However, despite such detailed investigations, the resistance gene H1 has not been identified. Furthermore, this candidate region is approximately 120kb long, and since the length that can be amplified by conventional PCR is only a few hundred to 1kb, it was not practical to use this region to determine the presence or absence of resistance or to introduce the resistance gene. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Finkers-Tomczak, A., et al, Theor Appl Genet., 2011, 122(3):595-608. [Overview of the project] [Problems that the invention aims to solve]

[0007] Given this background, the method used to confer resistance to the target plant has been to cross a variety susceptible to the cyst nematode with a resistant variety, thereby conferring resistance to the susceptible variety. However, in this case, desirable traits of the original susceptible variety are lost, or additional effort is required to select individuals that possess those traits, making the conferral of resistance inefficient.

[0008] Furthermore, at the molecular level, resistance has been detected using methods based on DNA markers that are highly likely to be linked to unidentified resistance genes. However, even if the DNA markers are highly likely to be linked, it cannot be definitively concluded that individuals in which the DNA markers are detected necessarily possess resistance to the cyst nematode in question. Therefore, the only way to confirm resistance is ultimately to inoculate the individuals with cyst nematodes, which requires a period of at least two months.

[0009] Therefore, the object of the present invention is to make potato cyst nematode disease resistance genes available, and to provide a method for producing resistant varieties having such genes and a simple method for determining resistance. [Means for solving the problem]

[0010] To solve the above problems, the inventors conducted intensive research and discovered that an unidentified potato cyst nematode disease resistance gene exists outside the expected region. This invention is based on this new finding and provides the following:

[0011] [1] A disease control agent for potato cyst nematode (Globodera rostochiensis) in target plants comprising a nucleic acid containing a base sequence encoding a polypeptide consisting of any one of the following (a) to (c): (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence or a fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. [2] The cyst nematode disease control agent according to [1], wherein the base sequence consists of any one of the following (1) to (4): (1) the base sequence shown in Sequence ID No. 2 or a fragment thereof; (2) a base sequence in which one or more bases are added, deleted, and / or substituted in the base sequence shown in Sequence ID No. 2 or a fragment thereof; (3) a base sequence or a fragment thereof having 90% or more sequence identity with the base sequence shown in Sequence ID No. 2; or (4) a base sequence or a fragment thereof that can hybridize with a base sequence complementary to the base sequence shown in Sequence ID No. 2 under highly stringent conditions. [3] A disease control agent according to [1] or [2], wherein the target plant is a plant of the genus Solanum. [4] A disease control agent as described in [3], wherein the target plant is potato. [5] Transformants expressing a polypeptide containing any one of the following (a) to (c) and exhibiting resistance to potato cyst nematode disease: (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence or a fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. A potato cyst nematode disease control composition comprising a disease control agent described in any of [6][1] to [4]. A method for producing potato cyst nematode-resistant plants, comprising an introduction step of introducing a disease control agent described in any of [7][1] to [4] into the cells of a target plant that does not exhibit resistance to potato cyst nematode disease. [8] A method for producing a genome-edited plant, comprising an introduction step of introducing a nucleic acid encoding a polypeptide having one of the following amino acid sequences (a) to (c) into the cells of a target plant: (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. A method for controlling potato cyst nematode disease, comprising an introduction step of introducing a disease resistance gene transfer construct containing a disease control agent described in any of [9][1] to [4] into the cells of a target plant that does not exhibit resistance to potato cyst nematode disease.

[10] A potato cyst nematode resistance determination kit comprising a binding molecule capable of binding to a polypeptide consisting of any one of the following (a) to (c), and / or a primer and / or probe capable of detecting nucleic acids containing a base sequence encoding the polypeptide: (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence or a fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[11] The kit according to

[10] wherein the primer and / or probe comprises a nucleotide sequence containing 18 or more consecutive bases in any one of the following nucleotide sequences (1) to (4): (1) the nucleotide sequence shown in SEQ ID NO: 2 or a fragment thereof; (2) a nucleotide sequence in which one or more bases are added, deleted, and / or substituted in the nucleotide sequence shown in SEQ ID NO: 2 or a fragment thereof; (3) a nucleotide sequence having 90% or more sequence identity with the nucleotide sequence shown in SEQ ID NO: 2 or a fragment thereof; or (4) a nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 under highly stringent conditions.

[12] A potato cyst nematode resistance determination device comprising a binding molecule capable of binding to a polypeptide consisting of any one of the following (a) to (c), and / or a primer and / or probe capable of detecting nucleic acids including a base sequence encoding the polypeptide: (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence or a fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[13] A method for determining resistance to potato cyst nematodes, comprising a detection step of detecting a polypeptide consisting of any one of the following (a) to (c) and / or a nucleic acid containing a base sequence encoding the polypeptide in a sample obtained from a target plant, and a determination step of determining that the target plant is resistant to cyst nematodes if the presence of the nucleic acid and / or polypeptide is detected: (a) the amino acid sequence shown in Sequence ID No. 1 or a fragment sequence thereof; (b) the amino acid sequence shown in Sequence ID No. 1 in which one or more amino acids are added, deleted, and / or substituted or a fragment sequence thereof; (c) the amino acid sequence shown in Sequence ID No. 1 having 90% or more sequence identity or a fragment sequence thereof.

[14] Potato cyst nematode disease inhibitors comprising a polypeptide having an amino acid sequence described in any one of (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; (b) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in SEQ ID NO: 1 or a fragment thereof; or (c) an amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO: 1. [Effects of the Invention]

[0012] According to the present invention, a novel disease control agent based on a potato cyst nematode disease resistance gene can be provided.

[0013] According to the disease control method of the present invention, disease control can be performed without changing other traits.

[0014] According to the resistance determination method of the present invention, the presence or absence of resistance to potato cyst nematode disease can be easily determined in a short period.

Brief Description of Drawings

[0015] [Figure 1] It is a diagram showing the configuration of a gene modification vector containing three guide RNAs used in Example 3. [Figure 2] It is a diagram showing the insertion or deletion mutation status in ORF45 of a transformant genome-edited by vector d45_dX in Example 3. Figure 2A shows the mutation site of transformant d45_dX_1, Figure 2B shows the mutation site of transformant d45_dX_2, and Figure 2C shows the mutation site of transformant d45_dX_3. In the figure, the underline indicates the target sequence of the guide RNA, and it is shown together with the name of the guide RNA targeting that sequence and the number of inserted (ins) or deleted (del) bases. [Figure 3] It is a diagram showing the insertion or deletion mutation status in ORFX of a transformant genome-edited by vector d45_dX in Example 3. Figure 3A shows the mutation site of transformant d45_dX_1, Figure 3B shows the mutation site of transformant d45_dX_2, and Figure 3C shows the mutation site of transformant d45_dX_3. In the figure, the underline indicates the target sequence of the guide RNA, and it is shown together with the name of the guide RNA targeting that sequence and the number of inserted (ins) or deleted (del) bases. [Figure 4] It is a diagram showing the insertion or deletion mutation status in ORF45 of a transformant genome-edited by vector d45_X in Example 3. Figure 4A shows the mutation site of transformant d45_X_1, Figure 4B shows the mutation site of transformant d45_X_2, and Figure 4C shows the mutation site of transformant d45_X_3. In the figure, the underline indicates the target sequence (complementary sequence) of the guide RNA, and it is shown together with the name of the guide RNA targeting that sequence and the number of inserted (ins) or deleted (del) bases. [Figure 5]This figure shows the cyst formation observed in the transformant d45_dX_2, which was genome-edited with the vector d45_dX in Example 3. In the figure, the dashed circles indicate the location of the formed cysts. [Modes for carrying out the invention]

[0016] 1. Potato cyst nematode disease control agent 1-1. Overview A first aspect of the present invention is a potato cyst nematode disease control agent. The disease control agent of the present invention contains a nucleic acid containing a base sequence encoding a specific polypeptide as an active ingredient, and controls target plants from potato cyst nematode disease. The disease control agent of the present invention can be used as an active ingredient in disease control compositions and can also be used in methods for producing disease-resistant plants, etc.

[0017] 1-2.Definition The terms used in this specification are defined below. A "cyst nematode" is a type of nematode that parasitizes plant roots and other structures. It is characterized by the formation of cysts on the plant surface by adult female nematodes that have grown larger by absorbing nutrients within the roots, and which contain eggs inside their bodies. The eggs inside the cysts hatch in response to substances secreted by the host plant's roots, and the larvae emerge from the cysts and invade the roots to parasitize them.

[0018] Potato cyst nematode (Globodera rostochiensis; hereinafter often referred to as "Gr") is a cyst nematode belonging to the genus Globodera that parasitizes plants. It is a disease and pest that infests the underground parts of Solanaceae plants such as potatoes, including the roots and tubers. Typically, the adult female is yellow to golden in color. When Gr absorbs nutrients from the host plant, symptoms such as wilting, yellowing and discoloration of leaves appear, and in severe cases, the plant dies. Examples of Gr prototypes include Ro1 (R1A), Ro2 (R2A), Ro3 (R3A), Ro4 (R1B), and Ro5, but Gr in this specification encompasses all of them. For example, Ro1 (R1A) and Ro4 (R1B) can be preferred targets.

[0019] In this specification, "potato cyst nematode disease" includes any disease caused by the potato cyst nematode. In this specification, "potato cyst nematode disease control" refers to reducing the occurrence or severity of potato cyst nematode disease.

[0020] In this specification, "potato cyst nematode disease resistance" refers to the ability to prevent or suppress damage and / or parasitism (infection) of host plants by potato cyst nematodes. The resistance of plants to potato cyst nematodes can be tested using methods known to those skilled in the art. Examples include inoculation tests in fields, pots, or cups. For example, a certain number (e.g., 200) of potato cyst nematode larvae are inoculated into the culture medium of a test plant, and the infection status is evaluated after a certain period (e.g., 2 months). The evaluation can be performed by observing the presence and degree of symptoms exhibited by the plant, such as yellowing, discoloration, and death of leaves; the presence and number of cysts (typically granular female adults about 0.4-0.7 mm in size) on the roots and tubers of the plant; and the presence and number of potato cyst nematode egg masses on the surface of the roots and tubers and / or potato cyst nematode egg masses. Alternatively, for example, the soil containing potato cyst nematodes and the target plant can be placed in a transparent container, stored in darkness for approximately 50 days, and then the presence and number of cysts can be observed from the bottom and sides of the container (Narabe et al., 2014, Kyoto University Press, pp. 183-184). The observation method is not particularly limited, but for example, visual inspection, magnification with a magnifying glass or stereomicroscope can be used.

[0021] In this specification, "whole" of a plant means the entire region that constitutes a living plant. "Part" of a plant means a part of a living plant, specifically an organ (for example, roots, stems, leaves, flowers, epidermis, or combinations thereof, or pollen, egg cells, or seeds, etc.), a tissue or part thereof consisting of morphologically and / or functionally differentiated cell groups, or a cell.

[0022] In this specification, "multiple items" means, for example, 2 to 100 items, 2 to 90 items, 2 to 80 items, 2 to 70 items, 2 to 60 items, 2 to 50 items, 2 to 40 items, 2 to 30 items, 2 to 20 items, 2 to 15 items, 2 to 10 items, 2 to 7 items, 2 to 5 items, 2 to 4 items, or 2 to 3 items (several items).

