WO2009119915A1

WO2009119915A1 - Plant disease resistance inducer

Info

Publication number: WO2009119915A1
Application number: PCT/JP2009/056922
Authority: WO
Inventors: 能年義輝; 白須賢
Original assignee: 独立行政法人理化学研究所
Priority date: 2008-03-28
Filing date: 2009-03-27
Publication date: 2009-10-01

Abstract

The object aims to establish a comprehensive and efficient screening method for a plant disease resistance inducer, and provide a novel plant disease resistance inducer by employging the screening method. Specifically disclosed is a plant disease resistance inducer comprising a compound capable of inhibiting the salicylic acid glucosyltransferase activity of a protein selected from the following proteins (a) and (b): (a) a protein comprising the amino acid sequence depicted in SEQ ID NO:2; and (b) a protein which comprises an amino acid sequence having the deletion, substitution or addition of one or several amino acid residues in the amino acid sequence depicted in SEQ ID NO:2 and has a salicylic acid glucosyltransferase activity.

Description

Plant disease resistance inducer

Technical field

The present invention relates to a plant disease resistance inducer obtained by a comprehensive and efficient screening method for plant disease resistance inducers. Light

Background art

Rice field

When a plant is infected with a pathogen, it synthesizes salicylic acid, which is a plant hormone, to induce a defense gene group to exert disease resistance (Non-patent Document 1). BTH (benzo (l, 2, 3) thiadiazole-7-carbothioic acid S-methyl ester) (Non-Patent Document 2 and Patent Document 1) and INA ( 2, 6-dichloroi sonicot inic acid) (Non-patent Document 3) is known, and it is known that these have a salicylic acid-like action.

Other disease resistance inducers include olizemate (active ingredient name: probenazole; patent documents 2 to 6), probenazole active substance BIT (patent document 7), biget (active ingredient name thiazinyl). ) and so on. These pharmacological actions are not mentioned. Recently, plant disease resistance inducers including isothiazoles are in the upper stage (Patent Document 8). Further similar compounds include benzoisothiazolin derivatives (Patent Documents 9, 10), NCI (N-cyanomethyl-2-chloroi sonicot inaraide) (Non-Patent Document 4), CMPA (3-chrolo-l-methyl-lH-pyrazole-5-carboxyl aci'd) (patent document 11, non-patent document 5) exists.

While drugs that kill disease-causing microorganisms have a risk of emergence of resistant varieties, drugs aimed at activating disease resistance of the plant itself are effective in disease control. The range of indications is limited, and the development of new drugs is expected. However, development at this stage is limited to the modification of existing compounds, and for innovative progress, a comprehensive search for compounds with new structures is required. Search is essential. Methods other than the above-mentioned development methods include a method using a protective substance possessed by plants (Patent Document 12) and a disease resistance addition technology using a plant symbiotic microorganism (Patent Document 13). In addition, a resistance imparting technique using a drug that inhibits the synthesis of abscisic acid that acts antagonistically on salicylic acid has been reported (Patent Document 14).

(Patent Document 1) Schurter et al., (1987) EU Patent 0313-512.

(Patent Document 2) JP-B 49 ― 3 7 1 4 7

(Patent Document 3) Japanese Patent Publication No. 45 1 1 2 1 5 7

(Patent Document 4) JP-B 45 ― 3 8 0 8 0

(Patent Document 5) JP-B 45 ― 3 8 3 5 6

(Patent Document 6) JP-B 47 ― 3 8 9 6 7

(Patent Document 7) JP 05-0 0 5 9 0 2 4

(Patent Document 8) Japanese Patent Laid-Open No. 20-3-1 1 3 1 6 7

(Patent Document 9) Japanese Patent Application Laid-Open No. 2007-7-9 1 5 9 6

(Patent Document 10) JP 20 0 7 ― 1 8 6 509

(Patent Document 1 1) Japanese Patent Laid-Open No. 08-81 0 1 2 5 10

(Patent Document 1 2) Japanese Patent Laid-Open No. 20 0 6 ― 3 2 7 995

(Patent Document 1 3) Japanese Patent Laid-Open No. 20 0 2 ― 2 2 3 747

(Patent Document 14) JP 20 06 1 1 1 7 608

(Non-Patent Document 1) Metraux et al., (1990) Science, 250: 1004-1006.

(Non-Patent Document 2) Friedrich et al., (1996) Plant J. 10: 6 to 70,

(Non-Patent Document 3) Vernooij et al., (1995) Mol. Plant-Microbe Interact. 8: 228-234.

(Non-patent document 4) Nakashita et al., (2002) Plant Cell Physiol. 43: 823-831 (Non-patent document 5) Yasuda et al., (2003) Biosci. Biotech. Biochem. 67: 2614-2620. Invention Disclosure of

If you expect innovative progress in the development of plant disease resistance inducers, It is necessary to isolate new compounds not known in In the conventional method, a candidate compound is added to a plant and its disease resistance is evaluated. In this case, a large amount of compound is required, and a large test is required to evaluate a large number of candidate compounds. Space was required and efficient processing was difficult. Therefore, an exhaustive search method such as screening of a compound library is effective for obtaining new compounds that do not have the conventional concept, but in order to do so, multiple samples can be processed quickly with the use of a small amount of compounds. A high-throughput screen system that can be used is essential.

Accordingly, an object of the present invention is to establish a comprehensive and efficient screening method for plant disease resistance inducers and to provide a novel plant disease resistance inducer using the screening method. is there.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have used hypersensitive cell death, which is one of disease resistance responses induced in cultured Arabidopsis cells as a result of infection with tomato bacterial leaf bacilli. As a result, we found that plant disease resistance inducers could be screened comprehensively and rapidly, and we established screening methods for plant disease resistance inducers by optimizing the screening conditions. And we succeeded in obtaining a new plant disease resistance inducer using the screening method. The present invention has been completed based on such findings.

