Plant parasitic nematode controlling agent

Abstract

A new gene that provides a difference in resistance between a plant parasitic nematode resistant rice variety ARC10313 and a plant parasitic nematode intolerance variety T65 is found and a plant parasitic nematode controlling agent based on the new gene is provided.　Provided is a plant parasitic nematode controlling agent comprising a polypeptide having any of amino acid sequences represented by: (a) the amino acid sequence represented by SEQ ID NO: 13; (b) an amino acid sequence resulting from deletion, substitution, or addition of one or more amino acids in the amino acid sequence represented by SEQ ID NO: 13; or (c) an amino acid sequence having an identity of 90% or more with the amino acid sequence represented by SEQ ID NO: 13, or a fragment of said polypeptide.

Description

Plant parasitic nematode control agent

[0001] The present invention relates to a plant-parasitic nematode control agent, a plant transformant, and a method for producing the plant transformant.

[0002] Plant-parasitic nematodes are known to have an extremely wide host range, parasitizing over 2,000 species of plants, including many agriculturally important crops, and the damage caused by these nematodes is reported to be as much as $150 billion worldwide.

[0003] Among plant-parasitic nematodes, three species are known to cause particularly severe damage to agriculture: root-knot nematodes, root-lesion nematodes, and cyst nematodes. For example, the sweet potato root-knot nematode (Meloidogyne incognita), a type of root-knot nematode, has a wide host range and parasitizes various plants worldwide, including agricultural crops, and has been reported to cause effects such as reduced yields, reduced quality, growth inhibition, and plant death. This is a global problem from the perspective of stabilizing food production, and countermeasures are urgently needed.

[0004] Agricultural damage caused by plant-parasitic nematodes is difficult to control because they generally progress while hidden in the soil. For this reason, spraying highly toxic chemicals (nematicides) has been an effective control technique for plant-parasitic nematodes. However, highly toxic chemicals such as nematicides can cause soil contamination, which is a problem. The use of methyl bromide, the main nematicide, has been banned since 2005 due to its ozone layer depletion. Therefore, there is a need for an effective nematode control technique that does not rely on nematicides.

[0005] The use of resistance genes against plant-parasitic nematodes is being promoted as a potential control technology to replace nematicides. In tomatoes, the Mi-1 gene has been found to confer resistance to the sweet potato root-knot nematode (Non-Patent Document 1). Tomatoes into which the Mi-1 gene has been introduced exhibit strong resistance to the sweet potato root-knot nematode, and tomatoes into which this gene has been introduced are widely used in agricultural fields. It has also been reported that lettuce into which the tomato-derived Mi-1 gene has been introduced acquires nematode resistance (Non-Patent Document 2).

[0006] However, the range of plant species to which the Mi-1 gene can be applied is limited, and no effective nematode resistance genes are known, particularly for monocotyledonous plants. Among monocotyledonous plants, agricultural damage caused by nematodes has been reported in succession for bananas, pineapples, and sugarcane, in addition to major grains. In Asia, the rice root-knot nematode (Meloidogyne graminicola) has caused severe damage to rice, and it has been reported that yields in infected areas can decrease by approximately 70% (Non-Patent Documents 3 and 4).

[0007] Therefore, there is a need for new resistance genes to plant-parasitic nematodes and new plant-parasitic nematode control agents based thereon.

[0008] Milligan, SB, et al., 1998, Plant Cell, 10:1307-1319.Zhang, L.-Y., et al., 2010, Plant Mol Biol Report, 28:204-211.De Waele, D. and Elsen, A., 2007, Annu. Rev. Phytopathol. 45:457-485.Bridge, J., Plowright, RA, and Peng, D., 2005, Plant parasitic nematodes in subtropical and tropical agriculture, pp.87-130.

[0009] WO2022 / 176971

[0010] The object of the present invention is to discover a novel gene that causes a difference in resistance between the plant-parasitic nematode-resistant rice variety ARC10313 and the plant-parasitic nematode-susceptible rice variety T65, and to provide a plant-parasitic nematode control agent based on the novel gene.

[0011] Previous research by the present inventors focused on the indica variety Kalo Dhan, which exhibits resistance to sweet potato root-knot nematodes. We generated 128 recombinant inbred lines (RILs) between this variety and the susceptible japonica variety Taichung No. 65 (T65), and performed QTL analysis and gene mapping based on the RILs (Patent Document 1). As a result, we found that the Os04g0112100 gene (herein referred to as ROOT KNOT NEMATODE RESISTANCE 1 or RKNR1) is one of the genes responsible for the difference in nematode resistance between the two varieties, Kalo Dhan and T65.

[0012] However, in a comparative study of the two varieties Kalo Dhan / T65 described in Patent Document 1, the contribution rate of the RKNR1 gene to nematode resistance in RILs produced between the two varieties was only 29.84%. Furthermore, although the nematode resistance of a transformed strain in which the RKNR1 gene from Kalo Dhan was introduced into a nematode-susceptible variety was enhanced, it did not reach the nematode resistance of Kalo Dhan itself. Therefore, RKNR1 alone could not explain the difference in nematode resistance between the two varieties Kalo Dhan / T65.

[0013] Although the present inventors attempted to identify additional resistance genes between the two cultivars, Kalo Dhan and T65, using the RIL lines, they were unable to map any resistance genes other than the RKNR1 gene in the RIL lines. Therefore, genes other than the RKNR1 gene that cause differences in nematode resistance between the two cultivars, Kalo Dhan and T65, have not been identified and remain unknown to this day.

[0014] To solve the above problems, the present inventors focused on a resistant cultivar, ARC10313, different from the cultivars described above and conceived the idea of ​​searching for a new nematode resistance gene different from that described above. Specifically, to search for a gene that contributes to the difference in resistance between the resistant cultivar ARC10313 and the susceptible cultivar T65, they created a new RIL line between the two cultivars, ARC10313 / T65. QTL analysis of the new RIL line was performed, and further gene mapping was performed using a chromosome segment substitution line (CSSL). As a result, the Os06g0621600 gene (herein referred to as ROOT MELOIDOGYNE INCOGNITA RESISTANCE 1 or RMIR1) was identified as a new gene associated with the difference in nematode resistance between the two cultivars, ARC10313 / T65. Furthermore, the inventors have found that the RMIR1 gene of a nematode-resistant variety can be introduced into a nematode-susceptible variety to produce a transformed strain, and have thus completed the present invention. The present invention is based on the above findings and provides the following:

[0015] (1) A plant-parasitic nematode control agent comprising an RMIR1 polypeptide having any of the amino acid sequences shown in (a) to (c) below, or a fragment thereof: (a) the amino acid sequence shown in SEQ ID NO: 13, (b) an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 13, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 13. (2) The plant-parasitic nematode control agent according to (1), further comprising an RKNR1 polypeptide having any of the amino acid sequences shown in (d) to (f) below, or a fragment thereof: (d) the amino acid sequence shown in SEQ ID NO: 1, (e) an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, or (f) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1. (3) A plant-parasitic nematode control agent comprising an RMIR1 polynucleotide encoding the RMIR1 polypeptide according to (1) or a fragment thereof. (4) The agent for controlling plant parasitic nematodes according to (3), wherein the RMIR1 polynucleotide comprises any of the following nucleotide sequences (g) to (j): (g) the nucleotide sequence shown in SEQ ID NO: 14, (h) a nucleotide sequence in which one or more nucleotides have been deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 14, (i) a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 14, or (j) a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 14. (5) The agent for controlling plant parasitic nematodes according to (3) or (4), further comprising an RKNR1 polynucleotide encoding the RKNR1 polypeptide or a fragment thereof according to (2). (6) The agent for controlling plant parasitic nematodes according to (5), wherein the RKNR1 polynucleotide comprises any of the following nucleotide sequences (k) to (n):(k) the nucleotide sequence shown in SEQ ID NO: 2, (l) a nucleotide sequence in which one or more nucleotides are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 2, (m) a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 2, or (n) a nucleotide sequence that hybridizes under highly stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2. (7) An agent for controlling a plant-parasitic nematode, comprising an expression vector containing the RMIR1 polynucleotide according to (3) or (4). (8) The agent for controlling a plant-parasitic nematode according to any of (1) to (7), wherein the plant-parasitic nematode is a root-knot nematode. (9) The agent for controlling a plant-parasitic nematode according to (8), wherein the root-knot nematode is selected from the group consisting of Meloidogyne oryzae, Meloidogyne incognita, Meloidogyne northernis, and Meloidogyne javanica. (10) A plant transformant having resistance to plant parasitic nematodes, comprising the plant parasitic nematode control agent according to any one of (1) or (2) or (3) to (9), or a progeny thereof harboring the polynucleotide or the expression vector. (11) The plant transformant according to (10) or a progeny thereof, which is a monocotyledonous plant. (12) The plant transformant according to (11) or a progeny thereof, wherein the monocotyledonous plant is a grass family plant. (13) The plant transformant according to (12) or a progeny thereof, wherein the grass family plant is selected from the group consisting of rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, and sorghum. (14) The plant transformant according to any one of (10) to (13) or a progeny thereof, which is a genetically modified plant. (15) A method for producing a plant transformant having resistance to a plant parasitic nematode, comprising the steps of: introducing the expression vector according to (7) into a plant; and selecting a plant into which the expression vector has been introduced. This specification incorporates the disclosure of Japanese Patent Application No. 2023-097058, from which the present application claims priority.