[0023] "Amino acid identity" refers to the percentage of identical amino acid residues in the total number of amino acid residues when two polypeptide sequences being compared are aligned by inserting gaps as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences for calculating amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW. For example, amino acid sequence identity can be easily calculated using genetic information processing software GENETYX (https: / / www.genetyx.co.jp / ) or the NCBI-provided BLAST server (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). "Base identity" is calculated similarly for base sequences. While not limited to specific algorithms, available algorithms for nucleic acids include the aforementioned BLAST and FASTA, as well as MUMmer.

[0024] In this specification, "(amino acid) substitution" refers to substitutions within a group of conserved amino acids that have similar properties such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that make up natural proteins. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp). Amino acid substitutions within these groups are preferred because they are known to cause little change in the properties of polypeptides.

[0025] In this specification, "stringent conditions" mean conditions under which nonspecific hybrids are less likely to form and hybridize to the target sequence to a greater extent than with other sequences (e.g., a measurement of the mean of the background measurement + the standard error of the background measurement × 2 or more). "Highly stringent conditions" mean conditions under which nonspecific hybrids are less likely to form, or not formed at all. Generally, the lower the salt concentration and the higher the temperature of the reaction conditions, the more stringent the conditions. For example, washing after hybridization is performed at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C with 0.1 × SSC and 0.1% SDS. In addition, the stringency of hybridization can be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

[0026] 1-3. Structure The disease control agent of the present invention consists of nucleic acids containing a base sequence encoding a specific polypeptide as an essential component. This will be explained in detail below.

[0027] 1-3-1. Active Ingredients (A) Polynucleotide encoding polypeptide The potato cyst nematode control agent according to this embodiment consists of nucleic acids encoding a specific polypeptide or a fragment thereof.

[0028] The polypeptide in this embodiment (hereinafter often referred to as "the polypeptide of the present invention") consists of an amino acid sequence described in any one of the following (a) to (c): (a) The amino acid sequence or fragment thereof shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having sequence identity of 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in Sequence ID No. 1.

[0029] In this specification, "fragment sequence" of an amino acid sequence refers to a fragment of the amino acid sequence of the present invention having any length, wherein the polypeptide having that sequence has potato cyst nematode control activity. Furthermore, "fragment sequence" of a nucleotide sequence refers to a nucleotide sequence that encodes such an amino acid fragment sequence. The length of the sequence is not particularly limited, but it should be, for example, a continuous region of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or 1100 amino acids from the target polypeptide, or a region encoding such a region (for example, at least 150, 300, 450, 600, 750, 900, 1050, 1200, 1350, 1500, 1650, 1800, 1950, 2100, 2250, 2400, 2550, 2700, 2850, 3000, or 3300 bases).

[0030] For example, the variant sequences and fragment sequences described in (b) and (c) retain the amino acid sequence of the amino acid region shown in SEQ ID NO: 1, selected from the group consisting of the amino acid regions at positions 479-719, 345-417, 497-587, 497-519, 497-612, 498-588, 496-589, 820-848, and 487-573, for example, the amino acid sequence of the region at positions 479-719 and / or 497-519. Furthermore, for example, the variant sequences and fragment sequences described in (b) and (c) are the amino acid sequences shown in SEQ ID NO: 1, specifically positions 376-848, 479-848, 479-612, 479-589, 479-588, 479-587, 479-573, 479-519, 487-848, 487-719, 487-612, 487-589, 487-588, 487-587, 487-573, and 487-519. It retains the amino acid sequences of positions 496-848, 496-719, 496-612, 496-588, 496-587, 496-573, 496-519, 497-848, 497-719, 497-589, 497-588, 497-573, 498-848, 498-719, 498-612, 498-589, 498-587, 498-573, and 498-519.

[0031] The mutant sequences and fragment sequences described in (b) and (c) preferably have an activity of 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the potato cyst nematode control activity of the polypeptide having the amino acid sequence shown in Sequence ID No. 1, or an activity equivalent to or greater thereto. For example, if a gene region located near the gene region encoding the additional polypeptide of the present invention (ORF45) (e.g., a gene region adjacent to ORF45) encodes the mutant sequence described in (b) or (c), it can be presumed that the mutant sequence satisfies the above-mentioned potato cyst nematode control activity. Base sequences, gene regions, the amino acid sequences encoded therein, and their positional relationships are readily available from public databases, etc.

[0032] The polypeptide of the present invention may be provided as a disease inhibitor for potato cyst nematodes.

[0033] The base sequence of the nucleic acid encoding the polypeptide of the present invention (hereinafter often referred to as "the nucleic acid of the present invention") is not particularly limited as long as it encodes the polypeptide of the present invention.

[0034] Examples of such base sequences include the base sequences shown in any one of the following (1) to (4): (1) The base sequence or fragment thereof shown in Sequence ID No. 2; (2) A nucleotide sequence or fragment thereof in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 2; (3) A nucleotide sequence or a fragment thereof having 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2; or (4) A nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2 under highly stringent conditions.

[0035] The base sequence of the nucleic acid of the present invention may be a base sequence that is codon-optimized to match the frequency of codon use in the cell into which the nucleic acid is introduced.

[0036] The nucleic acid of the present invention may be DNA or RNA such as mRNA. If the nucleic acid of the present invention is mRNA, its base sequence may be an mRNA that includes a coding region in which thymine (T) is replaced with uracil (U) in any of the base sequences exemplified above. The mRNA corresponding to the polynucleotide of the present invention may also include, in addition to the coding region, a 5' cap structure, a 3' polyA chain, a 5' untranslated region upstream of the start codon (5' UTR), and / or a 3' untranslated region downstream of the stop codon (3' UTR). The 5' UTR and / or 3' UTR may include sequences for regulating the amount of translation from the mRNA.

[0037] The nucleic acids of the present invention can be prepared using any method known in the art. For example, they can be prepared by cleaving a gene cassette with a suitable restriction enzyme and ligating it to restriction enzyme sites or multi-cloning sites of a suitable vector that forms the backbone. Alternatively, they may be prepared using techniques such as in-fusion cloning, TA cloning, or double crossover.

[0038] (B) A vector containing polynucleotides The disease control agent according to this embodiment may contain the nucleic acid of the present invention in the form of an expression vector.

[0039] The type of vector of the present invention is not particularly limited. Examples include plasmids (bacterial plasmids, yeast plasmids, etc.), autonomously replicating vectors such as bacmid, viral vectors, artificial chromosomes, or vectors capable of homologous recombination within chromosomes, or combinations thereof. The vector may also be a shuttle vector capable of replicating within other bacteria. Furthermore, the vector of the present invention may be an expression vector capable of expressing the polypeptide of the present invention, or it may be a vector that cannot express the polypeptide of the present invention on its own (for example, a recombinant vector).

[0040] An expression vector is a vector containing the aforementioned polynucleotides in an expressionable state. In this specification, "expressionable state" means that the gene to be expressed is located in the downstream region of the promoter, which is under the control of the promoter.

[0041] The following explanation uses an expression vector as an example, but is not limited to this. In the case of an expression vector, the essential components include a promoter in addition to the nucleic acid of the present invention.

[0042] In the case of plasmid-based vectors, while not limited to specific plasmids, examples include pPZP, pSMA, pUC (pUC57, pUC18, pUC19, pUC9, etc.), pLC (pLC41, etc.), pAL (pAL51, pAL156, etc.), pBR, pBluescript (Agilent Technologies), and pTriEX. TM You can use binary vectors from systems such as TaKaRa, pBI systems (pBI121, pBI101, pBI221, pBI2113, pBI101.2, etc.), pRI systems, or pGW systems, or intermediate vector systems (pLGV23Neo, pNCAT, etc.).

[0043] In the case of expression vectors using viruses, viruses such as cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV), and tobacco mosaic virus (TMV) can be used.

[0044] Furthermore, when using the Agrobacterium method, expression vectors suitable for the Agrobacterium method, such as binary vectors, or modified vectors thereof, can also be used. Examples of such expression vectors include pBI121, pBIN19, pSMAB704, pCAMBIA, and pGreen. In addition, when introducing into plant cells using the Agrobacterium method, it is desirable to include right-boundary sequences (RB) and left-boundary sequences (LB) derived from the Agrobacterium T-DNA sequence.

[0045] Various types of promoters can be used, such as overexpression promoters, constitutive promoters, site-specific promoters, time-specific promoters, and / or inducible promoters. The origin of the promoter is not particularly limited, as long as it can induce transcription in plant cells. For example, it may be plant-derived or non-plant-derived. Specific examples of constitutive promoters that can be overexpressed and actuated in plant cells include the isopentenyltransferase (IPT) gene promoter (e.g., the Agrobacterium tumefaciens-derived IPT gene promoter), the cauliflower mosaic virus (CaMV)-derived 35S promoter, its modified promoters (e.g., the 2x35S promoter, the El2-35S omega promoter), the nopaline synthase (NOS) gene promoter (e.g., the Ti plasmid-derived NOS gene promoter Pnos), the ubiquitin promoter (e.g., the parsley ubiquitin (PcUbi) promoter, the maize ubiquitin promoter), the actin promoter (e.g., the rice actin promoter), the alcohol dehydrogenase (ADH) gene promoter, the tobacco-derived infection-specific protein (PR protein) promoter, the ribulose diphosphate carboxylase (RuBisco) promoter, and the U6 promoter (e.g., the Arabidopsis thaliana U6 (AtU6) promoter). Various plant species of RuBisco small subunit (RuBisco ssu) promoters or histone promoters can also be used. Examples of inductive promoters include heat shock promoters that can be controlled by temperature, and tetracycline-responsive promoters that can be controlled by the presence or absence of tetracycline.

[0046] Expression vectors may also include terminators, enhancers, poly(A) addition signals, 5'-UTR (untranslated region) sequences, intron sequences, ribosome-binding sequences, labeling or selection marker genes, multicloning sites, nuclease recognition sequences, and / or replication origins. The specific types of these elements are not particularly limited, as long as they can exert their function within the host cell. Appropriate selections should be made based on the plant cells or plant host to which the expression vector is introduced, and those known in the relevant field.

[0047] Examples of terminators include the Pea3A terminator, the heat shock protein (HSP) gene terminator, the nopaline synthase (NOS) gene terminator, the octopin synthase (OCS) gene terminator, the CaMV 35S terminator, the 3' terminator of E. coli lipopolyprotein lpp, the trp operon terminator, the amyB terminator, and the ADH1 gene terminator. There are no particular limitations as long as the sequence can terminate the transcription of the gene transcribed by the promoter.

[0048] Examples of enhancers include enhancer regions containing upstream sequences within the CaMV 35S promoter. The enhancer is not particularly limited as long as it can enhance the expression efficiency of nucleic acids encoding active peptides.

[0049] Examples of nuclease recognition sequences include restriction enzyme recognition sequences, loxP sequences recognized by Cre recombinant enzymes, sequences targeted by artificial nucleases such as ZFNs and TALENs, sequences targeted by the PPR system, or sequences targeted by the CRISPR / Cas9 system. The SV40 replication origin sequence is an example of a replication origin sequence.

[0050] The selection marker gene is not particularly limited and can be any gene capable of selecting cells into which the vector of the present invention has been introduced. Examples of selection marker genes include drug resistance genes (e.g., tetracycline resistance gene, ampicillin resistance gene, kanamycin resistance gene, hygromycin resistance gene, spectinomycin resistance gene, chloramphenicol resistance gene, dihydrofolate reductase gene, or neomycin resistance gene), fluorescent or luminescent reporter genes (e.g., luciferase, β-galactosidase, β-glucuronidase (GUS), or green fluorescein protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase.

