KR20180052221A - Pesticidal composition for controlling bacterial leaf blight of rice - Google Patents

Abstract

The present invention relates to a composition for controlling bacterial leaf blight of rice including a compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof. Specifically, the controlling composition inhibits the activity of peptide deformylase (PDF) derived from Xanthomonas campestris pv. oryzae (Xoo) and controls plant diseases caused by Xoo. Therefore, the controlling composition can be useful in agricultural field.

Description

[0001] The present invention relates to a composition for controlling bacterial leaf blight of rice,

The present invention relates to a composition for controlling rice blight blight disease prevention, and more particularly, to a composition for controlling rice blight blight disease using a compound inhibiting activity of PDF (Peptide Deformylase) as an active ingredient.

Bacterial blight and bacterial leaf streak of rice are the first pathogens of rice, the first crop in Korea. Xanthomonas oryzae pv . oryzae ; Xoo) is currently being applied to three kinds of disinfectants, namely, Hanajin Wetting Agent (Kyongin Fenazin, Young Il Hanazin), Teram Wetting Agent (Shiragen, Sekong Toran, Kyungong Tiram, Chumina), Capopamid Liquid Wetting Agent Although the item is registered, it is a synthetic substance based on nonspecific mechanism, and its effectiveness is insufficient. So far, there are no effective sicknesses.

Although the entire genome (about 4.9 Mbps) of the KACC10331 strain of rice blast fungus was determined by the Rural Development Administration in 2005 as the world's first research, the current research is focused on finding a resistance gene to rice blast fungus in rice, Research on the mechanism by which rice blast fungus recognizes the host rice and induces virulence is insufficient.

Actually, many studies have been carried out on rice blast fungus, but the detection and transmission of external signals known to be a target of the development of a sickle pharmaceutical agent, adhesion proteins to host plants, host plant degrading enzymes, production of polysaccharides and secretory proteins , Toxin production and secretion proteins, iron-harboring proteins, and effector protein secretion proteins are difficult to express in normal tissues.

The International Rice Research Institute (IRRI) of France and the Philippines is investigating the resistance of rice blight to blight resistance to rice blight and to investigate the resistance of rice blight to temperature. In addition, Shizuoka University and Seed Company in Japan are conducting research to genetically explain the relationship between pathogenicity and resistance by searching for pathogenic genes from rice blast fungus and confirming the presence or absence of various Xoo race genes in the genes. The Institute of Agricultural Research is studying the pathogenicity mechanism by studying the expression of pathogenic genes when causing rice disease in rice blast fungus. In the United States and France, the Institute for Agronomy and Poultry Management, The identification of Xoo strain and race is monitored to monitor the spread of pathogens.

1. Korean Patent No. 10-1313783

An object of the present invention is to provide a composition for controlling blight of blight of blight of white blossom comprising a compound represented by the following general formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

It is still another object of the present invention to provide a method for controlling blight of blight of brown rice bladder comprising the step of treating the composition for controlling blight.

In order to accomplish the above object, the present invention provides a composition for preventing blight of blight of blight of white blossom comprising a compound represented by the following formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

In one embodiment of the present invention, the rice blast fungus is selected from the group consisting of Xanthomonas campestris pv . oryzae : Xoo).

In one embodiment of the present invention, the compound may inhibit the activity of PDF (Peptide Deformylase).

In one embodiment of the present invention, the PDF (Peptide Deformylase) is obtained from Xanthomonas campestris pv . oryzae : Xoo).

In addition, the present invention provides a method for controlling blight of blight of brown rice bladder comprising the step of treating the composition for controlling blight.

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

In addition, the present invention provides a method for preparing a protein, comprising: contacting a candidate substance to a peptide deformylase (PDF); And selecting candidates for which the activity of the PDF is inhibited. The present invention also provides a method for screening a rice blast fungus preventing agent.

In one embodiment of the present invention, the PDF (Peptide Deformylase) is obtained from Xanthomonas campestris pv . oryzae : Xoo).

