JP7742993B2 - Plant parasitic nematode control agent - Google Patents

Description

The present invention relates to a plant parasitic nematode control agent, a plant transformant, and a method for producing a plant transformant.

Plant-parasitic nematodes are said to infest over 2,000 species of plants, and are known to have an extremely wide host range. Potential host plants include many agriculturally important crops, and damage caused by them has been reported to amount to $150 billion worldwide.

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 a variety of plants worldwide, including agricultural crops, and has been reported to cause effects such as reduced yields, reduced quality, growth inhibition, and plant death. This poses a global problem from the perspective of stabilizing food production, and countermeasures are urgently needed.

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 potential. Therefore, there is a need for effective nematode control techniques that do not rely on nematicides.

The use of resistance genes against plant-parasitic nematodes is being promoted as a promising control technology to replace nematocides. 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 containing this gene have therefore become 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).

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 not only for major grains but also for bananas, pineapples, and sugarcane. In Asia, the rice root-knot nematode (Meloidogyne graminicola), a type of root-knot nematode, 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).

Therefore, there is a need for new resistance genes to plant-parasitic nematodes and new plant-parasitic nematode control agents based on these genes.

Milligan, S. B., 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, R. A., and Peng, D., 2005, Plant parasitic nematodes in subtropical and tropical agriculture, pp.87-130.

The object of the present invention is to provide a plant-parasitic nematode control agent based on a new plant-parasitic nematode resistance gene.

To address the above-mentioned challenges, the present inventors focused on two rice cultivars, one susceptible and the other resistant to the root-knot nematode (M. incognita). The japonica cultivar Taichung 65 is susceptible to M. incognita, while the indica cultivar Kalo Dhan is resistant to M. incognita. To identify the gene responsible for the difference in nematode resistance between these two rice cultivars, they created 128 recombinant inbred lines (RILs) using the two rice cultivars as breeding parents. They then performed QTL analysis based on the RILs and mapping using residual heterozygous lines (RHLs) obtained during the RIL creation process. As a result, they found that the Os04g0112100 gene (referred to herein as ROOT KNOT NEMATODE RESISTANCE 1 or RKNR1) is the gene responsible for the difference in nematode resistance between the rice cultivars. Furthermore, the inventors have found that the RKNR1 gene of Kalo Dhan can be introduced into a nematode-susceptible variety, Nipponbare, to produce a transformed strain, which can confer nematode resistance, leading to the completion of the present invention.

(1) A plant-parasitic nematode control agent comprising a polypeptide or a fragment thereof comprising any one of the amino acid sequences shown in (a) to (c) below:
(a) the amino acid sequence shown in SEQ ID NO: 1;
(b) an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1 (2) (1) A plant-parasitic nematode control agent comprising a polynucleotide encoding the polypeptide or a fragment thereof.
(3) The plant-parasitic nematode control agent according to (2), wherein the polynucleotide comprises any one of the base sequences shown in (a) to (d) below:
(a) the base sequence shown in SEQ ID NO: 2;
(b) a base sequence in which one or more bases are deleted, substituted or added in the base sequence shown in SEQ ID NO: 2;
(c) a base sequence having 90% or more identity with the base sequence shown in SEQ ID NO: 2, or (d) a base sequence that hybridizes under highly stringent conditions with a base sequence complementary to the base sequence shown in SEQ ID NO: 2. (4) A plant-parasitic nematode control agent comprising an expression vector containing the polynucleotide described in (2) or (3).
(5) The plant-parasitic nematode control agent according to any one of (1) to (4), wherein the plant-parasitic nematode is a root-knot nematode.
(6) The plant-parasitic nematode control agent according to (5), wherein the root-knot nematode is selected from the group consisting of Meloidogyne oryzae, Meloidogyne incognita, Meloidogyne northernis, and Meloidogyne javanica.
(7) A plant transformant having resistance to plant parasitic nematodes, comprising the polynucleotide described in (2) or (3), or the expression vector described in (4), or its progeny harboring the polynucleotide or the expression vector.
(8) The plant transformant or its progeny according to (7), which is a monocotyledonous plant.
(9) The plant transformant or its progeny according to (8), wherein the monocotyledonous plant is a grass plant.
(10) The plant transformant or its progeny according to (9), wherein the grass plant is selected from the group consisting of rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, and sorghum.
(11) The plant transformant or its progeny according to any one of (7) to (10), which is a genetically modified plant.
(12) A method for producing a plant transformant that is resistant to a plant parasitic nematode, the method comprising the steps of introducing the expression vector described in (4) into a plant and selecting a plant into which the expression vector has been introduced.
This specification includes the disclosure of Japanese Patent Application No. 2021-024793, from which this application claims priority.