[0051] The nucleic acids and vectors of the present invention can be prepared using common techniques such as DNA recombination technology, PCR, and automated DNA / RNA synthesis.

[0052] DNA recombination techniques and PCR methods can be used, for example, those described in Ausubel et al., *Current Protocols in Molecular Biology*, John Willey & Sons, US (1993); Sambrook et al., *Molecular Cloning: A Laboratory Manual*, Cold Spring Harbor Laboratory Press, US (1989).

[0053] DNA can be chemically synthesized using an automated DNA synthesizer. The phosphoamidite method is commonly used for this synthesis, allowing for the automated synthesis of single-stranded DNA up to approximately 100 base pairs. Automated DNA synthesizers are commercially available from companies such as Polygen, ABI, and Thermo Fishers. Alternatively, DNA can be fabricated using cDNA cloning.

[0054] 1-3-2. Additional active ingredients The disease control agent in this embodiment may include nucleic acids encoding additional polypeptides. In this case, the additional polypeptides are not particularly limited and may be polypeptides involved in disease control or polypeptides that are not involved.

[0055] Specifically, for example, the present invention may include nucleic acids (hereinafter often referred to as "the additional nucleic acids of the present invention") that include a base sequence encoding an additional polypeptide consisting of any one of the amino acid sequences described in (d) to (f) below (hereinafter often referred to as "the additional polypeptide of the present invention"): (d) The amino acid sequence or fragment thereof shown in Sequence ID No. 3; (e) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 3; or (f) An amino acid sequence or a fragment thereof having 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence shown in Sequence ID No. 3.

[0056] Examples of fragment sequences include polypeptide fragments consisting of the amino acid sequence described in any one of the following (g) to (i): (g) The amino acid sequence shown in Sequence ID No. 22, consisting of positions 1 to 147 in the amino acid sequence shown in Sequence ID No. 3; (h) an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 22; or (i) Examples include amino acid sequences that have sequence identity of 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence shown in Sequence ID No. 22.

[0057] The specific nucleotide sequences encoding the additional polypeptides of the present invention are not particularly limited, but examples include the nucleotide sequences shown in any one of the following (5) to (8): (5) The base sequence or fragment sequence shown in Sequence ID No. 4; (6) A nucleotide sequence or fragment thereof in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 4; (7) A nucleotide sequence or fragment thereof having 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 4; or (8) A nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 4 under highly stringent conditions.

[0058] The specific nucleotide sequence encoding the polypeptide fragment consisting of any one of the amino acid sequences described in (g) to (i) above is not particularly limited, but examples include the nucleotide sequences shown in any one of (9) to (12) below: (9) The base sequence or fragment thereof shown in Sequence ID No. 23; (10) A nucleotide sequence or fragment thereof in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 23; (11) A nucleotide sequence or fragment thereof having 80% or more, 82% or more, 85% or more, 87% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 23; or (12) A nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 23 under highly stringent conditions.

[0059] In another embodiment, a disease control agent for potato cyst nematodes (Globodera rostochiensis) in target plants is provided, comprising a nucleic acid containing a base sequence encoding a polypeptide consisting of any one of the amino acid sequences described in (d) to (f) above. In yet another embodiment, a disease control agent for potato cyst nematodes (Globodera rostochiensis) in target plants is provided, comprising a nucleic acid containing a base sequence encoding a polypeptide fragment consisting of an amino acid sequence described in any one of the amino acid sequences described in (g) to (i) above. In yet another embodiment, a disease inhibitor for potato cyst nematodes is provided, comprising a polypeptide consisting of an amino acid sequence described in any one of the amino acid sequences described in (d) to (f) above or a polypeptide fragment consisting of an amino acid sequence described in any one of the amino acid sequences described in (g) to (i) above.

[0060] The additional nucleic acids of the present invention may be included in the form of a vector containing them. In that case, the configuration of the vector will be the same as described above for the vector containing the nucleic acids of the present invention.

[0061] 1-3-3. Other ingredients The potato cyst nematode disease control agent according to this embodiment may be provided as a potato cyst nematode disease control composition, comprising additional components in addition to the above-mentioned active ingredients.

[0062] Furthermore, the potato cyst nematode disease inhibitor containing the polypeptide of the present invention may be provided as a potato cyst nematode disease inhibitory composition containing additional components in addition to the above-mentioned active ingredient.

[0063] The amount of active ingredient contained per unit amount in a composition depends on various conditions such as the dosage form, the type of target plant, the application site, and the application method when used for disease control or disease suppression. In the case of disease control compositions, it is preferable that the composition contains a sufficient amount of nucleic acid, which is the active ingredient, for introduction into the target plant. In the case of disease suppression compositions, it is preferable that the composition contains a sufficient amount of polypeptide, which is the active ingredient, for introduction into or application to the target plant. Therefore, within the scope of common technical knowledge in the art, the amount of the agent contained in the composition of the present invention should be determined by considering each condition so that the amount of the agent becomes effective after application.

[0064] The compositions of the present invention may contain agriculturally acceptable carriers. In this specification, "agriculturally acceptable carriers" means substances that do not substantially affect the activity of the disease control agent of the present invention and that, when applied to plant cultivation, have little to no harmful effects on the environment such as soil and water quality, or have little to no harmful effects on animals, especially humans. Examples include solvents, auxiliaries, excipients, emulsifiers, dispersants, surfactants, and the like.

[0065] The compositions of the present invention may contain other pharmacologically active ingredients, namely nematode miticides, herbicides, and fertilizers (e.g., urea, ammonium nitrate, superphosphate), to the extent that they do not affect the activity of the active ingredient.

[0066] 1-3-4. Dosage Form The dosage form of the composition of the present invention may be any form, as long as the disease control agent of the present invention can penetrate into the plant body to which it is applied, or the peptide can be retained in the plant body, its surface, or the surrounding soil. For example, it can be a liquid formulation or a solid formulation. In the case of a liquid formulation, examples include a solution formulation in which the active ingredient is suspended in a suitable solution, an oily dispersion liquid formulation, an emulsion formulation, and a suspension formulation. In the case of a solid formulation, there are no particular limitations as long as the active ingredient can act on the plant to which it is applied. For example, examples include a powder formulation, a spray formulation, a paste formulation, and a gel formulation.

[0067] 1-4. Target Plants The target plants in this specification are not particularly limited to plants that may exhibit diseases caused by potato cyst nematodes. For example, they may be host plants of potato cyst nematodes and / or white potato cyst nematodes.

[0068] For example, any plant belonging to the Chenopodiaceae or Solanaceae families is acceptable. Specifically, examples include the genus Chenopodium in the Chenopodiaceae family, and the genus Solanum, Datura (e.g., Datura tatula, Datura ferox), Hyoscyamus (e.g., Hyoscyamus niger), Nicotiana (e.g., Nicotiana acuminata), Physalis (e.g., Physalis longifolia, Physalis philadelphica), Physochlaina (e.g., Physochlaina orientalis), Salvellus, Saracha (e.g., Saracha jaltomata), and / or the genus Lycopersicon (e.g., Lycopersicon esculentum, Lycopersicon pimpinellifolium).

[0069] Examples of species in the Solanum genus include: Solanum acaule, Solanum americanum, Solanum aviculare, Solanum cardiophyllum, Solanum ehrenbergii, Solanum gilo, Solanum indicum, (Solanum marginatum), Solanum mauritianum, Solanum melongena, Solanum muricatum, Solanum nigrum, Solanum oplocense, Solanum quitoense, Solanum sarrachoides, and Solanum Solanum scabrum), Solanum spegazzinii, Solanum tuberosum, Solanum aethiopicum, Solanum ajanhuiri, Solanum ajuscoense, Solanum alandiae, Solanum alatum, Solanum anomalocalyx, Solanum antipoviczii, Solanum armatum, Solanum ascasabi, Solanum auriculatum, Solanum asperum (Solanum Solanum asperum, Solanum berthaultii, Solanum blodgettii, Solanum boergeri, Solanum brevidensSolanum brevidens), Solanum brevimucronatum, Solanum bukasovii, Solanum bulbocastanum, Solanum calcense, Solanum calcense × Solanum cardenasii, Solanum caldasii, Solanum canasense, Solanum capsicibaccatum, Solanum capsicoides, Solanum carolinense, Solanum chacoense, Solanum chaucha chaucha), Solanum chenopodioides, Solanum chloropetalum, Solanum citrillifolium, Solanum coeruleiflorum, Solanum commersonii, Solanum curtilobum, Solanum curtipes, Solanum demissum, Solanum demissum × Solanum tuberosum, Solanum dulcamara, Solanum durum, Solanum eleagnifolium (Solanum Solanum elaeagnifolium), Solanum famatinae, Solanum fraxinifolium, Solanum fructo-tecto, Solanum garciaeSolanum garciae), Solanum gibberulosum, Solanum giganteum, Solanum gigantophyllum, Solanum glaucophyllum, Solanum goniocalyx, Solanum gourlayi, Solanum gracile, Solanum heterophyllum, Solanum heterodoxum, Solanum hirtum, Solanum hispidum, Solanum integrifolium, Solanum intusum (Solanum Solanum intrusum), Solanum jamesii, Solanum jujuyense, Solanum juzepczukii, Solanum kesselbrenneri, Solanum kurtzianum, Solanum lanciforme, Solanum lapazense, Solanum lechnoviczii, Solanum leptostygma, Solanum ligustrinum, Solanum longipedicellatum, Solanum luteum Solanum luteum), Solanum maculae, Solanum macrocarpon, Solanum maglia, Solanum malinchense, Solanum mamiriferum (SolanumSolanum mamilliferum), Solanum miniatum, Solanum mochiquense, Solanum multidissectum, Solanum neocardenasii, Solanum nitidibaccatum, Solanum ochroleucum, Solanum okadae, Solanum ottonis, Solanum pampasense, Solanum parodii, Solanum penelli, Solanum photeinocarpum, Solanum phleha (Solanum Solanum phureja), Solanum pinnatum, Solanum pinnatisectum, Solanum platense, Solanum platypterum, Solanum polyacanthos, Solanum polyadenium, Solanum prinophyllum, Solanum radicans, Solanum raphanifolium, Solanum rostratum, Solanum rybinii, Solanum salamanii, Solanum sartense (Solanum saltense), Solanum sambucinum, Solanum sanctae-rosae, Solanum schenkii, Solanum sickii (SolanumSolanum schickii), Solanum semidemissum, Solanum simplicifolium, Solanum sinaicum, Solanum sisymbrifolium, Solanum sodomaeum, Solanum soukupii, Solanum sparsipilum, Solanum stenotomum, Solanum stoloniferum, Solanum suaveolens, Solanum subandigenum, Solanum scurense (Solanum Solanum sucrense), Solanum tarijense, Solanum tenuifilamentum, Solanum tlaxcalense, Solanum tomentosum, Solanum toralopanum, Solanum triflorum, Solanum vallis-mexicae, Solanum vernei, Solanum verrucosum, Solanum villosum, Solanum violaceimarmoratum, Solanum wittmackie Solanum wittmackii), Solanum wittonense, Solanum xanti, Solanum yabari, Solanum zukkagnianum (SolanumExamples include *Zuccagnacea*, etc. Suitable target plants in this specification include, for example, eggplant, potato, or their hybrids.

