KR20040052947A - Pepper esterase as a biocontrol agent - Google Patents

Abstract

PURPOSE: Pepper esterase as a biocontrol agent is provided, thereby effectively inducing a defense response within plant cells and inhibiting permeation of fungi into the plant cells. CONSTITUTION: The biological control material comprises 0.05 to 0.2 mg/ml of pepper esterase(PepEST) which has the amino acid sequence set forth in SEQ ID NO: 1, releases cutin monomer, 9-octadecenamide, produced by hydrolysis of cuticle from unmatured pepper fruits, and induces expression of PR gene to protect plants from anthrax bacteria and rice blast disease, wherein cDNA encoding the pepper esterase(PepEST) has the nucleotide sequence set forth in SEQ ID NO: 2; the protection of plant from anthrax bacteria is produced by hydrolysis of cellular wall of anthrax bacteria using 1,2-dithian-4,5-diol; and the germination of fungi spores can be inhibited when the concentration of pepper esterase is low and fungi permeation into the pepper fruits can be inhibited when the concentration of pepper esterase is high.

Description

Pepper esterase as a biocontrol agent

FIELD OF THE INVENTION The present invention relates to a pepper esterase (PepEST) recombinant protein as a biological control substance, and more particularly to a biological control substance capable of eliciting a protective response in plant cells and a direct control of fungal penetration into plant cells. A recombinant protein as a fungal inhibitor.

Plants have evolved an array of cellular mechanisms that defend themselves against invading pathogens. Recognizing pathogens, plants activate a complex set of defensive reactions to defeat pathogen attacks. This often includes acute defense at the infection site by induction of immunity at the plant terminal part. Development of resistance at local and terminal sites is partially related to expression of anti-microbial proteins. This includes glucanase, chitinase and lysozyme deposited in plants that exhibit increased resistance to various microorganisms. Recently, anti-microbial proteins have been introduced into crop plants to control plant-pathogens. In particular, antifungal polypeptides can be applied directly on sensitive plants to provide protection against fungi.

Colletotrichum gloeosporioides (Penz.) Is the casual substance of anthrax, the most destructive disease in pepper growing worldwide. Now anthrax fungi are controlled primarily by the application of substantial amounts of chemicals. There is an urgent need to establish pepper varieties that are resistant to anthrax to limit pesticides in crop harvesting. However, because there is no useful genetic resource for disease resistance, traditional breeding cannot be applied to the development of resistance lines. At present all commercial pepper lines are still susceptible to anthrax.

The plant response to the development of fungal forms in red pepper depends on the stage of ripening of the red pepper fruit. Signs of disease develop in immature fruit but are not infected with anthrax fungi in ripe fruit. To obtain genetic resources important for resistance, some genes were first isolated from ripening fruits that interact with anthrax. Among them, the pepper esterase gene ( PepEST ), which encodes a member of the esterase family, showed antifungal activity against anthrax fungi and rice blast fungi. In the present invention, the form of enzymatic action of the PepEST protein has been described to gain insight into the molecular mechanisms involved in pepper and fungal resistance reactions. PepEST protein has been applied externally to induce a protective response in immature fruit and to block fungal penetration into the fruit epidermis.

Genetic engineering provides resuscitation from fungal pathogens through the development of fungal regulatory systems based on the PepEST gene, isolated by the inventors and disclosed in US Pat. No. 6,018,038. The first is the development of novel fungicides using genetically engineered proteins. The second form of application is the application of proteins to plants to induce defense responses such as H ₂ O ₂ production and PR gene expression and to block fungal penetration into plant cells.