The present invention provides a plant-parasitic nematode control agent based on a new plant-parasitic nematode resistance gene.

This figure shows the results of evaluating the resistance of 77 rice varieties and RKNR1 transformed lines to sweet potato root-knot nematodes. A lower evaluation value (EV) indicates higher resistance to nematodes. "S type" and "L type" shown below the graph indicate varieties with S-type and L-type RKNR1 genes, respectively. Error bars indicate standard error. 1 shows the results of evaluating the resistance of rice cultivars carrying the S-type and L-type RKNR1 genes and RKNR1 transformed lines to root-knot nematodes. Error bars indicate standard error. Figure 1 shows the results of comparing the resistance of T65 and Kalo Dhan to root-knot nematodes. (A) The results of measuring the attractive activity of rice root apices to root-knot nematodes. * indicates P<0.05 (Student's t-test). (B) The results of measuring the number of root-knot nematodes migrating to the root apices of rice plants. (C) The results of evaluating the size of galls induced by root-knot nematodes (gall width) as the ratio (relative gall width) of the width of the non-gall area (length of the solid line in the left panel) to the width of the gall area (length of the dotted line). (D) The results of evaluating the growth rate of nematodes based on the body width of nematodes that have invaded the roots. Error bars indicate standard error. Figures depict giant cells induced in roots by nematodes. (A) Giant cells in T65 7 days after nematode inoculation. The scale bar represents 100 μm. (B) Giant cells in T65 21 days after nematode inoculation. The scale bar represents 100 μm. (C) Giant cells in Kalo Dhan 7 days after nematode inoculation. The scale bars represent 100 μm (left panel) and 50 μm (right panel). (D) Giant cells in Kalo Dhan 21 days after nematode inoculation. The scale bars represent 100 μm (left panel) and 50 μm (right panel). "N" in the figures indicates nematodes. Figure 1 shows the results of QTL analysis for sweet potato root-knot nematode resistance. (A) This figure shows the results of identifying qRKNR1 on chromosome 4 and qRKNR2 on chromosome 6 as major QTLs. (B) This figure shows the results of classifying RILs into four groups based on the genotypes of S4-693908 and S6-25039213 (T65 type or Kalo Dhan type), and plotting the evaluation scores of RILs for each group. S4-693908 and S6-25039213 were used as markers closest to the peaks representing qRKNR1 and qRKNR2, respectively. This diagram shows the region to which qRKNR1 is mapped on chromosome 4. The root-knot nematode resistance gene was mapped to the 1.3 Mb region between IDK0401 and IDK0405 (region indicated by double arrows). ** indicates P<0.01 (Student's t-test). Error bars indicate standard error. FIG. 1 shows the structure of the RKNR1 gene in Nipponbare, T65, N22, Kalo Dhan, Bei Khe, Naba, and Akage.

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 a nematode-resistant RKNR1 polypeptide or a fragment thereof, or comprises a polynucleotide encoding either of them, or an expression vector comprising the same. The plant-parasitic nematode control agent of the present invention has a control effect against plant-parasitic nematodes.

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.

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. Second-stage larvae move through the soil, invade 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 the Java root-knot nematode (Meloidogyne javanica), the sweet potato root-knot nematode (Meloidogyne incognita; often referred to as "Mi" in this specification), the northern root-knot nematode (Meloidogyne hapla), the apple root-knot nematode (Meloidogyne mali), and the arenaria root-knot nematode (Meloidogyne arenaria).

Examples of species belonging to the root-lesion nematode family 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).

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

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).

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

As used herein, plants are not particularly limited as long as they are 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, hyacinths, muscari, 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 plant). Root-knot nematodes have been reported to infect over 2,000 plant species, and although the host species they infect 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, causing plant diseases in a variety of agricultural crops. Examples of host plant species for root-knot nematodes include tomatoes, bell peppers, melons, potatoes, sweet potatoes, eggplants, carrots, burdock, spinach, Swiss chard, garland chrysanthemums, onions, ginger, peas, kidney beans, cowpeas, and rice.

Rice cultivars are classified as Asian rice (Oryza sativa) and African rice (Oryza glaberrima). Asian rice is divided into two subspecies, Indica and Japonica, and Japonica is further 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 the Temperate Japonica category include Taichung65 (T65), 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 cultivars belonging to 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. Also, NERICA is a hybrid between Oryza sativa (Oryza sativa) and Oryza sativa (Oryza sativa). Examples of cultivars belonging to NERICA include NERICA 1, NERICA 2, NERICA 4, NERICA 6, NERICA L20, and NERICA L41 (in Table 1, NERICA-related cultivars, including NERICA cultivars, are listed as "NERICA-related"). Hybrids not classified into any of the above groups (shown 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 (shown as "Unknown" in Table 1) include Basmati370, IRAT109, LTH, IR24, Kinandang Patong, and Silewah. Regarding the classification of rice cultivars, see Kojima et al., 2005, Breeding Science, 55, 431-440 and Yonemaru et al., 2014, Plant Cell Physiol., 55 (1), e9.