[0070] 1-5. Effects The disease control agent of the present invention can confer resistance to potato cyst nematodes to plants susceptible to potato cyst nematodes. Furthermore, the disease control agent of the present invention can enhance resistance to potato cyst nematodes in plants that are resistant to potato cyst nematodes.

[0071] The potato cyst nematode disease control agent of the present invention can suppress nematode attraction activity, nematode migration into the roots, nodule formation, nodule maturation, nematode growth within the roots, and / or induction of giant cells in the host plant in plants to which the agent is applied.

[0072] 2. Plant Transformants 2-1. Overview A second aspect of the present invention is a transformant exhibiting resistance to potato cyst nematode disease. The transformant of the present invention expresses the polypeptide described in the first aspect and exhibits resistance to potato cyst nematode disease. The plant transformant or its offspring of the present invention have suppressed nematode attraction activity, nematode migration into the roots, nodule formation, nodule maturation, nematode growth in the roots, and / or induction of megacells in the host plant, and are resistant to potato cyst nematode disease.

[0073] 2-2. Composition In this specification, “transformed plant” refers to a plant host that has been genetically modified to acquire resistance to potato cyst nematode disease. This genetic modification includes both genetic recombination and genome editing. Preferably, transformation is performed by genome editing. In this specification, “genome editing” means a technique for introducing modifications to specific base sequences on genomic DNA.

[0074] The transformant of the present invention has a disease resistance gene and expresses the polypeptide of the present invention as described in the first embodiment. Here, "disease resistance gene" refers to the gene encoding the polypeptide of the present invention.

[0075] For example, the transformant of the present invention includes the nucleic acid of the present invention as described in the first embodiment, or a vector containing the nucleic acid of the present invention. It may also include additional nucleic acids, such as additional nucleic acids of the present invention or vectors containing them, as needed.

[0076] The host plant species to be transformed in this invention is not particularly limited. The host plant is any plant species that can serve as a host for the potato cyst nematode. The specific type is not particularly limited, but for example, the plants exemplified as target plants in the first embodiment can be used. For example, plants of the Solanaceae family, plants of the Solanum genus, or potatoes can be suitably used.

[0077] The plant transformants of the present invention include clones having the same genetic information. The transformants of the present invention also include parts of the plant collected from the first generation of transformants, such as plant tissues like epidermis, phloem, parenchyma, xylem, or vascular bundles; plant organs like leaves, petals, stems, roots, or seeds; or clones obtained from plant cells by plant tissue culture, cuttings, grafting, or layering; or new clones newly generated from vegetative reproductive organs obtained asexually from the first generation of transformants, such as rhizomes, tubers, corms, or runners; and somatic embryos induced by dedifferentiation from the first generation of transformants or clones derived therefrom. The transformant of the present invention may be, for example, a genetically modified organism.

[0078] In this specification, "later generation" means a host plant that is a sexually reproduced offspring of the first generation of the transformant, expressing the polypeptide of the present invention or harboring the nucleic acid of the present invention or a vector containing it, and exhibiting resistance to potato cyst nematode disease. The later generation is irrelevant.

[0079] 3. Method for creating potato cyst nematode-resistant plants 3-1. Overview A third aspect of the present invention relates to a method for producing potato cyst nematode-resistant plants. According to the method of the present invention, potato cyst nematode-resistant transformants described in the second aspect can be produced from potato cyst nematode-infected plants.

[0080] 3-2.Process The manufacturing method of the present invention includes an introduction step as an essential step, and a callus formation step, a regeneration step, and a selection step as optional steps. Each step will be described in detail below.

[0081] 3-2-1.Introduction process In this embodiment, the "introduction step" is the step of introducing a disease resistance gene introduction construct into the cells of a target plant. This step can be performed before, after, and / or simultaneously with the callus formation step. If this step is performed after the callus formation step, this step is the step of introducing the disease resistance gene introduction construct into the callus formed in the callus formation step.

[0082] The plant cells used in this process may be plant tissue fragments (e.g., leaf, stem, or root sections), callus, or protoplast.

[0083] The disease resistance gene transfer construct in this embodiment only needs to be capable of expressing the polypeptide of the present invention in the cells after the transfer, and its specific composition is not particularly limited. For example, the disease control agent described in the first embodiment, or the nucleic acid of the present invention or a vector containing the same can be used as a suitable construct. The composition of the disease control agent and the nucleic acid of the present invention or a vector containing the same is the same as described in the first embodiment. In addition, additional nucleic acids, such as additional nucleic acids of the present invention or vectors containing the same, may be included as needed.

[0084] Methods for introducing the expression vector include those known in the field, such as the Agrobacterium method, PEG-calcium phosphate method, electroporation method, liposome method, particle gun method, microinjection method, whisker method, plasma method, laser injection method, cell membrane permeable peptide method, etc. The introduced polynucleotide may be integrated into the host's genomic DNA, or it may exist in the introduced polynucleotide state (for example, remaining contained in the foreign vector). Furthermore, the introduced polynucleotide may be maintained within the host cell, as if integrated into the host's genomic DNA, or it may be retained transiently.

[0085] When using the Agrobacterium method, the nucleic acid or partial sequence thereof of the present invention is inserted into an expression vector suitable for the Agrobacterium method, and then introduced into a suitable Agrobacterium, such as Agrobacterium tumefaciens, by electroporation or the like. This strain is then inoculated into plant cells, callus, or cotyledon sections to infect them. Suitable Agrobacterium species include, but are not limited to, GV3101, C58, and C58C1Rif. (R) Strains such as EHA101, EHA105, AGL1, and LBA4404 can be used.

[0086] The target plant species is not particularly limited, as long as it does not express the polypeptide of the present invention. For example, plants that do not exhibit resistance to potato cyst nematode disease, i.e., plants susceptible to potato cyst nematode disease, can be suitably used. Specific plant species are not particularly limited, but for example, the plants exemplified as target plants in the first embodiment can be used. For example, plants of the Solanaceae family, plants of the Solanum genus, or potatoes can be suitably used.

[0087] The host sample to be introduced may be a section of a plant leaf or similar, or a protoplast may be prepared and used (Christou P, et al., Bio / technology (1991) 9: 957-962). For example, in the particle gun method, a gene transfer device (e.g., PDS-1000 (BIO-RAD)) can be used according to the manufacturer's instructions to inject metal particles coated with the nucleic acid or vector of the present invention into the host sample, thereby introducing it into plant cells and obtaining transformed plant cells. The operating conditions are usually a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

[0088] 3-2-2. Callus Formation Process The callus formation step is a step of culturing plant cells to form callus. This step can be performed before, after, and / or simultaneously with the introduction step. When this step is performed after the introduction step, this step is a step of culturing plant cells into which a disease resistance gene introduction construct (a disease control agent described in the first embodiment, or the nucleic acid of the present invention or a vector containing the same, etc.) has been introduced in the introduction step to form callus.

[0089] The plant cells used in this process may be plant tissue fragments (e.g., leaf, stem, or root sections) or protoplasts.

[0090] The callus formation process may include culturing plant cells in a medium containing plant hormones, particularly auxin and cytokinin.

[0091] In this specification, "auxin" refers to indoleacetic acid (IAA), indolebutyric acid (IBA), naphthaleneacetic acid, naphthoxyacetic acid, phenylacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). In this specification, "cytokinin" refers to zeatin, benzyladenine, and thidiazurone. In this specification, "culture medium" may be any culture medium commonly used for plant tissue culture, such as basic media like MS medium (Murashige and Skoog medium), LS medium (Linsmaier and Skoog medium), Gamborg B5 medium, White medium, Niche medium, KNUDSON C medium, SB medium, R2 medium, N6 medium, and Tuleeke medium. The culture medium may also contain sugars, vitamins, etc. The culture medium may be a solid medium or a liquid medium.

[0092] In the callus formation process, the amounts of auxin and cytokinin in the culture medium can be appropriately adjusted according to the type of plant and are not particularly limited. For example, the auxin concentration can be 1 mg / L to 10 mg / L, 1 mg / L to 5 mg / L, or 1 mg / L to 3 mg / L, and the cytokinin concentration can be, for example, 0.1 mg / L to 5 mg / L, 0.1 mg / L to 1 mg / L, or 0.3 mg / L to 0.7 mg / L. The culture conditions in the callus formation process are not particularly limited, but can be static or shaking culture at 15 to 35°C in or without light.

[0093] In the callus formation process, the formed callus may be subcultured to maintain and proliferate it in an undifferentiated state. While not particularly limited, subculturing of the callus is typically performed every 1 to 4 weeks.

[0094] 3-2-3.Regeneration process The regeneration process involves cultivating callus to regenerate the plant body. The callus used in this process may be the callus obtained in the callus formation process, or it may be the callus into which a disease resistance gene introduction construct (the disease control agent described in the first embodiment, or the nucleic acid of the present invention or a vector containing the same, etc.) has been introduced in the introduction process.

[0095] The regeneration process may include culturing callus in a medium containing plant hormones, particularly auxin and / or cytokinin. The concentrations of auxin and / or cytokinin in the medium should be appropriately determined depending on the tissue to be redifferentiated, the type of plant, etc.

[0096] For example, when differentiating stems and leaves from callus, a culture medium containing cytokinin but not auxin can be used. In this case, the concentration of cytokinin in the medium can be, for example, 0.1 mg / L to 5 mg / L, 0.1 mg / L to 1 mg / L, or 0.3 mg / L to 0.7 mg / L.

[0097] In this process, the culture medium may be appropriately changed according to the growth stage. For example, callus may be cultured in a medium containing plant hormones (particularly auxin and / or cytokinin) to regenerate the plant body, and then the regenerated plant body may be transferred to a medium that does not contain plant hormones.

[0098] 3-2-4. Selection Process In this embodiment, the "selection step" is the step of selecting plants on which the structure has been introduced. This step can be carried out by methods known in the art after introducing the construct into the host using the method described above. For example, transformants can be selected by utilizing the activity of proteins encoded by selection marker genes or reporter genes in the construct. Alternatively, for example, nucleic acid amplification can be performed on the nucleic acid region containing the nucleic acid of the present invention, using nucleic acid extracted from a plant as a template. The success of the construct introduction can be determined by determining the base sequence of the resulting amplification product by sequencing or by restrictive enzyme treatment of the amplification product. Alternatively, for example, various detection methods exemplified in the detection step of the fifth embodiment may be used.

[0099] Furthermore, for example, transformed plants may be selectively regenerated by performing a callus formation step and / or regeneration step in a selective medium in accordance with a plant tissue culture method on plant cells or cotyledon sections into which the nucleic acid of the present invention or a vector containing the same has been introduced.

[0100] 3-3.Applications The method of this embodiment is carried out to confer the effects of the polypeptide of the present invention to a plant that does not possess it, in order to create a transformant, particularly a genome-edited plant. The effects in this case may be any effects of the polypeptide of the present invention and are not particularly limited, but for example, potato cyst nematode disease resistance.

[0101] The method according to this embodiment can be used, for example, as a method for controlling potato cyst nematode disease.

[0102] 3-4. Effects According to the method of this embodiment, resistance to potato cyst nematode disease can be conferred to the target plant without impairing other traits (for example, without damaging the genes related to those traits).

[0103] 4. Potato cyst nematode disease resistance determination kit and device 4-1. Overview A fourth aspect of the present invention relates to a kit and device for determining resistance to potato cyst nematode disease. The kit and device of the present invention make it possible to easily determine whether or not a target plant is resistant to potato cyst nematode disease.