[0016] According to the present invention, it is possible to provide a plant-parasitic nematode control agent based on the RMIR1 gene that confers resistance to plant-parasitic nematodes.

[0017] The results of evaluating the resistance of 77 rice varieties to the sweet potato root-knot nematode are shown. A lower evaluation value (EV) indicates higher resistance to the nematode. Error bars indicate standard error. Gene mapping results for sweet potato root-knot nematode resistance are shown. (A) Nine SNP markers on chromosome 6 and their evaluation values ​​(EV) are shown for each chromosome segment substitution line (CSSL line). The SNP markers in the figure are indicated by "A" for the T65 type and "B" for the ARC10313 type. ** indicates P<0.01 (Student's t-test). Error bars indicate standard error. (B) The location of each SNP marker on chromosome 6 is shown. The double arrow indicates the genomic region to which the RMIR1 gene is mapped. The structures of the polypeptides encoded by the RMIR1 gene in ARC10313, N22, Kalo Dhan, T65, and Nipponbare are shown. The results of evaluating the resistance of RMIR1 transformed lines to the root-knot nematode are shown. ** indicates P<0.01 (Student's t-test). Error bars indicate standard error. The results of evaluating the resistance of 77 rice varieties, RKNR1 transformed lines, and RMIR1 transformed lines to the root-knot nematode are shown. Error bars indicate standard error.

[0018] 1. Plant-parasitic nematode control agent 1-1. Overview A first aspect of the present invention is a plant-parasitic nematode control agent. The plant-parasitic nematode control agent of the present invention consists of or contains a nematode-resistant RMIR1 polypeptide or a fragment thereof, or contains a polynucleotide encoding either of them, or an expression vector containing the polynucleotide. The plant-parasitic nematode control agent of the present invention has a control effect against plant-parasitic nematodes.

[0019] 1-2. Definition of Terms As used herein, the term "plant-parasitic nematode" is not particularly limited as long as it is a nematode that can parasitize plants. Known plant-parasitic nematodes include root-knot nematodes (Meloidogyne), root-lesion nematodes (Pratylenchus), cyst nematodes (six genera of cyst nematodes are known: Afenestrata, Cactodera, Dolichodera, Globodera, Heterodera, and Punctodera), leaf nematodes (Aphelenchoides), and stem nematodes (Ditylenchus). The plant-parasitic nematode that is the target of control in the present invention is preferably a root-knot nematode.

[0020] Root-knot nematodes parasitize plant roots, deriving nutrients from the protoplasm of plant cells and forming nodules on the plant's roots. Root-knot nematode larvae molt once inside the egg and hatch as second-stage (J2) larvae. The second-stage larvae move through the soil, penetrate the tissue near the root tip of the plant's root, settle near the vascular bundle, ingest nutrients, then molt a second time to become adults. Adult body lengths are approximately 0.5 to 1 mm, and female adults excrete egg sacs into which they lay approximately 400 to 1,500 eggs. Examples of species belonging to the genus Meloidogyne include Meloidogyne oryzae, Meloidogyne javanica, Meloidogyne incognita (often referred to as "Mi" in this specification), Meloidogyne hapla, Meloidogyne mali, and Meloidogyne arenaria.

[0021] Examples of species belonging to the genus Pratylenchus include the northern root-lesion nematode (Pratylenchus penetrans), the southern root-lesion nematode (Pratylenchus coffeae), the wheat root-lesion nematode (Pratylenchus neglectus), the sawtooth root-lesion nematode (Pratylenchus crenatus), the walnut root-lesion nematode (Pratylenchus vulnus), and the channel root-lesion nematode (Pratylenchus loosi).

[0022] Examples of species belonging to the cyst nematode include the potato cyst nematode (Globodera rostochiensis), the soybean cyst nematode (Heterodera glycines), and the clover cyst nematode (Heterodera trifolii).

[0023] Examples of species belonging to the genus Aphelenchoides include the peel nematode (Aphelenchoides ritzemabosi), the strawberry nematode (Aphelenchoides fragariae), and the rice root nematode (Aphelenchoides besseyi).

[0024] Examples of species belonging to the stem nematode include the potato leaf nematode (Dithlenchus destructor) and the common stem nematode (Ditylenchus dipsaci).

[0025] As used herein, the term "plant" refers to a plant species that can be parasitized by plant-parasitic nematodes, and may be either angiosperms or gymnosperms. Angiosperms also include both dicotyledonous and monocotyledonous plants. Representative examples include plants that are important in agriculture, particularly in the seed and floriculture industries, such as crop plants such as grains, flowers, vegetables, and fruits. Specifically, monocotyledonous plants include species belonging to the Poaceae family (e.g., rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, sorghum, and sorghum), species belonging to the Musaceae family (e.g., banana and musa), and species belonging to the Bromeliaceae family (e.g., pineapple). Dicotyledonous plants include species belonging to the Brassicaceae family (e.g., cabbage, radish, Chinese cabbage, rapeseed), species belonging to the Fabaceae family (e.g., soybean, peanut, pea, kidney bean, adzuki bean, broad bean, sweet pea), species belonging to the Solanaceae family (e.g., tomato, eggplant, potato, tobacco, bell pepper, chili pepper, petunia), species belonging to the Convolvulaceae family (e.g., sweet potato, water spinach), species belonging to the Rosaceae family (e.g., strawberry, rose, apple, pear, peach, loquat, almond, plum, plum, cherry), and species belonging to the Orchidaceae family (e.g., Cymbidium, Phalaenopsis, Cattleya, Dendrobium), species belonging to the Liliaceae family (e.g., lilies, tulips), Asparagaceae family (e.g., hyacinths, muscari), Amaryllidaceae family (e.g., leeks, onions, garlic), Rutaceae family (e.g., mandarins, oranges, grapefruit, lemons, yuzu), species belonging to the Vitaceae family (e.g., grapes), species belonging to the Asteraceae family (e.g., lettuce, chrysanthemums, dahlias, daisies, sunflowers), species belonging to the Caryophyllaceae family (e.g., carnations, baby's breath), and species belonging to the Theaceae family (e.g., sasanqua, tea plants). Root-knot nematodes have been reported to parasitize (infect) over 2,000 species of plants. Although the hosts they parasitize vary depending on the nematode species, they have a wide host range, including Solanaceae, Poaceae, Brassicaceae, Fabaceae, Cucurbitaceae, Convolvulaceae, Liliaceae, Asteraceae, Chenopodiaceae, Apiaceae, Araceae, Zingiberaceae, and Malvaceae, and parasitize a variety of agricultural crops, causing plant diseases.Examples of host plant species for root-knot nematodes include tomatoes, bell peppers, gourds, potatoes, sweet potatoes, eggplants, carrots, burdock, spinach, Swiss chard, garland chrysanthemums, leeks, ginger, peas, kidney beans, cowpeas, and rice.

[0026] Rice cultivars are classified into Asian rice (Oryza sativa) and African rice (Oryza glaberrima). Asian rice is divided into two subspecies, Indica and Japonica, and Japonica is divided into Temperate Japonica (shown as "Temperate Japonica" in Table 1) and Tropical Japonica (shown as "Tropical Japonica" in Table 1). Examples of cultivars belonging to Temperate Japonica include Taichung65 (T65), Ginbouzu, Nipponbare, Kinmaze, Hinohikari, Yukihikari, Aikoku, Kameji, Kyotoasahi, Akage, and Dianyu1. Examples of varieties belonging to the tropical japonica include Ma Sho, Khao Nok, Jaguary, Khau Mac Kho, Padi Perak, Rexmont, Senshou, Kahei, etc. Indica can be classified into Indica (shown as "Indica" in Table 1) and Aus (shown as "Aus" in Table 1). Examples of varieties belonging to the Indica include Bei Khe, Naba, Pulik Arang, Ryou Suisan Koumai, Jinguoyin, Keiboba, Qingyu, Deng Pao Zhai, Milyang23, Karahoushi, etc. Examples of varieties belonging to the Aus include Kasalath, Jena035, Muha, Jhona2, Nepal8, Jarjan, Kalo Dhan, Anjana Dhan, Shoni, Surjamukhi, ARC7291, ARC5955, ARC7047, ARC11094, Badari Dhan, Nepal555, Kaluheenati, DV85, ARC10313, and N22. NERICA is a hybrid between Asian rice and African rice.NERICA varieties include NERICA 1, NERICA 2, NERICA 4, NERICA 6, NERICA L20, and NERICA L41 (in Table 1, NERICA-related varieties, including NERICA varieties, are listed as "NERICA related"). Hybrids not classified into any of the above groups (listed as "Admixture" in Table 1) include Davao1, Asu, IR58, Co13, Vary Futsi, Shwe Nang Gyi, Pinulupot1, Local Basmati, Basilanon, Khau Tan Chiem, Tima1, and Tupa729. Varieties of unknown classification (listed as "Unknown" in Table 1) include Basmati370, IRAT109, LTH, IR24, Kinandang Patong, and Silewah. For classification methods of rice cultivars, see Kojima et al., 2005, Breeding Science, 55, 431-440 and Yonemaru et al., 2014, Plant Cell Physiol., 55 (1), e9.