Rice varieties can be classified into varieties susceptible to plant parasitic nematodes and varieties resistant to plant parasitic nematodes. For example, an evaluation value (EV) can be calculated using the evaluation method described in the Examples of this 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 a certain value can be classified as nematode-resistant varieties.

As used herein, "resistance to plant-parasitic nematodes" refers to the ability to prevent or inhibit damage to and/or parasitism (infection) of a host plant by plant-parasitic nematodes. Resistance of plants 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 medium of a test plant, and the infection status is evaluated after a certain period of time (e.g., two months). Evaluation is preferably performed 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.

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 in host plants.

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 these. As used herein, the term "RKNR1 gene" encompasses 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), as well as the L-type RKNR1 gene and S-type RKNR1 gene described below.

The RKNR1 gene sequence may differ between rice cultivars. For example, the presence or absence of a 1,754-bp sequence region in the RKNR1 ORF that stretches from the center of the NB-ARC domain to near the C-terminus of the LRR domain in the RKNR1 amino acid sequence found in Nipponbare and T65 rice cultivars differs between rice cultivars. Herein, rice RKNR1 genes are classified into S-type and L-type RKNR1 genes based on the presence or absence of this 1,754-bp region. In other words, the S-type RKNR1 gene lacks a 1,754-bp region compared to the L-type RKNR1 gene (the L-type RKNR1 gene has a 1,754-bp region inserted compared to the S-type RKNR1 gene).

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

Examples of L-type RKNR1 genes include the RKNR1 genes of some varieties belonging to the temperate japonica species (e.g., Akagei and Ginbozu), as well as varieties belonging to the indica, tropical japonica, Ausu, and NERICA species. 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.

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" encompasses wild-type and mutant RKNR1 polypeptides derived from any biological species (referred to as "wild-type RKNR1 polypeptide" and "mutant RKNR1 polypeptide," respectively), as well as the L-type RKNR1 polypeptide and S-type RKNR1 polypeptide described below.

Wild-type rice RKNR1 polypeptides are classified as S-type RKNR1 polypeptides encoded by S-type RKNR1 genes and L-type RKNR1 polypeptides encoded by L-type RKNR1 genes. 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 the 1754-bp region is not 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.

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

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 residues. 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 gaps inserted as needed into one or both sequences 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 similarly.

As used herein, "(amino acid) substitution" refers to a 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 make up naturally occurring proteins. Examples include substitutions within the 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 alter the properties of polypeptides.

As used herein, "stringent conditions" refers to conditions under which nonspecific hybrids are unlikely to form. "Highly stringent conditions" refer to conditions under which nonspecific hybrids are unlikely to form or are 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 post-hybridization washing, for example, conditions include washing with 0.1x SSC and 0.1% SDS at 50°C to 70°C, 55°C to 68°C, or 65°C to 68°C. Additionally, the stringency of hybridization can be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

1-3. Constitution The plant-parasitic nematode control agent of the present invention contains, as an active ingredient, (1) a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof, (2) a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof, or (3) an expression vector comprising a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof.

1-3-1. Active ingredient (1) Plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof In one embodiment, the plant-parasitic nematode control agent of the present invention consists of or comprises a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof.

As used herein, "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-parasitic nematode-resistant plant.

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 of these 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, Badari Wild-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.

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 is 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 identical 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.

In one 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.

In one 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 are 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.

As used herein, a "fragment" of a 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, such as a fragment that 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 RKNR1 polypeptide, or an activity equivalent to or greater than these. Examples include polypeptide fragments containing the LRR domain of the plant-parasitic nematode-resistant RKNR1 polypeptide. 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.

(2) Polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 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 RKNR1 polypeptide or a fragment thereof.

The polynucleotide of the present invention encodes the above-mentioned plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof. The polynucleotide of the present invention is not particularly limited in its nucleotide sequence, as long as it encodes a 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).

In one embodiment, the polynucleotide of the present invention comprises any of the following: (a) a base sequence shown in SEQ ID NO: 2; (b) a base sequence in which one or more bases are deleted, substituted, or added in the base sequence shown in SEQ ID NO: 2; (c) a base 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 base sequence shown in SEQ ID NO: 2; or (d) a base sequence that hybridizes under highly stringent conditions to a base sequence complementary to the base sequence shown in SEQ ID NO: 2.

