KR20200045712A - Composition for preventing plant diseases comprising decitabine - Google Patents

Abstract

The present invention relates to a composition for controlling plant diseases, comprising decitabine represented by chemical formula 1, wherein the decitabine can control plant diseases by inhibiting the activity of UDP-glucose pyrophosphorylase (UGPase) in a strain of genus Xanthomonas, thereby being usefully used in agriculture.

Description

Composition for preventing plant diseases comprising decitabine}

The present invention relates to a composition for controlling plant diseases comprising decitabine.

Bacterial blight and bacterial leaf streak of rice is the first pathogen of rice, the first crop in Korea, and causes damage of up to 100 billion won in Korea alone. The fungicides applied to rice white leaf blight ( Xanthomonas oryzae pv . There are 7 items of 3 types of mid-liquid repellent (solar), but they are synthetic compounds based on a non-specific mechanism, and the control effect is insufficient.

Xanthomonas oryzae pv. Oryzae is a gram-negative aerobic bacterium with monopolar hairs and rod-shaped. A yellow colony is formed on the solid medium, the growth rate is rather slow, and the optimum temperature for growth is 26-30 ℃. It is known that pathogenic bacteria that remain in dead plants that have dried up on the diseased mass or can die for more than 8 months, but die in a short period of time in a high temperature and humid environment.

UTP: α-D-glucose-1 phosphate uridylyltransferase (EC2.7.7.9) is commonly called UDP-glucose pyrophosphorylase, and is also used briefly as UGPase. UGPase is known to be an enzyme involved in the synthesis of UDP-glucose (uridine diphosphate glucose) from D-glucose 1-phosphate and UTP (uridine diphosphate glucose) in Mg ^{2+ -dependent} reversible reactions and release pyrophosphate (Biochimica et biophysica acta 2017, 1865 (11 Pt A): 1348-1357). The main product of UGPase, UDP-glucose, is very essential in various processes of carbohydrate metabolism. In particular, in prokaryotes, bacteria such as capsules and lipopolysaccharide (LPS), which are the virulence factors of many microorganisms, It plays an essential role in the synthesis of the main components of the envelope. Although these UGPases play a variety of roles in many organisms, bacterial UGPase and eukaryotic UGPase have no genetic homology and can be considered targets for antibiotics (Proceedings of the National Academy of Sciences) of the United States of America 2002, 99 (3): 1420-1425).

Korean Registered Patent No. 10-1620013

An object of the present invention is to provide a composition for controlling plant diseases comprising decitabine represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

.

.

Another object of the present invention is to provide a method for controlling plant diseases comprising the step of treating the composition with a plant.

Another object of the present invention is to contact a candidate substance with UGPase (UDP-glucose pyrophosphorylase); And selecting a candidate substance for inhibiting the activity of the UGPase.

In order to achieve the above object, the present invention provides a composition for controlling plant diseases comprising decitabine represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

.

.

In one embodiment of the present invention, the plant disease may be rice white leaf blight.

In one embodiment of the present invention, the rice white leaf blight may be a plant disease caused by a strain of the genus Xanthomonas, and the strain of the genus Xanthomonas is ant. It may be a duck ( Xanthomonas oryzae pv. Oryzae ).

In one embodiment of the present invention, the decitabine (decitabine) may be to inhibit the activity of UGPase (UDP-glucose pyrophosphorylase) protein.

In addition, the present invention provides a plant disease control method comprising the step of treating the composition to the plant.

In one embodiment of the present invention, the treatment may be to treat rice, rice seeds or rice cultivated soil.

In addition, the present invention comprises the steps of contacting a candidate substance with UGPase (UDP-glucose pyrophosphorylase); And selecting a candidate substance for which the activity of the UGPase is inhibited.

Decitabine according to the present invention can be usefully used in the agricultural field by inhibiting the activity of UGPase (UDP-glucose pyrophosphorylase) protein to control plant diseases caused by the strain of genus Xanthomonas.