[0104] 4-2. Composition The kits and devices of the present invention include, as essential components, binding molecules capable of binding to the polypeptide of the present invention, and / or primers and / or probes capable of detecting the nucleic acids of the present invention. Each step will be described in detail below.

[0105] 4-2-1. Binding molecules The binding molecule may consist of a peptide, nucleic acid, small molecule compound, or a combination thereof. Furthermore, the binding molecule in this embodiment may optionally include additional elements, such as a labeling substance.

[0106] In this specification, "labeled substance" refers to a substance that emits a signal that can be detected. Examples of labeled substances include fluorescent molecules, luminescent labeling substances that emit light under specific conditions such as chemiluminescent substances, sound-emitting labeling substances that emit sound waves such as photoacoustic effect probes, and radioactive labeling substances. Examples of fluorescent molecules, though not limited to them, include fluorescent proteins, fluorescein and its derivatives, pyrene and its derivatives, and quantum dots. Examples of chemiluminescent substances include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP). Examples of radioactive labeling substances include, for example 14 C, 3 H, 125 Reagents containing I, etc., are examples. The photoacoustic effect is a phenomenon in which thermoelastic waves are generated by adiabatic expansion accompanying light absorption, and these thermoelastic waves can be detected as acoustic waves. Examples of photoacoustic effect probes include indocyanine green or its derivatives, curcumin derivatives, or choline derivatives. If the absorbance characteristics of the binding molecule and labeling substance used are known, it is not always necessary to use a labeling substance for the photoacoustic effect; for example, a luminescent labeling substance may be detected based on the photoacoustic effect.

[0107] If necessary, the invention may include binding molecules that can be bound to the polypeptide of the present invention, such as additional polypeptides of the present invention.

[0108] (A) Peptide When the binding molecule is composed of a peptide, the specific peptide used is not particularly limited. Examples include antibodies, peptide aptamers, and receptor or ligand proteins.

[0109] (i) Antibody The antibodies that can be used as binding molecules refer to antibodies that can immunologically and specifically bind to the polypeptide of the present invention as an antigen, and in this specification, this also includes fragments thereof that have antigen-binding ability.

[0110] The species from which the antibodies originate is not particularly limited. They can be derived from animals, including mammals and birds. Examples of such animals include mice, rats, guinea pigs, rabbits, goats, donkeys, sheep, camels, horses, chickens, or humans.

[0111] Any type of antibody may be used, including polyclonal antibodies, monoclonal antibodies, recombinant antibodies, synthetic antibodies, or combinations thereof.

[0112] A "polyclonal antibody" refers to a group of multiple immunoglobulins that recognize and bind to different epitopes of the same antigen. Polyclonal antibodies can be obtained from the serum of an animal after immunizing it with a target molecule (in this case, the polypeptide of the present invention) as an antigen.

[0113] A "monoclonal antibody" refers to a group of antibodies consisting of a clone of a single immunoglobulin. Each immunoglobulin constituting a monoclonal antibody contains a common framework region (FR) and a common complementarity determining region (CDR), allowing them to recognize and bind to the same epitope of the same antigen. Monoclonal antibodies can be obtained from hybridomas derived from single cells.

[0114] When the antibody is a polyclonal or monoclonal antibody, immunoglobulin molecules are known to belong to the classes IgG, IgM, IgA, IgE, and IgD, but the antibody of the present invention may belong to any of these classes. For example, IgG can be suitably used.

[0115] The specific method for producing a hybridoma that recognizes and binds to the polypeptide of the present invention is not particularly limited and can be produced in accordance with antibody production methods known in the art.

[0116] In this specification, "recombinant antibody" includes chimeric antibodies, humanized antibodies, or multispecific antibodies. A "chimeric antibody" is an antibody produced by combining the amino acid sequences of antibodies derived from different animals, in which the V region of one antibody is replaced with the V region of another antibody. A "multispecific antibody" is a polyvalent antibody, that is, an antibody having multiple antigen-binding sites within a single molecule, in which each antigen-binding site binds to a different epitope. For example, in the case of an antibody having two antigen-binding sites, such as IgG, a bispecific antibody is an example in which each antigen-binding site specifically binds to the same or different markers described in the first embodiment.

[0117] In this specification, "synthetic antibody" refers to an antibody synthesized by chemical methods or recombinant DNA methods. For example, this includes a monomeric polypeptide molecule or a polymer polypeptide thereof, obtained by artificially linking one or more VLs and one or more VHs of a specific antibody via a linker peptide of appropriate length and sequence. Specific examples of such polypeptides include single-chain Fv (scFv: single-chain Fragment of variable region) (see Pierce Catalog and Handbook, 1994-1995, Pierce Chemical Co., Rockford, IL), scFv-Fc, sc(Fv)2, Fv, diabody, triabody, or tetrabody.

[0118] In immunoglobulin molecules, VL and VH are usually located on separate polypeptide chains (L chain and H chain). "Single-chain Fv" is a synthetic antibody fragment that has a structure in which the V regions on these two polypeptide chains are linked by a sufficiently long flexible linker and incorporated into a single polypeptide chain.

[0119] "Diabody" is a molecule based on a single-chain Fv dimeric structure and possessing two functional antigen-binding sites (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-6448). "Triabody" and "Tetrabody" have trimer and tetramer structures based on the single-chain Fv structure, similar to diabody. They are trivalent and tetravalent antibody fragments, respectively, and may also be multispecific antibodies.

[0120] In the case of diabodies and other multivalent antibodies, each antigen-binding site may bind to the same epitope, or each may recognize and specifically bind to different epitopes, exhibiting multispecificity.

[0121] In this specification, "the active fragment" refers to a partial fragment of the polyclonal or monoclonal antibody described above, which is a polypeptide chain or complex thereof having substantially equivalent activity to the antigen-specific binding activity of the antibody. For example, this includes an antibody portion containing at least one antigen-binding site, i.e., a polypeptide chain having at least one pair of VL and VH, or a complex thereof. Specific examples include numerous well-characterized antibody fragments produced by cleaving immunoglobulins with various peptidases. More specific examples include Fab, F(ab')2, Fab', etc. All of these antibody fragments contain antigen-binding sites and have the ability to specifically bind to target molecules that are antigens.

[0122] (ii) Peptide aptamers An "aptamer" is a ligand molecule that has the ability to bind strongly and specifically to a target substance due to its three-dimensional structure. Aptamers can be broadly classified into nucleic acid aptamers and peptide aptamers depending on the type of molecule that makes up the aptamer.

[0123] A "peptide aptamer" is an aptamer composed of amino acids that, like antibodies, recognizes the surface structure of a target molecule and can specifically bind to the target substance based on its three-dimensional structure. Peptide aptamers can be prepared using methods known in the field. For example, one can refer to Whaley, SR, et al., 2000, Nature, 405, 665-668, which specifically describes methods such as phage display and cell surface display.

[0124] (B) Nucleic acid When the binding molecule is composed of nucleic acids, specific examples, though not limited to them, include nucleic acid aptamers.

[0125] A "nucleic acid aptamer" refers to an aptamer composed of nucleic acids. The nucleic acids constituting a nucleic acid aptamer may be DNA, RNA, or a combination thereof. If necessary, chemically modified nucleic acids such as PNA, LNA / BNA, methylphosphonate DNA, phosphorothioate DNA, and 2'-O-methyl RNA may also be included.

[0126] Nucleic acid aptamers can be prepared based on the target molecule using methods known in the field. For example, RNA aptamers can be prepared by in vitro selection using the SELEX (systematic evolution of ligands by exponential enrichment) method. The specific method of the SELEX method can be carried out, for example, in accordance with Pan et al. (Proc. Natl. Acad. Sci. 1995, USA, 92:11509-11513).

[0127] 4-2-2. Primers and / or probes The kit or device of the present invention comprises a probe that hybridizes to the base sequence of the nucleic acid of the present invention, and optionally includes a set of primers for amplifying the nucleic acid of the present invention as a component.

[0128] The nucleotide sequences of the primers and probes in the kit of the present invention are not particularly limited as long as they perform the functions described above, but examples include the following polynucleotides: (i) A polynucleotide consisting of 15 or more consecutive bases selected from the base sequence of the nucleic acid of the present invention (for example, the base sequence shown in Sequence ID No. 2); (ii) A polynucleotide having one or more base deletions, substitutions, additions or insertions in the base sequence of the nucleic acid in (i); (iii) Polynucleotides having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, and 99.9% or more base identity with respect to the base sequence of the nucleic acid of (i); (iv) A polynucleotide that hybridizes under high-stringent conditions with a polynucleotide consisting of 15 or more consecutive bases selected from a base sequence complementary to the base sequence of the nucleic acid of the present invention (for example, the base sequence shown in SEQ ID NO: 2); and (v) A polynucleotide consisting of a base sequence complementary to the base sequences of the polynucleotides (i) to (iv).

[0129] Furthermore, if necessary, the invention may include additional nucleic acids, or primers and / or probes capable of detecting additional nucleic acids. The base sequence is not particularly limited, but examples include polynucleotides corresponding to (i) to (v) for the additional nucleic acids.

[0130] In this specification, "probe" includes polynucleotides capable of specifically recognizing and detecting DNA, RNA produced by gene expression, or polynucleotides derived therefrom, complementary polynucleotides, and aptamers.

[0131] In this specification, "primer" includes polynucleotides capable of specifically recognizing and amplifying DNA, RNA produced by gene expression, or polynucleotides derived therefrom, and complementary polynucleotides.

[0132] "Complementary" refers to a relationship in which nucleic acid bases can form base pairs with each other via hydrogen bonds. This includes so-called Watson-Crick base pairs (natural base pairs) or Hoogsteen base pairs.

[0133] The length of the bases of the primer and probe in this embodiment is not particularly limited as long as it is 15 or more consecutive bases. For example, it can be 17 or more bases, 18 or more bases, 19 or more bases, 20 or more bases, 21 or more bases, etc.

[0134] When used as a primer, it is preferable to include the aforementioned consecutive bases on the 3' end, which is in the extension direction. Furthermore, it is preferable to include a base sequence region consisting of a base sequence that is 100% identical to the target base sequence, and a base sequence region consisting of a base sequence that has a certain level or higher of identity with the target base sequence, starting from the 3' end. The length of the base sequence region consisting of a base sequence identical to the target base sequence is not particularly limited, but for example, it may be 2 or more bases, 3 or more bases, 5 or more bases, 10 or more bases, 15 or more bases, 17 or more bases, or 19 or more bases.

[0135] In this case, the 5' end can include any additional base sequence. The total number of bases in the primer is not particularly limited, but it can be, for example, 50 bases or less, 40 bases or less, or 30 bases or less.

[0136] The kit of the present invention may include probes and / or primers in any combination, but typically the primers are included as a primer pair consisting of a forward primer and a reverse primer.

[0137] When used with already amplified nucleic acids, the kit of the present invention does not need to include primers.

[0138] The primers and / or probes in this embodiment may be DNA, RNA, or a mixture thereof, and may optionally include chemically modified nucleic acids or labeling substances. Specific details of these are as described in the descriptions of the binding molecules and nucleic acid aptamers in this embodiment.

[0139] 4-2-3. Other Components The kit and device of the present invention may further include means for collecting a sample, a container for recovering the sample, a reagent for purifying the sample, a dilution or reaction buffer containing components necessary for measurement, a washing solution, a colorimetric reagent, a reaction vessel, and the like.