In one embodiment, the base sequence of the polynucleotide of the present invention may be a base sequence that has been codon-optimized to match the codon usage frequency in the cell into which the polynucleotide is introduced.

The polynucleotide of the present invention may be DNA or RNA such as mRNA.
When the polynucleotide of the present invention is an 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). In addition to the coding region, the mRNA corresponding to the polynucleotide of the present invention may also contain 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.

(3) Expression vector containing a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 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 a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof.

The expression vector of the present invention contains, in an expressible state, a polynucleotide encoding the plant-parasitic nematode-resistant RKNR1 polypeptide of the present invention or a fragment thereof. As used herein, "expressible state" means that the gene to be expressed is located downstream of the promoter under the control of the promoter.

The expression vector of the present invention includes, as essential components, a promoter and a polynucleotide described in "(2) Polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof."

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

In the case of expression vectors that use plasmids (hereinafter often referred to as "plasmid expression vectors"), the plasmids that can be used include, but are not limited to, pPZP, pSMA, pUC, pBR, pBluescript (Agilent Technologies), pTriEXTM (TaKaRa), or binary vectors such as pBI, pRI, or pGW.

In the case of expression vectors that use 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).

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.

Various promoters can be used, including overexpression promoters, constitutive promoters, site-specific promoters, stage-specific promoters, and/or inducible promoters. Specific examples of overexpression constitutive promoters that can function in plant cells include the 35S promoter derived from cauliflower mosaic virus (CaMV), the Pnos promoter of the Ti plasmid-derived nopaline synthase gene, the ubiquitin promoter derived from maize, the rice actin promoter, and the tobacco PR protein promoter. 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.

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

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, and the ADH1 gene terminator. There are no particular limitations on the sequence, as long as it is capable of terminating transcription of the gene transcribed by the promoter.

An example of an enhancer is an enhancer region containing an upstream sequence within the CaMV 35S promoter. There are no particular limitations on the enhancer, as long as it can enhance the expression efficiency of a nucleic acid encoding an active peptide.

Examples of nuclease recognition sequences include restriction enzyme recognition sequences, loxP sequences recognized by Cre recombinase, sequences that are targets of artificial nucleases such as ZFN and TALEN, and sequences that are targets of the CRISPR/Cas9 system.
An example of the replication origin sequence is the SV40 replication origin sequence.

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 fluorescent protein (GFP)), and enzyme genes such as neomycin phosphotransferase II (NPT II) and dihydrofolate reductase.

The selectable marker gene that can be included 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 selectable marker genes include drug resistance genes such as the ampicillin resistance gene, kanamycin resistance gene, tetracycline resistance gene, chloramphenicol resistance gene, neomycin resistance gene, puromycin resistance gene, and hygromycin resistance gene.

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

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.

The plant-parasitic nematode control agent of the present invention may contain an agriculturally acceptable carrier. As used herein, "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 is no or only slightly harmful to animals, particularly humans. Examples include solvents, adjuvants, excipients, emulsifiers, dispersants, surfactants, etc.

The plant-parasitic nematode control agent of the present invention may 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.

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.

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.

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 within the roots, and/or the induction of giant cells in the host plant in the plants to which the agent is applied.

2. Plant transformant or its progeny having resistance to plant parasitic nematodes 2-1. Overview A second aspect of the present invention is a plant transformant or its progeny having resistance to plant parasitic nematodes. The plant transformant of the present invention or its progeny comprises 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 its progeny has suppressed nematode-attracting 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.

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.

The plant transformant of the present invention comprises a polynucleotide encoding the RKNR1 polypeptide or a fragment thereof that is resistant to plant parasitic nematodes described in the first aspect, or an expression vector containing the polynucleotide.

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), and species belonging to the Bromeliaceae family (e.g., pineapple). Host plants are preferably plant species susceptible to plant-parasitic nematodes or plant species with weak resistance to plant-parasitic nematodes (e.g., rice varieties with an S-type RKNR1 gene or plant species lacking an L-type RKNR1 gene).

The plant transformant of the present invention includes clones having the same genetic information. Also included in the plant transformant of the present invention are 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 newly 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 transformed plant of the present invention may be a genetically modified plant.

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 a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof, or an expression vector containing the polynucleotide, and that is resistant to plant-parasitic nematodes. The progeny generation is not important.

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.

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.

3-2-1. Introduction step In this embodiment, the "introduction step" is a step of introducing an expression vector containing a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof into a host plant. The configuration of the expression vector introduced in this step is similar to that described in "(3) Expression vector containing a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof" in the first embodiment.

