Plant parasitic nematode control agent

Abstract

Provided is a plant parasitic nematode controlling agent based on a new plant parasitic nematode resistant gene.　The plant parasitic nematode controlling agent comprises: (a) an amino acid sequence represented by SEQ ID NO. 1; (b) an amino acid sequence resulting from deletion, substitution, or addition of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO. 1; or (c) a polypeptide having any one of amino acid sequences indicating an identity of 90% or more with respect to the amino acid sequence represented by SEQ ID NO. 1, or a fragment of said polypeptide.

Description

[Technical Field]

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

[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 a variety of plants worldwide, including agricultural crops, and has been reported to cause effects such as reduced yields, reduced quality, growth inhibition, and withering. 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 powerful control technology to replace nematocides. In tomatoes, the Mi-1 gene has been found to be a resistance gene against 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 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 areas infected with the nematode 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. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Milligan, SB, et al., 1998, Plant Cell, 10:1307-1319. [Non-patent document 2] Zhang, L.-Y., et al., 2010, Plant Mol Biol Report, 28:204-211. [Non-patent document 3] De Waele, D. and Elsen, A., 2007, Annu. Rev. Phytopathol. 45:457-485. [Non-patent document 4] Bridge, J., Plowright, RA, and Peng, D., 2005, Plant parasitic nematodes in subtropical and tropical agriculture, pp.87-130. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a plant-parasitic nematode control agent based on a new plant-parasitic nematode resistance gene. [Means for solving the problem]

[0010] To solve the above-mentioned problems, the present inventors focused on two rice cultivars, one susceptible and the other resistant to nematodes. The japonica cultivar Taichung 65 is susceptible to nematodes, while the indica cultivar Kalo Dhan is resistant to nematodes. To identify the gene responsible for the difference in nematode resistance between these two rice cultivars, we created 128 recombinant inbred lines (RILs) using the two rice cultivars as breeding parents. We then performed QTL analysis based on the RILs and mapping using residual heterozygous lines (RHLs) obtained during the RIL creation process. As a result, we 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.

[0011] (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) A plant-parasitic nematode control agent, comprising a polynucleotide encoding the polypeptide or a fragment thereof according to (1). (3) The plant-parasitic nematode control agent according to (2), wherein the polynucleotide comprises any one of the nucleotide 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 nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 2, or (d) a nucleotide sequence that hybridizes under highly stringent conditions with a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2 (4) A plant-parasitic nematode control agent, comprising an expression vector containing the polynucleotide according to (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 family 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 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. [Effects of the Invention]

[0012] According to the present invention, it is possible to provide a plant-parasitic nematode control agent based on a new plant-parasitic nematode resistance gene. [Brief explanation of the drawings]

[0013] [Figure 1] 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. [Figure 2] 1 shows the results of evaluating resistance to the sweet potato root-knot nematode in rice varieties carrying the S-type and L-type RKNR1 genes, and RKNR1 transformed lines. Error bars indicate standard error. [Figure 3] Figure 1 shows the results of comparing the resistance of T65 and Kalo Dhan to rhizomeloid nematodes. (A) Results of measuring the attractive activity of rice root apices to rhizomeloid nematodes. * indicates P<0.05 (Student's t-test). (B) Results of measuring the number of rhizomeloid nematodes migrating to the rhizomes of rice root apices. (C) Results of evaluating the size of galls induced by rhizomeloid nematodes (gall width) as the ratio (relative gall width) of the width of the gall region (length of the dotted line) to the width of the non-gall region (length of the solid line in the left panel). (D) 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. [Figure 4]Figures showing giant cells induced in roots by nematodes. (A) Giant cells in T65 7 days after nematode inoculation. The scale bar indicates 100 μm. (B) Giant cells in T65 21 days after nematode inoculation. The scale bar indicates 100 μm. (C) Giant cells in Kalo Dhan 7 days after nematode inoculation. The scale bar indicates 100 μm (left panel) and 50 μm (right panel). (D) Giant cells in Kalo Dhan 21 days after nematode inoculation. The scale bar indicates 100 μm (left panel) and 50 μm (right panel). "N" in the figures indicates nematodes. [Figure 5] Figures 1A and 1B show 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 RILs classified into four groups based on the genotypes of S4-693908 and S6-25039213 (T65 type or Kalo Dhan type), and plots the evaluation scores of the RILs for each group. S4-693908 and S6-25039213 were used as markers closest to the peaks representing qRKNR1 and qRKNR2, respectively. [Figure 6] This figure shows the region to which qRKNR1 is mapped on chromosome 4. The sweet potato root-knot nematode resistance gene was mapped to the 1.3 Mb region between IDK0401 and IDK0405 (region indicated by double arrow). ** indicates P<0.01 (Student's t-test). Error bars indicate standard error. [Figure 7] FIG. 1 shows the structure of the RKNR1 gene in Nipponbare, T65, N22, Kalo Dhan, Bei Khe, Naba, and Akage. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1. Plant parasitic nematode control agent Overview A first aspect of the present invention is an agent for controlling plant parasitic nematodes. The agent for controlling plant parasitic nematodes 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 polynucleotide. The agent for controlling plant parasitic nematodes of the present invention has a control effect against plant parasitic nematodes.