[0027] Rice varieties can be classified into plant parasitic nematode-susceptible varieties and plant parasitic nematode-resistant varieties. For example, an evaluation value (EV) can be calculated using the evaluation method described in the Examples of the present specification, and rice varieties with an evaluation value equal to or greater than a certain value can be classified as nematode-susceptible varieties, while rice varieties with an evaluation value less than the certain value can be classified as nematode-resistant varieties.

[0028] As used herein, "resistance to plant-parasitic nematodes" refers to the ability to prevent or suppress damage to and / or parasitism (infection) of a host plant by plant-parasitic nematodes. Resistance of a plant to plant-parasitic nematodes, such as root-knot nematodes, can be tested using methods known to those skilled in the art. For example, a certain number (e.g., 200) of root-knot nematode J2 larvae is inoculated into the culture soil of a test plant, and the infection state is evaluated after a certain period (e.g., two months). The evaluation is preferably carried out by counting the number of nodules on the roots of the plant and / or the number of root-knot nematode egg masses. The number of nodules can be counted visually.

[0029] As used herein, "control of plant-parasitic nematodes" refers to the action of preventing or suppressing damage to and / or parasitism (infection) of plant-parasitic nematodes on host plants.

[0030] As used herein, the term "RMIR1 (Root Meloidogyne Incognita Resistance 1) gene" refers to the Os06g0621600 gene in the Nipponbare genome, which is associated with resistance to plant parasitic nematodes in rice; an orthologous gene corresponding to the Os06g0621600 gene in any rice cultivar; an orthologous gene corresponding to the Os06g0621600 gene in any plant species; or a mutant gene derived from any of these. The RMIR1 gene is designated as the Os06g0621600 gene in the RAP-db, but is designated as the LOC_Os06g41670.1 gene in the Rice Genome Annotation Project (RGAP). In this specification, when the term "RMIR1 gene" is used simply, it includes wild-type and mutant RMIR1 genes derived from any biological species (referred to as "wild-type RMIR1 gene" and "mutant RMIR1 gene", respectively), as well as the L-type RMIR1 gene and S-type RMIR1 gene described below.

[0031] The RMIR1 gene sequence may differ between rice varieties. For example, the RMIR1 gene of ARC10313 encodes a protein consisting of 1,154 amino acid residues, while the RMIR1 gene of T65 encodes a deletion-type protein consisting of 579 amino acid residues in which the C-terminal region is deleted. Herein, rice RMIR1 genes are classified into L-type RMIR1 genes and S-type RMIR1 genes depending on the presence or absence of the C-terminal region. That is, S-type RMIR1 genes encode deletion-type proteins in which the C-terminal region is deleted compared to the L-type RMIR1 gene.

[0032] Examples of L-type RMIR1 genes include the RMIR1 genes of ARC10313, N22, and Kalo Dhan. The nucleotide sequences of the RMIR1 genes of ARC10313, N22, and Kalo Dhan are all shown in SEQ ID NO: 14, and are 100% identical.

[0033] Examples of the S-type RMIR1 gene include the RMIR1 genes of T65 and Nipponbare, etc. The nucleotide sequences of the RMIR1 genes of T65 and Nipponbare are both shown in SEQ ID NO: 16 and are 100% identical.

[0034] As used herein, the term "RMIR1 polypeptide" refers to a polypeptide encoded by the Os06g0621600 gene on the Nipponbare genome, which is associated with resistance to plant parasitic nematodes in rice (i.e., a polypeptide encoded by the RMIR1 gene), its corresponding ortholog in any rice variety, its corresponding ortholog in any plant species, or a mutant polypeptide derived from either of them. As used herein, the term "RMIR1 polypeptide" includes wild-type and mutant RMIR1 polypeptides derived from any biological species (referred to as "wild-type RMIR1 polypeptide" and "mutant RMIR1 polypeptide," respectively), and also includes the L-type RMIR1 polypeptide and S-type RMIR1 polypeptide described below.

[0035] Wild-type rice RMIR1 polypeptides are classified into L-type RMIR1 polypeptides encoded by L-type RMIR1 genes and S-type RMIR1 polypeptides encoded by S-type RMIR1 genes. L-type RMIR1 polypeptides are NB-LRR proteins containing a nucleotide-binding domain (NB-ARC domain) and 13 leucine-rich repeat (LRR) domains. In contrast, S-type RMIR1 polypeptides lack 11 C-terminal LRR domains in L-type RMIR1 polypeptides because the position corresponding to the serine residue at position 580 in L-type RMIR1 polypeptides is a stop codon. An example of the amino acid sequence of an L-type RMIR1 polypeptide is SEQ ID NO: 13. The amino acid sequences of RMIR1 polypeptides in ARC10313, N22, and Kalo Dhan are all shown in SEQ ID NO: 13 and are 100% identical. An example of the amino acid sequence of an S-type RMIR1 polypeptide is SEQ ID NO: 15. The amino acid sequences of the RMIR1 polypeptides of T65 and Nipponbare are both shown in SEQ ID NO: 15 and are 100% identical. Compared to the RMIR1 polypeptides of ARC10313, N22, and Kalo Dhan, the RMIR1 polypeptides of T65 and Nipponbare lack 575 amino acid residues at the C-terminus, and also have an alanine residue at position 138 substituted with a threonine residue and an arginine residue at position 548 substituted with a leucine residue (Figure 3).

[0036] As used herein, the term "RKNR1 (ROOT KNOT NEMATODE RESISTANCE 1) gene" refers to the Os04g0112100 gene on the Nipponbare genome, which is associated with resistance to plant parasitic nematodes in rice, an orthologous gene corresponding to the Os04g0112100 gene in any rice cultivar, an orthologous gene corresponding to the Os04g0112100 gene in any plant species, or a mutant gene derived from either of them. As used herein, the term "RKNR1 gene" includes wild-type and mutant RKNR1 genes derived from any biological species (referred to as the "wild-type RKNR1 gene" and the "mutant RKNR1 gene," respectively), and also includes the L-type RKNR1 gene and the S-type RKNR1 gene described below.

[0037] The RKNR1 gene sequence may differ between rice varieties. For example, it is known that the presence or absence of a sequence region in the RKNR1 ORF that extends from the center of the NB-ARC domain to the C-terminus of the LRR domain in the RKNR1 amino acid sequence differs between rice varieties. Herein, rice RKNR1 genes are classified into L-type RKNR1 genes and S-type RKNR1 genes based on the presence or absence of this sequence region.

[0038] Examples of L-type RKNR1 genes include the RKNR1 genes of some varieties belonging to the temperate japonica group (e.g., Akagei and Ginbozu), as well as varieties belonging to the indica, tropical japonica, Ausu, and NERICA groups. Examples of the nucleotide sequences of L-type RKNR1 genes include SEQ ID NO: 2 (N22 and Kalo Dhan), SEQ ID NO: 5 (Naba), SEQ ID NO: 6 (Bei Khe), and SEQ ID NO: 7 (Akagei). The nucleotide sequences of the RKNR1 genes are 100% identical between N22 and Kalo Dhan, and both are shown in SEQ ID NO: 2.

[0039] Examples of the S-type RKNR1 gene include the RKNR1 genes of some temperate Japonica varieties (e.g., T65, Nipponbare, Kinmaku, Hinohikari, Yukihikari, Aikoku, Kameji, Kyoto Asahi, and Dianyu1). Examples of the nucleotide sequences of the S-type RKNR1 gene include SEQ ID NO: 3 (T65) and SEQ ID NO: 4 (Nipponbare).

[0040] As used herein, the term "RKNR1 polypeptide" refers to a polypeptide encoded by the Os04g0112100 gene on the Nipponbare genome, which is associated with resistance to plant parasitic nematodes in rice (i.e., a polypeptide encoded by the RKNR1 gene), its corresponding ortholog in any rice variety, its corresponding ortholog in any plant species, or a mutant polypeptide derived from any of them. As used herein, the term "RKNR1 polypeptide" includes wild-type and mutant RKNR1 polypeptides derived from any biological species (referred to as "wild-type RKNR1 polypeptide" and "mutant RKNR1 polypeptide," respectively), and also includes the L-type RKNR1 polypeptide and S-type RKNR1 polypeptide described below.