Methods for introducing expression vectors can be known in the art, such as the Agrobacterium method, 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 within the host cell, as if it were integrated into the genomic DNA of the host, or may be maintained transiently.

When using the Agrobacterium method, a polynucleotide encoding the RKNR1 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 introduced into an appropriate Agrobacterium, such as Agrobacterium tumefaciens, by electroporation or other methods. This strain is then inoculated into plant cells, callus, cotyledon segments, or the like for infection. Suitable Agrobacterium strains that can be used include, but are not limited to, GV3101, C58, C58C1Rif(R), EHA101, EHA105, AGL1, and LBA4404.

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 is typically performed under pressure of approximately 450-2000 psi and at a distance of approximately 4-12 cm.

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.

This step can be carried out by methods known in the art after introducing the expression vector into the host using the method described above. For example, transformants can be selected using the activity of a protein encoded by a selectable marker gene or reporter gene in the expression vector.

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

Example 1: Examination of resistance of various rice varieties to sweet potato root-knot nematode
(the purpose)
Various rice varieties will be examined for resistance to the sweet potato root-knot nematode (Mi).

(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.

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

Specifically, rice seeds were soaked in a disinfectant solution (a 1:1000 diluted solution of Kao Corporation kitchen bleach) at 26°C for three days (water absorption period) to kill mold and bacteria and induce germination. After germination, the seeds were sown in paper pouches (CYG Seed Germination Pouch, Mega International, USA), two seeds per pouch. The pouches containing the germinated seeds were placed in a dark place at 26°C for three days. Next, 10 pouches were sandwiched between wooden boards, secured with spring clamps, and grown in a 12-hour light (26°C)/12-hour dark (24°C) environment for eight 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).

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, 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 after completely removing all moisture from the roots, they were weighed. The number of egg masses per unit dry root weight was calculated from the obtained dry root weight and number of egg masses. The standardized value, using the T65 value as the reference value, was used as the evaluation value (EV) for each variety.

Rice varieties with an evaluation value of 0.6 or higher were classified as Mi-susceptible varieties, and rice varieties with an evaluation value of less than 0.6 were classified as Mi-resistant varieties.

(result)
The evaluation values obtained for each variety are shown in Figures 1 and 2 and in Table 1 below.

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

The indica variety Kalo Dhan showed an evaluation value of approximately 1% compared to T65 (EV=0.01), indicating that it is one of the varieties with the highest resistance to Mi. Based on this result, Kalo Dhan was used as a Mi-resistant variety in the following examples.

Example 2: Comparison of T65 and Kalo Dhan
(the purpose)
Resistance to sweet potato root-knot nematode (Mi) will be compared between the japonica variety T65 and the indica variety Kalo Dhan.

(Methods and Results)
(1) Nematode attractant activity
The ability of T65 and Kalo Dhan to attract nematodes to the root apex was examined.
Measurement of nematode attraction was performed using a Pluronic F-127-based matrix as described previously (Wang, C., Lower, S., and Williamson, V.M., 2009, Nematology, 11:453-464). Twenty thousand J2-stage Mi were mixed in 3.5 mL of medium (1.5 mL ddH2O, ₂ mL 50% [w/v] Pluronic F-127 [Sigma P2443]) in a 60 mm dish at 4°C, and the medium was allowed to solidify. Two rice roots were placed on each dish and incubated at 26°C in the dark. After overnight incubation, root-knot nematodes attracted to the rice roots were recorded using a DP74 camera (Olympus). The attraction index was calculated using the following formula, using the method described in the literature (Tsai, A., et al., 2019, Mol Plant 12:99-112).
Attraction index = [attracted population - background population] / [total population]
(In the formula, the number of attracted individuals and the number of background individuals represent the number of individuals attracted to rice or water, respectively, and the total number represents the sum of these numbers.)
The mean ± standard error obtained from N = 8 experiments is shown in Figure 3A. The results showed that the nematode attractant activity of Kalo Dhan was significantly lower than that of T65.

(2) Number of migrating nematodes
Mi was directly inoculated into the root apex of rice plants, and a 3 cm area of the root apex was harvested 1 to 21 days later. The samples were treated with fuchsinic acid and the number of nematodes that had invaded the roots was counted.
The results are shown in Figure 3B. Measurement of the number of migrating nematodes revealed that 7 days after inoculation, the number of migrating nematodes in T65 was 8.35 per root apex, compared with 2.42 in Kalo Dhan. Furthermore, 14 to 21 days after inoculation, the number of migrating nematodes in T65 was 8 to 10 per root apex, compared with only 1 to 3 in Kalo Dhan. This indicates that the number of migrating nematodes in Kalo Dhan was lower than that in T65.