That is, the present invention includes the following inventions.

(1) A plant disease resistance inducer comprising a compound that inhibits the darcosyl salicylate transferase activity of the following protein (a) or (b):

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2

(b) a protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having salicylate darcosyltransferase activity

(2) The plant disease resistance inducer according to (1), wherein the compound that inhibits salicylate darcosyltransferase activity is a compound represented by the following general formula (I):

[Chemical 1]

(In the formula, X—Y represents one CH = CH— or one CO—Ο—, and! ^ ¹ to! ^ ⁹ are independently a hydrogen atom, a hydroxyl group, a halogen atom, a nitro group, An amino group, an alkyl group having 1 to 4 carbon atoms or an alkoxy group, or an alkenyl group having 2 to 4 carbon atoms or an alkynyl group is represented.)

(3) A compound that inhibits salicylate darcosyltransferase activity has the general formula

The plant disease resistance inducer according to (1), which is a compound represented by (II).

(In the formula, R ^{1 Q} and R ¹¹ are each independently substituted with a hydrogen atom, an alkylcarbonyl group having 1 to 4 carbon atoms which may be substituted with a halogen atom, or an alkoxy group having 1 to 4 carbon atoms. The benzyl group that may be present, or R ^{1 G} , R ¹ \ and the N atom to which they are bonded together represents a phthalimide group.)

(4) The compound that inhibits salicylate dalcosyltransferase activity is a compound represented by the following formula (la) ヽ (lb), (Ic) (IIa), (IIb), (lie), or (lid): The plant disease resistance inducer according to (1).

[Chemical 3]

(5) A plant disease resistance inducer comprising a compound represented by the following formula (式), (IV), or (V):

[Chemical 4]

(6) A method for controlling plant diseases, comprising using the plant disease resistance inducer according to any one of (1) to (5).

(7) A plant cell infected with a tomato spotted bacterial pathogen (Pseudomonas syringae pv. Tomato DC3000 strain) having an AvrRpml protein is cultured with the test compound, and the test compound is hypersensitive cell death in the plant cell. A method of screening a compound for inducing resistance to plant diseases, comprising: determining whether or not to enhance hypersensitivity cell death, and selecting a compound determined to enhance hypersensitive cell death. Brief Description of Drawings

Figure 1 shows the results of examination of the infection index for Pst in cultured Arabidopsis cells. Figure 2 shows the detection results of Evans blue staining of cultured Arabidopsis cells that have undergone hypersensitive cell death due to Pst infection.

Figure 3 shows the morphology of Pst-infected Arabidopsis plants.

Fig. 4 shows the hypersensitive cell death-inducing activity of cultured Arabidopsis cells for each Pst strain.

Figure 5 shows the effect of known cell death inhibitors on hypersensitive cell death of cultured Arabidopsis cells by Pst infection. Figure 6 shows the effect of known plant disease resistance inducers on hypersensitive cell death of cultured Arabidopsis cells by Pst infection.

Figure 7 shows the effect of DMS0 concentration on hypersensitive cell death in Arabidopsis cultured cells.

FIG. 8 shows the procedure of a screening method for a plant disease resistance-inducing compound. Figure 9 shows how the sample was applied to the microtiter plate used in the screening method.

FIG. 10 shows the structure of a plant disease resistance-inducing compound isolated by screening.

Figure 11 shows the concentration dependence of the hypersensitive cell death-promoting activity of the isolated plant disease resistance-inducing compound (0, 125, 100, 75, 50, 25, 10, 0 μ Μ).

Figure 12 shows the results of PR1 gene expression test in Arabidopsis cultured cells using the isolated plant disease resistance-inducing compound.

Fig. 13 shows the results of PR1 gene expression test in Arabidopsis cultured cells with the isolated plant disease resistance-inducing compound.

Figure 14 shows the results of investigating the dependency of the PR1 gene expression ability of the isolated plant disease resistance-inducing compound on the salicylic acid synthesis pathway.

FIG. 15 shows changes in salicylic acid content of cultured Arabidopsis cells by addition of an isolated plant disease resistance-inducing compound.

Figure 16 shows the type and activity of Arabidopsis thaliana salicylate darcosyltransferase.

Figure 17 shows the results of thin-layer chromatography detection of the reaction product of salicylic acid dalcosyltransferase derived from Arabidopsis thaliana.

Figure 18 shows the effect of isolated plant disease resistance-inducing compounds on salicylate dalcosyltransferase activity.

FIG. 19 shows the effect of isolated plant disease resistance-inducing compounds on salicylate dalcosyltransferase activity. Figure 20 shows the structures (A) of CB_8, CB_9 and their similar compounds, and their hypersensitive cell death promoting activity (B) (in each compound, 0, 250, 100, 75, 50 from the left side). , 25, 10, 0 μΜ).

Figure 21 shows the effect of CB-8, CB_9, CB-11, and similar compounds on salicylate dalcosyltransferase activity.

Figure 22 shows the structures (A) of CB_11, CB_12, and their similar compounds, and their hypersensitive cell death-promoting activity (B) (in each compound, 0, 250, 100, 75, 50, 25, 10, 0 / ζ Μ).

Figure 23 shows the effect of thiazinyl and probenazole on salicylate darcosyltransferase activity.

Figure 24 shows changes in the number of plants after administration of a plant disease resistance-inducing compound in Arabidopsis leaves infected with Pst. [From the left, CB_6 (200 Μ), CB_7 (200 μ Μ), CB_8 (100 μ Μ ), CB 9 (100 μΜ), CB 10 (200 μΜ), CB 11 (100 μΜ), SA (200; u M)].

