Glycosyltransferase inhibitor, plant disease resistance inducer containing the same, and plant disease control method using the same

Abstract

To provide a salicylic acid xylosylating enzyme inhibitor that is suitable as a resistance inducer for inhibiting many plant diseases and has high activity and less phytotoxicity.SOLUTION: A xylosylating enzyme inhibitor contains a compound with an IC50 of 5 μM or less to a salicylic acid xylosylating enzyme, as an active ingredient.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a glycosyltransferase inhibitor having high inhibitory activity against salicylic acid glycosyltransferase, a plant disease resistance inducer containing such a glycosyltransferase inhibitor, and a plant disease control method using the same. [Background technology]

[0002] Fungicides that directly suppress the growth of pathogens are generally used to control plant diseases. However, the effectiveness of these materials is often defeated by the emergence of pathogens that are resistant to the fungicide. On the other hand, resistance inducers that exert disease control effects by activating the disease resistance of the plant itself are also in practical use. These have a low risk of the emergence of resistant bacteria, and have little impact on microorganisms other than pathogens, so they have a low environmental impact. Therefore, resistance inducers have the potential to contribute to the realization of sustainable development.

[0003] When plants sense infection by a parasitic pathogen, they synthesize the plant hormone salicylic acid, which induces the expression of defense-related genes and thus exerts disease resistance (Non-Patent Document 1). Functional analogs of salicylic acid, such as BTH (benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester) (Non-Patent Document 2 and Patent Document 1) and INA (2,6-dichloroisonicotinic acid) (Non-Patent Document 3), are known to confer disease resistance to plants.

[0004] Other resistance inducers include oryzemate (active ingredient: probenazole; Patent Documents 2-5), saccharin (BIT), the active ingredient of probenazole (Patent Document 6), V-get (active ingredient: tiadinil), Stout (active ingredient: isotianil) (Patent Document 7), and Boon (active ingredient: diclobenthiazox) (Non-Patent Document 4). Other examples include benzisothiazoline derivatives (Patent Documents 8 and 9), NCI (N-cyanomethyl-2-chloroisonicotinamide) (Non-Patent Document 5), and CMPA (3-chloro-1-methyl-1H-pyrazole-5-carboxylic acid) (Patent Document 10, Non-Patent Document 6). Furthermore, a resistance-inducing effect has been detected with validamycin A, a known fungicidal pesticide (Non-Patent Document 7). Recently, resistance-inducing activity has also been confirmed for some rare sugars (Patent Document 11). Some of these compounds have been found to enhance the salicylic acid-related signal transduction mechanism (Non-Patent Document 8), but the details of their action have not been clarified.

[0005] Other methods include those using plant defense substances and plant symbiotic microorganisms to confer resistance (Patent Documents 12 and 13). Another technique for conferring resistance is the use of an abscisic acid synthesis inhibitor, which acts antagonistically against salicylic acid (Patent Document 14).

[0006] Due to their usefulness, the use of resistance inducers is expected to expand in agriculture in the future, but the application of the pesticides currently in practical use is mainly limited to rice blast disease (Non-Patent Document 7). Some pesticides have been attempted to be used on dicotyledonous crops, but since phytotoxicity is severe when commercially available pesticides are sprayed on the leaves, it is necessary to devise application methods to mitigate this. Since existing commercially available pesticides all have similar chemical structures, their mechanisms of action and phytotoxicity are thought to be similar.

[0007] A comprehensive search for pesticides with novel structures and actions is effective for developing pesticides suitable for new crops and target diseases. Previously, resistance inducers were generally discovered by treating plants with a candidate pesticide, infecting the pathogen, and evaluating the degree of disease suppression. However, this method requires large amounts of pesticide, a large testing space, and a long time, making it difficult to process efficiently and unsuitable for comprehensive searches. Meanwhile, because the mechanisms of action of existing resistance inducers are unknown, screening using drug target proteins, which is commonly used in pharmaceutical drug discovery, has not been performed in the agricultural field.

[0008] To address these issues, several high-throughput screening methodologies for resistance inducers have been developed (Patent Document 15, Non-Patent Documents 9, 10, 11, and 12). These methods utilize transgenic plants carrying genes in which the promoters of salicylic acid-responsive genes are linked to reporters. The promoter activity is then used as an indicator to screen for potential resistance inducers after adding candidate drugs to seedlings. Constitutive activation of the salicylic acid pathway produces strong disease suppression effects, but it often results in growth inhibition, hindering practical application. In fact, the pesticide registration of BTH, a salicylic acid analog, has been revoked in Japan for the same reasons. Other methodologies include those that use the yellowing of Arabidopsis seedling leaves upon infection or the generation of reactive oxygen species as indicators, and active compounds have been isolated (Non-Patent Document 13 and Patent Document 16). Numerous natural products and synthetic compounds with resistance inducer activity have also been reported, but only a limited number have been put to practical use (Non-Patent Document 14). Reasons why they have not yet been put to practical use include the difficulty of chemically synthesizing the substances and their low activity. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] European Patent Application Publication No. 313512 [Patent Document 2] Special Publication No. 12157-1973 [Patent Document 3] Special Publication No. 45-38080 [Patent Document 4] Special Publication No. 45-38356 [Patent Document 5] Special Publication No. 47-38967 [Patent Document 6] Japanese Patent Publication No. 5-59024 [Patent Document 7] JP 2003-113167 A [Patent Document 8] Patent Publication No. 2007-91596 [Patent Document 9] Patent Publication No. 2007-186509 [Patent Document 10] Japanese Patent Publication No. 8-12510 [Patent Document 11] WO2010 / 21121A1 [Patent Document 12] Patent Publication No. 2006-327995 [Patent Document 13] Patent Publication No. 2002-223747 [Patent Document 14] JP 2006-117608 A [Patent Document 15] JP 2007-151492 [Patent Document 16] Special table number 2013-538039 [Patent Document 17] WO2009 / 119915A1 [Non-patent literature]

[0010] [Non-Patent Document 1] Metraux et al., (1990) Science 250:1004-1006. [Non-patent document 2] Friedrich et al., (1996) Plant J 10:61-70. [Non-patent document 3] Vernooij et al., (1995) Mol Plant-Microbe Interact 8:228-234.