[0140] The kit and device of the present invention may include a container. The material of the container is not limited as long as it is made of a material that does not contaminate the contents or is not contaminated by the contents. Examples include plastics such as polypropylene and polystyrene, glass, or paper with a special surface coating.

[0141] If detection equipment is included, there are no particular limitations on its type. The detection method may be appropriately selected according to the labels and reagents used, and the properties of the sample, and the equipment necessary for that detection method may be used. For example, if a fluorescent dye or luminescent substance is used, it can be detected, for example, by visual inspection, using a microscope (e.g., a fluorescence microscope), using a detector (e.g., a photometer, a spectrophotometer), or a combination thereof.

[0142] The kits and devices of the present invention may, if necessary, include instructions for use, as well as application methods such as syringes, droppers, and micropipettes, and storage methods.

[0143] In the kit and device of the present invention, the above-mentioned binding molecules may be immobilized on a substrate. The "substrate" is a solid-phase support for immobilizing the binding molecules.

[0144] The substrate material should be one that allows binding molecules to be directly or indirectly immobilized on its surface. While not limited, it is preferable that the material be water-insoluble. Examples include plastics, glass, metals, ceramics, natural resins (e.g., natural rubber or lacquer), natural or chemical fibers or aggregates thereof (e.g., paper, nonwoven fabrics, filters), polysaccharide polymers (e.g., agar), gelling proteins (e.g., gelatin, collagen), or mixtures thereof.

[0145] The shape of the substrate can be appropriately determined depending on the application of the kit and device of the present invention. Examples of shapes include plates (including rectangular ones such as 96-well microtiter plates), dishes, tubes, sticks, beads, plates, or test pieces. When solidifying on the surface of beads, the substrate can be a sphere with a diameter of approximately 1 μm to approximately 1 cm. Furthermore, for example, if the kit and device of the present invention are used as a sensor chip for an SPR measuring sensor, the substrate should be shaped to fit the SPR measuring sensor being used.

[0146] The substrate can also be a multilayer structure composed of two or more materials. For example, a substrate in which a thin gold film is laminated on a glass surface is one such example. When the substrate has a multilayer structure in this way, at least the layers constituting the substrate surface are made of a material that can solidify binding molecules.

[0147] "Immobilization on the substrate surface" refers to fixing binding molecules to a substrate surface that can directly contact a sample taken from the subject. The method of immobilization is not particularly limited. Examples include chemical adsorption, physical adsorption, affinity, or a combination thereof. Chemical adsorption includes chemical bonds such as covalent bonds or ionic bonds. Physical adsorption includes van der Waals forces.

[0148] The kit and device of this embodiment may further include additional components used for determination, as needed. The specific additional components are not limited, but may include, for example, multiple types of the aforementioned binding molecules, primers, probes, etc.

[0149] 5. Method for determining resistance to potato cyst nematodes 5-1. Overview A fifth aspect of the present invention relates to a method for determining resistance to potato cyst nematodes. According to the method of production of the present invention, it is possible to easily determine whether or not a target plant has resistance to potato cyst nematode disease.

[0150] 5-2.Process The determination method of the present invention includes a detection step and a determination step as essential steps, and a sample preparation step as an optional step. Each step will be described in detail below.

[0151] 5-2-1. Detection process This step is an essential step of the method according to this embodiment, and involves detecting the polypeptide and / or nucleic acid of the present invention in a sample obtained from a target plant.

[0152] The sample may be used directly in the method of this embodiment after being collected from the target plant, or it may be subjected to additional processing. Specific processing is not particularly limited, but examples include storage at low temperatures, concentration, dilution, addition of any chemicals, removal of unwanted components, or combinations thereof.

[0153] In this process, additional components such as additional polypeptides and additional nucleic acids of the present invention may be detected as needed.

[0154] (A) In the case of polypeptides When making a determination based on the polypeptide of the present invention, the measurement can be performed using known methods for quantifying proteins, and is not particularly limited. Examples include immunological detection methods, aptamer analysis methods, gel filtration HPLC methods, mass spectrometry, or combinations thereof.

[0155] (i) Immunological detection methods "Immunological detection methods" are the most common methods for detecting and quantifying target molecules by forming an immune complex with an antibody or a fragment thereof that specifically binds to a target molecule, using the target molecule as an antigen. In the present invention, since the polypeptide of the present invention corresponds to the target molecule, measurement can be performed using an antibody that can specifically bind to the polypeptide of the present invention. Note that the antibody here includes an antigen-binding fragment. The antibody is described in the fourth embodiment.

[0156] Specific immunological detection methods include, for example, enzyme immunoassay, fluorescence immunoassay, luminescence immunoassay, surface plasmon resonance (SPR), quartz crystal microbalance (QCM), radioimmunoassay (RIA), immunoturbidimetry, latex agglutination immunoassay, latex turbidimetry, particle agglutination reaction, gold colloid method, capillary electrophoresis, Western blotting, or immunohistochemistry (immunostaining). All of these methods are well-known and, in principle, should be carried out in accordance with the usual methods in the field. For example, Current protocols in Protein Sciences, 1995, John Wiley & Sons Inc.; Current protocols in Immunology, 2001, John Wiley & Sons Inc.; Green & Sambrook, Molecular Cloning, 2012, Fourth Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; Japanese Society of Clinical Pathology (ed.), "Special Issue No. 53 of Clinical Pathology, Immunoassays for Clinical Testing - Techniques and Applications," Clinical Pathology Publication Association, 1983; Ishikawa, Eiji et al. (eds.), "Enzyme Immunoassays," 3rd edition, Igaku-Shoin, 1987; Kitagawa, Tsunehiro et al. (eds.), "Protein, Nucleic Acid, and Enzyme Supplement No. 31 Enzyme Immunoassays," Kyoritsu Shuppan, 1987; Irie, Minoru (ed.), "Radioimmunoassays," Kodansha Scientific, 1974; Irie, Minoru (ed.), "Continued Radioimmunoassays," The methods described can be found in Kodansha Scientific, 1979; Kazuhiro Nagata and Hiroshi Handa (eds.), "Real-time Analysis Experimental Methods for Biomolecular Interactions," Springer-Verlag Tokyo, 1988; and Toyosaka Moriizumi and Takamichi Nakamoto, "Sensor Engineering," Shokodo, 1997, among others.

[0157] (ii) Aptamer analysis method "Aptamer analysis" is a method for quantifying target molecules using nucleic acid aptamers or peptide aptamers. Essentially, it can be performed by replacing the antibody in the aforementioned immunological detection method with an aptamer that can specifically bind to the target molecule.

[0158] (iii) Mass spectrometry method Mass spectrometry is a method of analyzing substances in a sample by ionizing the sample under high vacuum and then electromagnetically separating the ions. When the target molecule to be detected in the sample is known, the target molecule (in this case, the polypeptide of the present invention) can be used as a standard, and by comparing the mass spectrum of the sample with that of the target molecule, the target molecule can be detected and quantified.

[0159] The specific mass spectrometry methods used are not limited to high-performance liquid chromatography-mass spectrometry (LC-MS), high-performance liquid chromatography-tandem mass spectrometry (LC-MS / MS), gas chromatography-mass spectrometry (GC-MS), gas chromatography-tandem mass spectrometry (GC-MS / MS), capillary electrophoresis-mass spectrometry (CE-MS), and intercellular plasma chromatography (ICP-MS).

[0160] (B) In the case of nucleic acids When making a determination based on the nucleic acid of the present invention, the measurement can be performed using known nucleic acid quantification methods, and is not particularly limited. Examples include PCR (including RT-PCR, qPCR, real-time PCR, etc.), hybridization methods (including Northern hybridization, Southern hybridization, Northern blotting, Southern blotting, nucleic acid arrays, etc.), or combinations thereof. The specific procedures and conditions for these methods should basically follow methods well known in the art.

[0161] When using the PCR method, the amount of nucleic acid of the present invention can be detected and measured by using the primers described in the fourth aspect. Specifically, for example, the procedure can be followed. First, the PCR method is performed using a pair of primers capable of amplifying the target nucleic acid region with DNA prepared from a sample as a template, and the resulting double-stranded DNA is detected. Methods for detecting double-stranded DNA include performing the above PCR using primers that have been previously labeled with a radioactive isotope or fluorescent substance, performing electrophoresis of the PCR product on an agarose gel and staining the double-stranded DNA with ethidium bromide or the like for detection, and transferring the produced double-stranded DNA to a nylon membrane or the like according to a conventional method and hybridizing it with a labeled probe for detection.

[0162] Furthermore, for example, when using real-time PCR, the amount of nucleic acid can be measured by the increase in the signal derived from the label accompanying the generation of the amplified product, using the primers and probes described in the fourth aspect. In this case, the amount of nucleic acid is generally calculated by substituting the obtained Ct value (Threshold Cycle value) into a predetermined calibration formula. Here, the Ct value refers to the number of cycles at which the amplified product reaches a certain amount. The calibration formula refers to the relationship between the amount of amplified product and the Ct value of each serially diluted sample, obtained by performing the same amplification reaction using the primer set used for measurement, with nucleic acids extracted from serially diluted samples with known gene copy numbers as a template. The specific calibration formula is not particularly limited.

[0163] For example, when using hybridization methods, particularly blotting techniques, the amount of the target nucleic acid can be detected and measured by using the probe described in the fourth embodiment. Specifically, this can be done, for example, by following the procedure below. First, the probe (complementary strand) is mixed with a radioactive isotope ( 32 P, 33 P, 35Label them using substances such as S or fluorescent substances. Next, transfer the nucleic acid in the sample to a nylon membrane or the like. Apply the labeled probe to this membrane to hybridize the labeled probe with the nucleic acid. Finally, detect and measure the signal derived from the label in the formed double strand using a radiation detector or a fluorescence detector or the like.

[0164] When using nucleic acid array analysis, use an RNA chip or a DNA chip in which the probe described in the fourth aspect is immobilized on a solid phase such as a substrate. In this method, nucleic acid molecules obtained from a sample are applied to the chip, and the presence or absence of the target nucleic acid is detected by detecting the signal of the chip.

[0165] Those in which nucleic acid is immobilized on a substrate are generally called by names such as nucleic acid chip, nucleic acid array, microarray, etc. Also, DNA or RNA arrays include DNA or RNA macroarrays and DNA or RNA microarrays. In this specification, when referring to a chip, it shall include all of them.

[0166] The method for measuring the signal of the chip is not limited, but for example, a method of detecting and measuring the signal derived from the label of the detection composition using an image detector (such as Typhoon 9410 (GE Healthcare), 3D-Gene (R) scanner (Toray Industries, Inc.) etc.) can be exemplified.

[0167] 5-2-2. Determination step This step is an essential step of the method of this aspect, and when the presence of the nucleic acid and / or polypeptide of the present invention is detected, it is a step of determining that the target plant has potato cyst nematode resistance.

[0168] The determination criteria used in this step may be any that reflect the presence or absence of the nucleic acid and / or polypeptide of the present invention, and are not particularly limited. For example, the determination can be made based on the comparison between the measured value obtained in the detection step and a predetermined reference value, or based on the comparison with a control sample in which the presence or absence of the nucleic acid and / or polypeptide of the present invention is known.