[0015] 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 to be controlled by the present invention is preferably a root-knot nematode.

[0016] 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 Meloidogyne javanica, Meloidogyne incognita (often referred to as "Mi" in this specification), Meloidogyne hapla, Meloidogyne mali, and Meloidogyne arenaria.

[0017] 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).

[0018] 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).

[0019] Examples of species belonging to the genus Nematode include the peel nematode (Aphelenchoides ritzemabosi), the strawberry nematode (Aphelenchoides fragariae), and the rice root nematode (Aphelenchoides besseyi).

[0020] Examples of species belonging to the stem nematode include the potato leaf nematode (Dithlenchus destructor) and the common stem nematode (Ditylenchus dipsaci).

[0021] As used herein, the term "plant" refers to any 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 important plants in agriculture, particularly in the seed and floriculture industries, such as crop plants including 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 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, grapefruits, 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 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, Leguminosae, Cucurbitaceae, Convolvulaceae, Liliaceae, Asteraceae, Chenopodiaceae, Umbelliferae, 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.

[0022] 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 the Temperate Japonica species 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, and Kahei. 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, and Karahoushi. 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 that do not fall 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.

[0023] 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.

[0024] 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 are inoculated into the culture soil of a test plant, and the infection state is evaluated after a certain period of time (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.

[0025] 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.

[0026] 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.

[0027] The RKNR1 gene sequence may differ between rice varieties. For example, the presence or absence of a 1,754-bp sequence region in the ORF of the RKNR1 gene, which extends 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, differs between rice varieties. Herein, rice RKNR1 genes are classified into S-type RKNR1 genes and L-type RKNR1 genes based on the presence or absence of this 1,754-bp region. That is, the S-type RKNR1 gene lacks a 1,754-bp region compared to the L-type RKNR1 gene (the L-type RKNR1 gene has an insertion of a 1,754-bp region compared to the S-type RKNR1 gene).

[0028] 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).

[0029] Examples of L-type RKNR1 genes include the RKNR1 genes of some varieties belonging to the temperate japonica group (e.g., Akage and Ginbozu), as well as those of varieties belonging to the indica, tropical japonica, Aus, 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 (Akage). The nucleotide sequences of the RKNR1 genes are 100% identical between N22 and Kalo Dhan, and both are shown in SEQ ID NO: 2.

[0030] 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 either 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.

[0031] Wild-type rice RKNR1 polypeptides are classified into 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.

[0032] 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 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 group of morphologically and / or functionally differentiated cells, or a cell.

[0033] 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. 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 to be 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.

[0034] 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.

[0035] 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. Washing after hybridization is performed, for example, 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. Additionally, the stringency of hybridization can be increased by appropriately combining other conditions such as probe concentration, probe base length, and hybridization time.

[0036] 1-3.Configuration 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.

[0037] 1-3-1. Active ingredients (1) RKNR1 polypeptide or a fragment thereof that is resistant to plant parasitic nematodes 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.

[0038] 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 that is resistant to plant-parasitic nematodes.

[0039] 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 Timal, 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.

[0040] 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.

[0041] 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.

[0042] 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 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.

[0043] 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.

[0044] (2) 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 a polynucleotide encoding a plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof.

[0045] 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 the plant-parasitic nematode-resistant RKNR1 polypeptide or a fragment thereof. For example, the polynucleotide may be a polynucleotide encoding 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 a polynucleotide encoding 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).

[0046] In one embodiment, the polynucleotide of the present invention comprises any of (a) the nucleotide sequence shown in SEQ ID NO: 2, (b) the nucleotide sequence shown in SEQ ID NO: 2 in which one or more nucleotides have been deleted, substituted, or added, (c) 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 (d) a nucleotide sequence that hybridizes under highly stringent conditions to a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO: 2.

[0047] 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 a cell into which the polynucleotide is introduced.

[0048] 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.

[0049] (3) an expression vector containing a polynucleotide encoding the RKNR1 polypeptide or a fragment thereof that confers resistance to plant-parasitic nematodes; 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.

[0050] The expression vector of the present invention comprises a polynucleotide encoding the plant-parasitic nematode-resistant RKNR1 polypeptide of the present invention or a fragment thereof in an expressible state. As used herein, the term "expressible state" refers to the placement of a gene to be expressed downstream of a promoter under its control.