Non-licensed Document 4

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Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

Non-licensed Document 11

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Non-licensed Document 15

Non-licensed Document 16

[0011] As a result of extensive research aimed at solving the above-mentioned problems, the present inventors discovered that resistance inducers can be screened by using hypersensitive cell death, a disease resistance response induced in Arabidopsis thaliana cultured cells by infection with Bacillus speciosa, as an indicator. By optimizing the screening conditions, they established a method for comprehensively and rapidly screening for resistance inducers that do not induce a resistance response by themselves but have a priming effect that accelerates or strengthens the strength of the plant's innate defense responses (Patent Document 17, Non-Patent Documents 15 and 16). Using this method, they then screened a compound library and successfully isolated and identified several plant immune priming agents, demonstrating their disease control effects (Patent Document 17, Non-Patent Document 15). Further research revealed the mechanism of action of some of these agents: they exert their disease control effects by targeting and inhibiting glycosyltransferases that metabolize salicylic acid (Non-Patent Document 15). When plants are infected with diseases, they biosynthesize salicylic acid, which is inactivated by glycosylation with salicylic acid glycosyltransferase, terminating the defense response. When glycosylation of salicylic acid is inhibited, the rate of free salicylic acid accumulation increases, resulting in an immune priming effect. However, the disease control effect of these plant immune priming agents has not always been sufficient.

[0012] The present invention has been made to solve the above-mentioned problems, and aims to provide a salicylic acid glucosyltransferase inhibitor that is highly active and causes little phytotoxicity, and can be suitably used as a resistance inducer or the like to suppress many plant diseases. [Means for solving the problem]

[0013] Since the target of the plant defense inducer was revealed in the above study, the inventors separately conducted a comprehensive search for salicylic acid glycosyltransferase inhibitors and succeeded in obtaining a new plant defense inducer. 50 It has been found that a high disease control effect can be achieved by using a compound having a specific value or less. The present invention has been completed based on this finding.

[0014] The above problem is solved by IC 50 The problem is solved by providing a glycosyltransferase inhibitor containing, as an active ingredient, a compound having an IC value of 5 μM or less for 2,4-dihydroxybenzoic acid glycosyltransferase and indole-3-acetic acid glycosyltransferase. 50 It is also preferable that the compound is at least one selected from the group consisting of compounds represented by the following formulas (I) to (VII) and pharmaceutically acceptable salts and solvates thereof:

[0015] [ka]

[0016] A plant disease resistance inducer comprising a glycosyltransferase inhibitor is a preferred embodiment of the present invention. In this case, it is preferred that the plant to which the resistance inducer is applied is a plant belonging to the Brassicaceae, Poaceae, Solanaceae, Cucurbitaceae, Fabaceae, Cruciferae, Rosaceae, Moraceae, Malvaceae, Umbelliferae, Asteraceae, Vitaceae or Amaranthaceae. The plant diseases to which the resistance inducer is applied are preferably rice blast fungus, rice sheath blight fungus, bacterial seedling blight fungus, Helminthosporium oryzae fungus, sheath blight fungus, rice bacterial leaf blight fungus, potato powdery scab fungus, potato Phytophthora infestans, potato black spot fungus, potato scab fungus, barley powdery mildew fungus, wheat Fusarium head blight fungus, wheat snow blight fungus, wheat leaf rust fungus, wheat powdery mildew fungus, and wheat. Diseases caused by wheat root rot fungus, soybean downy mildew fungus, soybean purpura fungus, pea mycosis fungus, corn smut fungus, sweet potato fusarium wilt fungus, melon fusarium wilt fungus, lettuce root rot fungus, tomato wilt fungus, tomato verticillate fungus, tomato anthracnose fungus, tomato bacterial spot fungus, spinach wilt fungus, cruciferous clubroot fungus, cucumber seedling damping-off fungus, or strawberry Botrytis cinerea are also preferred.

[0017] A method for controlling plant diseases using the plant disease resistance inducer is also a preferred embodiment of the present invention. [Effects of the Invention]

[0018] IC for salicylic acid glycosyltransferase 50The glycosyltransferase inhibitors of the present invention, which contain compounds with extremely low oxidative stress as active ingredients, have high resistance-inducing activity against bacterial spot disease and sheath blight, making them suitable for use as resistance inducers for suppressing many plant diseases. Furthermore, due to their mechanism of action, they do not directly induce plant immunity but rather exert a priming effect that enhances the plant's inherent resistance response, thereby causing no phytotoxicity (growth inhibition). Among these, compounds with plant disease resistance-inducing activity obtained by a specific screening method have extremely high activity in suppressing bacterial spot disease and sheath blight. Since the resistance-inducing activity of these compounds was detected in a model infection system for evaluating disease responses via the salicylic acid pathway, glycosyltransferase inhibitors containing these compounds as active ingredients are expected to exhibit similar disease control activity against many plant diseases. Furthermore, because there are few resistance inducers that are effective against sheath blight pathogens, this compound may serve as a new strategy for controlling sheath blight. [Brief explanation of the drawings]