Figure 25 shows the effects of plant disease resistance-inducing compounds on the growth of Arabidopsis plants. This application claims priority of Japanese Patent Application No. 2008-088491 filed on Mar. 28, 2008, and includes the contents described in the specification of the patent application. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

1. Screening method for compounds that induce plant disease resistance

The method for screening a compound for inducing resistance to plant diseases according to the present invention comprises: a plant cell infected with a stalk fungus (£ _seudomona svringae pv. Tomato DC3000 strain) having an AvrRpral protein; And test compound, determining whether or not the test compound enhances hypersensitive cell death in the plant cell, and selecting a compound determined to enhance hypersensitive cell death Here, hypersensitive cell death refers to cell death pre-programmed in the plant itself by the plant's self-protection mechanism against disease. This method [Pseudomonas syringae pv. Tomato DC3000 strain] (hereinafter referred to as “Pst”) has the AvrRpml protein in the strongest and most sensitive cell death in a short time. preferably a strain, for example, is Pseudomonas syringae pv. tomato DC3000 avrRpml strain force ^s preferred (Rere for this.

Plant cells to be infected with the above-mentioned diseases include Arabidopsis cultured cell awakening strain (Menges and Murray, (2002) Plant J. 30: 203-212.) And T87 strain (Axelos, et al., (1992) Plant Physiol Biochem. 30: 123-128.) Can be used, but the MMl strain is preferred because it is easier to prepare the medium than the T87 strain and its suspension is high and easy to handle. .

As a specific procedure, for example, plant cells and Pst were cultured in a 96-well microtiter plate in the presence or absence of a test compound, and then hypersensitive cell death of plant cells due to Pst infection was detected by Evans Blue staining. To detect. The color change due to Evans Blue staining may be observed visually, but the effect of the test compound on hypersensitive cell death can be quantified by measuring the absorbance with a microtiter plate reader. Further, dimethyl sulfoxide (DMS0) is used as a solvent for the test compound, but it is preferably used at a final concentration of 0.25 to 1.0%.

In the Evans blue staining after the above culture, when the test compound is toxic to plant cells, it may be detected as positive. Therefore, in this disk re-learning methods to eliminate this false positive, simultaneous parallel manner conducted experiments with the addition of only the test compound without addition of p _s t. By this operation, it becomes possible to exclude those that cause hypersensitive cell death only with the test compound.

When performing quantification by Evans Blue staining, a simple device combining a 96-well deep plate, a 96-well Chu Black, a gel preparation plate for electrophoresis, and the like can be used. In Evans Blue Staining, it is necessary to remove the excess dye after washing by washing, but in this device, by setting the lower limit to reach the electric pipette, without removing the settled cultured cells after staining, A certain amount of washing supernatant can be collected. This process can also be automated using laboratory robots (such as Biomek (Beckman Coulter)).

There are no particular restrictions on the test compound to be screened. For example, heaven However, single compounds such as compounds, organic compounds, inorganic compounds, proteins, peptides, and compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermented microorganism products, plant extracts, etc. However, it is not limited to these.

2. Plant disease resistance inducer and plant disease control method

The plant disease resistance inducer of the present invention contains a compound obtained by the screening method of 1 above as an active ingredient.

The compound includes a compound that inhibits darcosyltransferase activity of salicylate, and examples of the compound include compounds represented by the following general formula (I).

[Chemical 5]

(Where X—Y represents one CH = CH— or one CO—O—, and scales ¹ to! ^ ⁹ are each independently a hydrogen atom, a hydroxyl group, a halogen atom, a nitro group, an amino group, Group, an alkyl group having 1 to 4 carbon atoms or an alkoxy group, or an alkyl group having 2 to 4 carbon atoms or an alkynyl group.)

Examples of the compound that inhibits salicylic acid darcosyltransferase activity also include compounds represented by the following general formula (式).

[Chemical 6]

(In the formula, R ^{1 Q} and R ¹¹ are each independently substituted with a hydrogen atom or an alkylcarbonyl group having 1 to 4 carbon atoms which may be substituted with a halogen atom or an alkoxy group having 1 to 4 carbon atoms. The benzyl group which may be bonded, or R ^{1 Q} , scale ¹¹ and the N atom to which they are bonded together represent a phthalimide group.)

In the definition of each symbol in the above general formula, “halogen atom” is fluorine atom, chlorine atom, bromine atom, iodine atom; “alkyl group” having 1 to 4 carbon atoms is methyl, ethyl, n-propyl , Isopropinole, n-butinole, isobutinole, sec-butyl, tert-butyl; “alkoxy groups” having 1 to 4 carbon atoms include methoxy, ethoxy, propoxy, isopropoxy, butoxy, tert —Butoxy group: “Alkenyl group” having 2 to 4 carbon atoms includes bur, n-propenyl, isopropeninole, n-buteninole, isobuteninole, sec-buteninole, tert-buteninore; carbon number 2 to 4 “Alkynyl group” includes ethynyl, n-propiel (1-propynyl), isopropynyl (2-propynyl), n-butynyl, isobu Interview Le, sec - butynyl, tert- Buchuru.

In particular, among the compounds represented by the general formulas (1) and (II), the following formulas (la), (Ib), (Ic), (I la), (lib), (lie), or (lid ) Is preferred.

[Chemical 7]

Here, salicylic acid dalcosyltransferase refers to Arabidopsis At2g43840 (UGT74Fl) having the activity of transferring glucose to salicylic acid (SA) to produce salicylic acid 2-0-j3-D-darcoside (SAG). It has the amino acid sequence shown in 2.

UGT74F1 may be a mutant protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 as long as the enzyme activity is maintained. Here, the number of amino acids that may be deleted, substituted, and / or added is preferably 1 to several. The number of “several” is not particularly limited. For example, it means 20 or less, preferably 10 or less, more preferably 7 or less, and further preferably 5 ^: or less. The “mutation” here means a mutation artificially introduced mainly by a known mutant protein production method, but may be a similar naturally occurring mutation. Protein The homology search can be performed by using a program such as FASTA or BLAST for the DNA Databank of Japan (DDBJ), for example.