Plant cuticles are physiological barriers composed of polyesters of C ₁₆ and C ₁₈ hydroxy fatty acids. Infection of C. gloesporioides is achieved through conidia germination and the formation of appresorium necessary for subepithelial infiltration. Penetration of fungi into plants involves cutinase that hydrolyzes plant cuticles. The degradation products of plant cuticles by cutinases act as chemical signals to activate fungal cutinase genes. The cutin monomer may also act to induce plant resistance to fungal infections. Spray application of the C ₁₈ family of curtin monomers was performed by phytopathogenic fungi, Erysiphe gramis f. sp. Barley and rice were protected against Hordei and Magnaporthe grisea infections. In particular kyutin monomers are reported as inductors and reinforcing agents of H ₂ O ₂ in H ₂ O ₂ in a plant. In the present study, we assume that exogenous treatment of pepper esterase liberates the cutin monomers that can induce the production of H ₂ O ₂ . Plant defense genes induced by H ₂ O ₂ are then developed for plant protection against fungi.

In turn, the application of PepEST protein may provide an agriculturally relevant level of disease control on pepper harvests without adverse side effects to contribute to more substantial agricultural practices.

The technical problem to be achieved in the present invention is to provide a pepper esterase (PepEST) recombinant protein as a biological control material, more specifically a biological control material that can elicit a protective response in plant cells and fungi into plant cells Provided are recombinant proteins as direct fungal inhibitors that can block penetration.

1 shows SDS-PAGE analysis of recombinant PepEST protein produced in E. coli carrying pGEX6p-1 / PepEST, (B) size of soluble protein from E. coli carrying pGEX6p-1 / PepEST Post-classification fractions Nos. 9 to 26 and (C) show Western blots of the same fractions in B representing the PepEST protein band.

FIG. 2 shows GC-Mass analysis of hydrolyzed material from immature pepper fruit after PepEST treatment (0.1 mg / ml). (A) GC profile; (B) mass spectrum of peak 2 at 22.57 minutes; (C) True 9-octadecenamid (C).

Figure 3 shows the generation of hydrogen peroxide by PepEST protein exogenous treatment in red pepper. (A) Epidermal cells of immature fruit were stained for H ₂ O ₂ with diamino-benzidine (DAB) after 1 μg of PepEST treatment; (B) Time course of H ₂ O ₂ accumulation in immature fruit after 1 μg PepEST treatment.

Figure 4 shows the induction of PR gene expression in immature fruit after 1 μg of PepEST protein treatment.

Figure 5 shows the induction of PR gene expression by 1 μg PepEST treatment in immature fruit infected with anthrax fungi.

Figure 6 shows the loss of fungal viability after PepEST protein treatment.

Figure 7 shows the abnormal growth of fungal development by various concentrations of PepEST protein. (A) Numbers indicate protein amount (μg). (B) Inhibition of anthrax fungi on immature red pepper fruits after PepEST treatment.

FIG. 8 shows (A) FITC-labeling of PepEST recombinant protein and (B) adhesion of labeled protein on the surface of fungi.

Figure 9 shows the surface damage of fungi after PepEST protein treatment. (A) Ultrafine structure of fungal surface degraded by PepEST treatment of FITC-labeled Concavanalin A and (B) 1 μg bound to sugar residues on cell surface.

FIG. 10 shows GC-mass analysis of hydrolyzed material from anthrax fungi after PepEST treatment. (A) GC profile; (B) mass spectrum of peak 2 at 12.46 minutes; (C) Mass spectrum of true 1,2-dithiane-4,5-diol.

Figure 11 shows protection of pepper cells from anthrax fungi by PepEST protein. Inoculated fruits were photographed 24 h after infection (HAI) and 110 h after infection (HAI), focusing on fungal and capsicum epidermis, respectively.

Figure 12 shows inhibition of fungal development by a formula containing PepEST protein. Formula includes PepEST fraction in soluble protein of E. coli containing DTT and pGEX6p-1 / PepEST.