[0019] [Figure 1] 1 is a graph showing the ratio of the expression level of the rpoD gene of Pst to the CBP20 gene of Arabidopsis thaliana injected with Saglutins 1 to 7 (compounds I to VII) in Example 2. [Figure 2] In Example 2, the disease index of each inoculated leaf that was sprayed with Saglutins 1 to 7 (compounds I to VII) and then inoculated with a mycelial plug of Rhizoctonia solani is shown. [Figure 3] 1 shows photographs of PDA media in Example 3, each containing Saglutin 1 to 7 (compounds I to VII) dissolved therein, inoculated with a mycelial plug of Rhizoctonia solani, and cultured statically in a dark place at 25° C. for 24 hours. DETAILED DESCRIPTION OF THE INVENTION

[0020] The glycosyltransferase inhibitor of the present invention has an IC 50 The compound contains as an active ingredient a compound with an IC of 5 μM or less. 50means the 50% inhibitory concentration, and is the IC 50 The IC50 for salicylic acid glycosyltransferase is the concentration of a compound required to inhibit the activity of salicylic acid glycosyltransferase by 50%, i.e., the activity of the enzyme to glycosylate salicylic acid by 50%. 50 Since compounds with an IC of 5 μM or less exhibit high plant disease resistance induction activity, it is believed that the glycosyltransferase inhibitors of the present invention containing such compounds as active ingredients will similarly exhibit disease control activity against many crop diseases. 50 is preferably 3.5 μM or less, more preferably 2 μM or less, even more preferably 1 μM or less, even more preferably 0.6 μM or less, particularly preferably 0.3 μM or less, and most preferably 0.2 μM or less. 50 The IC of the compound against salicylic acid glycosyltransferase is usually 0.0001 μM or more, preferably 0.001 μM or more, and more preferably 0.01 μM or more. 50 is measured by the method described in the Examples below.

[0021] In the glycosylation reaction of salicylic acid by salicylic acid glycosyltransferase, the inhibition constant (Ki) of the compound against the substrate salicylic acid is preferably 5 μM or less, more preferably 2 μM or less, even more preferably 1 μM or less, even more preferably 0.5 μM or less, particularly preferably 0.15 μM or less, and most preferably 0.1 μM or less. On the other hand, the inhibition constant (Ki) is usually 0.0001 μM or more, preferably 0.001 μM or more, and more preferably 0.01 μM or more. The inhibition constant (Ki) of the compound against each substrate is measured by the method described in the Examples below.

[0022] In the glycosylation reaction of salicylic acid catalyzed by salicylic acid glycosyltransferase, the inhibition constant (Ki) of the compound against the substrate UDP-glucose (uridine diphosphate glucose) is preferably 5 μM or less, more preferably 3.5 μM or less, even more preferably 2 μM or less, even more preferably 1 μM or less, particularly preferably 0.8 μM or less, and most preferably 0.4 μM or less. On the other hand, the inhibition constant (Ki) is usually 0.0001 μM or more, preferably 0.001 μM or more, and more preferably 0.01 μM or more.

[0023] The type of salicylic acid glycosidase is not particularly limited as long as it is present in plants, but it is preferably UGT76B1 or UGT74F1, which are salicylic acid glycosidases of Arabidopsis thaliana. 50 In the Examples described below, compounds having an IC value within the above range have been shown to have high resistance-inducing activity in both monocotyledonous and dicotyledonous plants, and to have high resistance-inducing activity against multiple types of diseases, and thus have resistance-inducing activity against many plant diseases. It is more preferable that the salicylic acid glycosyltransferase is UGT76B1 and UGT74F1. The IC values ​​for UGT76B1 and UGT74F1 are 50 If both of these fall within the above-mentioned ranges, the compound also has resistance induction activity against a greater number of plant diseases.

[0024] IC of the compounds against 2,4-dihydroxybenzoic acid glycosyltransferase and indole-3-acetic acid glycosyltransferase 50 It is preferable that the IC values ​​for 2,4-dihydroxybenzoic acid glycosyltransferase and indole-3-acetic acid glycosyltransferase are both greater than 20 μM. 50 A high IC indicates a small effect on these enzymes. 50 and IC for 2,4-dihydroxybenzoic acid glycosyltransferase and indole-3-acetic acid glycosyltransferase 50A compound with a high IC value for 2,4-dihydroxybenzoate glycosidase and indole-3-acetic acid glycosidase can be said to have high specificity for the salicylic acid glycosidase, and by using such a compound, it is possible to induce disease resistance while further reducing phytotoxicity. The types of 2,4-dihydroxybenzoate glycosidase and indole-3-acetic acid glycosidase are not particularly limited as long as they are present in plants, but the 2,4-dihydroxybenzoate glycosidase is preferably UGT72B1, a 2,4-dihydroxybenzoate glycosidase from Arabidopsis thaliana, and the indole-3-acetic acid glycosidase is preferably UGT84B1, an indole-3-acetic acid glycosidase from Arabidopsis thaliana. The IC values ​​of the compounds for 2,4-dihydroxybenzoate glycosidase and indole-3-acetic acid glycosidase are 50 is measured by the method described in the examples.