The composition for controlling blight of blight of blight of white blossom according to the present invention is useful for controlling Xanthomonas campestris pv . oryzae : Xoo) was inhibited and the activity of Xanthomonas campestris pv . oryzae : Xoo) can be used in the field of agriculture.

Figure 1 shows the bacterial protein synthesis pathway.
Figure 2a shows the purified PDF (Xoo1075) identified by SDS-PAGE.
Figure 2b shows the crystallization of XoPDF.
3A shows the overall structure of XoPDF.
Figure 3b shows the active site of XoPDF in detail.
Figure 4 is a schematic representation of a VLS experiment to search for antagonist drugs for a particular target protein.
Figure 5 shows the structure of a selected compound derived from VLS and docking combined with a target protein.

The present invention provides a composition for controlling blight of blight of blight of white blossom comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

[Chemical Formula 1]

The controlling composition of the present invention may further comprise an agriculturally acceptable carrier.

The carrier may be inorganic or organic and of synthetic or natural origin, and the active composition may be mixed with or formulated with the carrier to facilitate the storage, movement and / or handling of the plant, seed, soil or other treated subject, Of the substance. In general, any material that can be routinely used as a carrier in pesticides, herbicides or antimicrobial compositions is suitable for use in the present invention. The control composition of the present invention may be used alone or in combination with the solid and / or liquid dispersible carrier vehicle and / or other compatible compatible excipients, especially other insecticides, siccatives, biting agents, In the form of a mixture with a plant protection agent such as a fungicide, a bactericide, a rodent scavenger, a herbicide, a fertilizer, a growth regulator or the like, or, if desired, a specific dosage form for a particular application made therefrom, Emulsions, suspensions, powders, pastes, and granules. The control composition of the present invention may optionally contain conventional inert (plant compatible or herbicidally inert) insecticide diluents or swelling agents of the type available for conventional insecticidal agents or compositions, for example, conventional gas, solutions , Emulsions, suspensions, emulsifiable concentrates, spray powders, pastes, soluble powders, dust collectors, granules, foams, pastes, tablets, aerosols, natural and synthetic materials incorporated with active compounds, microcapsules, seeds And foamable cans, as well as preparations for use with burning equipment, such as ULV cold air fog and hot air fog formulation, as well as other coating compositions and foaming cartridges. The carrier is preferably adjusted depending on the formulation of the composition, the application site and the application method.

The present invention provides a rice blight blight control product (for example, an insecticide) comprising the above composition.

The controlled product may be one which is formulated in an appropriate form according to the purpose of use and use, and may be, for example, a granule, a powder, a liquid, an aerosol, a spray, an extract, a paste, a fluid extract, The emulsifier, the fumigant, the fumigant or the aerosol are most preferably formulated to suitably control the volatility of the insecticide composition, which may be a liquid, a liquid wettable powder, a granule wettable powder, a wettable powder, a powder, a fine particle, an oil, a gel broth, Do.

The present invention provides a method for controlling rice blight blight disease comprising applying a pharmaceutically effective amount of a controlling composition to a crop.

The composition for controlling blight of rice bladder blight of the present invention may be used in formulations such as an aerosol, a pump spray, a liquid agent, a suspension agent, a spray agent, a capsule agent, a coating agent or a gel agent, 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. The spraying agent for the aerosol formulation may be at least one selected from liquefied natural gas and dimethyl ester, compressed air, compressed nitrogen, wherein the ratio of composition to propellant is in the range of 65: 35 to 40:60 by volume But is not limited thereto.

The composition for controlling blight of rice blight of the present invention may use a volatile solvent in combination with the plant extract. The volatile solvent is preferably ethanol or methanol, but is not limited thereto.

The composition for controlling blight of rice blight of the present invention can be used both as a food-borne or as a contact agent, and is preferably used as a contact agent in order to enhance the insecticidal effect.

In order to prevent such a phenomenon, the insect pest has a protective system for preventing blight of rice blight of the present invention. In order to prevent such a phenomenon, the insect pest has a protective system which prevents water droplets from flowing by using surface tension when a liquid component touches the surface thereof, It is preferable to use a penetrating agent in combination.