(3) Gall width: Mi was directly inoculated into the root apex of rice plants as described in (2) above, and gall width was measured in the roots of T65 and Kalo Dhan. Gall width was evaluated by measuring the width of the non-gall area and the width of the gall area 28 days after Mi inoculation (Fig. 3C, left), and calculating the ratio as the relative gall width.
The results are shown in the right side of Figure 3C. The relative nodule widths for T65 and Kalo Dhan were 2.86 ± 0.07 and 1.44 ± 0.04, respectively, indicating that Kalo Dhan was resistant to nodule maturation. The nodule counts for T65 and Kalo Dhan were 301 and 46, respectively, indicating that Kalo Dhan formed fewer nodules than T65.

(4) Nematode growth rate As in (2) above, Mi was inoculated directly into the root apex of rice plants, and the body width of nematodes that had invaded the roots was measured 14 to 21 days after inoculation.
The results are shown in Figure 3D. Kalo Dhan showed a significant inhibition of nematode growth compared to T65.

(5) Giant cell formation
To observe Mi-induced giant cell formation, roots were harvested 7 and 21 days after Mi inoculation. After harvest, root samples were immediately placed in FAA buffer (60% ethanol, 7.5% acetic acid, 2.5% formalin) and fixed for 20 hours. They were then dehydrated in ethanol and embedded in Technovit 7100 epoxy resin (Kulzer Friedrichsdorf). Five-micrometer sections were prepared and stained with 2.5% [w/v] toluidine blue staining solution before microscopic examination.

Figure 4 shows the appearance of giant cells in the roots of T65 and Kalo Dhan. In Kalo Dhan nodules, giant cell enlargement was significantly suppressed compared to T65 nodules. Furthermore, cell division was suppressed in the surrounding cells in Kalo Dhan nodules.

These results indicate that, compared to T65, Kalo Dhan strongly suppresses nematode attractant activity, nematode migration to roots, initiation of nodule formation, nodule maturation, nematode growth within roots, and giant cell induction.

Example 3: Mapping and identification of the Mi resistance gene
(the purpose)
The Mi resistance gene will be identified by QTL analysis and positional cloning.
(Methods and Results)
(1) Analysis of RIL
We generated 128 recombinant inbred lines (RILs) by crossing T65 and Kalo Dhan, and performed quantitative trait loci (QTL) analysis using 2,144 SNPs. Two major QTLs were identified on chromosomes 4 and 6, named qRKNR1 (qROOT KNOT NEMATODE RESISTANCE 1) and qRKNR2, respectively (Fig. 5A). The contributions of qRKNR1 and qRKNR2 were 29.84% and 14.47%, respectively.

Next, the RILs were classified into four groups based on the genotypes (T65 or Kalo Dhan) of S4-693908 and S6-25039213, the markers closest to the peaks on chromosomes 4 and 6 (Figure 5B). The results showed that the group with S4-693908 and S6-25039213 as Kalo Dhan and T65 had lower scores and higher Mi resistance than the group with S4-693908 and S6-25039213 as T65 and Kalo Dhan. These results indicated that qRKNR1 has the strongest effect on Mi resistance, and further mapping of qRKNR1 was conducted.

(2) Analysis of RHL
For further mapping of qRKNR1, 43 lines derived from residual heterozygous lines (RHLs) obtained during the RIL production process were obtained, and the correlation between the genotypes of indel markers (IDK0401, IDK0404, IDK0405, and IDK0407) and the evaluation scores was examined.
The results are shown in Figure 6. From the results of line #29267-39 (EV = 0.45) and line #29267-63 (EV = 0.17), the resistance gene was mapped to the 1.3 Mb region between IDK0401 and IDK0404.

(3) Identification of the RKNR1 gene. The genome sequences of Nipponbare, which is Mi susceptible like T65, and N22, which is Mi resistant like Kalo Dhan, were compared for the region identified in (2) above. Sequence differences were found between Nipponbare and N22 at the Os04g0112100 locus. Genomic sequencing of this locus in T65 and Kalo Dhan revealed that the Os04g0112100 gene sequence was completely identical to the sequences of Nipponbare and N22, respectively. The Os04g0112100 gene was designated RKNR1 (ROOT KNOT NEMATODE RESISTANCE 1).