[0025] The compound is preferably at least one selected from the group consisting of compounds represented by the following formulas (I) to (VII) and pharmaceutically acceptable salts and solvates thereof. These compounds were identified from a library of 210,560 low-molecular-weight compounds using a screening method developed by the present inventors, and have IC activity against salicylic acid glycosyltransferase. 50 The activity of these compounds is extremely low, and they also have little effect on other enzymes such as 2,4-dihydroxybenzoic acid glycosyltransferase and indole-3-acetic acid glycosyltransferase. Therefore, by using a glycosyltransferase inhibitor containing at least one compound selected from the group consisting of compounds represented by the following formulas (I) to (VII) and pharmaceutically acceptable salts and solvates thereof as an active ingredient, the inhibitory effect on salicylic acid glycosyltransferase is further enhanced, and phytotoxicity is further reduced. Moreover, these compounds are easy to synthesize.

[0026] [ka]

[0027] The pharmaceutically acceptable salt is not particularly limited, but examples thereof include salts with inorganic acids such as sulfates, hydrochlorides, and phosphates; salts with organic acids such as formates, acetates, maleates, citrates, and malates; salts with alkali metals such as sodium and potassium; salts with alkaline earth metals such as calcium and magnesium; ammonium salts, etc. The solvent that forms the pharmaceutically acceptable solvate is not particularly limited, but examples thereof include water, isopropyl alcohol, xylene, cyclohexane, methylnaphthalene, etc.

[0028] The compound is more preferably at least one selected from the group consisting of compounds represented by the above formulas (I) to (IV) and pharmaceutically acceptable salts and solvates thereof, more preferably at least one selected from the group consisting of compounds represented by the above formulas (I) and (II) and pharmaceutically acceptable salts and solvates thereof, and even more preferably at least one selected from the group consisting of compounds represented by the above formula (I) and pharmaceutically acceptable salts and solvates thereof.

[0029] In the present invention, the above-mentioned compound may be used as a glycosyltransferase inhibitor directly, or the glycosyltransferase inhibitor may contain, together with the compound, one or more solid carriers, liquid carriers, surfactants, and other formulation adjuvants commonly used in the formulation of pesticides. That is, the glycosyltransferase inhibitor may be in a variety of formulation forms prepared by mixing the compound with other formulation adjuvants. The formulation of the glycosyltransferase inhibitor is not particularly limited, and the glycosyltransferase inhibitor may be in any form, such as granules, dusts, liquids, emulsions, wettable powders, water-soluble agents, oil solutions, aerosols, or flowables. Examples of carriers used in formulation include solid carriers such as talc, bentonite, clay, kaolin, diatomaceous earth, white carbon, vermiculite, calcium carbonate, slaked lime, silica sand, ammonium sulfate, and urea, and liquid carriers such as isopropyl alcohol, xylene, cyclohexane, and methylnaphthalene.

[0030] The content of the above-mentioned compound as an active ingredient in the glycosyltransferase inhibitor of the present invention can be appropriately set as needed, but is usually 0.1 to 80% by mass. When the glycosyltransferase inhibitor is in the form of a dust or granule, the content is usually 0.1 to 50% by mass, and when it is in the form of an emulsifiable concentrate or wettable powder, the content is usually 5 to 80% by mass.

[0031] The glycosidase inhibitor of the present invention may be used by any of spraying, dusting, spraying, dipping, dressing, painting, fumigation, smoking, irrigation, etc. Specific modes of use include a method of spraying or applying the glycosidase inhibitor to a plant body, a method of soaking plant seeds in a liquid containing the glycosidase inhibitor, a method of spraying the glycosidase inhibitor in a field where a disease has occurred or where there is a risk of the disease occurring, and a method of mixing the glycosidase inhibitor into soil.

[0032] The amount of the glycosyltransferase inhibitor of the present invention to be used may be appropriately determined depending on the type of disease, the type of target plant, the growth stage of the target plant, the type of formulation, shape, form, application method, application time, application period, etc., and is not particularly limited. 2 When used in liquid form such as an emulsifiable concentrate or wettable powder, the concentration of the active ingredient is usually 0.1 to 10,000 ppm, preferably 10 to 3,000 ppm.

[0033] A plant disease resistance inducer comprising the glycosyltransferase inhibitor is a preferred embodiment of the present invention. In the present invention, the term "resistance inducer" refers to a drug for inducing or activating plant disease resistance and controlling plant diseases. Since the plant disease resistance inducer of the present invention is intended to prevent disease, it is preferably applied before the onset of disease, but it may also be applied after, before, or after the onset of disease.

[0034] Plants that can be treated with the plant disease resistance inducer of the present invention include all cultivated plants, and may be either monocotyledonous or dicotyledonous plants. Examples of such plants include plants belonging to the Brassicaceae family (Arabidopsis thaliana, cabbage, rapeseed, etc.), Poaceae family (rice, corn, barley, wheat, wheatgrass, etc.), Solanaceae family (tomato, eggplant, potato, tobacco, etc.), Cucurbitaceae family (cucumber, melon, pumpkin, etc.), Fabaceae family (soybean, pea, kidney bean, alfalfa, peanut, etc.), Brassicaceae family (radish, Chinese cabbage, cabbage, etc.), Rosaceae family (strawberry, apple, pear, etc.), Moraceae family (mulberry, etc.), Malvaceae family (cotton, etc.), Apiaceae family (carrot, parsley, celery, etc.), Asteraceae family (burdock, sunflower, chrysanthemum, lettuce, etc.), Vitaceae family (grape, etc.), and Amaranthaceae family (spinach, etc.), but are not limited to these plants.