[0041] Wild-type rice RKNR1 polypeptides are classified into L-type RKNR1 polypeptides encoded by the L-type RKNR1 gene and S-type RKNR1 polypeptides encoded by the S-type RKNR1 gene. In rice cultivars other than Bei Khe and Naba, L-type RKNR1 polypeptides are NB-LRR proteins containing a nucleotide-binding domain (NB-ARC domain) and seven leucine-rich repeat (LRR) domains. In contrast, S-type RKNR1 polypeptides are L-type RKNR1 polypeptides lacking part of the nucleotide-binding domain and most of the LRR domain. Note that the RKNR1 polypeptides of Bei Khe and Naba are classified as L-type RKNR1 polypeptides because they do not contain the 1754-bp region deleted in their gene sequences. However, they are shortened due to a stop codon generated by a frameshift. Examples of amino acid sequences of S-type RKNR1 polypeptides include SEQ ID NO: 8 (T65) and SEQ ID NO: 9 (Nipponbare). Examples of the amino acid sequences of L-type RKNR1 polypeptides include SEQ ID NO: 1 (N22 and Kalo Dhan), SEQ ID NO: 10 (Naba), SEQ ID NO: 11 (Bei Khe), and SEQ ID NO: 12 (red hair). The amino acid sequences of the RKNR1 polypeptides are 100% identical between Kalo Dhan and N22, both of which are represented by SEQ ID NO: 1.

[0042] As used herein, the term "all" of a plant refers to all regions constituting a living plant. Furthermore, the term "part" of a plant refers to a partial region constituting a living plant, specifically, an organ (e.g., roots, stems, leaves, flowers, epidermis, or a combination thereof, or pollen, egg cells, seeds, etc.), a tissue or part thereof consisting of a morphologically and / or functionally differentiated cell group, or a cell.

[0043] As used herein, "multiple" refers to, for example, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 15, 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3 amino acids. Furthermore, "amino acid identity" refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues when the amino acid sequences of two polypeptides being compared are aligned, with appropriate gaps inserted into one or both sequences as needed to maximize the number of identical amino acid residues. Alignment of two amino acid sequences to calculate amino acid identity can be performed using known programs such as Blast, FASTA, and ClustalW. "Nucleotide identity" is calculated in a similar manner.

[0044] As used herein, "(amino acid) substitution" refers to substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity, among the 20 amino acids that constitute 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 be less likely to cause changes in the properties of polypeptides.

[0045] As used herein, "stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form. "Highly stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form or are not formed at all. In general, the lower the salt concentration and the higher the temperature of the reaction conditions, the more stringent the conditions. For washing after hybridization, for example, washing 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.

[0046] 1-3. Configuration The plant-parasitic nematode control agent of the present invention contains, as an active ingredient, (1) a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, (2) a polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, or (3) an expression vector comprising a polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof.

[0047] 1-3-1. Active ingredient (1) Plant-parasitic nematode-resistant RMIR1 polypeptide or fragment thereof In one embodiment, the plant-parasitic nematode control agent of the present invention consists of or contains a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof.

[0048] As used herein, "a plant-parasitic nematode-resistant RMIR1 polypeptide" refers to an RMIR1 polypeptide that confers resistance to plant-parasitic nematodes to a host plant and / or enhances the host plant's resistance to plant-parasitic nematodes, and refers to a wild-type or mutant RMIR1 polypeptide derived from a plant that is resistant to plant-parasitic nematodes.

[0049] Wild-type RMIR1 polypeptides resistant to plant-parasitic nematodes include wild-type L-type RMIR1 polypeptides. Examples of wild-type L-type RMIR1 polypeptides include the wild-type RMIR1 polypeptides of ARC10313, N22, and Kalo Dhan shown in SEQ ID NO: 13, and their wild-type L-type RMIR1 orthologs of other rice varieties or other plant species.

[0050] Examples of mutant RMIR1 polypeptides that are resistant to plant parasitic nematodes include amino acid sequences in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of any of the above-mentioned wild-type RMIR1 polypeptides that are resistant to plant parasitic nematodes, or polypeptides containing an amino acid sequence that has 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the amino acid sequence of any of the above-mentioned wild-type RMIR1 polypeptides that are resistant to plant parasitic nematodes. Examples of such polypeptides include an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 13, or a polypeptide comprising an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the amino acid sequence shown in SEQ ID NO: 13. Preferably, the plant-parasitic nematode-resistant mutant RMIR1 polypeptide has 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the plant-parasitic nematode-resistant wild-type RMIR1 polypeptide, or an activity equivalent to or greater than that.

[0051] In one embodiment, the RMIR1 polypeptide resistant to plant-parasitic nematodes consists of a polypeptide or a fragment thereof comprising either (a) the amino acid sequence shown in SEQ ID NO: 13, (b) the amino acid sequence shown in SEQ ID NO: 13 in which one or more amino acids have been deleted, substituted or added, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 13.

[0052] As used herein, a "fragment" of a plant-parasitic nematode-resistant RMIR1 polypeptide refers to a fragment of the above-mentioned plant-parasitic nematode-resistant RMIR1 polypeptide that has the activity of conferring resistance to plant-parasitic nematodes to a host plant and / or enhancing the resistance of a host plant to plant-parasitic nematodes, for example, a fragment having 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the plant-parasitic nematode-resistant RMIR1 polypeptide, or an activity equivalent to or greater than these. Examples include polypeptide fragments of the plant-parasitic nematode-resistant RMIR1 polypeptide that contain one, two, or three or more LRR domains. The amino acid length of the polypeptide constituting this fragment is not particularly limited, but may be, for example, a region of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 consecutive amino acids in the RMIR1 polypeptide that is resistant to plant parasitic nematodes.

[0053] In a further embodiment, the plant-parasitic nematode control agent further comprises a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof in addition to a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof.

[0054] As used herein, the term "RKNR1 polypeptide resistant to plant-parasitic nematodes" refers to a RKNR1 polypeptide that confers resistance to plant-parasitic nematodes to a host plant and / or enhances the resistance of a host plant to plant-parasitic nematodes, and refers to a wild-type or mutant RKNR1 polypeptide derived from a plant that is resistant to plant-parasitic nematodes. Patent Document 1 (WO2022 / 176971) discloses that the RKNR1 gene is a gene that can enhance the resistance of a plant to plant-parasitic nematodes, and also discloses that the RKNR1 gene can suppress the nematode-attracting activity of the plant, nematode migration into the roots, gall formation, gall maturation, nematode growth in the roots, and / or the induction of giant cells in the host plant.