The disease resistance induced by the compounds represented by the above formulas (la), (lb), (Ic), (IIa), (lib), (lie), or (lid) is dalcosyl salicylate. ZE

This is based on a new mechanism of action of inhibiting the enzyme activity of (UGT74F1). Therefore, a gene encoding salicylate darcosyltransferase (UGT74F1) (hereinafter,

It is possible to confer disease resistance to plants by deleting the function of “TUGT74F1 gene”. Examples of methods of loss of function include a method of expressing an antisense sequence of the gene and an RNAi sequence in a plant, a method of controlling the gene with a disease-inducible promoter, and suppressing its expression only at the time of disease infection, etc. Is mentioned.

The UGT74F1 gene according to the present invention is a gene encoding salicylate darcosyltransferase having the amino acid sequence shown in SEQ ID NO: 2, and has the base sequence shown in SEQ ID NO: 1. The UGT74F1 gene may be a homologous gene, and as such a homologous gene, it hybridizes with a DNA consisting of a base sequence shown in SEQ ID NO: 1 and a DNA consisting of a complementary base sequence under stringent conditions. And a gene encoding the protein having the enzyme activity described above.

Here, the stringent condition means a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, a nucleic acid having high homology, i.e., a nucleic acid comprising a nucleotide sequence having a homology of 80% or more, preferably 85% or more, more preferably 90% or more, and most preferably 95% or more with the base sequence shown in SEQ ID NO: 1. In which the complementary strands of the nucleic acid are hybridized and the phase-trapping strand of the nucleic acid consisting of a base sequence having a lower homology is not hybridized. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 raM, more preferably 300 to 750 raM, the temperature is 25 to 70 ° C, preferably 50 to 70 ° C, more preferably 55. The condition is ˜65 ° C. and the formamide concentration is 0-50%, preferably 20-50%, more preferably 35-45%. Furthermore, under stringent conditions, the filter washing conditions after hybridization are usually such that the sodium salt concentration is 15-600 mM, preferably 50-600 mM, more preferably 300-600 mM, and the temperature is 50- 70 ° C, preferably 55-70 ° (:, more preferred 60 to 65 ° C.

Those skilled in the art can refer to Molecular Cloning (Sambrook, J. et al., Olecular Cloning: a Laboratory Manual 2nd ed., Cold Spring Harbor Laboratory Press, 10 Skyline Drive Plainview, NY (1989)), etc. Such homologous genes can be easily obtained. Similarly, the homology of the above base sequences can be determined by FASTA search or BLAST search.

The compound obtained by the above screening method 1 also includes a compound represented by the following formula (III), (IV), or (V).

[Chemical 8]

In the present invention, the “plant disease resistance inducer” refers to an agent for inducing resistance to plant diseases and controlling plant diseases.

As the plant disease resistance inducer of the present invention, the above-mentioned compounds may be used as they are, but solid carriers, liquid carriers, surfactants, and other formulation aids used for formulating general agricultural chemicals for the above-mentioned compounds. Preparations of various dosage forms may be prepared by mixing agents. As the types of drug forms, for example, any form such as granules, powders, liquids, emulsions, wettable powders, water solvents, oils, aerosols, flowables and the like may be used. Examples of carriers used for formulation include talc, bentonite, clay, kaolin, and diatom. Examples thereof include solid carriers such as soil, white carbon, vermiculite, calcium carbonate, slaked lime, silica sand, ammonium sulfate and urea, and liquid carriers such as isopropyl alcohol, xylene, cyclohexane and methylnaphthalene.

The content of the above-mentioned compound, which is an active ingredient in the plant disease resistance inducer of the present invention, can be appropriately set as necessary. However, in the case of powders or granules, 0.1 to 50% (weight), or emulsion In the case of a wettable powder, 5 to 80% (weight) can be exemplified.

Since the plant disease resistance inducer of the present invention is intended to prevent diseases, it is preferably applied before the time when the disease occurs. The method for using the plant disease resistance inducer of the present invention may be any of spraying, dusting, dipping, powdering, application, fumigation, smoking, irrigation, and the like. Specific uses include: spraying and applying chemicals to plants, dipping plant seeds in liquids containing chemicals, and chemicals in fields where disease is or is likely to occur The method of spraying, the method of mixing chemicals into soil, etc. are mentioned.

The amount used of the plant disease resistance inducer of the present invention may be appropriately set according to the kind of plant, the target plant, the growth stage of the target plant, the type of dosage form, the application method, the application time, etc. However, for example, per 1 000 Om ² is usually 1 to 5000 g, preferably 5 to 1 000 g, as an active ingredient. When used in the form of a liquid, such as an emulsion or wettable powder, the concentration of the active ingredient is from 0. 1 pm pm to 10.000 p pm, preferably from 10 to 3, OOO p pm.