[0035] When the plant disease resistance inducer of the present invention is applied in the form of the glycosyltransferase inhibitor as described above, for example, plant diseases caused by filamentous fungi, bacteria and viruses can be effectively controlled. For example, Magnaporthe oryzae, Burkholderia plantarii, Cochliobolus miyabeanus, Rhizoctonia solani, Xanthomonas oryzae, Spongospora subterranea, Phytophthora infestans, Rhizoctonia solani, Streptomyces scab, Eryshiphe graminis f. sp. hordei, Gibberella zeae, Sclerotinia sclerotioides, and Sclerotinia sclerotioides. borealis), wheat leaf rust (Puccinia recondita), wheat powdery mildew (Erysiphe graminis), wheat root rot (Rhizoctonia solani), soybean downy mildew (Peronospora manshurica), soybean purple spot (Cercospora kikuchii), pea ascochyta (Mycosphaerella pinodes), corn smut (Ustilago maydis), sweet potato fusarium wilt (Fusarium oxysporum f. sp. batatas), melon fusarium wilt (Fusarium oxysporum f. sp. melonis), lettuce root rot (Fusarium oxysporum f. sp. lactucae), tomato wilt (Fusarium oxysporum f. sp. lycopersici), tomato half wilt (Verticillium dahliae), tomato anthracnose (Colletotrichum phomoides), spinach wilt (Fusarium oxysporum f. sp.These include, but are not limited to, diseases caused by Plasmodiophora spinaciae, Plasmodiophora brassicae, Pythium debaryanum, and Botrytis cinerea.

[0036] The plant disease resistance inducer of the present invention can also be used in combination with one or more other agricultural chemicals such as herbicides, fungicides, insecticides, fertilizers, plant growth regulators, soil conditioners, etc.

[0037] The plant disease resistance inducer of the present invention exhibits a high resistance induction effect in both monocotyledonous and dicotyledonous plants. Furthermore, because the plant's own resistance is enhanced, the desired effect is achieved regardless of the type of disease. Furthermore, due to its mechanism of action, the plant disease resistance inducer of the present invention does not directly induce plant immunity but instead exhibits a priming effect that enhances the plant's inherent resistance response. In other words, it is characterized by not causing phytotoxicity (growth inhibition), which has been a problem in the practical application of immunity-inducing resistance inducers. Therefore, the plant disease resistance inducer of the present invention can be suitably used to control many plant diseases and can also be used as a research tool to elucidate the role of salicylic acid glycosylation in various plants and crops. Furthermore, a method for controlling plant diseases using the plant disease resistance inducer is also a preferred embodiment of the present invention. [Example]

[0038] The present invention will be explained in more detail below with reference to examples, but these examples are not intended to limit the present invention.

[0039] IC for salicylic acid glycosylation by UGT76B1 50 Measurement of Glycosyltransferase (UGT76B1, final concentration 3.3 μg / ml), 0.17 mM salicylic acid, 0.17 mM UDP-glucose, 2 mM DTT (dithiothreitol), 10 mM magnesium chloride, 50 mM MES (2-morpholinoethanesulfonic acid), and water were added with glycosyltransferase inhibitors [Compound I (0.025, 0.05, 0.075, 0.1, 0.2, 0.25 μM), Compound II (0.05, 0.1, 0.2, 0.3, 0.4 μM), Compound III (0.1, 0.2, 0.3, 0.4, 0.5, 1.0 μM), Compound IV (0.1, 0.2, 0.4, 0.5, 1.0, 1.5 ... 50 μL mixtures containing compound V (0.5, 1.0, 1.5, 2.0, 3.0, 4.0 μM), compound VI (0.25, 0.5, 0.75, 1.0, 1.5, 2.0 μM), and compound VII (2.0, 3.0, 4.0, 5.0, 7.0 μM) were incubated at 30°C for 30 minutes, and then 8 μL of 50% (V / V) trichloroacetic acid was added to terminate the reaction. The reaction mixtures were analyzed by reverse-phase HPLC (elution: acetonitrile / water (containing 0.1% trifluoroacetic acid) = 45 / 55 (volume ratio), 1.3 mL / min, 4 minutes, 40°C). Salicylic acid and salicylic acid glucoside were analyzed by absorption or fluorescence at 296 nm (excitation wavelength: 295 nm, emission wavelength: 370 nm). The inhibitor candidate compound concentration (μM) at which the enzyme activity is 50% of the enzyme activity without the inhibitor candidate compound is plotted against the inhibitor rate (%). The IC 50 was requested as follows.

[0040] IC for salicylic acid glycosylation by UGT74F1 50 Measurement of The glycosyltransferase used in the reaction was changed to UGT74F1, and the concentrations of the glycosyltransferase inhibitors added were changed to [Compound I (0.01, 0.025, 0.05, 0.1, 0.2, 0.25 μM), Compound II (0.1, 0.2, 0.3, 0.4, 0.5 μM), Compound III (0.2, 0.3, 0.4, 0.5, 0.75, 1.0 μM), Compound IV ( Compound V (0.25, 0.5, 0.75, 1.0, 1.5, 2.0 μM), Compound V (0.25, 0.5, 0.75, 1.0, 1.5, 2.0 μM), Compound VI (0.5, 0.75, 1.0, 1.5, 2.0, 4.0 μM), Compound VII (1.0, 2.0, 4.0, 5.0, 7.0, 10.0 μM) were used. The IC 50 When the type of salicylic acid glycosyltransferase or glycosyltransferase inhibitor was changed to one other than those mentioned above, the IC was calculated in the same manner as above, except that the type and the concentration of the glycosyltransferase inhibitor were changed appropriately. 50 can be obtained.