The present invention consists of an effective amount of pepper esterase (PepEST) having the amino acid sequence of SEQ ID NO: 1, wherein the effective amount of pepper esterase is 0.05 to 0.2 mg per milliliter to provide a biological control substance . The present invention relates to the use of proteins with resistant activation in plants. Use in this context means that a formula containing PepEST protein with pathogenic regulatory action has been applied locally to the plant. PepEST protein breaks down the cuticle components on the pepper fruit. The cutin monomer, 9-octadecenamide, was released from the cuticle. Hydrogen peroxide was then produced in immature fruit where the defense-related genes were subsequently expressed. Thus, the pepper esterase has a function of protecting the plant by inducing a defense reaction. In this case the plant is red pepper. Moreover, the pepper esterase dissolved the fungal wall, releasing the dithian compound from the fungus. Moreover, formulas and protein fractions containing dithiothreitol (DTT) inhibit germination of fungal spores depending on the concentration of the esterase; Ii) showed a direct antifungal function of inhibiting fungal penetration into the pepper fruit.

For the application of pepper esterases the fungus is Colletotrichum gloeosporioides .

The invention also provides a formula comprising a partially purified capsicum esterase produced by transforming E. coli comprising a vector comprising a DTT and a cDNA fragment of SEQ ID NO: 2.

Carboxylesterases are enzymes that catalyze the hydrolysis of compounds including ester bonds. Tobacco esterase and two Arabidopsis lipase genes in plant-microbial interactions were isolated in hypersensitivity to plant-pathogens. Highly expressed pepper esterases were cloned from pepper during incompatible interactions between ripened fruit and C. gloeosporioides . Recombinant PepEST protein expressed in E. coli (FIG. 1A) hydrolyzed p-nitrophenyl ester but showed no detectable level of lipase activity.

Little is known about the physiological role of proteins in the resistance mechanism of ripened pepper berries, since no natural substances for plant esterase have yet been identified. Identification of the compounds produced by PepEST will clarify the role of plant esterases in the defense system. Therefore, the effect of PepEST protein by exogenous treatment was observed in immature pepper fruits in relation to resistance induction. In the current study, the PepEST protein was found to be sufficient to stimulate the complex defense response in pepper cells, consisting of release of cutin monomers, H ₂ O ₂ production and defense-related gene activation. The results indicate that the PepEST gene product is involved in the defense mechanism against fungal infiltration of outer-epidermal cells of ripened fruit.

Active oxygen species (AOS) have been shown to play many important roles in the defense response during plant-pathogen interactions. In addition to the direct anti-microbial effects of AOS, they can function as messengers to the activation of protective response genes. Immature pepper fruit was used to correlate biochemically directed inducer experiments. The cutin monomer, 9-octadecenamide, was released from the fruit's cuticles when PepEST protein was applied locally on immature pepper fruit (FIG. 2).

In addition to cutin monomer dissolution, H ₂ O ₂ induction was also increased by PepEST protein. H ₂ O ₂ production in immature berries doubled at apparent time-points (6 h) (FIG. 3). As suggested by Iiyama et al (1994), expression of defense-related genes was measured in peppers after treatment because they could function as activators of defense-related genes. Some pepper PR genes were induced by exogenous application of PepEST protein on immature fruit (FIG. 4). Expression patterns could lead to increased disease resistance without significant negative effects. A particular aspect of this view is the observation that H ₂ O ₂ regulation is also activated by PepEST treatment during infiltration into pepper cells (FIG. 5). The PR gene was induced locally within the infection site by contiguous application of PepEST protein with fungal spores on immature fruit. Together these results indicate that the PepEST gene as an esterase is involved in the defense response mechanism against ripening fruit against pathogens.

On the contrary, even if the fungal mycelium still elongated, the viability of the fungus was greatly impaired by PepEST treatment (FIG. 6). Eventually, PepEST protein was able to inhibit fungal development with weak fungicide activity, which completely blocked fungal colony formation from the initial stage of infection (FIG. 7B). To demonstrate the important role of PepEST protein in fungal morphogenesis, the walls from the germinated conidia of anthrax fungi treated with PepEST recombinant protein were analyzed (FIG. 8). The surface of the fungus was observed using FITC-labeled Concanavalin A (Con A) which binds to sugar residues on the cell wall (FIG. 9A). Unlike normal fungi, the surface of PepEST-treated cell walls was disturbed when probed with ConA-FITC. Next, the wall structure was observed using a scanning electron microscope to determine whether PepEST hydrolyzes the cell wall (FIG. 9B). The surface of the fungal cell wall was covered with undefined mucus in distilled water, but the skeleton of the cell wall structure was exposed by the enzymatic action of PepEST protein. The results strongly indicate that the PepEST protein can hydrolyze the material that covers the fungal wall. Finally, the dithiane compound, 1,2-dithiane-4,5-diol, was detected as glycerol in solution from PepEST-treated fungal cell walls (FIG. 10).