Examples of plants targeted by the plant disease resistance inducer of the present invention include all cultivated plants, and may be monocotyledonous plants or dicotyledonous plants. For example, Brassicaceae

(Arabidopsis, cabbage, rape, etc.), Gramineae (rice, corn, barley, wheat, etc.), Solanaceae (tomato, eggplant, potato, tobacco, etc.), Cucurbitaceae

(Such as cucumbers, melons, cabotillas), legumes (soybean, gendo, bean beans, alfa alfa, laccasei, etc.), cruciferous plants (radish, Chinese cabbage, cabbage, etc.), Rosaceae (strawberry, apple, pear, etc.) , Cucumber family (such as cucumber), cereal family (such as moth), celery family (carrot, parsley, celery etc.), asteraceae (burdock, sunflower, chrysanthemum, lettuce, etc.) Plants are included, but are not limited to these plants. The plant disease resistance inducer of the present invention can control plant diseases caused by filamentous fungi, bacteria and viruses by the above-mentioned application form. For example, rice blast fungus

(Magnaporthe gri sea, Rice ¾ 丙 (Burkholderia plantari i) (Spongospora subterranea), Phytophthorainf estans, Rhizoctonia solani, Streptomycesscabies, Eryshiphe gramini s f. Sp. Hordei ¾1 Red power and ί 丙丙-(Gibberel la zeae), wheat, Sclerotinia boreal is, wheat red rust; / Puccinia recondita, com = Tsudonko; s), Peridospora manshurica, Guiz purpura; / n- (Cercospora kikuchn), Mycosphaerel lapinodes, Ust i lago maydi s, Satsuma vine damage ¹ J fungus (Fusarium oxysporum f .sp. batatas), melon vine harm ij fungus (Fusarium oxysporum f. sp. meloni s), lettuce root rot fungus

(Fusarium oxysporum f. Sp. Lactucae), Fusarium oxysporum f. Sp. Lycopers ici, Vertici ll ium dahl iae, Col letotrichumphomoides), Fusarium oxysporum f. Examples include diseases caused by (Botrytis cinerea), but are not limited thereto.

The plant disease resistance inducer of the present invention can be used by mixing with other herbicides, fungicides, insecticides and other agricultural chemicals, fertilizers, plant growth regulators, soil conditioners and the like. EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but these examples do not limit the present invention.

(Example 1) Examination of screening conditions ''

(1) Examination of infection index for Pst in Arabidopsis cultured cells

卜マ卜] The mechanism of the infection of Pseudomonas syringae pv. Tomato DC3000 (Pst) to Shironunazuna is best understood as a model experimental system. (Belkhadir et al., (2004) Curr. Op in. Plant Biol. 7: 391-399.). In Arabidopsis cultured cells, Pst infection induces a characteristic phenomenon in the disease resistance response as in the case of plants. Phenomena that plant cells induce during pathogen infection include activation of MAP kinase, production of reactive oxygen species, bioluminescence, increase in salicylic acid content, accumulation of force rosin, accumulation of autofluorescent substances, and PR1 gene. Induction of the expression of various defense genes, hypersensitive cell death, and the like. Among these phenomena, we examined which one is suitable as an index for quantifying the disease resistance response of plants. As a result, hypersensitive cell death is a phenomenon that is positioned at the final stage of the resistance response in infected cells, and the measurement using a microtiter plate reader is used by an inexpensive and easy method called Evans blue staining. From the viewpoint of being able to quantify, it was judged that it is optimal to use hypersensitive cell death as an index (Fig. 1).

(2) Examination of types of tomato spotted bacterial disease strains

Seed Arabidopsis cultured cells MM1 strain (Menges and Murray, (2002) Plant J. 30: 203-212.) And T87 strain (Axelos, et al., (1992) Plant Physiol. Biochem. 30: 123-128.) Tomato DC3000, Pseudomonas syringae pv. Tomato DC3000 avrRpml) was co-cultured and infected, and stained with eno blue. Cell death was observed specifically in bacterial strains carrying the AvrRpml effector protein (Figure 2). Moreover, when Arabidopsis thaliana was infected with tomato spotted bacterial disease ¾ (Pseudomonas syringae DV. Tomato D and 3000, Pseudomonas svringae pv. Tomato DC3000 avrRpml), cell death was specifically induced in the bacterial strain carrying the AvrRpml effector. Observed as atrophy (Figure 3). On the other hand, bacterial strains without AvrRpml did not cause cell death and continued bacterial growth in the leaves.

A comparison of four types of Pst strains, avrRpml, avrRpt2, avrRps4, and avrPphB, confirms that the avrRpml strain induces the strongest hypersensitive cell death in a short time (about 20 hours). (Fig. 4). The intensity of this response was similar to that of plants.

(3) Verification of the effectiveness of using hypersensitive cell death as an indicator of Pst infection

We investigated whether hypersensitive cell death caused by the interaction between MM1 and Pst in Arabidopsis cultured cells was actually inhibited by known cell death inhibitors. Known cell death inhibitors As a result of experiments using phosphatase inhibitors staurosporine and K252a, phosphatase inhibitor okadaic acid, NADPH oxidase inhibitor diphenol-lendonium (DPI), MM1 force S Pst infection On the other hand, it was confirmed that hypersensitive cell death was inhibited by any compound in a concentration-dependent manner (Fig. 5). In addition, thiazinyl (TDN), a known plant resistance inducer, and salicylic acid (SA), an endogenous resistance-inducing hormone, are used to induce hypersensitive cell death induced by MM1 force S Pst infection. When the effect was examined, it was confirmed that both compounds enhance hypersensitive cell death (Fig. 6). (4) Examination of solvent concentration

The effect of DMS0, a compound solvent, on cultured MM1 cells was examined, and up to 1% was confirmed to have no significant effect on hypersensitive cell death (Fig. 7).

(Example 2) Search and identification of candidate compounds for plant disease resistance inducers

(1) Screening method

Screening of plant disease resistance inducers was carried out according to the following procedure (Fig. 8). Arabidopsis cultured cells were swirled in VH medium (light conditions were 16 hours light / 8 hours dark; Maor et al., (2007) Molecular & Cellular Proteoraics 6: 60 to 610.). One week later, the cultured cells were dispensed into 1ml-volume 96-well deep-titer plates (nunc260252) in 58.5 μl units using an eight-pipetette (cut the tip of the tip). To the same titer plate, 2 wells of each test compound (0.5 · 1 μl) were added, and the plate was struck by the side and stirred. Two lanes of the plate were used as control lines and only DMS0 was added. After 1 hour, Pseudomonas syringae pv.