The RKNR1 alleles in N22 and Kalo Dhan contain a three-base insertion, corresponding to the insertion of one amino acid residue between positions 271 and 272 in the Nipponbare RKNR1 amino acid sequence (SEQ ID NO: 9) (N22, Kalo Dhan in Figure 7). Furthermore, at position 339 in the Nipponbare RKNR1 amino acid sequence (SEQ ID NO: 9), a single base difference results in a Thr residue in Nipponbare/T65, whereas an Arg residue in N22 and Kalo Dhan (Figure 7). Furthermore, compared to N22 and Kalo Dhan, the Nipponbare/T65 genome lacks a 1754-bp deletion in the RKNR1 amino acid sequence, spanning from the center of the NB-ARC domain to near the C-terminus of the LRR domain. As a result, Nipponbare and T65 lack most of the LRR domain in the RKNR1 amino acid sequence (Nipponbare, T65 in Figure 7). Hereinafter, the RKNR1 allele having the 1754 bp deletion found in the RKNR1 amino acid sequences of Nipponbare and T65 will be referred to as S type, and the RKNR1 allele without this deletion will be referred to as L type.

Example 4: Determination of RKNR1 gene structure in various rice varieties
(the purpose)
The structure of the RKNR1 gene will be determined for the rice varieties evaluated in Example 1, and its relationship with Mi resistance will be examined.

(Methods and Results)
The various rice varieties evaluated in Example 1 will be examined for the presence or absence of the 1754 bp deletion in the RKNR1 amino acid sequence found in Nipponbare/T65, and the RKNR1 gene will be classified into L-type or S-type.
The results of classifying the RKNR1 gene of each rice variety into L-type or S-type are shown at the bottom of Figure 1.

In Figure 1, among the rice varieties with an evaluation value of 0.6 or higher and susceptible to Mi, the RKNR1 gene was found to be S-type in all varieties except Akage, Bei Khe, and Naba. On the other hand, in the Mi-resistant varieties with an evaluation value of less than 0.6, the RKNR1 gene was found to be L-type.

Next, the RKNR1 gene sequences were determined by sequencing in redheads, Bei Khe, and Naba. The results showed that in Bei Khe and Naba, there was a four-base deletion before the NB-ARC domain, resulting in a stop codon in the middle of the ORF due to a frameshift (Bei Khe and Naba in Figure 7). The RKNR1 gene sequence in redheads was found to contain a SNP in which the amino acid at position 1040 in the LRR domain of N22 was substituted from Leu to Glu (L1040Q in Akage in Figure 7). It was thought that this SNP altered the function of the LRR domain in redheads.

Example 5: Effect of introduction of Kalo Dhan-type RKNR1 gene on Mi resistance trait
(the purpose)
We will create a transformed strain by introducing the genomic region containing the RKNR1 gene of Kalo Dhan into Nipponbare, and verify whether Mi resistance can be obtained.

(method)
The RKNR1 upstream sequence (region 1) from -3060 to -1307 bp in the Os04g0112100 gene was amplified from the wild-type Kalo Dhan genome using the attB1-OsMi4c1p-F (SEQ ID NO: 13) and attB2-OsMi4c1p-M3Fr (SEQ ID NO: 14) primer sets and PrimeSTAR Max DNA Polymerase (TaKaRa). PCR was then performed using PrimeSTAR Max with the attB1 and attB2 primers, and the PCR product was cloned into the pDONR221 vector using BP clonase II (Thermo Fisher Scientific).

Next, the RKNR1 upstream sequence (region 2) from -1324 to -560 bp in Os04g0112100 was synthesized and cloned into the pUC57-Amp vector (Genewiz, Kawaguchi).

The RKNR1 upstream sequence (region 3) from -569 to -1 bp and a portion of the coding region in Os04g0112100 were PCR amplified from the Kalo Dhan genome using the primer set pUC19-IF-OsMi4c1_r5s (SEQ ID NO: 15) and pUC19-IF-OsMi4c1p-L4-r (SEQ ID NO: 16) and SapphireAmp Fast PCR Master Mix (TaKaRa). PCR was then performed using the above primer set with PrimeSTAR Max, and the PCR product was cloned into the pUC19 Linearized vector (TaKaRa) using NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs).

The RKNR1 coding sequence was PCR amplified from the Kalo Dhan genome using the attB1-OsMi4c1-cDNA-F (SEQ ID NO: 17) and attB2-OsMi4c1-cDNA-R (SEQ ID NO: 18) primer sets and PrimeSTAR Max. PCR was then performed using PrimeSTAR Max with the attB1 and attB2 primers, and the PCR product was cloned into the pDONR221 vector using BP clonase II.

The RKNR1 3' sequence (3007 bp) was PCR amplified from the Kalo Dhan genome using the attB1-OsMi4c1t-F (SEQ ID NO: 19) and attB2-OsMi4c1t-R (SEQ ID NO: 20) primer sets and PrimeSTAR Max. PCR was then performed using PrimeSTAR Max with the attB1 and attB2 primers, and the PCR product was cloned into the pDONR221 vector using BP clonase II.