The composition for controlling the blight of blight of blight of white blossom may be diluted to an appropriate concentration at the time of use.

As the diluting solvent, at least one selected from the group consisting of water and a surfactant can be used, and for example, water can be used.

The method for controlling the blight of rice blight may be applied by directly spraying or applying the composition for controlling blight of blight of rice blast to the crop and / or soil in the facility.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are for further illustrating the present invention, and the scope of the present invention is not limited to these examples.

Example  1. Preparation of compounds

The compounds used in the present invention were purchased from ChemBridge Corporation (16981 Via Tazon, Suite G, San Diego, CA, 92127, USA).

Example  2. Xanthomona soryzae pv . oryzae (Xoo)  Derived XoPDF ( Xoo  Cloning, expression, purification, crystallization and crystal structure determination of Peptide Deformylase

2.1. Cloning

Bacteria need formylated Met as the first amino acid to be produced in the process of protein production, and it is necessary to remove the Formylated group again from newly formed protein peptide. PDF (Peptide Deformylase) is an enzyme that removes the Formylated group. The PDF enzyme can be an important target protein because it is essential for bacterial survival. Specifically, PDF (Peptide Deformylase) catalyzes the enzymatic reaction to formyl-L-methionyl peptide + H2O formate + methionyl peptide, which is an essential step in the protein synthesis of bacteria.

In particular, in the present invention, XoPDF (Xoo1075) derived from a bacterial cell (Xoo KACC10331 strain) genome was used, and the PDF coding sequence of XoPDF was amplified by polymerase chain reaction as a template. Oligonucleotide primer sequences have been designed based on previously reported gene sequences (Lee, BM, et al. (2005) Nucleic Acids Res 33 , 577-86). The forward and reverse primers are: 5'-GGG GGG CAT ATG ATT CGC GAC ATT ATC CGC ATG-3 'and 5'-GGG GGG GGA TCC CTA CAG ATC GTA AGA CAA GAC - 3 '. The Nde I and Bam HI restriction sites in the sequence are indicated in bold. The PCR amplicons were double-treated with Nde I and Bam HI and had a tobacco etch virus (TEV) protease cleavage site and an additional residue of 6xHis in the pET11a vector (Novagen) in front of the Nde I site to facilitate purification of the expressed protein Lt; RTI ID = 0.0 > pET11a < / RTI > vector (His-TEV-pET11a).

2.2. Over-expression and purification

The His-TEV-pET11a expression vector containing the coding sequence of Xoo1075 was transformed into E. coli BL21 (DE3). Cells were incubated at 37 ° C in Luria-Bertani medium containing 50 μg / ml ampicillin until OD ₆₀₀ reached 0.5. Protein expression was induced by adding 0.5 mM IPTG (Isopropyl-beta-D-thiogalactopyranoside) to the culture, and after induction, the cells were incubated for an additional 16 hours at 37 ° C. The cultured Escherichia coli was collected by centrifugation (Vision VS24-SMTi V5006A rotor) at 277 K for 20 minutes at 6,000 rpm, and then the collected E. coli was incubated with a temperature-decreasing buffer solution A [25 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM Imidazole], and disrupted by sonication on ice (Sonomasher, S & T Science, Korea). Then, to obtain a cell extract, the disruption was centrifuged at 277K and 12,000 rpm for 30 minutes (Vision VS24-SMTi V508A rotor). Only 10% of the entire expressed Xoo1075 was water soluble (not shown). To use the affinity purification method, the supernatant containing water-soluble XoPDF was loaded onto Ni-NTA resin (Novagen). The affinity purification was carried out at 4 캜 according to the manufacturer's protocol. The XoPDF protein with the 6xHis-tag was then eluted using a buffer for cleavage A containing 200 mM imidazole and treated with TEV protease at 4 ° C overnight to remove the 6XHis-tag. The protein solution thus treated was dialyzed at 4 ° C. for 6 hours using buffer solution B [25 mM Tris-HCl pH 7.5, 15 mM NaCl], and further purified using a UNO 6 Q column (Bio-Rad). The purity of the purified protein was analyzed using SDS-PAGE (Fig. 2). The purified protein was concentrated to 8 mg ml ^-1 and used for crystallization.