The final concentration of “Pst” is 0D ₆ . . 0.24 VH medium adjusted to a final MES concentration of 14 mM was added to 40.5 1 to a final volume of 100 1. 1 hole does not contain Pst

VH medium was added. Tap on the side of the plate, stir, and co-culture for 21 hours in a swirl incubator, then add 5 μ 1 of 1% Evans Blue aqueous solution and stir, occasionally stirring

It was left to stand for 30 minutes (Fig. 9).

Next, lml of water was added with 8 electric pipettes and allowed to stand for 10 minutes to precipitate cultured cells, and lml of the supernatant was sucked up. This washing operation was repeated three times. Eluent (50% methanol,

(0. 1% SDS) 400 μ 1 was taken, floated in a 55 ° C water bath for 20 minutes, and taken into dead cells Blues dye was eluted. The absorption was measured 0D ₅₉₅ with supernatant of each sample into a 96-well microtiter tarp rate The eluate 160 mu 1 40 // added 1 (5-fold dilution) microplate reader.

The average Evans blue concentration of the control is 100%, and the value is 120% or more by the addition of the test compound, and the value when Pst is not added does not change from the control. Those that did not induce sensitive cell death) were selected as positive.

(2) Search and identification of compounds that enhance hypersensitive cell death

The compound library used was 10,000 compounds (0.1 mg / 20 / z 1DMS0 dissolved) of DIVERSet (NovaCore NQ612) commercially available from ChemBridge. Screening was repeated three times by the method (1) above. As a result, seven compounds were selected as candidate compounds that reproducibly enhance hypersensitive cell death [5160360 (CB_6), 6297908 (CB_7), 6380249 (CB—8), 7724984 (CB_9), 7725027 (CB — 10), 7732575 (CB—11), 7726438 (CB—12)] were isolated (FIG. 10).

When these candidate compounds were examined for their effects on Pst-induced hypersensitive cell death at concentrations of 125, 100, 75, 50, 25, 10 // M, respectively, concentration dependence was observed (Fig. 11). CB_11 and CB_12 are expected to have the same mechanism of action due to their structural similarity, and since CB_11 is oily, only CB_11 was used in the subsequent experiments.

(Example 3) Examination of the action of a hypersensitivity cell-enhancing compound as a disease resistance inducer Searched in Example 2 · To investigate the action of the identified candidate compound (hypersensitive cell death-enhancing compound), each compound (Final concentration 50 μΜ) was added to Arabidopsis thaliana MM1 cultured cells.

After 24 hours, RNA was extracted (PureLink RNA purification kit; Invitrogen). each

Synthesis of cDNA based on RNA (Superscriptlll first strand synthesis kit;

Invitrogen), various markers (PR1, Actin, PDF1.2) RT-PCR experiments were performed using gene-specific primers. As a result, it was confirmed that these compounds induce the expression of PR1, a protective gene, in cultured cells of S. thaliana (Fig.

1 2). On the other hand, there was no change in the expression of PDF1.2, an injury-inducing gene. Furthermore, for the purpose of confirming whether each compound acts on the plant body, Arabidopsis seedlings were soaked in each compound of ΙΟΟμΜ, and RNA after 1 and 3 days was extracted (PureLink RNA purification kit; Invitrogen) _Q ¾ -CDNA was synthesized from RNA ¾r (Superscriptlll first strand synthesis kit; Invitrogen), and RT-PCR experiments were performed using PRl and Actin gene-specific primers. As a result, all the compounds induced the expression of the PR1 gene, which is a marker gene of resistance reaction, in Arabidopsis plants (FIG. 13). These results show that each compound does not act on pathogens but induces cell death activation by inducing the disease resistance response of Shiro nunazuna, and each compound exhibits plant disease resistance inducing activity. It means to have.

(Example 4) Examination of the action point of a hypersensitive cell enhancing compound

(1) Salicylic acid dependence

Salicylic acid, a plant hormone, plays a major role in plant disease resistance responses. Therefore, each of the Arabidopsis mutants sid2 lacking salicylic acid (mutants of the enzyme that synthesizes isochorismate, a precursor of salicylic acid from chorismate; Wildermuth et al., Nature (2001) 414: 562-565.) The ability of compounds to induce PRl genes was examined. Each compound was treated with Arabidopsis seedlings (wild type and sid2 mutant) at a concentration of ΙΟΟμΜ, and RNA was extracted after 1 and 3 days (PureLink RNA purification kit; Invitrogen). CDNA was synthesized based on each RNA (Superscriptlll first strand synthesis kit; Invitrogen), and RT-PCR experiments were performed using PRl and Actin gene-specific primers. As a result, four types of compounds, CB_8, CB_9, CB_10, and CB_11, did not induce PRl gene expression in the sid2 mutant. From these results, it was found that the two effects of CB_6 and CB_7 are not dependent on salicylic acid, and the remaining five are dependent on salicylic acid (Fig. 14).

(2) Salicylic acid amount increasing action

It was examined whether the salicylic acid content was changed by the addition of each compound. Each compound was added to Arabidopsis thaliana cultured cell MM1 at a concentration of lOO w M, and the salicylic acid content after 24 hours was quantified by LC-MS. As a result, it was found that CB_8, CB_10, and CB-11 significantly increased salicylic acid content (Fig. 15). (3) Discovery of the mechanism of action of compounds that increase the amount of salicylic acid

The hypothesis that each compound inhibits salicylic acid-inactivating enzyme as a mechanism for increasing the amount of salicylic acid by the addition of each compound was conducted and a verification experiment was conducted. The inactivation mechanism of salicylic acid is dalcosylation, and three types of darcosyltransferases that can use salicylic acid as a substrate have been reported: At2g43840 (UGT74Fl), At2 g43820 (UGT74F2), Atlg05560 (UGT75B1) ( Lim et al., JBC (2002) 277: 586-592.). There are two types of inactive glycosylated salicylic acid, salicylic acid 2-0- J3-D-darcoside (SAG) and darcosylsalicylic acid (GS), depending on the sugar transfer site. UGT74F1 is SAG, UGT75B1 is GS and UGT74F2 have activity to produce both SAG and GS (Lira et al., Ibid: Fig. 16).