Next, from the entry clone containing region 1, a region 1 fragment was PCR amplified together with the Gateway attL1 sequence using the primer set pUC19-IF-M13F (SEQ ID NO: 29) and OsMi4c1p-M3Fr (SEQ ID NO: 14) and PrimeSTAR Max. From the vector containing region 1, a region 2 fragment was PCR amplified using the primer set OsMi4c1p-M3F (SEQ ID NO: 21) and OsMi4c1p-r5-r (SEQ ID NO: 23) and KOD One PCR Master Mix -Blue- (TOYOBO). From the vector containing region 3, a region 3 fragment was PCR amplified using the primer set OsMi4C1_r5 (SEQ ID NO: 24) and OsMi4c1-IF-ATG-pro-R (SEQ ID NO: 25) and PrimeSTAR Max. The RKNR1 coding region was PCR-amplified from a vector containing the RKNR1 coding region using the primer set OsMi4c1-ATG-F (SEQ ID NO: 26) and OsMi4C1-TAA-R (SEQ ID NO: 27) and PrimeSTAR Max. From an entry clone containing the RKNR1 3' sequence, the RKNR1 3' sequence was PCR-amplified along with the Gateway attL2 sequence using the primer set OsMi4c1-IF-TAA-Ter-F (SEQ ID NO: 28) and pUC19-IF-M13R (SEQ ID NO: 30) and PrimeSTAR Max. The five PCR products were then integrated into the pUC19 linearized vector using NEBuilder. The entry clone containing the RKNR1 genomic sequence was transferred to the pGWB1 vector (Nakagawa T., et al., 2007, J. Biosci. Bioeng. 104:34-41) using LR clonase II (Thermo Fisher Scientific). The sequences of the primers used in this example are shown in Table 2 below.

The binary vector was introduced into Agrobacterium tumefaciens strain EHA105 by electroporation. 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. They were then cultured on N6D medium containing 50 mg/L hygromycin at 32°C under continuous light for 2 weeks, after which plantlets were regenerated and collected. The resulting transformants were tested for Mi resistance.

(result)
The transformants, which were transformed with the genomic region containing the RKNR1 gene from Kalo Dhan, showed a significantly lower evaluation score than the control line, which was transformed with the vector alone (Fig. 2). These results demonstrated that the RKNR1 gene is responsible for the Mi resistance trait, and that introducing the RKNR1 gene from a Mi-resistant cultivar into a Mi-susceptible cultivar confers Mi resistance.
All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (12)

A plant-parasitic nematode control agent comprising a polypeptide containing any of the amino acid sequences shown in (a) to (c) below:
(a) the amino acid sequence shown in SEQ ID NO: 1;
(b) an amino acid sequence in which 1 or 2 to 100 amino acids are deleted, substituted, or added in the amino acid sequence shown in SEQ ID NO: 1, or (c) an amino acid sequence having 90% or more identity with the amino acid sequence shown in SEQ ID NO: 1. An agent for controlling plant-parasitic nematodes, comprising a polynucleotide encoding the polypeptide of claim 1. The plant-parasitic nematode control agent according to claim 2, wherein the polynucleotide comprises the following base sequence (a) or (b) :
(a) the base sequence shown in SEQ ID NO: 2, or
( b ) A nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 2 A plant-parasitic nematode control agent comprising an expression vector containing the polynucleotide of claim 2 or 3. The plant-parasitic nematode control agent according to any one of claims 1 to 4, wherein the plant-parasitic nematode is a root-knot nematode. The plant-parasitic nematode control agent according to claim 5, wherein the root-knot nematode is selected from the group consisting of Meloidogyne oryzae, Meloidogyne incognita, Meloidogyne northernis, and Meloidogyne javanica. A transformed plant having resistance to plant parasitic nematodes, comprising the polynucleotide of claim 2 or 3, or the expression vector of claim 4, or its progeny harboring said polynucleotide or said expression vector. The transformed plant or its progeny according to claim 7, which is a monocotyledonous plant. The transformed plant or its progeny according to claim 8, wherein the monocotyledonous plant is a grass plant. The transformed plant or its progeny according to claim 9, wherein the grass plant is selected from the group consisting of rice, wheat, barley, rye, corn, sugarcane, foxtail millet, millet, barnyard millet, and sorghum. The plant transformant or its progeny described in any one of claims 7 to 10, which is a genetically modified organism. 1. A method for producing a plant transformant having resistance to plant-parasitic nematodes, comprising:
A method comprising the steps of: introducing the expression vector according to claim 4 into a plant; and selecting a plant into which the expression vector has been introduced.