[0055] Wild-type RKNR1 polypeptides resistant to plant-parasitic nematodes include L-type RKNR1 polypeptides other than the RKNR1 polypeptides of Bei Khe, Naba, and Akage. Examples of L-type RKNR1 polypeptides other than the RKNR1 polypeptides of Bei Khe, Naba, and Akage include the wild-type RKNR1 polypeptide of N22 shown in SEQ ID NO: 1, the wild-type RKNR1 polypeptide of Kalo Dhan shown in SEQ ID NO: 1, and wild-type RKNR1 orthologs thereof of other rice varieties or other plant species. For example, the wild-type RKNR1 polypeptide of Ma Sho, the wild-type RKNR1 polypeptide of Khao Nok, the wild-type RKNR1 polypeptide of Jaguary, the wild-type RKNR1 polypeptide of Khau Mac Kho, the wild-type RKNR1 polypeptide of Padi Perak, the wild-type RKNR1 polypeptide of Rexmont, the wild-type RKNR1 polypeptide of Senshou, the wild-type RKNR1 polypeptide of Kahei, the wild-type RKNR1 polypeptide of Puluik Arang, the wild-type RKNR1 polypeptide of Ryou Suisan Koumai, the wild-type RKNR1 polypeptide of Jinguoyin, the wild-type RKNR1 polypeptide of Keiboba, the wild-type RKNR1 polypeptide of Qingyu, and the wild-type RKNR1 polypeptide of Deng Pao. Wild-type RKNR1 polypeptide from Zhai, wild-type RKNR1 polypeptide from Milyang23, wild-type RKNR1 polypeptide from Karahoushi, wild-type RKNR1 polypeptide from Kasalath, wild-type RKNR1 polypeptide from Jena035, wild-type RKNR1 polypeptide from Muha, wild-type RKNR1 polypeptide from Jhona2, wild-type RKNR1 polypeptide from Nepal8, wild-type RKNR1 polypeptide from Jarjan, wild-type RKNR1 polypeptide from Anjana Dhan, wild-type RKNR1 polypeptide from Shoni, wild-type RKNR1 polypeptide from Surjamukhi, wild-type RKNR1 polypeptide from ARC7291, wild-type RKNR1 polypeptide from ARC5955, wild-type RKNR1 polypeptide from ARC7047, wild-type RKNR1 polypeptide from ARC11094, BadariWild-type RKNR1 polypeptide from Dhan, wild-type RKNR1 polypeptide from Nepal555, wild-type RKNR1 polypeptide from Kaluheenati, wild-type RKNR1 polypeptide from DV85, wild-type RKNR1 polypeptide from ARC10313, wild-type RKNR1 polypeptide from WAB56-50, wild-type RKNR1 polypeptide from WAB56-104, wild-type RKNR1 polypeptide from NERICA 1, wild-type RKNR1 polypeptide from NERICA 2, wild-type RKNR1 polypeptide from NERICA 4, wild-type RKNR1 polypeptide from NERICA 6, wild-type RKNR1 polypeptide from NERICA L20, wild-type RKNR1 polypeptide from NERICA L41, wild-type RKNR1 polypeptide from CG14, wild-type RKNR1 polypeptide from WK18, wild-type RKNR1 polypeptide from Davao1, wild-type RKNR1 polypeptide from Asu, wild-type RKNR1 polypeptide from IR58, wild-type RKNR1 polypeptide from Co13, Vary Examples of such polypeptides include the wild-type RKNR1 polypeptide from Futsi, the wild-type RKNR1 polypeptide from Shwe Nang Gyi, the wild-type RKNR1 polypeptide from Pinulupot1, the wild-type RKNR1 polypeptide from Local Basmati, the wild-type RKNR1 polypeptide from Basilanon, the wild-type RKNR1 polypeptide from Khau Tan Chiem, the wild-type RKNR1 polypeptide from Tima1, the wild-type RKNR1 polypeptide from Tupa729, the wild-type RKNR1 polypeptide from Basmati370, the wild-type RKNR1 polypeptide from IRAT109, the wild-type RKNR1 polypeptide from LTH, the wild-type RKNR1 polypeptide from IR24, the wild-type RKNR1 polypeptide from Kinandang Patong, and the wild-type RKNR1 polypeptide from Silewah.

[0056] Examples of mutant RKNR1 polypeptides that are resistant to plant parasitic nematodes include amino acid sequences in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence of any of the above-mentioned wild-type RKNR1 polypeptides that are resistant to plant parasitic nematodes, or polypeptides containing an amino acid sequence that has 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the amino acid sequence of any of the above-mentioned wild-type RKNR1 polypeptides that are resistant to plant parasitic nematodes. Examples of such polypeptides include an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, or a polypeptide comprising an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the amino acid sequence shown in SEQ ID NO: 1. Preferably, the plant-parasitic nematode-resistant mutant RKNR1 polypeptide has 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the plant-parasitic nematode-resistant wild-type RKNR1 polypeptide, or an activity equivalent to or greater than that.

[0057] In a further embodiment, the plant-parasitic nematode-resistant mutant RKNR1 polypeptide has an amino acid residue other than Gly (e.g., Asp) at position 315 in the amino acid sequence shown in SEQ ID NO: 12, an amino acid residue other than Asp (e.g., Glu) at position 505 in the amino acid sequence shown in SEQ ID NO: 12, an amino acid residue other than Val (e.g., Ala) at position 745 in the amino acid sequence shown in SEQ ID NO: 12, and / or an amino acid residue other than Gln (e.g., Leu) at position 1040 in the amino acid sequence shown in SEQ ID NO: 12. The plant-parasitic nematode-resistant mutant RKNR1 polypeptide preferably has an amino acid residue other than Gln (e.g., Leu) at position 1040 in the amino acid sequence shown in SEQ ID NO: 12.

[0058] In a further embodiment, the plant-parasitic nematode-resistant RKNR1 polypeptide consists of a polypeptide or a fragment thereof comprising either (a) the amino acid sequence shown in SEQ ID NO: 1, (b) the amino acid sequence shown in SEQ ID NO: 1 in which one or more amino acids have been deleted, substituted, or added, or (c) an amino acid sequence having 90% or more identity to the amino acid sequence shown in SEQ ID NO: 1.

[0059] As used herein, a "fragment" of the plant-parasitic nematode-resistant RKNR1 polypeptide refers to a fragment of the above-mentioned plant-parasitic nematode-resistant RKNR1 polypeptide that has the activity of conferring resistance to plant-parasitic nematodes to a host plant and / or enhancing the resistance of a host plant to plant-parasitic nematodes, for example, a fragment having 50% or more, 60% or more, 70% or more, 80% or more, or 90% or more of the activity of the plant-parasitic nematode-resistant RKNR1 polypeptide, or an activity equivalent to or greater than these. For example, a polypeptide fragment comprising the LRR domain of the plant-parasitic nematode-resistant RKNR1 polypeptide is exemplified. The amino acid length of the polypeptide constituting this fragment is not particularly limited, but may be, for example, a region of at least 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or 1000 consecutive amino acids in a plant parasitic nematode-resistant RKNR1 polypeptide.

[0060] (2) RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof In one embodiment, the plant-parasitic nematode control agent of the present invention comprises or consists of a polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof (referred to herein as an "RMIR1 polynucleotide").

[0061] The RMIR1 polynucleotide encodes the above-mentioned plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof. The nucleotide sequence of the RMIR1 polynucleotide is not particularly limited, as long as it encodes the plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof. For example, it is a polynucleotide encoding the wild-type RMIR1 polypeptide of ARC10313 consisting of the amino acid sequence shown in SEQ ID NO: 13 (for example, a polynucleotide that is the wild-type RMIR1 gene of ARC10313 and consists of the nucleotide sequence shown in SEQ ID NO: 14).

[0062] In one embodiment, the RMIR1 polynucleotide comprises any of (g) the base sequence shown in SEQ ID NO: 14, (h) a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 14, (i) a base sequence having 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the base sequence shown in SEQ ID NO: 14, or (j) a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 14.

[0063] In a further embodiment, the plant-parasitic nematode control agent of the present invention further comprises, in addition to a polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof (referred to in this specification as an "RKNR1 polynucleotide").

[0064] The RKNR1 polynucleotide encodes the above-mentioned plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof. The RKNR1 polynucleotide is not particularly limited in its nucleotide sequence, as long as it encodes the plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof. For example, the polynucleotide may encode the wild-type RKNR1 polypeptide of N22 consisting of the amino acid sequence shown in SEQ ID NO: 1 (e.g., a polynucleotide that is the wild-type RKNR1 gene of N22 and consists of the nucleotide sequence shown in SEQ ID NO: 2), or the RKNR1 polypeptide of Kalo Dhan consisting of the amino acid sequence shown in SEQ ID NO: 1 (e.g., a polynucleotide that is the wild-type RKNR1 gene of Kalo Dhan and consists of the nucleotide sequence shown in SEQ ID NO: 2).

[0065] In a further embodiment, the RKNR1 polynucleotide comprises any of: (k) the nucleotide sequence shown in SEQ ID NO: 2; (l) a nucleotide sequence in which one or more bases are deleted, substituted, or added in the nucleotide sequence shown in SEQ ID NO: 2; (m) a nucleotide sequence having 60% or more, 65% or more, 70% or more, 75% or more, 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 identity to the nucleotide sequence shown in SEQ ID NO: 2; or (n) a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2.

[0066] In one embodiment, the base sequence of the RMIR1 polynucleotide and / or the RKNR1 polynucleotide may be a base sequence that has been codon-optimized to match the codon usage frequency in a cell into which the polynucleotide is introduced.

[0067] The RMIR1 polynucleotide and / or the RKNR1 polynucleotide may be DNA or RNA such as mRNA. When the RMIR1 polynucleotide and / or the RKNR1 polynucleotide is mRNA, the base sequence may be an mRNA containing, as a coding region, a base sequence in which thymine (T) in any of the base sequences exemplified above is substituted with uracil (U). The mRNA corresponding to the polynucleotide of the present invention may contain, in addition to the coding region, a cap structure at the 5' end, a poly(A) tail at the 3' end, a 5' untranslated region (5' UTR) upstream of the start codon, and / or a 3' untranslated region (3' UTR) downstream of the stop codon. The 5' UTR and / or 3' UTR may contain a sequence for regulating the amount of translation from the mRNA.

[0068] (3) Expression vector containing an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof In one embodiment, the plant-parasitic nematode control agent of the present invention comprises or consists of an expression vector containing an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof.

[0069] The expression vector of the present invention comprises, in an expressible state, an RMIR1 polynucleotide encoding the plant-parasitic nematode-resistant RMIR1 polypeptide of the present invention or a fragment thereof. As used herein, the term "expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter.