We cloned these three types of darcosyltransferase genes and performed an enzyme activity detection experiment using proteins expressed by Escherichia coli. First, cDNA of 3 genes, At2g43840 (UGT74F1), At2g43820 (UGT74F2) and Atlg05560 (UGT75B1) was cloned, incorporated into pET28 vector, and a protein with histidine tag was expressed and purified in E. coli. Next, purified protein (1.6, 3.2, 8.0, 16.0 / xg), 50 mM Tris-HCl (pH 7.0), 14raM 2-mercaptoethanol, 5raM UDP-Darcose, ΙΟΟμΜ salicylic acid (7- "C-labeled ： American Radiolabeled Chemicals Inc.) was added to the reaction solution of 200 1 for 30 minutes at 30 ° C. 25 1 of 5raM salicylic acid (ethanol solution) was added to the reaction solution to stop the reaction. After centrifugation for 5 minutes, the supernatant 10/1 was developed by thin layer chromatography (Whatman LK6F (4866-820), developing solvent: 1-butanol / acetic acid / water = 4: 1: 1). After the layer chromatography plate was air-dried, “C-labeled salicylic acid was detected with an imaging plate and an imaging analyzer (FLA-70000). The detection method of glycosylated salicylic acid by thin-layer chromatography was according to the literature (Lee and Raskin, Phytopathology (1998) 692-697).

As a result, the SAG production activity of UGT74F1 was detected most strongly (Fig. 17).

(Lira et al., JBC (2002) 277: 586-592.). In the plant

Since the amount of GS is small and the amount of SAG increases rapidly with SA when the pathogen is infected (Lee and Raskin, ibid.), The main inactive form of SA is SAG. Thought. In addition, UGT74F2 has been reported to use anthralic acid as a substrate (Quiel and Bender, JBC (2003) 278: 6275-6281.), And the main enzyme responsible for inactivation of salicylic acid is UGT74F1. I concluded.

Next, using UGT74F1, which shows the strongest activity, the effect on enzyme activity when each compound was added to the above reaction solution at a high concentration was examined. As a result, the salicylate darcosyltransferase activity of UGT74F1 was inhibited at least at 500 / M by CB_8, CB_9, and CB_11 (FIG. 18). On the other hand, since it is not inhibited by acetyl salicylic acid (SANa), it has been shown that the substrate specificity can be evaluated. UGT74F1 activity of darcosyl salicylate transferase was also inhibited by CB_8, CB_9, and CB_11 even at 50 μΜ (Figure 19). In addition, since the effective concentration that inhibits the enzyme activity coincided with the effective concentration that promotes hypersensitive cell death of plant cultured cells (Figure 19), the pharmacological actions of CB_8, CB_9, and CB_11 are due to the inhibition of UGT74F1. It was concluded that the accumulation of active SA occurred early because SA produced by pathogen infection could not be inactivated.

On the other hand, CB_6 and CB_10 did not inhibit UGT74F1 activity (Figure 18). For CB_6 and CB-7, the PR1 gene expression induction experiment using the sid2 mutant ((1) above) revealed that the action does not depend on salicylic acid, so the compound itself has salicylic acid-like activity. Therefore, it can be judged that it has the ability to induce the expression of the defense gene group (bion type). In addition, CB_10 is thought to have an effect of activating salicylic acid synthesis (probenazole, thiazinyl type), as clearly shown in Fig. 15.

(Example 5) Structure-activity relationship of CB_8, CB-9, CB_11, CB-12

(1) CB_8 and CB_9

Since CB_8 and CB_9 were similar in structure, we searched for compounds with the common structure from the CheraCupid database (Namiki Shoji). NS- 00227 obtained from search results

354 (Pharmeks) was examined for its effects on hypersensitive cell death. As a result, NS-

00227354, like CB_8 and CB_9, was found to enhance hypersensitive cell death (FIG. 20). It was also confirmed that this compound, like CB-8 and CB_9, inhibited salicylate darcosyltransferase activity (Fig. 21). (2) CB_11 and CB_12

Since structural similarities were also found for CB_11 and CB_12, compounds with the common structure were searched from the ChemCupid database (Namiki Corporation). The compounds NS-02286103 (Pharmeks) and NS-020023475 (Pharmeks) obtained from the search results were examined for their effects on hypersensitive cell death. As a result, it was confirmed that these compounds also have hypersensitive cell death promoting activity (FIG. 22). In addition, at least NS-0002347 was confirmed to inhibit salicylic acid darcosyltransferase activity as in CB_11 (FIG. 21).

(Example 6) Effects of known plant disease resistance inducers on darcosyl salicylate transferase activity

The effects of commercially available probenazole and thiazyl on salicylate dalcosyltransferase activity were examined. As a result, thiazinyl, its active substance SV-03 and probenazole, and its active substance BIT (benzisothiazole) do not inhibit salicylic acid darcosyltransferase activity at effective concentrations for each hypersensitivity cell death enhancement. It became clear (Fig. 23). This indicates that the pharmacological action of CB_8, CB_9, and CB_11 is a novel mechanism for inducing resistance.

(Example 7) Demonstration of plant disease resistance inducing ability of CB_6, CB_7, CB_8, CB—9, CB_10, CB—11

Although it has been shown that the isolated hypersensitivity cell death promoting compound can induce the expression of PR1, which is one of the disease resistance marker genes, in cultured cells and plants. The following method was used to determine whether disease resistance could be induced.