1 is Xanthomonas orijae pv. This is a result of measuring the MIC value of decitabine for Orijae ( Xanthomonas oryzae pv. Oryzae, Xoo ).

The present invention provides a composition for controlling plant diseases comprising decitabine represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Formula 1]

The composition for control of the present invention may further include an agriculturally acceptable carrier.

The carrier may be of inorganic or organic and synthetic or natural origin, and the active composition may be mixed or formulated with the carrier to facilitate plant, seed, soil or other processing targets, or storage, transfer and / or handling thereof. It means the substance that makes it. In general, any material that can be conventionally used as a carrier in a pesticide, herbicide or antimicrobial composition is suitable for use in the present invention. The control composition of the present invention may be used alone or in the solid and / or liquid dispersible carrier vehicle and / or other known compatible active agents, in particular other insecticides, insecticides, acaricides, nematodes. , In the form of a mixture with plant protection agents such as fungicides, bactericides, rodent removers, herbicides, fertilizers, growth regulators, etc., or if desired, for specific applications prepared therefrom, in specific dosage forms, eg solutions, It can also be used in ready-to-use forms such as emulsions, suspensions, powders, pastes and granules. The control composition of the present invention is, optionally, a conventional pesticidal agent or a conventional inert (plant compatible or herbicidal inert) type of pesticide diluent or swelling agent, e.g., conventional gas, solution Used in natural and synthetic substances, microcapsules, seeds mixed with active compounds, emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, soluble powders, dust collectors, granules, foaming agents, pastes, tablets, aerosols For coating compositions and flame retardant cartridges, foamable cans and foamable coils, as well as formulations used with burning equipment such as ULV cold and mist formulations. It is preferable to adjust the carrier according to the formulation of the composition, the place of application and the method of application.

The present invention provides a plant disease control product (eg, pesticide) comprising the above composition.

The control product may be formulated in an appropriate form according to the purpose and use of the use, for example, granules, powders, liquids, aerosols, sprays, ex-agents, pastes, fluid extracts, emulsions, suspensions, capsules Agents, liquid hydrating agents, granulating hydrating agents, hydrating agents, powders, particulates, oils, gels, smokers, fumigants, and the like, and formulations that can adequately control the volatility of the pesticide composition are the most preferred emulsions, smokers, fumigants or aerosols Do.

The present invention provides a method for controlling plant diseases comprising the step of applying a composition for controlling pharmaceutically effective amounts to a plant.

The composition for controlling plant diseases of the present invention may be used as a formulation such as aerosol, pump spray, liquid, suspension, spray, capsule, paint or gel, but is not limited thereto. As the spraying agent, LPG, n-butane, isobutane, propane, carbon dioxide, freon, HCFC, HFC, nitrogen and oxygen may be used alone or in combination. As the propellant for the aerosol formulation, one or more components selected from liquefied natural gas, dimethyl ester, compressed air, and compressed nitrogen may be used, wherein the composition to propellant ratio is 65:35 to 40:60 in volume ratio. It is preferred, but is not limited thereto.

The composition for controlling plant diseases of the present invention may use a solvent having good volatility in a plant extract, and ethanol or methanol is preferred as the solvent having good volatility, but is not limited thereto.

The composition for controlling plant diseases of the present invention can be used as a feeding poison or a contact poison, and is preferably used as a contact poison to enhance the insecticidal effect.

Pests have a natural defense system that prevents them from penetrating into the body by allowing the water droplets to flow down by using surface tension when the liquid component comes into contact with the surface, to prevent such a phenomenon, for controlling plant diseases of the present invention It is preferred to use a mixture of penetrants in the composition.

The composition for controlling plant diseases may be diluted in an appropriate concentration when used.

As the diluting solvent, one or more selected from the group consisting of water and surfactant may be used, for example, water may be used.

The method for controlling plant diseases may be applicable as a method of directly spraying or applying a composition for controlling plant diseases to crops and / or soil in a facility.