[0070] The expression vector of the present invention contains, as essential components, a promoter and the polynucleotide described in "(2) RMIR1 polynucleotide encoding an RMIR1 polypeptide or a fragment thereof conferring resistance to plant-parasitic nematodes."

[0071] In a further embodiment, the expression vector of the present invention further comprises, in addition to the RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, an RKNR1 polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof in an expressible state.

[0072] Vectors that can be used as the expression vector of the present invention include, for example, plasmid- or virus-based expression vectors. As used herein, the term "expression vector" encompasses recombinant vectors.

[0073] In the case of an expression vector using a plasmid (hereinafter often referred to as a "plasmid expression vector"), the plasmid may be, but is not limited to, a pPZP series, pSMA series, pUC series, pBR series, pBluescript series (Agilent Technologies), pTriEXTM series (TaKaRa), or a pBI series, pRI series, or pGW series binary vector.

[0074] In the case of expression vectors that utilize viruses (hereinafter often referred to as "viral expression vectors"), viruses that can be used include cauliflower mosaic virus (CaMV), bean golden mosaic virus (BGMV), and tobacco mosaic virus (TMV).

[0075] When the Agrobacterium method is used, an expression vector suitable for the Agrobacterium method, such as a binary vector, or a modified vector thereof can also be used. Examples of such expression vectors include pBI121, pBIN19, pSMAB704, pCAMBIA, and pGreen.

[0076] Various promoters can be used, including overexpression promoters, constitutive promoters, site-specific promoters, time-specific promoters, and / or inducible promoters. Specific examples of overexpression constitutive promoters operable in plant cells include the 35S promoter derived from cauliflower mosaic virus (CaMV), the Pnos promoter of the nopaline synthase gene derived from Ti plasmid, the ubiquitin promoter derived from maize, the actin promoter derived from rice, and the PR protein promoter derived from tobacco. The small subunit (Rubisco ssu) promoter of ribulose bisphosphate carboxylase from various plant species or histone promoters can also be used. Examples of inducible 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.

[0077] The expression vector may also contain a terminator, an enhancer, a poly(A) addition signal, a 5'-UTR (untranslated region) sequence, an intron sequence, a ribosome binding sequence, a tagging or selection marker gene, a multicloning site, a nuclease recognition sequence, and / or a replication origin. The type of each component is not particularly limited as long as it can perform its function in the host cell. Any component known in the art may be appropriately selected depending on the plant cell or plant host to be introduced.

[0078] Examples of terminators include the nopaline synthase (NOS) gene terminator, the octopine synthase (OCS) gene terminator, the CaMV 35S terminator, the 3' terminator of Escherichia coli lipopolyprotein lpp, the trp operon terminator, the amyB terminator, the ADH1 gene terminator, etc. There are no particular limitations on the sequence as long as it is capable of terminating transcription of the gene transcribed by the promoter.

[0079] The enhancer may be, for example, an enhancer region containing an upstream sequence in the CaMV 35S promoter, and is not particularly limited as long as it can enhance the expression efficiency of a nucleic acid encoding an active peptide.

[0080] Examples of nuclease recognition sequences include restriction enzyme recognition sequences, loxP sequences recognized by Cre recombinase, sequences targeted by artificial nucleases such as ZFN and TALEN, and sequences targeted by the CRISPR / Cas9 system. Examples of replication origin sequences include the SV40 replication origin sequence.

[0081] Examples of selectable 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 fluorescent protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase.

[0082] The selectable marker gene that can be contained in the expression vector of the present invention is a selectable marker gene that allows selection of cells into which the expression vector of the present invention has been introduced. Specific examples of the selectable marker gene include drug resistance genes such as an ampicillin resistance gene, a kanamycin resistance gene, a tetracycline resistance gene, a chloramphenicol resistance gene, a neomycin resistance gene, a puromycin resistance gene, or a hygromycin resistance gene.

[0083] The reporter gene that can be contained in the expression vector of the present invention is a gene encoding a reporter that can identify cells into which the expression vector of the present invention has been introduced. Examples of reporter genes include genes encoding fluorescent proteins such as GFP and RFP, and luciferase genes.

[0084] 1-3-2. Other Components The plant-parasitic nematode control agent of the present invention may consist solely of the active ingredients described in "1-3-1. Active ingredients," but may also contain other ingredients as needed.

[0085] The plant parasitic nematode control agent of the present invention may contain an agriculturally acceptable carrier. As used herein, the term "agriculturally acceptable carrier" refers to a substance that does not substantially affect the activity of the plant parasitic nematode control agent of the present invention, and that has no or little harmful effect on the environment, such as soil and water quality, when applied to plant cultivation, or has no or little harmful effect on animals, particularly humans. Examples of such a carrier include solvents, adjuvants, excipients, emulsifiers, dispersants, surfactants, etc.

[0086] The plant-parasitic nematode control agent of the present invention can also contain other ingredients with pharmacological action, i.e., nematicides, herbicides, and fertilizers (e.g., urea, ammonium nitrate, and superphosphate), to the extent that the activity of the active ingredient is not affected.

[0087] 1-3-3. Dosage Form The dosage form of the plant parasitic nematode control agent of the present invention may be in any state as long as it is capable of penetrating into the plant body to which it is applied, and may be, for example, a liquid formulation in a liquid state or a solid formulation in a solid state. In the case of a liquid formulation, examples include a solution in which the active ingredient is suspended in an appropriate solution, an oil dispersion, an emulsion, and a suspension. In the case of a solid formulation, there are no particular limitations as long as the active ingredient is in a state that can act on the plant to which it is applied. Examples include dusts, powders, pastes, and gels.

[0088] 1-4. Effects The plant-parasitic nematode control agent of the present invention can confer resistance to plant-parasitic nematodes to plants susceptible to plant-parasitic nematodes. Furthermore, the plant-parasitic nematode control agent of the present invention can enhance the resistance to plant-parasitic nematodes in plants that are resistant to plant-parasitic nematodes.

[0089] The plant-parasitic nematode control agent of the present invention can suppress nematode attractant activity, nematode migration into the roots, nodule formation, nodule maturation, nematode growth in the roots, and / or the induction of giant cells in the host plant in the plant to which the agent is applied.

[0090] 2. Plant transformant or progeny thereof having resistance to plant parasitic nematodes 2-1. Overview A second aspect of the present invention relates to a plant transformant or progeny thereof having resistance to plant parasitic nematodes. The plant transformant of the present invention or progeny thereof comprises the plant parasitic nematode control agent described in the first aspect, for example, the polynucleotide or expression vector described in the first aspect, and has resistance to plant parasitic nematodes such as root-knot nematodes. The plant transformant of the present invention or progeny thereof has suppressed nematode attractant activity, nematode migration into roots, nodule formation, nodule maturation, nematode growth in roots, and / or giant cell induction in the host plant, and is resistant to plant parasitic nematodes.

[0091] 2-2. Constitution The term "transformed plant" as used herein refers to a plant host that has been genetically modified to acquire resistance to plant-parasitic nematodes.

[0092] In one embodiment, the transformed plant of the present invention comprises an RMIR1 polynucleotide encoding the RMIR1 polypeptide or a fragment thereof conferring resistance to plant parasitic nematodes described in aspect 1, or an expression vector comprising the polynucleotide. More specifically, the transformed plant of the present invention has been transformed with an RMIR1 polynucleotide encoding the RMIR1 polypeptide or a fragment thereof conferring resistance to plant parasitic nematodes described in aspect 1, or an expression vector comprising the polynucleotide.

[0093] In a further embodiment, the plant transformant of the present invention comprises an RMIR1 polynucleotide encoding the RMIR1 polypeptide or fragment thereof conferring resistance to plant parasitic nematodes described in the first aspect, or an expression vector comprising said polynucleotide, and an RKNR1 polynucleotide encoding the RKNR1 polypeptide or fragment thereof conferring resistance to plant parasitic nematodes described in the first aspect, or an expression vector comprising said polynucleotide, and is, for example, transformed with the above two types of polynucleotides or expression vectors.

[0094] The host plant species to be transformed in the present invention is not limited. The host plant may be a monocotyledonous or dicotyledonous plant, with monocotyledonous plants being particularly preferred. Monocotyledonous plants may include species belonging to the Poaceae family (e.g., rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, sorghum, and sorghum), species belonging to the Musaceae family (e.g., banana and musa), species belonging to the Amaryllidaceae family (e.g., leek, onion, garlic, and chive), or species belonging to the Bromeliaceae family (e.g., pineapple). Preferred host plants are plant species susceptible to plant-parasitic nematodes or plant species with weak resistance to plant-parasitic nematodes, such as rice varieties having an S-type RMIR1 gene and / or an S-type RKNR1 gene, or plant species lacking an L-type RMIR1 gene and / or an L-type RKNR1 gene. Examples of rice varieties susceptible to plant parasitic nematodes include rice varieties that show an evaluation value of 1.0 or more in FIG. 1 or rice varieties that show an evaluation value of 0.8 or more.