[0041] [K for salicylic acid substrate in salicylic acid glycosylation by UGT76B1 i Measurement of Glycosyltransferase (UGT76B1, final concentration 3.3 μg / ml), 0.17 mM UDP-glucose, 2 mM DTT (dithiothreitol), 50 mM MES (2-morpholinoethanesulfonic acid), and water were treated with glycosyltransferase inhibitors [Compound I (0.025, 0.05, 0.075, 0.1 μM), Compound II (0.1, 0.2, 0.3, 0.4 μM), Compound III (0.1, 0.2, 0.3, 0.4 μM), Compound IV (0.2, 0.4, 0.5, 1.0 μM), Compound V (0.25, 0.5, 0.75, 1.0 μM), Compound VI (0.5, 1.0, 1.5, 2.0 μM), Compound VII (0.5, 1.0, 1.5, 2.0 μM), Compound VIII ... A 50 μL mixture containing salicylic acid (0.09, 0.17, 0.34 mM for compounds I, II, III, VI, and VII; 0.09, 0.13, 0.34 mM for compound IV; and 0.09, 0.24, 0.34 mM for compound V) was incubated at 30°C for 30 minutes, and then 8 μL of 50% (V / V) trichloroacetic acid was added to terminate the reaction. The reaction mixture was analyzed by reverse-phase HPLC (acetonitrile / water (containing 0.1% trifluoroacetic acid) = 45 / 55 (volume ratio), 1.3 mL / min, 4 minutes, 40°C). Salicylic acid and salicylic acid glucoside were analyzed by absorption or fluorescence at 296 nm (excitation wavelength 295 nm, emission wavelength 370 nm). For each result, a graph (Dixon plot) was prepared showing the reciprocal of the rate of salicylic acid glucoside formation versus the inhibitor concentration, and the K was calculated from the intersection of the graphs for three different salicylic acid substrate concentrations. i When this graph does not intersect due to uncompetitive inhibition, a graph of (substrate concentration) / (salicylic acid glucoside production rate) versus inhibitor concentration is created, and the K is calculated from the intersection of the graphs. i was calculated.

[0042] [K for UDP-glucose substrate in salicylic acid glycosylation by UGT76B1 i Measurement of The K reaction was carried out in the same manner as above, except that 1.7 mM salicylic acid was added to the reaction and the initial concentration of UDP-glucose substrate was varied (0.045, 0.17, and 0.67 mM for compounds I and II; 0.045, 0.09, and 0.17 mM for compounds III and V; and 0.025, 0.045, and 0.09 mM for compounds IV, VI, and VII). i asked for.

[0043] [K for salicylate substrate in salicylate glycosylation by UGT74F1 i Measurement of Glycosyltransferase (UGT74F1, final concentration 3.3 μg / ml), 0.17 mM UDP-glucose, 2 mM DTT (dithiothreitol), 50 mM MES (2-morpholinoethanesulfonic acid), and water were mixed with glycosyltransferase inhibitors [Compound I (0.025, 0.05, 0.075, 0.1 μM), Compound II (0.1, 0.2, 0.3, 0.4 μM), Compound III (0.1, 0.2, 0.3, 0.4 μM), Compound IV (0.2, 0.4, 0.5, 1.0 μM), Compound V (0.25, 0.5, 0.75, 1.0 μM), Compound VI ... Compound VI (0.5, 1.0, 1.5, 2.0 μM), compound VII (1.0, 2.0, 3.0, 4.0 μM), and salicylic acid (0.09, 0.17, 0.28 mM for compounds I, II, and VII, and 0.09, 0.13, 0.28 mM for compounds III, VI, V, and VI) were added to a 50 μl mixture and kept at 30° C. for 30 minutes. Then, the amounts of salicylic acid and salicylic acid glucoside were analyzed by the above method, and the K values ​​were calculated from the Dixon plot. i was calculated.

[0044] [K for UDP-glucose substrate in salicylic acid glycosylation by UGT74F1 i Measurement of The K was prepared in the same manner as above, except that 1.7 mM salicylic acid was added to the reaction and the initial concentration of UDP-glucose substrate was varied (0.08, 0.17, and 0.33 mM for compounds I, II, and III, and 0.04, 0.08, and 0.17 mM for compounds IV, V, VI, and VII). i asked for.

[0045] [IC for 2,4-dihydroxybenzoic acid glycosylation by UGT72B1 50 Measurement of Five μl of a mixture containing 2,4-dihydroxybenzoic acid glycosyltransferase (UGT72B1, final concentration 1.0 μg / ml), 0.2 mM 2,4-dihydroxybenzoic acid, 0.1 mM UDP-glucose, 2 mM DTT (dithiothreitol), 10 mM magnesium chloride, 50 mM MES (2-morpholinoethanesulfonic acid), 0.02% Triton X-100, and water, plus glycosyltransferase inhibitors (0, 0.1, 0.5, 2.0, 10.0, and 20.0 μM) was incubated at room temperature for 120 minutes. Subsequently, 5 μl of a solution containing 0.1 M Tris-HCl (pH 7.5), 0.1 mM ATP, 2 U / ml nucleoside diphosphate kinase, 0.01% bovine serum albumin, and 0.02% Triton X-100 was added and incubated at room temperature for 60 minutes. Further, 10 μl of a solution containing 0.1 M Tris-HCl (pH 7.5), 2 mM glucose, 2 U / ml ADP-hexokinase, 2 U / ml glucose-6-phosphate dehydrogenase, 2 U / ml diaphorase, 0.2 mM nicotinamide adenine dinucleotide phosphate, 0.1 mM resazurin, 0.01% bovine serum albumin, 0.02% Triton X-100, and 20 mM N-ethylmaleimide was added and incubated at room temperature for 60 minutes. The fluorescence value of the solution (excitation wavelength 540 nm, fluorescence wavelength 590 nm) was measured using a microplate reader, and this was taken as the amount of UDP produced by the glycosylation reaction. The inhibition rate (%) of the candidate drug was calculated from the ratio to the fluorescence intensity when no drug was added, and the concentration (μM) of the candidate inhibitor compound at which the enzyme activity was 50% of the enzyme activity when no inhibitor candidate compound was added was defined as the IC 50 was requested as follows.