[0169] The "predetermined reference values" used in this process are not particularly limited, as long as they can distinguish between the presence or absence of nucleic acids and / or polypeptides. They can be appropriately determined based on the type of sample used, the molecular species to be detected, sequence information, the detection method used in the detection process, etc. Both empirical and objective criteria can be used as the predetermined reference values ​​in this process. As objective criteria, for example, a cutoff value can be used.

[0170] In this specification, "cutoff value" refers to a value that allows for the determination of the presence or absence of the target molecule. Preferably, the cutoff value exhibits sufficiently high sensitivity and specificity. Generally, it is derived using known methods from ROC curves drawn based on a direct comparison between the non-existent group and the present group, but is not limited to this. For example, the cutoff value may be set based on, for example, the detection limit, without using an ROC curve.

[0171] The method of comparison with the reference value is not particularly limited, but examples include visual inspection and the use of software. Examples of software-based methods include using calculation software such as Excel, using automated processing by calculation software, and using known software capable of classification based on the reference value.

[0172] For example, if a reference value is set as the minimum value for determining whether a sample has been detected, and the measured value in a sample obtained from the target plant is equal to or greater than the reference value, it can be determined that the nucleic acid and / or polypeptide of the present invention is present in the target plant and that the target plant is resistant to potato cyst nematodes. Also, if a similar reference value is used, for example, if the measured value in a sample obtained from the target plant is less than the reference value, it can be determined that the nucleic acid and / or polypeptide of the present invention is not present in the target plant and that the target plant is not resistant to potato cyst nematodes (i.e., is susceptible to potato cyst nematodes).

[0173] When making a judgment based on a comparison with a control sample, for example, a judgment can be made based on statistically significant differences.

[0174] In this specification, "statistically significant" means that when the difference between the measured value of the subject and the control value is statistically analyzed, there is a significant difference between the two. For example, when the significance level of the obtained value is small, specifically less than 5% (p<0.05), less than 1% (p<0.01), or less than 0.1% (p<0.001). The "p(value)" shown here represents the probability that the test statistic happens to be that value within the distribution based on the null hypothesis in a statistical test. Therefore, the smaller "p" is, the lower the probability that the test statistic is that value, and the more likely the null hypothesis is to be rejected. The statistical testing method may be any known test method that can determine the presence or absence of significance, and is not particularly limited. For example, Student's t-test, paired Student's t-test, Welch's t-test, Wilcoxon rank-sum test, analysis of variance, Tukey post-hoc test, etc., can be used, but are not particularly limited.

[0175] In this specification, when a determination is made based on statistical significance, the determination is made based on a comparison with a control value similarly calculated for a control sample.

[0176] When using a sample known to contain the nucleic acid and / or polypeptide of the present invention (positive control sample) as a control sample, if there is no statistically significant difference between the value measured in the sample obtained from the target plant and the positive control value similarly measured in the positive control sample, it can be determined that the nucleic acid and / or polypeptide of the present invention is present in the target plant and that the target plant is resistant to potato cyst nematodes. Conversely, if there is a statistically significant difference between the value measured in the sample obtained from the target plant and the positive control value similarly measured in the positive control sample, it can be determined that the nucleic acid and / or polypeptide of the present invention is not present in the target plant and that the target plant is not resistant to potato cyst nematodes (is susceptible to potato cyst nematodes).

[0177] Furthermore, when using a sample known to be free of the nucleic acids and / or polypeptides of the present invention (negative control sample) as a control sample, if there is a statistically significant difference between the value measured in the sample obtained from the target plant and the negative control value similarly measured in the negative control sample, it can be determined that the nucleic acids and / or polypeptides of the present invention are present in the target plant and that the target plant is resistant to potato cyst nematodes. Conversely, if there is no statistically significant difference between the value measured in the sample obtained from the target plant and the negative control value similarly measured in the negative control sample, it can be determined that the nucleic acids and / or polypeptides of the present invention are not present in the target plant and that the target plant is not resistant to potato cyst nematodes (is susceptible to potato cyst nematodes).

[0178] The measurement and calculation of control values ​​may be performed simultaneously with or separately from the detection and measurement of samples obtained from the target plants. For example, the determination may be made using previously measured and detected values ​​provided in the form of a database, or based on a comparison with values ​​that can be used to determine statistical significance, such as confidence intervals, or based on a comparison with results measured at the same facility and / or at the same time as the measurement of samples obtained from the target plants.

[0179] The determination in this step may further be based on whether additional components, such as additional polypeptides or additional nucleic acids of the present invention, have been detected.

[0180] 5-3. Effects Until now, it was only possible to determine whether or not a potato plant was resistant to potato cyst nematode disease after at least 50 days of cultivation.

[0181] However, according to the method of this embodiment, the presence or absence of resistance to potato cyst nematode disease can be easily determined in a short period of time. [Examples]

[0182] <Example 1. Search for candidate disease resistance gene sequences> (the purpose) Based on fragmented information about previously reported candidate sequences, we conducted an integrated analysis to search for candidate sequences of disease resistance genes.

[0183] (Methods and Results) 1. Determination and analysis of the distal starting sequence We focused on DNA marker N146 (Asano, et al, 2012, doi:10.1270 / jsbbs.62.142), which is located distal to the H1 gene on the chromosome and has been reported to be highly linked to the H1 gene, an unidentified potato cyst nematode resistance gene, as a distal starting sequence. We amplified the DNA marker N146 in the DNA of the potato variety 'Sayaka', which exhibits Gr resistance and is suggested to possess the H1 gene, using the following primer pair: Fw primer for N146: 5'-AAGCTCTTGCCTAGTGCTC-3' (SEQ ID NO: 5); Rv primer for N146: 5'-AGGCGGAACATGCCATG-3' (SEQ ID NO: 6).

[0184] The amplification reaction was carried out for 30 cycles at an annealing temperature of 55°C, using the enzyme TaKaRa Ex Taq. (R) (Takara Bio Corporation) was used.

[0185] For sequencing, the amplification product is processed using pCR. TM 4-TOPO TM The DNA was cloned into a vector (Invitrogen) according to the manufacturer's recommended protocol, and the base sequence of the DNA marker N146 (SEQ ID NO: 7: 506 bases) was determined using contract analysis services provided by Eurofins Genomics, Inc.

[0186] Next, to identify the chromosomal location of this DNA marker, we searched for the sequence of DNA marker N146 in the H1 gene vicinity sequence (accession number: HQ223091; version HQ223091.1) reported for the potato cyst nematode-resistant potato line SH83-92-488 and the tetraploid genomic DNA sequence (annotation version: v2.0 (updated January 11, 2022); URL: https: / / spuddb.uga.edu / phased_tetraploid_potato_download.shtml) of the potato cyst nematode-resistant potato variety Atlantic. Homology searches were performed using GENETYX (ver. 15).

[0187] As a result, the sequence of the DNA marker N146 was found to be located at positions 213,095 to 213,600 in the sequence near the H1 gene. In the Atlantic genome sequence, it was found in the sequence fragment (chr05_2) of chromosome 5, and was located at positions 45,409,010 to 45,409,515.

[0188] 2. Determination and analysis of the proximal starting sequence We focused on the DNA marker 57R_1P (Meiyalaghan et al, 2018, doi:10.1007 / s11032-018-0832-z), which has been reported to be highly linked to the H1 gene as a proximal starting sequence and is located proximal to the H1 gene on the chromosome. The sequence of this DNA marker 57R_1P (SEQ ID NO: 8: 145 base pairs) has been reported.

[0189] To identify the chromosomal location of this DNA marker, the sequence of the DNA marker 57R_1P was searched in the sequence near the H1 gene and in the tetraploid genomic DNA sequence of the potato variety Atlantic, as well as in the distal starting sequence.

[0190] As a result, the 57R_1P sequence was found to be located at positions 95,786 to 95,928 of the sequence near the H1 gene (accession number: HQ223091; version HQ223091.1). In the Atlantic genome sequence, it was found in the sequence fragment of chromosome 5 (chr05_2), and four candidate locations were obtained: positions 44,991,517 to 44,991,659, 45,011,584 to 45,011,726, 45,126,250 to 45,126,392, and 45,300,375 to 45,300,517.

[0191] Of these, the sequences found in the vicinity of the H1 gene, based on the surrounding sequences, were found to be at positions 45,300,375 to 45,300,517.

[0192] 3. Identification of candidate genes Based on the two starting sequences mentioned above, the H1 gene is located distal to the DNA marker 57R_1P and proximal to the DNA marker N146. In other words, it can be said to be located at positions 95,928 to 213,095 of the sequence near the H1 gene (accession number: HQ223091; version HQ223091.1). SH1 and SH2, among others, have been mapped as candidate regions for the H1 gene in the SH83-92-488 lineage, and the sequence region (SH) containing all of these is from position 45,244,717 onwards in the Atlantic genome sequence. Since SH1 corresponds to positions 199,785 to 289,082 (89,298 base pairs) of the H1 gene vicinity sequence mentioned above, and SH2 corresponds to positions 92,925 to 199,776 (106,852 base pairs), the predicted regions of the H1 gene identified in this study correspond to positions 2,862 to 106,852 in the SH2 sequence and positions 1 to 13,816 in the SH1 sequence.

[0193] In the tetraploid genomic DNA sequence of the potato variety Atlantic (annotation version: v2.0 (updated January 11, 2022); URL: https: / / spuddb.uga.edu / phased_tetraploid_potato_download.shtml), the ORF present in this region is Soltu.Atl.05_2G020330 (ORF32: Soltu.Atl_v3.05_2G022380.1), Soltu.Atl.05_2G020340(ORF39:Soltu.Atl_v3.05_2G022400.1), Sol tu.Atl.05_2G020360(ORF45), Soltu.Atl.05_2G020370(ORF49:Soltu.Atl_v3.05_2G022430.1) were found. These ORF numbers are based on the information in Supplementary Table 2 of Finkers-Tomczak et al, 2011, DOI:10.1007 / s00122-010-1472-9. For those where annotation is provided in the tetraploid genomic DNA sequence of the potato variety Atlantic (annotation version: v3 (updated February 17, 2023); URL:https: / / spuddb.uga.edu / ATL_v3_download.shtml), the annotation number for v3 is also included.

[0194] Furthermore, a sequence not included in the aforementioned H1 gene vicinity sequence (ORFX: Sequence ID 2) was used as a comparison ORF. This ORFX corresponds to the gene region that would be additionally included if the candidate sequence of 57R_1P, specifically positions 45,126,250 to 45,126,392, one position proximal to the 57R_1P sequence, were the true sequence of 57R_1P. Note that there was no corresponding ORF listed in Supplementary Table 2 of Finkers-Tomczak et al, 2011, DOI:10.1007 / s00122-010-1472-9.

[0195] <Example 2. Identification of resistance genes in silico> (the purpose) In silico analysis is performed to identify resistance genes from a list of candidate resistance genes.

[0196] (method) Regarding ORF32, ORF39, ORF45, and ORF49, which were identified as candidates, we decided to further narrow down the candidate genes essential for resistance using in silico. RNA-seq data (8 hours after cyst nematode infection) for the potato cyst nematode-resistant variety "SW93-1015" and the susceptible variety "Desiree" are publicly available (Walter et al, 2018, doi:10.1007 / s10658-018-1474-z). Therefore, we performed a BLAST search using the 500-base sequence from the 5' end of each ORF in the nucleotide sequences of each of these varieties.