[0095] The plant transformant of the present invention encompasses clones having the same genetic information. The plant transformant of the present invention also includes parts of plants collected from the first generation of plant transformants, such as plant tissues such as the epidermis, phloem, parenchyma, xylem, or vascular bundles, plant organs such as leaves, petals, stems, roots, or seeds, or clones obtained from plant cells by plant tissue culture, cuttings, grafting, or layering; new clones generated from vegetative propagation organs obtained by asexual reproduction from the first generation of plant transformants, such as rhizomes, tuberous roots, corms, and runners; and somatic embryos induced by dedifferentiation treatment from the first generation of plant transformants or clones derived therefrom. In one embodiment, the plant transformant of the present invention may be a genetically modified plant.

[0096] As used herein, the term "progeny" refers to a host plant that is a descendant of the first generation of the plant transformant through sexual reproduction, that harbors an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, or an expression vector containing the polynucleotide, and that is resistant to plant-parasitic nematodes, regardless of the progeny generation.

[0097] 2-3. Effects Plant transformants containing an RMIR1 polynucleotide encoding the plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof of the present invention, or an expression vector containing the polynucleotide (hereinafter referred to as "RMIR1 polypeptide, etc.") exhibit resistance to plant parasitic nematodes. Furthermore, plant transformants containing the plant-parasitic nematode-resistant RMIR1 polypeptide, etc. of the present invention and an RKNR1 polynucleotide encoding the plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof, or an expression vector containing the polynucleotide (hereinafter referred to as "RKNR1 polypeptide, etc.") have further enhanced nematode resistance compared to transformants containing only either the RMIR1 polypeptide, etc. or the RKNR1 polypeptide, etc.

[0098] 3. Method for Producing a Plant Transformant Resistant to Plant Parasitic Nematodes 3-1. Overview A third aspect of the present invention relates to a method for producing a plant transformant resistant to plant parasitic nematodes. According to the production method of the present invention, a resistant plant transformant can be produced from a plant susceptible to plant parasitic nematodes.

[0099] The method of the present invention for producing a plant transformant having resistance to plant-parasitic nematodes essentially comprises an introduction step and a selection step. Each step will be specifically described below.

[0100] 3-2-1. Introduction Step In this embodiment, the "introduction step" is a step of introducing into a host plant an expression vector containing an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof.

[0101] In a further embodiment, the introduction step is a step of introducing into a host plant an expression vector comprising an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof, in addition to an expression vector comprising an RKNR1 polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof.

[0102] The structure of the expression vector introduced in this step is as described in "(3) Expression vector comprising an RMIR1 polynucleotide encoding a plant-parasitic nematode-resistant RMIR1 polypeptide or a fragment thereof" in the first embodiment.

[0103] Methods known in the art can be used to introduce expression vectors, such as the Agrobacterium method, the PEG-calcium phosphate method, electroporation, liposome method, particle gun method, and microinjection method. The introduced polynucleotide may be integrated into the genomic DNA of the host, or may exist in the form of an introduced polynucleotide (e.g., as contained in a foreign vector). Furthermore, the introduced polynucleotide may be maintained continuously in the host cell, as if it were integrated into the genomic DNA of the host, or may be maintained transiently.

[0104] When the Agrobacterium method is used, a polynucleotide encoding the RMIR1 polypeptide or a fragment thereof that confers resistance to plant parasitic nematodes is inserted into an expression vector suitable for the Agrobacterium method, and then the polynucleotide is introduced into an appropriate Agrobacterium, such as Agrobacterium tumefaciens, by electroporation or other methods, and this strain is then inoculated into and infected with plant cells, callus, cotyledon segments, etc. Suitable Agrobacterium strains that can be used include, but are not limited to, GV3101, C58, C58C1Rif(R), EHA101, EHA105, AGL1, and LBA4404.

[0105] The host sample to be introduced may be a section of a plant leaf or the like, 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 introduction device (e.g., PDS-1000 (BIO-RAD)) is used according to the manufacturer's instructions to bombard such a sample with metal particles coated with the expression vector or DNA construct of the present invention, thereby introducing the vector into plant cells and obtaining transformed plant cells. The operation conditions are typically a pressure of about 450 to 2000 psi and a distance of about 4 to 12 cm.

[0106] 3-2-2. Selection Step In this embodiment, the "selection step" is a step of selecting a plant into which the expression vector has been introduced.

[0107] This step may be carried out by a method known in the art after introducing the expression vector into a host by the method described above. For example, transformants can be selected by utilizing the activity of a protein encoded by a selection marker gene or reporter gene in the expression vector.

[0108] In one embodiment, plant cells or cotyledon segments into which the expression vector or polynucleotide of the present invention has been introduced are cultured on a selective medium according to plant tissue culture methods, and the surviving callus is then cultured on a regeneration medium (containing appropriate concentrations of plant hormones (auxin, cytokinin, gibberellin, abscisic acid, ethylene, brassinolide, etc.)), thereby regenerating transformed plants. Transformants can be selected in this manner.

[0109] Example 1: Examination of resistance of various rice varieties to Meloidogyne incognita> (Objective) Various rice varieties are examined for resistance to Meloidogyne incognita (Mi).

[0110] (Method) (1) Rice Varieties The rice varieties evaluated in this example were 11 varieties belonging to the temperate japonica group, 8 varieties belonging to the tropical japonica group, 20 varieties belonging to the Aus group, 10 varieties belonging to the Indica group, 12 hybrid varieties, 8 varieties belonging to NERICA-related lines, and 8 varieties of unknown classification. The above varieties were obtained from Genbank of the National Agriculture and Food Research Organization (NARO) and Nagoya University.

[0111] (2) Method for evaluating Mi resistance Evaluation of Mi resistance was performed according to the method described in the present inventors' report (Sunohara, H., Kaida, S., and Sawa, S., 2020, Plant Biotechnol., 37, 343-347).

[0112] Specifically, rice seeds were soaked in a disinfectant solution (a 1:1000 dilution of Kao Corporation kitchen bleach) at 26°C for 3 days (water absorption period) to kill mold and bacteria and induce germination. After germination, seeds were sown in paper pouches (CYG Seed Germination Pouch, Mega International, USA), two seeds per pouch. The pouches containing germinated seeds were then placed in a dark place at 26°C for 3 days. Next, 10 pouches were sandwiched between wooden boards, secured with spring clamps, and grown under a 12-hour light (26°C) / 12-hour dark (24°C) regime for 8 days. The purpose of sandwiching the pouches between wooden boards was to remove excess moisture from the paper pouches, as high moisture reduces nematode infection efficiency. After 14 days of imbibition, 2 mL of a solution containing 400 J2-stage root-knot nematodes per mL (800 total nematodes) was applied along the roots (nematode inoculation). After inoculation, the pouches were placed horizontally between wooden boards in a dark place for 3 days, then placed upright in the dark at 28°C for 12 hours and 26°C for 12 hours. Watering was maintained throughout the growth period, and each plant was treated with 2 mL of liquid fertilizer (described in Nishiyama H. ​​et al., 2015, Nematol. Res. 45:45-49) once a week (7, 14, 21, and 28 days after inoculation).

[0113] The number of egg masses was counted 48 days after imbibition (34 days after inoculation). The entire root system was immersed in 50 ng / μL erioglossin for at least 15 minutes to stain the roots, and the number of blue-stained egg masses was counted. After counting the number of egg masses, the root system was placed in a 50°C incubator for at least 5 days, and the roots were weighed after completely removing all moisture. The number of egg masses per unit dry root weight was calculated from the obtained dry root weight and the number of egg masses. The standardized value, using the T65 value as the reference value, was used as the evaluation value (EV) for each cultivar.

[0114] Rice varieties with an evaluation score of 0.6 or higher were classified as Mi susceptible varieties, and those with an evaluation score of less than 0.6 were classified as Mi resistant varieties.

[0115] (Results) The evaluation values ​​obtained for each variety are shown in FIG. 1 and Table 1 below.

[0116]

[0117] In temperate japonica, cultivars other than Ginbouzu (T65, Nipponbare, Kinnamipu, Hinohikari, Yukihikari, etc.) were susceptible to Mi. On the other hand, in tropical japonica, Aus, indica, and NERICA cultivars, all cultivars other than Bei Khe and Naba were resistant to Mi.

[0118] The Aus variety ARC10313 showed an evaluation value of approximately 11.7% (EV=0.117) compared to T65, indicating that it is one of the varieties with strong resistance to Mi. Based on this result, ARC10313 was used as a Mi-resistant variety in the following examples.

[0119] Example 2: Mapping and identification of the Mi resistance gene (Objective) The Mi resistance gene is identified by QTL analysis using RIL lines and gene mapping using CSSL lines.