[0051] The expression vector of the present invention contains, as essential components, a promoter and the polynucleotide described in "(2) Polynucleotide encoding the RKNR1 polypeptide or a fragment thereof conferring resistance to plant-parasitic nematodes."

[0052] 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.

[0053] In the case of an expression vector that uses a plasmid (hereinafter often referred to as a "plasmid expression vector"), the plasmid that can be used includes, but is not limited to, pPZP, pSMA, pUC, pBR, pBluescript (Agilent Technologies), pTriEXTM (TaKaRa), or pBI, pRI, or pGW binary vectors.

[0054] 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).

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 1-3-2.Other ingredients 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.

[0065] 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.

[0066] 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.

[0067] 1-3-3. Dosage form The formulation of the plant parasitic nematode control agent of the present invention may be in any form as long as it can penetrate into the plant to which it is applied, and can 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 form that can act on the plant to which it is applied. Examples include dusts, powders, pastes, and gels.

[0068] 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, and can also enhance the resistance to plant-parasitic nematodes in plants that are resistant to plant-parasitic nematodes.

[0069] 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.

[0070] 2. Transformed plants or their progeny that are resistant to plant-parasitic nematodes 2-1. Overview A second aspect of the present invention relates to a plant transformant or its progeny that is resistant 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 is resistant to plant parasitic nematodes such as root-knot nematodes. The plant transformant of the present invention or its progeny is resistant to plant parasitic nematodes because nematode-attracting activity, nematode migration into roots, nodule formation, nodule maturation, nematode growth in roots, and / or giant cell induction in the host plant are suppressed.

[0071] 2-2.Configuration As used herein, the term "transformed plant" refers to a plant host that has been genetically modified to acquire resistance to plant-parasitic nematodes.

[0072] The plant transformant of the present invention comprises a polynucleotide encoding the RKNR1 polypeptide or a fragment thereof conferring resistance to plant-parasitic nematodes according to the first aspect, or an expression vector comprising the polynucleotide.

[0073] 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). Preferred host plants are 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).

[0074] The plant transformant of the present invention encompasses 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 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.

[0075] 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 the RKNR1 polypeptide or a fragment thereof that is resistant to plant parasitic nematodes, or an expression vector containing the polynucleotide, and that is resistant to plant parasitic nematodes, regardless of the progeny generation.

[0076] 3. Method for producing a plant transformant resistant to plant-parasitic nematodes Overview A third aspect of the present invention relates to a method for producing a plant transformant that is resistant to plant parasitic nematodes. According to the production method of the present invention, a resistant plant transformant can be produced from a plant that is susceptible to plant parasitic nematodes.

[0077] 3-2. Method The method of the present invention for producing a plant transformant having resistance to plant-parasitic nematodes comprises an introduction step and a selection step as essential steps. Each step will be specifically described below.

[0078] 3-2-1.Introduction process 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.

[0079] Methods known in the art, such as the Agrobacterium method, PEG-calcium phosphate method, electroporation, liposome method, particle gun method, and microinjection method, can be used to introduce an expression vector. The introduced polynucleotide may be integrated into the genomic DNA of the host, or may exist in the state of the introduced polynucleotide (e.g., as contained in a foreign vector). Furthermore, the introduced polynucleotide may be maintained continuously within the host cell, as when integrated into the genomic DNA of the host, or may be maintained transiently.

[0080] 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 the polynucleotide is 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, etc., for infection. Suitable Agrobacterium strains that can be used include, but are not limited to, GV3101, C58, C58C1Rif(R), EHA101, EHA105, AGL1, and LBA4404.

[0081] 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 in accordance with 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 about 450 to 2000 psi and at a distance of about 4 to 12 cm.

[0082] 3-2-2. Selection process In this embodiment, the "selection step" is a step of selecting a plant into which the expression vector has been introduced.

[0083] 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.

[0084] 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. [Example]

[0085] <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).

[0086] (method) (1) Rice varieties The rice varieties evaluated in this study 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. These varieties were obtained from the National Agriculture and Food Research Organization (NARO) Genbank and Nagoya University.

[0087] (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).

[0088] Specifically, rice seeds were soaked in a disinfectant solution (a 1:1000 dilution 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 under a 12-hour light (26°C) / 12-hour dark (24°C) regime 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 individuals total) 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 fertilized 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).

[0089] 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 egg masses stained blue 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 the water from the roots. 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 variety.

[0090] 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.

[0091] (result) The evaluation values ​​obtained for each variety are shown in Figures 1 and 2 and in Table 1 below.

[0092] [Table 1] TIFF0007742993000002.tif222148

[0093] In temperate japonica varieties, 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 varieties, all cultivars except Bei Khe and Naba were resistant to Mi.