[0197] (result) Of the four ORFs identified, only ORF45 showed a 100% identical sequence, expressed in the resistant SW93-1015 strain but not in the susceptible Desiree strain. Therefore, ORF45 (SEQ ID NO: 4) was considered a candidate for the H1 gene. Furthermore, the 500-base sequence from the 5' end of ORFX also showed similar expression to ORF45.

[0198] <Example 3. Creation of resistance gene deletion strains and confirmation of the presence or absence of resistance to potato cyst nematodes> (the purpose) We will create strains lacking ORF45 and investigate whether these strains possess resistance to potato cyst nematodes.

[0199] (method) 1. Vector Design Two vectors (d45_dX and d45_X) were designed by inserting three insertion / deletion mutation target sequences, which are generated by Cas9 DNA cleavage, into pPZP200 based on the ORF45 genomic DNA sequence (Figure 1). The guide RNA sequences used in each vector are shown in Table 1.

[0200] [Table 1]

[0201] Of the three guide RNAs (45X_g1, 45X_g2, and 45X_g3) used in vector d45_dX, 45X_g1 and 45X_g2 also have target sequences in ORFX. On the other hand, all three guide RNAs (45_g1, 45_g2, and 45_g3) used in vector d45_X have target sequences only in ORF45.

[0202] 2. Introduction of Vectors Each vector was introduced into Agrobacterium radiobacter (formerly Agrobacterium tumefaciens) strain GV3101 MP90. The Agrobacterium cells into which the vectors were introduced were cultured in YEB liquid medium at 28°C for 12 hours with shaking to prepare an infectious bacterial suspension. The YEB liquid medium used was prepared by adding 1 g / L yeast extract, 5 g / L beef extract, 5 g / L peptone, 5 g / L sucrose, 2 mM magnesium sulfate (pH 7.2), and 100 ppm spectinomycin to distilled water.

[0203] Potato transformation and regeneration were carried out according to Umemoto et al. (Plant Genome Editing Experimental Protocols, 2022, Kagaku Dojin, pp. 137-143). As the potato varieties used, 'Sassy' or 'Sayaka', which exhibit resistance to potato cyst nematode disease, were used.

[0204] The presence or absence of the kanamycin resistance gene was confirmed by PCR in shoots that rooted from transformed plants, and plants possessing the kanamycin resistance gene were isolated as transformants. The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 minutes, denaturation at 95°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 1 minute, with 30 cycles of amplification followed by maintenance at 72°C for 10 minutes. The following sequences were used as primers: U1353 (Fw primer): 5'-CTCACCTTGCTCCTGCCGAGA-3' (SEQ ID NO: 15) U1354 (Rv primer): 5'-CGCCTTGAGCCTGGCGAACAG-3' (SEQ ID NO: 16).

[0205] Two lines of potato transformants (d45_dX_1, d45_dX_2) were obtained from "Sayaka" and one line (d45_dX_3) was obtained from "Sassy" using the vector d45_dX. Three lines of potato transformants (d45_X_1, d45_X_2, d45_X_3) were obtained from "Sassy" using the vector d45_X.

[0206] 3. Checking the status of modifications The extent of genome editing modifications in the obtained individuals was confirmed by amplifying the genomic DNA using PCR and then sequencing it.

[0207] The ORF45 and ORFX genes were amplified and sequenced using the following primers, respectively. The amplification reaction was carried out for 35 cycles at an annealing temperature of 60°C, and the enzyme used was TaKaRa Ex Taq. (R) (Takara Bio Inc.) was used. Sequencing was performed using contract analysis services provided by Eurofins Genomics Inc.

[0208] ORF45 gene (d45_dX) Fw primer: U1325: 5'-GTAATGACGAAATGTCAAATGAGATTC-3' (SEQ ID NO: 17) Rv primer: U1326: 5'-CAGAGCCATAACAGAAGTTTCATA-3' (SEQ ID NO: 18) Sequence Primer: U1325 ORF45 gene (d45_X) Fw primer: U1492: 5'-TGGCGAGTTTCACAACTCAA-3' (SEQ ID NO: 19) Rv primer: U1326 Sequence Primer: U1326 ORFX gene (d45_dX) Fw primer: U1501: 5'-GTAATGACGAAATGTCTTGCATATCC-3' (SEQ ID NO: 20) Rv primer: U1502: 5'-CAGAAGTAGATTATCTAGCTTGGAGTGAAT-3' (SEQ ID NO: 21) Sequence Primer: U1502 The sequencing results are shown in Table 2 and Figures 2-4.

[0209] [Table 2]

[0210] In all transformants, it was confirmed that frameshift mutations were introduced, specifically through insertions or deletions of a total number of base pairs that were not multiples of 3 in each ORF.

[0211] 4. Checking for resistance Resistance testing was conducted based on the description in Narabe et al., 2007, Hokkaido Agricultural Research Center Report, pp. 103-106, etc. The general procedure is as follows:

[0212] First, the transgenic plants were grown to about 10 cm in MS medium containing 3% sucrose to obtain in vitro plants. The in vitro plants were acclimatized for 5 days in transparent plastic cups containing sterilized culture medium "Hanazanmai" (Sakata Seed Co., Ltd.). After acclimatization, the plants were transplanted into another plastic cup containing a soil mixture of sterilized culture medium and soil contaminated with cyst nematodes (potato cyst nematode: pathogen type Ro1) in a 3:1 ratio. After 41-50 days of cultivation, the presence or absence of cysts on the extended roots was observed from the outside of the plastic cup, and after 2.5 months, the soil was opened to further check the condition of the cysts formed on the roots. All cultivation was carried out under conditions of 22°C, 6700 lux, and a day length of 16 hours. Light from the sides was blocked by placing the plastic cups in paper cups.

[0213] As a control, in vitro plants of 'Sayaka', 'Sassy', and 'May Queen' that had not undergone transformation were grown in the same manner and cultivated using the same procedures.

[0214] (result) In the control groups, the potato cyst nematode-resistant varieties 'Sayaka' and 'Sassy' were cultivated, and no cyst formation was observed in either variety. On the other hand, cyst formation was observed in the control group, the potato cyst nematode-susceptible variety 'May Queen'. This confirms that the experimental results appropriately reflect the presence or absence of resistance.

[0215] Surprisingly, in d45_X_1, d45_X_2, and d45_X_3, where d45_X was introduced and a frameshift mutation was introduced only in ORF45, cyst formation was not observed in any of them. This indicates that loss of ORF45 function does not result in the loss of potato cyst nematode resistance, and that ORF45 is not the main component of the resistance gene H1.

[0216] On the other hand, surprisingly, when d45_dX was introduced, significant cyst formation occurred in all three strains—d45_dX_1, d45_dX_2, and d45_dX_3—in which the frameshift mutation was introduced in both ORF45 and ORFX (Figure 5), confirming that the mutation resulted in the loss of resistance to potato cyst nematodes.

[0217] We used MOTIF (GenomeNet; URL: https: / / www.genome.jp / tools / motif / ) to search for amino acid sequence motifs of known domains in the ORFX protein domain. As a result, the following amino acid sequences were identified in Sequence ID No. 1: the NB-ARC domain from positions 479 to 719, the Rx N-terminal domain from positions 345 to 417, the AAA domain from positions 497 to 587, the STAND NTPase 3 domain from positions 497 to 519, the AAA domain from positions 497 to 612, the AAA domain from positions 498 to 588, the AAA ATPase domain from positions 496 to 589, the ATP-grasp-like domain from positions 820 to 848, and the DnaB-like helicase C-terminal domain from positions 487 to 573. In particular, the NB-ARC domain and the STAND NTPase 3 domain have been reported to be involved in plant resistance to diseases, including bacterial diseases.

[0218] Based on the above, it was suggested that ORFX is the true identity of the cyst nematode resistance gene H1.

Claims

1. A disease control agent for potato cyst nematode (Globodera rostochiensis) in target plants, comprising a nucleic acid containing a base sequence encoding a polypeptide consisting of any one of the amino acid sequences described in (a) to (c) below: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

2. The cyst nematode disease control agent according to claim 1, wherein the base sequence consists of any one of the following (1) to (4): (1) The base sequence or fragment sequence shown in Sequence ID No. 2; (2) A nucleotide sequence or fragment thereof in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 2; (3) A nucleotide sequence or a fragment thereof having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2; or (4) A nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2 under highly stringent conditions.

3. The disease control agent according to claim 1, wherein the target plant is a plant of the genus Solanum.

4. The disease control agent according to claim 3, wherein the target plant is potato.

5. A transformant expressing a polypeptide containing any one of the amino acid sequences described in (a) to (c) below, and exhibiting resistance to potato cyst nematode disease: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

6. A potato cyst nematode disease control composition comprising the disease control agent described in any one of claims 1 to 4.

7. A method for producing a potato cyst nematode-resistant plant, comprising an introduction step of introducing a disease control agent according to any one of claims 1 to 4 into the cells of a target plant that does not exhibit resistance to potato cyst nematode disease.

8. A method for producing a genome-edited plant, comprising an introduction step of introducing a nucleic acid encoding a polypeptide having the amino acid sequence described in any one of (a) to (c) below into the cells of a target plant: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

9. A method for controlling potato cyst nematode disease, comprising an introduction step of introducing a disease-resistant gene-transformed construct containing the disease control agent described in any one of claims 1 to 4 into the cells of a target plant that does not exhibit resistance to potato cyst nematode disease.

10. A potato cyst nematode resistance testing kit comprising a binding molecule capable of binding to a polypeptide consisting of any one of the amino acid sequences described in (a) to (c) below, and / or a primer and / or probe capable of detecting nucleic acids containing a base sequence encoding the polypeptide: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

11. The kit according to claim 10, wherein the primer and / or probe comprises a nucleotide sequence containing 18 or more consecutive nucleotides in any one of the following nucleotide sequences (1) to (4) or a nucleotide sequence complementary thereto: (1) The base sequence or fragment sequence shown in Sequence ID No. 2; (2) A nucleotide sequence or fragment thereof in which one or more nucleotides are added, deleted, and / or substituted in the nucleotide sequence shown in Sequence ID No. 2; (3) A nucleotide sequence or a fragment thereof having 90% or more sequence identity with the nucleotide sequence shown in Sequence ID No. 2; or (4) A nucleotide sequence or a fragment thereof that can hybridize with a nucleotide sequence complementary to the nucleotide sequence shown in Sequence ID No. 2 under highly stringent conditions.

12. A potato cyst nematode resistance determination device comprising a binding molecule capable of binding to a polypeptide consisting of any one of the amino acid sequences described in (a) to (c) below, and / or a primer and / or probe capable of detecting nucleic acids containing a base sequence encoding the polypeptide: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

13. A method for determining resistance to potato cyst nematodes, A detection step in which a sample obtained from a target plant is detected for a polypeptide consisting of any one of the amino acid sequences described in (a) to (c) below, and / or a nucleic acid containing a base sequence encoding the polypeptide, and If the presence of the nucleic acid and / or polypeptide is detected, the determination step is to determine that the target plant has resistance to cyst nematodes. The determination method includes: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No.

1.

14. A potato cyst nematode disease inhibitor comprising a polypeptide having the amino acid sequence described in any one of (a) to (c) below: (a) The amino acid sequence or fragment sequence shown in Sequence ID No. 1; (b) an amino acid sequence or fragment thereof in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown in Sequence ID No. 1; or (c) An amino acid sequence or a fragment thereof having 90% or more sequence identity with the amino acid sequence shown in Sequence ID No. 1.

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