[0120] (Methods and Results) (1) QTL analysis using RILs We generated 113 recombinant inbred lines (RILs) by crossing T65 and ARC10313, and performed QTL (quantitative trait loci) analysis using 2,144 SNPs. As a result, one of the major QTLs was found on chromosome 6 and named qRMIR1 (qROOT MELOIDOGYNE INCOGNITA RESISTANCE 1).

[0121] (2) Gene Mapping Using CSSLs / Identification of the RMIR1 Gene In the qRMIR1 gene mapping, no RILs were found to be heterozygous for the genomic region containing the qRMIR1 gene. Therefore, it was not possible to narrow down the locus by obtaining additional recombinants. However, because chromosome segment substitution lines (CSSLs) between the two cultivars, T65 and ARC10313, were distributed by Oryzabase, we narrowed down the locus using these lines. The three CSSL lines used here (TACSSL-42, TACSL-45, and TACSL-47) possessed sequences derived from the resistant cultivar ARC10313 in a region between 0.596 and 29.229 Mb on chromosome 6 that differed between the two cultivars, but retained sequences derived from the susceptible cultivar T65 outside this region.

[0122] Three CSSL strains (TACSSL-42, TACSSSL-45, and TACSSSL-47) and their parent strains, T65 and ARC10313, were subjected to Mi resistance testing using the method described in Example 1. The Mi resistance scores for TACSSSL-42, TACSSSL-45, and TACSSSL-47 were 1.517 ± 0.035, 0.505 ± 0.015, and 1.236 ± 0.372, respectively (Fig. 2A). These results suggest that the Mi resistance gene on chromosome 6 is located in the 20.258-27.829 Mb region (approximately 7.6 Mb) derived from ARC10313 in TACSSSL-45.

[0123] Next, we searched for genes within the mapped region using the rice database RAP-db (https: / / rapdb.dna.affrc.go.jp / ). This database was created based on genome information from Nipponbare, and 449 genes were annotated in the approximately 7.6 Mb region described above. Among these 449 gene sequences, we identified the Os06g0621600 gene (hereafter referred to as the ROOT MELOIDOGYNE INCOGNITA RESISTANCE 1 gene or RMIR1 gene) as a gene containing the NB-ARC domain and exhibiting significant differences in amino acid sequence between resistant and susceptible cultivars. The Os06g0621600 gene in the RAP-db corresponds to the LOC_Os06g41670.1 gene in the Rice Genome Annotation Project (RGAP). In the following examples, the amino acid sequence based on the nucleotide sequence of the RGAP LOC_Os06g41670.1 gene was used as the sequence of the polypeptide encoded by the RMIR1 gene (hereinafter referred to as "RMIR1 polypeptide").

[0124] The structures of the polypeptides encoded by the RMIR1 gene in the resistant cultivar ARC10313 and the susceptible cultivar T65 are shown in FIG.

[0125] The RMIR1 gene of ARC10313 encodes a 1,154 amino acid long L-type RMIR1 polypeptide containing a nucleotide-binding domain (NB-ARC domain) and 13 leucine-rich repeat (LRR) domains. The nucleotide sequences of the RMIR1 genes of ARC10313, N22, and Kalo Dhan are all shown in SEQ ID NO: 14 and are 100% identical.

[0126] In contrast, the RMIR1 gene of T65 encodes a 579-amino acid S-type RMIR1 polypeptide in which serine at position 580 has been replaced with a stop codon, resulting in a deletion of 575 amino acid residues at the C-terminus, and in which alanine at position 138 has been replaced with threonine and arginine at position 548 has been replaced with leucine. Therefore, it was estimated that 11 of the 13 LRR domains in the RMIR1 gene of T65 have been deleted. Furthermore, the RMIR1 gene of T65 contains a 141-base insertion sequence downstream of the stop codon that is not present in ARC10313. The nucleotide sequences of the RMIR1 genes of T65 and Nipponbare are both set forth in SEQ ID NO: 16 and are 100% identical.

[0127] The amino acid identity between the RMIR1 polypeptide of ARC10313 (SEQ ID NO: 13) and the RKNR1 polypeptide of Kalo Dhan (SEQ ID NO: 1) is 62.64%.

[0128] Example 3: Effect of introduction of RMIR1 gene on Mi resistance trait (Objective) To create a transformed strain by introducing a genomic region containing the RMIR1 gene derived from Kalo Dhan, a nematode-resistant variety, into Nipponbare, and to verify whether Mi resistance can be obtained.

[0129] (Method) A 12-kb genomic region containing the Os06g0621600 gene (RMIR1 gene) of wild-type Kalo Dhan was PCR-amplified and cloned into a vector to create an RMIR1 transformation vector. The RMIR1 transformation vector or a control vector lacking the RMIR1 genomic region was electroporated into Agrobacterium tumefaciens strain EHA105. Rice transformation was performed according to the method described previously (Toki S., et al., Plant J. 47:969-976). Specifically, Nipponbare seeds were placed on 2N6 medium at 28°C for 7 days, immersed in an Agrobacterium suspension for several minutes, and then transferred to 2N6-AS medium. After co-cultivation for 3 days in the dark at 2°C, the seeds were washed with sterile water containing 25 mg / L meropenem (Wako) to remove the Agrobacterium. The plants were then cultured in N6D medium containing 50 mg / L of hygromycin at 32°C under continuous light for 2 weeks, followed by regeneration and recovery of plantlets. The RMIR1 transformant line transformed with the RMIR1 transformation vector and the vector control line transformed with the control vector were tested for Mi resistance using the same method as in Example 1.

[0130] (Results) The results are shown in Figures 4 and 5. The evaluation score of the RMIR1 transformants was calculated relative to the average evaluation score of the vector control. The evaluation score of the vector control strain was 1.003 ± 0.150, and the evaluation score of the RMIR1 transformants was 0.510 ± 0.056, and a t-test detected a significant difference at the 1% level. These results demonstrated that the RMIR1 gene is the causative gene for the Mi resistance trait and that introducing the RMIR1 gene of a Mi-resistant variety into a Mi-susceptible variety confers Mi resistance. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. A plant parasitic nematode control agent comprising an RMIR1 polypeptide or a fragment thereof having any of the amino acid sequences shown in (a) to (c) below: (a) the amino acid sequence shown in SEQ ID NO: 13, (b) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 13, or (c) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

13.

2. The plant parasitic nematode control agent according to claim 1, further comprising an RKNR1 polypeptide or a fragment thereof comprising any of the following amino acid sequences (d) to (f): (d) the amino acid sequence shown in SEQ ID NO: 1, (e) an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 1, or (f) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO:

1.

3. A plant parasitic nematode control agent comprising an RMIR1 polynucleotide encoding the RMIR1 polypeptide or a fragment thereof according to claim 1.

4. The plant parasitic nematode control agent according to claim 3, wherein the RMIR1 polynucleotide comprises any of the following base sequences (g) to (j): (g) a base sequence shown in SEQ ID NO: 14, (h) a base sequence in which one or more bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 14, (i) a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 14, or (j) a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO:

14.

5. The plant parasitic nematode control agent according to claim 3, further comprising a RKNR1 polynucleotide encoding the RKNR1 polypeptide or a fragment thereof according to claim 2.

6. The plant parasitic nematode control agent according to claim 5, wherein the RKNR1 polynucleotide comprises any of the following base sequences (k) to (n): (k) a base sequence shown in SEQ ID NO: 2, (l) a base sequence in which one or more bases have been deleted, substituted or added in the base sequence shown in SEQ ID NO: 2, (m) a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2, or (n) a base sequence which hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO:

2.

7. A plant parasitic nematode control agent comprising an expression vector containing the RMIR1 polynucleotide described in claim 3.

8. The plant parasitic nematode control agent according to claim 1, wherein the plant parasitic nematode is a root-knot nematode.

9. The plant parasitic nematode control agent according to claim 8, wherein the root-knot nematode is selected from the group consisting of Meloidogyne oryzae, Meloidogyne incognita, Meloidogyne northernis, and Meloidogyne javanica.

10. A plant transformant having resistance to plant parasitic nematodes, comprising the plant parasitic nematode control agent according to any one of claims 3 to 9, or a progeny thereof harboring said polynucleotide or said expression vector.

11. The transformed plant or its progeny according to claim 10, which is a monocotyledonous plant.

12. The transformed plant or its progeny according to claim 11, wherein the monocotyledonous plant is a grass plant.

13. The transformed plant or its progeny according to claim 12, wherein the Gramineae plant is selected from the group consisting of rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, and sorghum.

14. The plant transformant or its progeny according to claim 10, which is a genetically modified organism.

15. A method for producing a plant transformant having resistance to a plant parasitic nematode, comprising the steps of: introducing the expression vector described in claim 7 into a plant; and selecting a plant into which the expression vector has been introduced.

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