Arabidopsis seedlings were hydroponically cultivated with rock wool (short-day conditions (8 hours l ight / 16 hours dark)). Transfer plants grown for 3 weeks to pots, 100 μM or

Immerse in 200 μΜ of each compound suspension. In the control experiment, the plant was immersed in a solution containing only DMS0, which is a solvent for the compound. 0D ₆ after 4 days. . = 0.0002 concentration of Pst suspension (10 mM MgCl ₂ ) was injected into the apoplast from the back of the leaf using a 1 ml syringe without a needle. Immediately afterwards, the leaves were cut out with a cork borer with a diameter of 6 mm, and the 3 pastures were transferred to a 2 ml tube, 4 grains of zirconia balls (φ 3ιηπι) and 500 MgCl ₂ (lOmM) solution were added, and the crusher (QIAGEN TissueLyser) Crush the leaves for 3 minutes. Prepare 10-fold dilution series with MgCl ₂ , spot each 10 1 on LB agar medium (50ug / ml kanamycin, 50ug / ml rifampicin), grow at 28 ° C, count the number of colonies, “Number of colonies / cm ² ” was calculated. The process of counting the colonies on the LB agar medium from the crushing of the leaves was similarly performed 4 days later.

As a result of comparing the Pst growth rate in plant leaves, it was found that the growth of Pst was suppressed in Arabidopsis to which CB_6, CB_7, CB_8, CB_9, CB_10, and CB_11 were added (Fig. 24).

(Example 8) Effect of CB_6, CB-7, CB_8, CB_9, CB_10, CB-11 on plant growth The effect of the isolated plant disease resistance-inducing compound on plant growth was examined. As a method, sterilized Arabidopsis seeds were dispensed into 96-well plates, MS liquid medium in which 100 // M compound was dissolved was added, and cultured under long sunlight conditions to observe its growth. As a result, CB_6, CB_7, and CB_10 inhibited the germination and greening of Arabidopsis, and CB_8, CB_9, and CB-11 did not inhibit the growth (Fig. 25). Growth suppression occurs as a result of the induction of salicylic acid-inducible gene group expression by the accumulation of salicylic acid or activation of the signal transduction pathway, but the above results indicate that the amount of salicylic acid that is amplified by the addition of CB_8, CB_9, and CB_11 inhibits growth. It became clear that it was not so much. The phenomenon that CB—8, CB_9, and CB_11 do not inhibit growth is consistent with its pharmacological action. Since salicylic acid is synthesized in large quantities only after disease stimuli are added, CB_8, CB_9, and CB_11 are resistant by increasing the rate of accumulation of active salicylic acid by inhibiting the inactivation of salicylic acid that increases during infection. It is thought that it works to accelerate the induction of the reaction. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety. Industrial applicability

The present invention provides a high-throughput screening method for plant disease resistance inducers. By using this screening method, it is possible to process a large number of samples in a clear and rapid manner. In addition, by using this screening method, a practical and highly safe plant disease resistance inducer that does not inhibit plant growth and exhibits its effect specifically at the time of infection was obtained. In addition, some of the compounds with phytopathogenicity-inducing activity obtained by this screening method were newly discovered to inhibit salicylate darcosyltransferase activity, and the rate of accumulation of salicylic acid specifically during infection The mechanism for imparting resistance was clarified by speeding up the process. These findings suggest the possibility that disease resistance can be imparted to plants by artificially controlling this salicylic acid dalcosyltransferase gene.

Claims

Range of requests

1. A plant disease resistance inducer comprising a compound that inhibits darcosyl salicylate transferase activity of the following protein (a) or (b):

(a) a protein comprising the amino acid sequence shown in SEQ ID NO: 2

(b) a protein consisting of an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2 and having salicylate dalcosyltransferase activity

2. The plant disease resistance inducer according to claim 1, wherein the compound that inhibits salicylate darcosyltransferase activity is a compound represented by the following general formula (I):

[Chemical 1]

(Wherein X—Y represents one CH═CH— or one CO—O—, and R i R ⁹ each independently represents a hydrogen atom, a hydroxyl group, a halogen atom, a nitro group, an amino group, Represents an alkyl or alkoxy group having 1 to 4 carbon atoms, or an alkenyl or alkynyl group having 2 to 4 carbon atoms.)

3. The plant disease resistance inducer according to claim 1, wherein the compound that inhibits salicylic acid darcosyltransferase activity is a compound represented by the following general formula (Π).

[Chemical 2]

(In the formula, R ^{1 Q} and R ¹¹ are each independently substituted with a hydrogen atom, an alkylcarbonyl group having 1 to 4 carbon atoms which may be substituted with a halogen atom, or an alkoxy group having 1 to 4 carbon atoms. The benzyl group which may be, or R ^{1 ()} , R ¹ \ and the N atom to which they are bonded together represent a phthalimide group.)

4. The compound that inhibits salicylate darcosyltransferase activity is a compound represented by the following formula (Ia), (lb), (Ic), (IIa), (lib), (lie), or (lid) The plant disease resistance inducer according to claim 1.

[Chemical 3]

5. A plant disease resistance inducer comprising a compound represented by the following formula (III), (IV), or (V):

[Chemical 4]

6. A method for controlling plant diseases, comprising using the plant disease resistance inducer according to any one of claims 1 to 5.

7. A plant cell infected with a tomato spotted bacterial bacterium (Pseudomonas syringae v. Tomato DC3000 strain) having AvrRpral protein is cultured with the test compound, and the test compound causes hypersensitive cell death in the plant cell. A method for screening a compound that induces plant disease resistance, comprising determining whether or not to enhance and selecting a compound that has been determined to enhance 逼 -sensitive cell death.