[0094] The indica variety Kalo Dhan showed an evaluation value of approximately 1% compared to T65 (EV = 0.01), and was found to be one of the varieties with extremely high resistance to Mi. Based on this result, Kalo Dhan was used as a Mi-resistant variety in the following examples.

[0095] Example 2: Comparison of T65 and Kalo Dhan (the purpose) Resistance to sweet potato root-knot nematode (Mi) is compared between the japonica variety T65 and the indica variety Kalo Dhan.

[0096] (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 larval 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.

[0097] (2) Nematode migration population Mi was directly inoculated into the root apex of rice plants, and 1 to 21 days later, a 3 cm area of ​​the root apex was collected. 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 tip, compared with 2.42 in Kalo Dhan. Furthermore, 14 to 21 days after inoculation, the number of migrating nematodes in T65 was 8–10 per root tip, compared with only 1–3 in Kalo Dhan. This indicates that the number of migrating nematodes in Kalo Dhan was lower than that in T65.

[0098] (3) Lumbar width As in (2) above, Mi was directly inoculated into the root apex of rice plants, and the root nodule widths of T65 and Kalo Dhan were measured. 28 days after Mi inoculation, the root widths of the non-nodule region and the nodule region were measured (Fig. 3C, left), and the ratio was calculated as the relative nodule 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.

[0099] (4) Nematode growth rate As in (2) above, Mi was inoculated directly into the root apex of rice plants, and the body width of the 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.

[0100] (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.

[0101] The appearance of giant cells in the roots of T65 and Kalo Dhan is shown in Figure 4. 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.

[0102] These results indicate that Kalo Dhan strongly suppresses nematode attraction, nematode migration to roots, initiation of nodule formation, nodule maturation, nematode growth within roots, and giant cell induction compared to T65.

[0103] 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.

[0104] 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 group containing S4-693908 and S6-25039213, which were Kalo Dhan and T65, respectively, had lower scores and higher Mi resistance than the group containing S4-693908, T65, and S6-25039213, which were Kalo Dhan. These results indicated that qRKNR1 has the strongest effect on Mi resistance, and further mapping of qRKNR1 was performed.

[0105] (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 a 1.3 Mb region between IDK0401 and IDK0404.

[0106] (3) Identification of the RKNR1 gene We compared the genome sequences of Nipponbare, which is susceptible to Mi like T65, and N22, which is resistant to Mi like Kalo Dhan, 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).

[0107] The RKNR1 alleles in N22 and Kalo Dhan have 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 the position corresponding to position 339 in the Nipponbare RKNR1 amino acid sequence (SEQ ID NO: 9), a single base difference in the nucleotide sequence results in a Thr residue in Nipponbare / T65, whereas an Arg residue in N22 and Kalo Dhan (Figure 7). Furthermore, compared with 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.

[0108] <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.

[0109] (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 in the bottom of Figure 1.

[0110] In Figure 1, among the rice cultivars with an evaluation score of 0.6 or higher and susceptible to Mi, all cultivars except Akage, Bei Khe, and Naba were found to have the S-type RKNR1 gene. On the other hand, in all Mi-resistant cultivars with an evaluation score of less than 0.6, the RKNR1 gene was found to be the L-type.

[0111] Next, we determined the RKNR1 gene sequences of Akage, Bei Khe, and Naba by sequencing. As a result, Bei Khe and Naba had 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 of Akage 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). This SNP is thought to alter the function of the LRR domain in Akage.

[0112] 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.

[0113] (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 (ThermoFisher Scientific).

[0114] 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).

[0115] The upstream sequence of RKNR1 (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 sets 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 primer sets 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).

[0116] 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.

[0117] 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.

[0118] 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 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.

[0119] [Table 2]

[0120] 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 harvested. The resulting transformants were tested for Mi resistance.

[0121] (result) In Nipponbare transformed with the genomic region containing the RKNR1 gene from Kalo Dhan, the evaluation score was significantly lower than in the control line transfected with the vector alone (Fig. 2). These results demonstrated that the RKNR1 gene is the causative gene 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

1. 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

2. An agent for controlling plant-parasitic nematodes, comprising a polynucleotide encoding the polypeptide of claim 1.

3. 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

4. A plant-parasitic nematode control agent, comprising an expression vector containing the polynucleotide according to claim 2 or 3.

5. 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.

6. 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.

7. A plant transformant 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.

8. The transformed plant or its progeny according to claim 7, which is a monocotyledonous plant.

9. The transformed plant or its progeny according to claim 8, wherein the monocotyledonous plant is a grass plant.

10. 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.

11. The plant transformant or its progeny according to any one of claims 7 to 10, which is a genetically modified plant.

12. 1. A method for producing a plant transformant having resistance to plant-parasitic nematodes, comprising: Introducing the expression vector according to claim 4 into a plant; and a step of selecting a plant into which the expression vector has been introduced. A method comprising:

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