Hereinafter, the present invention will be described in more detail through examples. These examples are intended to illustrate the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. Gene cloning

The gene encoding the multi-drug resistant strain Acinetobacter baumannii UDP-glucose pyrophosphorylase (UGPase, hereinafter referred to as "AbUGPase") protein is a PCR (polymerase chain reaction) Was cloned by. Primer sequences were designed based on genomic sequences of other Acinetobacter Baumani strains retrieved from the NCBI website. Forward and reverse primers contain the Nde I and Bam HI restriction sites (in bold) and the sequence is as follows: forward: 5'-ccc ccc cat atg att aaa aaa gca gtt tta cca -3 '; reverse: 5'-ccc ccc gat cct tta taa ttt aag tcc cag aat -3 '. PCR products were double treated with Nde I and Bam HI restriction enzymes and inserted into a modified pET11a vector (His-TEV-pET11a). The vector was constructed to include 7xHis tag and tobacco etch virus (TEV) protease cleavage site in front of the Nde I site in the pET11a vector (Novagen).

1.2. Protein expression and purification

The recombinant vector, pET11a-AbUGPase, into which AbUGPase was inserted, was transformed by inserting it into E. coli strain BL21 (DE3) pLysS. Cells were cultured in LB (Luria-Bertani) medium containing 50 μg ml- ¹ ampicillin. The overexpression of AbUGPase was carried out at 310 K until 0.5 mM of IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to the culture medium until the OD ₆₀₀ value reached 0.6. After induction of overexpression, cells were cultured for an additional 8 hours. The cultured cells were obtained after centrifugation at 277 K for 6,000 xg (Supra 30K A1000S-4 rotors, Hanil, Seoul, Republic of Korea) for 20 minutes. The cell pellet was resuspended in cold lysis buffer [25 mM Tris-HCl pH 7.5, 300 mM NaCl, 35 mM Imidazole, 3 mM β-mercaptoethanol, 10% Glycerol], and ultrasound on ice (Sonomasher, S & T Science, Korea) ) And homogenized. The cell lysate was then centrifuged at 277 K at 21,000 xg (Vision VS24-SMTi V508A rotor) for 40 minutes. The supernatant containing soluble AbUGPase was loaded with Ni ²⁺ charged resin (Ni-NTA His · Bind® Resin, Biorad) pre-equilibrated with lysis buffer. Affinity purification was performed at 277 K according to the manufacturer's protocol. The lysis buffer was used to wash off non-specifically bound proteins. Histidine (His) tagged AbUGPase was eluted with elution buffer [25 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM Imidazole, 3 mM β-mercaptoethanol, 10% Glycerol]. The protein solution was dialyzed at 277 K with buffer A [25 mM Tris-HCl pH 7.5, 3 mM β-mercaptoethanol, 10% Glycerol], and the buffer concentration was 25 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM β It was carried out until -mercaptoethanol and 10% glycerol. Then, the protein was concentrated to 7 mg ml ^-1 in buffer [25 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM β-mercaptoethanol, 10% Glycerol] using Vivaspin 20 (10,000 MWCO, Vivascience).

1.3. Crystallization and structure determination

Crystallization was performed using Hydra II e-drop automated pipetting system (Matrix) using screening kits from Crystal screen Lite, Crystal screen Cryo, PEGRx (Hampton Research), Wizard precipitant Synergy (Emerald BioSystems) and MorpheusTM MD (Molecular dimensions Limited). Screening was initiated at 287 K by sitting drop vapor diffusion method in a 96-well intelli-plate (Art Robbins). However, the present inventors did not obtain crystals from AbUGPase in buffer [25 mM Tris-HCl pH 7.5, 10 mM NaCl, 3 mM β-mercaptoethanol, 10% Glycerol]. Thereafter, several different crystallization conditions were obtained by adding 15 mM MgCl ₂ , and after optimization with the conditions of 1.0 M Ammonium citrate tribasic pH 7.0, 0.1 M BIS-TRIS propane pH 7.0, hanging drop vapor diffusion method, 1.0 M Ammonium citrate The conditions of tribasic pH 8.0, 0.1 M BIS-TRIS propane pH 9.0 were found to be the best conditions for crystallization while maintaining a protein concentration of 7 mg ml ^-1 . Crystals were made under these conditions after 2 days. The complete crystals were frozen in liquid nitrogen after adding a cryoprotectant consisting of a reservoir solution and 30% (v / v) glycerol. X-ray diffraction data was collected from frozen crystals using a beamline 5C ADSC Quantum 315r CCD detector (Pohang Accelerator Laboratory, Pohang University of Science and Technology, South Korea).