[0046] [IC for indole-3-acetic acid glycosylation by UGT84B1 50 Measurement of The IC was performed in the same manner as above, except that indole-3-acetic acid was added instead of 2,4-dihydroxybenzoic acid and UGT72B1 was replaced with UGT84B1, an indole-3-acetic acid glycosyltransferase. 50 Even when the types of glycosyltransferases or glycosyltransferase inhibitors are changed to those other than those mentioned above, the IC values ​​were calculated in the same manner as above, except for changing the types. 50 can be obtained. Example 1 [Screening and identification of candidate compounds for plant disease resistance inducers] (1) Discovery and identification of compounds that enhance hypersensitive cell death UGT76B1, one of the salicylic acid glycosyltransferases of Arabidopsis thaliana, was expressed and purified in Escherichia coli, and an inhibitor screening system for this enzyme was established according to the method described in Non-Patent Document 17. 302 primary hit compounds were identified from the small molecule compound library (210,560 compounds) held by the Drug Discovery Center of the University of Tokyo, and 125 compounds were isolated as candidate inhibitor compounds after excluding false positives that inhibited the screening system. Furthermore, concentration dependency was examined at 0.1 to 20 μM, and reproducibility and concentration dependency were observed, with IC values ​​within this concentration range. 50 The 95 compounds for which values ​​could be calculated were identified as secondary hit compounds.

[0047] Furthermore, when these secondary hit compounds were examined for their effects on UGT74F1, another salicylic acid glycosyltransferase in Arabidopsis thaliana, 91 compounds showed inhibitory effects. Since Arabidopsis thaliana has 107 glycosyltransferase genes (Non-Patent Document 18), in order to search for highly specific drugs, UGT72B1, a glycosyltransferase that uses 2,4-dihydroxybenzoic acid, similar to salicylic acid, as a substrate, and UGT84B1, a glycosyltransferase that uses indole-3-acetic acid (auxin) as a substrate, were used to examine the inhibitory effects of the secondary hit compounds on these enzymes, and 65 and 27 compounds, respectively, were found to exhibit inhibitory activity (IC 50 As a result, 30 drugs were identified as selective inhibitors of the Arabidopsis salicylate glycosyltransferases UGT76B1 and UGT74F1, and these were designated as the third hit compounds.

[0048] Furthermore, among the third hit compounds, IC 50 Thirteen compounds with a 50% inhibitory concentration (50% inhibitory concentration) of 10 μM or less were assayed for their ability to enhance hypersensitive cell death, a disease resistance response exhibited by cultured Arabidopsis cells against infection with the incompatible pathogen Pseudomonas syringae pv. tomato DC3000 avrRpm1 (Pst-avrRpm1), according to the method described in Non-Patent Document 16. As a result, 12 compounds were isolated that also exhibited hypersensitive cell death enhancement in cultured Arabidopsis cells. Furthermore, among these, the following seven compounds (named Saglutin: SA glucosyltransferase inhibitors) [Saglutin 1 (compound I), Saglutin 2 (compound II), Saglutin 3 (compound III), Saglutin 4 (compound IV), Saglutin 5 (compound V), Saglutin 6 (compound VI), and Saglutin 7 (compound VII)], which are available commercially in fixed amounts, were tested for their specific and potent inhibitory activity against two salicylic acid glycosyltransferases in Arabidopsis thaliana (IC500 for UGT76B1 and UGT74F1). 50 5 μM or less and IC for UGT72B1 and UGT84B1 50 These compounds were narrowed down to candidate compounds for resistance inducers that have been confirmed to have a resistance-inducing effect against disease.

[0049] [ka]

[0050] The IC values ​​of Saglutins 1-7 (compounds I-VII) for salicylic acid glycosyltransferases UGT76B1 and UGT74F1 in Arabidopsis thaliana 50 The inhibition constants (Ki) for these substrates, salicylic acid and UDP-glucose, were measured and the results are shown in Table 1.

[0051] [Table 1]

[0052] Example 2 [Demonstration of the resistance-inducing ability of Saglutin 1-7] Whether Saglutins 1 to 7 (compounds I to VII) can actually confer disease resistance to plants by administering them to plants was further examined by the following method.

[0053] (1) Study of the control effect of bacterial leaf spot disease on Arabidopsis thaliana We investigated the resistance induction effect of salicylic acid glycosyltransferase inhibitors (Saglutins 1–7) on Arabidopsis thaliana, a dicotyledonous plant in the Brassicaceae family, against the bacterial spot pathogen. Rosette leaves of Arabidopsis plants were sprayed with salicylic acid or other agents, and then inoculated with the bacterial spot pathogen by infiltration two days later. The bacterial growth rate within the leaves was evaluated based on the ratio of bacterial gene content to plant-derived gene content. The details are explained below. Arabidopsis plants were grown for three weeks under short-day conditions (8 hours of light / 16 hours of darkness) and then sprayed with a solution of 50 μM (Saglutins 1–5, 7 (Compounds I–V, VII)) salicylic acid glycosyltransferase inhibitors or 10 μM (Saglutin 6 (Compound VI)) in 0.04% Tween 20. DMSO, the solvent for the compound, was used as a negative control, and a solution containing sodium salicylate (100 μM) was used as a positive control. 600A suspension of Pseudomonas syringae pv. tomato DC3000 (Pst) in 10 mM MgCl2 at a concentration of 0.002 was injected into the apoplast from the underside of the leaf using a 1 ml syringe without a needle. Three days later, leaves were excised using a 6 mm diameter cork borer. Three leaves were transferred to a 2 ml tube, and four 3 mm zirconia balls were added and frozen in liquid nitrogen. The leaves were then crushed for 3 minutes in a grinder. RNA was extracted from the samples using the Invitrogen PureLink RNA purification kit, and cDNA was synthesized using each RNA using the Takara PrimeScript® RT reagent kit with gDNA Eraser. Quantitative RT-PCR experiments were performed using primers specific for the Arabidopsis CBP20 gene and the Pst rpoD gene.