2.3. Crystallization and X- Lay  Diffraction Data Collection

Crystallization of XoPDF was performed via the Hydra II e-drop (Matrix) automated pipetting system using several screening kits such as Crystal screen HT, Index HT, and Salt Rx HT (Hampton Research) high throughput (HT)] determination Screening started. Initially, hexagonal needle-like crystals were obtained from Hampton Crystal Screen condition No. 1. (0.05 M cadmium sulfate, 0.1 M HEPES (pH 7.5), and 2.0 M sodium acetate trihydrate) were obtained with 0.05 M cadmium sulfate, 0.1 M HEPES (pH 7.5) and 1.0 M sodium acetate trihydrate . They were reproduced using the hanging drops method, and crystals were made for a week. The grown crystals were frozen at 100 K in liquid nitrogen after addition of antifreeze 20% ( v / v ) glycerol to the crystallization conditions and used for data collection. Determination data were collected at 2.1 A resolution using an ADSC Quantum 270 CCD detector at Beamline 17A, Photon Factory, Japan. Data sets were integrated and calculated using DENZO and SCALEPACK (Otwinowski, Z. and Minor, W. (1997) Methods Enzymol 276 , 307-326.). Through the auto-indexing process, the crystal space group of P6122 and the unit cell parameter a = b = 59.0, c = 266.3 were deduced.

The crystal structure of XoPDF and its active site residues are shown in Fig. The entire structure of XoPDF is made up of a single round domain. The active site of XoPDF is located in the middle of the protein enzyme. The structure of the enzyme is composed of two alpha helices and eight beta strands. The enzyme active site has a divalent metal bonding site necessary for its activity. It was confirmed that a substrate recognition site composed of amino acids was present.

Example  3. target  enzyme XoPDF  About Virtual  Library Screening ( VLS ) And molecular docking process

Since XoPDF (Xoo1075) is one of the possible target genes for rice blight blight based on GSK (GlaxoSmithKline), it is antagonistic to the substrate binding site of XoPDF enzyme through high efficiency (HT) virtual library screening (VLS, The drug lead compound was searched.

A computer simulation VLS was performed using a database of 18 million compounds in the ICM Molsoft modeling suite for drug screening. The substrate of each flexible structure present in the compound database found in the Lipinski formula of the VLS was docked to the substrate bonded pocket grid of XoMCAT using ICM and assigned a score reflecting the quality of the complex. In order to check the regeneration of each substrate binding result, they were simultaneously run in parallel three times. 1991 compounds with top scores were selected and their complexes were visualized (quantified) by coefficients such as shape complementarity, hydrogen bonding network and ligand flexibility. Based on the above-mentioned final selection step, a compound represented by the formula (1) was obtained for characterization through an experimental analysis.

[Chemical Formula 1]

Therefore, the compound represented by the formula (1) binds to the substrate binding site of the XoPDF enzyme and binds to the Xanthomonas campestris pv . oryzae ) in the case of the present invention.

Claims (8)

1. A composition for controlling blight of rice blast fungus comprising a compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

The method according to claim 1,
The above-mentioned rice blotch blight disease is caused by Xanthomonas campestris pv . oryzae : Xoo). < / RTI > The method according to claim 1,
Wherein said compound inhibits the activity of PDF (Peptide Deformylase). The method of claim 3,
The above-mentioned PDF (Peptide Deformylase) is a strain of Xanthomonas campestris pv. oryzae : Xoo). < / RTI > A method for controlling blight of blight of blight of white blotch comprising treating the composition for controlling blight of claim 1. 6. The method of claim 5,
Characterized in that the treatment is carried out on rice, rice seed or cultivated soil of rice. Contacting the candidate material to PDF (Peptide Deformylase); And
And screening candidates for which the activity of the PDF is inhibited. ≪ Desc / Clms Page number 20 > 8. The method of claim 7,
The above-mentioned PDF (Peptide Deformylase) is a strain of Xanthomonas campestris pv. oryzae : Xoo). < / RTI >

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