1.4. MIC (minimum inhibitory concentration) measurement

MIC values were determined by the broth microdilution assay method. The method is a nucleotide analog inhibitor (nucleotide analog inhibitor) of decitabine (decitabine) xanthomonas ori pv. It was used to test sensitivity to Gram-negative bacteria such as Orijae ( Xanthomonas oryzae pv. Oryzae, Xoo ). Xoo was inoculated with 2 x 10 ^ 5 cfu in 100 μl medium, and then cultured in Nutrient broth medium. Inhibitors were diluted from 512 mg ml ^-1 to 2 mg ml ^-1 in 96-well plates. After incubation at 310 K for 18 hours using a thermo shaker (MB100-4P), the MIC value, which is the minimum concentration at which bacteria cannot grow, was measured.

Example 2. Acinetobacter baumani ( Acinetobacter baumannii ) UDP-glucose pyrophosphorylase (UDP-glucose pyrophosphorylase, UGPase) crystal structure

The present inventors confirmed the crystal structures of apo, substrate-bound (uridine triphosphate) and product-bound (Uridine diphosphate glucose) against UDP-glucose pyrophosphorylase (AbUGPase) derived from Acinetobacter baumani. In addition, a structure in which decitabine, a nucleotide analog inhibitor, was combined was confirmed to a resolution of 2.1Å.

Example 3. MIC measurement results

The present inventors of the nucleotide analogue inhibitor (nucleotide analog inhibitor) decitabine (decitabine) of xanthomonas orijae pv. To test the sensitivity to Orijae ( Xanthomonas oryzae pv. Oryzae, Xoo ), the MIC value of decitabine was measured. As a result, the MIC value of decitabine was determined to be 128 μg ml ⁻¹ (FIG. 1).

Therefore, decitabine (decitabine) binds to the substrate binding site of the UGPase protein, which is the cause of rice white blight disease, Xanthomonas orijae pv. Orijae ( Xanthomonas oryzae pv. Oryzae, Xoo ) was confirmed to have an effect of inhibiting or inhibiting.

Claims (12)

A composition for controlling plant diseases comprising decitabine represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof:
[Formula 1]

. According to claim 1,
The plant disease is a composition characterized in that the rice white leaf blight. According to claim 2,
The rice white leaf blight is a composition characterized in that the plant disease caused by a strain of genus Xanthomonas. The method of claim 3,
The strain of genus Xanthomonas is Xanthomonas orijae pv. Orijae ( Xanthomonas oryzae pv. Oryzae ) characterized in that the composition. According to claim 1,
The decitabine (decitabine) is a composition characterized in that to inhibit the activity of UGPase (UDP-glucose pyrophosphorylase) protein. A method for controlling plant diseases comprising the step of treating the composition of any one of claims 1 to 5 with a plant. The method of claim 6,
The plant disease is a method characterized in that the rice white leaf blight. The method of claim 7,
The rice white leaf blight disease is characterized in that the plant disease caused by a strain of genus Xanthomonas. The method of claim 8,
The strain of genus Xanthomonas is Xanthomonas orijae pv. A method characterized in that it is Orijae ( Xanthomonas oryzae pv. Oryzae ). The method of claim 6,
The treatment is characterized in that the treatment on rice, rice seeds or cultivated soil of rice. Contacting a candidate substance with UGPase (UDP-glucose pyrophosphorylase); And
A method for screening a plant disease control agent, comprising: selecting a candidate substance for which the activity of the UGPase is inhibited. The method of claim 11,
The plant disease is a method characterized in that the rice white leaf blight.

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