In conclusion, pepper esterase (PepEST) protects immature fruit susceptible to fungal infection by inducing H ₂ O ₂ production followed by expression of defense-related genes (FIG. 11). Pepper esterase has been shown to be able to hydrolyse plant cell parts as well as the surrounding cell layers of fungi. These results show that PepEST protein can be used for disease control as an effective biological control agent (FIG. 12). The application of PepEST protein in pepper plants will benefit the environment by reducing the amount of pesticides used as well as farmers by reducing harvesting costs.

1. Fungal Inoculum and Plant Material

Monoclonal isolate KG13 of C. gloeosporioides was incubated on potato dextrose agar (PDA) (Difco) for 7 days in dark at 28 ° C. Conidia were harvested and suspended in sterile distilled water. 10 μl of spore suspension (5 × 10 ⁵ spores / ml) was used for inoculum and in vivo drop inoculation, amended with 10 μl (final concentration 1 μg) of recombinant PepEST and applied to the cover glass It became. 1 μg of GST or sterile water was used as a control. The cover glass was incubated in a wet chamber at 25 ° C. dark conditions. The spore suspension was then stained with 0.1% (wt / vol) cotton blue in lactophenol. Spore germination and appresorium formation on the cover glass were observed under an optical microscope (Olympus).

Dose-response experiments of recombinant PepEST on fungal morphogenesis were performed. Inoculation tests with spores modified with recombinant PepEST were performed on healthy immature fruit as described previously (Oh et al., 1998). Fruits were placed in a plastic box (25x16x6 cm) containing a wet paper towel to maintain 100% relative humidity at 29 ° C dark conditions. After incubation the fruit was excised to 1 cm 2 at the drop-application site for fungi. Samples were frozen in liquid nitrogen.

Immature adult-green pepper berries were obtained from pepper plants grown under greenhouse conditions in Gwangju, Korea.

2. Dissolution of Surface Wax and Fungi from Red Pepper by PepEST

10 μl of PepEST protein (0.1 mg / ml) was applied locally on immature pepper berries. Spores were fertilized with 1 μg PepEST protein in 10 μl distilled water. After 24 hours treatment soluble fractions were collected, lyophilized and dissolved in chloroform. Wax extracts were analyzed by GC-mass spectrometry (Hewlett Packard).

3. Quantification of hydrogen peroxide

1 g of red pepper fruit was frozen in liquid vagina and powdered in mortar with cold 5% TCA and 45 mg of activated charcoal. The powder was centrifuged at 0 ° C. for 10 minutes at 18000 g and the supernatant was immediately filtered (Advantec 0.45 μm) by applying pressure with a syringe to remove traces of precipitated protein and activated charcoal. The filtrate was titrated to pH 8.4 with NH ₄ OH and then refiltered to remove the precipitate formed when the extract was neutralized. The amount of H ₂ O _{2 in} the obtained extract was determined by pipetting 100 μl of the test solution into 0.2 M NH ₄ OH (pH 9.5) so 100 μl of 0.5 mM luminol (3-aminophthalhydrazide, Sigma). Tested. The test tubes were placed in a measurement cuvette of the Aminco Chem Glow Photometer for the disclosed chemiluminescence (CL) release by injecting 100 μl of 1 mM K ₃ Fe (CN) ₆ (in 0.2 M NH ₄ OH) into the mixture. The emitted photons were counted for 5 seconds with a pulse integrator (Lumat LB9501). Furthermore, histochemical detection of H ₂ O ₂ is performed by an endogenous peroxidase-dependent staining procedure using DAB (3,3′-diaminobenzidine) as described by Thordal-Christensen et al. (1997). It became. Immature fruit treated with PepEST protein was placed in 1 mg / ml DAB solution for 8 hours.