[0054] Figure 1 is a graph showing the ratio of the expression level of the Pst rpoD gene to the CBP20 gene in Arabidopsis thaliana sprayed with Saglutins 1 to 7 (compounds I to VII). As shown in Figure 1, it was revealed that treatment with Saglutins 1, 3 to 7 suppressed the proliferation of Pst in Arabidopsis leaves.

[0055] (2) Study on the control effect of sheath blight fungus on wheatgrass We investigated the effect of Saglutins 1–7 (compounds I–VII) on the induction of resistance to sheath blight fungus in Brachypodium distachyon, a monocotyledonous grass. Brachypodium distachyon was grown for 3 weeks under long-day conditions (20 h light / 4 h dark). Cut leaves were placed on a Petri dish lined with water-moistened filter paper and sprayed with a solution containing 5 μM (Saglutins 1–3) or 50 μM (Saglutins 4–7) of each compound (Saglutins 2, 3, 4, and 7 dissolved in 0.2% methanol; Saglutins 1, 5, and 6 dissolved in 0.5% DMSO). Negative controls included the compound solvents (0.2% methanol or 0.5% DMSO), and positive controls included sodium salicylate (1 mM). After 24 hours, the plants were inoculated with mycelial plugs of Rhizoctonia solani AG-1 IA, which causes sheath blight.

[0056] Three days later, the severity of lesions on each inoculated leaf was assessed on a four-point scale (the higher the level, the greater the degree of infection) and expressed as a disease index. Figure 2 shows the disease index of each inoculated leaf that was sprayed with Saglutins 1 to 7 (compounds I to VII) and then inoculated with a mycelial plug of R. solanacearum. As shown in Figure 2, it was clear that the addition of Saglutins 1 to 7 suppressed the degree of R. solanacearum infection in wheatgrass.

[0057] Example 3 [Effect of Saglutin 1-7 on the growth of Rhizoctonia solani] The effects of the isolated resistance-inducing compounds on the growth of R. solani were investigated. Mycelial plugs of R. solani were inoculated onto PDA medium containing Saglutins 1–7 (compounds I–VII) dissolved to a final concentration of 50 μM (Saglutins 2, 3, 4, and 7 in 0.2% methanol, and Saglutins 1, 5, and 6 in 0.5% DMSO). The mycelial plugs were then incubated at 25°C in the dark for 24 hours, and growth was observed (Figure 3). As a control, PDA medium supplemented with the compound solvents (0.2% methanol, 0.5% DMSO, and the fungal antibiotic hygromycin) was used. The results showed that none of the Saglutins 1–7 (compounds I–VII) inhibited the growth of R. solani. From these results, it is thought that the inhibitory effect of Saglutin 1 to 7 (compounds I to VII) on sheath blight infection, as shown in the above section "(2) Investigation of the control effect against sheath blight fungus in Minato Kamoshi grass," is not due to a bactericidal effect on the sheath blight fungus (filamentous fungus), but rather to a resistance-inducing effect on the plant.

Claims

1. IC for salicylic acid glycosyltransferase ５０ 5 μM or less as an active ingredient, wherein the compound is at least one selected from the group consisting of compounds represented by the following formulas (I) to (III) and (V) to (VII), and pharmaceutically acceptable salts and solvates thereof: 【Chemical 1】 【change】

2. A plant disease resistance inducer comprising the glycosyltransferase inhibitor according to claim 1.

3. The plant disease resistance inducer according to claim 2, wherein the target plant of the resistance inducer is a plant belonging to the Brassicaceae, Poaceae, Solanaceae, Cucurbitaceae, Fabaceae, Brassicaceae, Rosaceae, Moraceae, Malvaceae, Umbelliaceae, Asteraceae, Vitaceae or Amaranthaceae family.

4. The plant diseases targeted by the resistance inducer are rice blast fungus, rice sheath blight fungus, bacterial seedling blight fungus, Helminthosporium leaf blight fungus, rice sheath blight fungus, bacterial leaf blight fungus, potato powdery scab fungus, potato Phytophthora infestans, potato black spot fungus, potato scab fungus, barley powdery mildew fungus, wheat head blight fungus, wheat snow blight fungus, wheat brown rust fungus, wheat powdery mildew fungus, wheat root rot fungus, 4. The plant disease resistance inducer according to claim 2 or 3, which is a disease caused by soybean downy mildew, soybean purpura, pea mycosis, corn smut, sweet potato fusarium wilt, melon fusarium wilt, lettuce root rot, tomato wilt, tomato verticillate, tomato anthracnose, tomato bacterial spot, spinach wilt, cruciferous clubroot, cucumber seedling damping-off, or strawberry Botrytis cinerea.

5. A method for controlling plant diseases, comprising using the plant disease resistance inducer according to any one of claims 2 to 4.

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