4. Gene Expression Essay

Total RNA was extracted using Trizol reagent (Gibco BRL) according to the manufacturer's instructions. Equal amounts of total RNA (10 μg per lane) were separated on 1.2% modified agarose gels in the presence of formaldehyde. RNA gel blotting, hybridization and washing were performed as described by the manufacturer of the positively charged nylon membrane (Hybond N ⁺ ; Amersham Pharmacia Biotech). Radioisotope labeled probes were prepared with [α- ³² P] dCTP (3000 Ci / mmol, PerkinElmer) using the redi-prime ^™ kit (Amersham Pharmacia Biotech). The membrane was washed twice at 65 ° C. with 2 × SSPE, 0.1% SDS and once at 65 ° C. with 0.5 × SSPE, 0.1% SDS.

Probe of defense-related genes such as PR3 (chitinase class II (CAChi2)), PR5 (Thaumatin-like protein (TLP)), PR10, PepThi was tested by Northern hybridization or RT-PCR. It was used to measure my gene expression. Primer pairs used for PCR analysis were PR3-5 '(ATG GAG TTC TCT GTA TCA CCA GTG G) (SEQ ID NO: 3), 3' (TCC GAA TGT CTA AAG TGG TAC AAG) (SEQ ID NO: 4) , PR5-5 '(ATG GGC TAT TTG AGA TCA TCT) (SEQ ID NO: 5), 3' (TCA CCT CTC TGC AAT CAA TAT) (SEQ ID NO: 6), PR10-5 '(CTG ACA AGT CCA CAG CCT CAG) (SEQ ID NO: 7), 3 '(GTT CTT TCC ATG ACA ACC AAT TG) (SEQ ID NO: 8), Pepthi-5' (GGG GGA TCC AAA ATG GCT CGT TCC) (SEQ ID NO: 9), 3 (CTC GGT ACC CTT TAT TTA ATT TTG TGT GAC ACT) (SEQ ID NO: 10).

5. Induction and Purification of Recombinant PepEST Protein

A single colony of E. coli containing the recombinant pGEX6P-1 plasmid was selected to prepare 5 ml of 2x YTA (16 g / l tryptone, 10 g / l yeast extract, 5 g / l NaCl, 100 mg / l) ampicillin. Inoculated and incubated at 37 ° C. for 12-15 hours with strong shaking. Cultures 1: 100 were diluted into fresh preheated 2x YTA medium and grown at 37 ° C until absorbance A ₆₀₀ reached 1.0. A final concentration of 0.2 mM IPTG was then added to the culture and incubated at 30 ° C. for an additional 3 hours. Finally, the culture was centrifuged at 4 ° C. for 10 minutes at 12,000 rpm to precipitate the cells. Supernatant was removed. The cell pellet was completely suspended by adding 50 μl of cold 1 × PBSfm per ml of culture medium. Suspended cells were ruptured using a sonicator with a short burst on ice. The lysate was bound to Glutathione Sepharose 4B according to the manufacturer's instructions and then the fusion protein was treated with 80 U Prescission protease to remove reduced GST from PepEST protein. Protein content was measured by the Braford method (1979). SDS-PAGE was performed on a 10% polyacrylamide gel.

Lysis was applied to a 120 cm gel-filtration column (Sephadex G-150, Sigma) to partially separate the PepEST protein. Separated proteins in fractions 9-26 were mixed, lyophilized and dissolved in 1 mM dithiothreitol (DTT) prior to use.

6. PepEST Labeling with FITC

Fluorescein isothiocyanate (FITC) was dissolved in DMSO at 10 mg / ml and 1 mg / ml of purified PepEST in 100 mM sodium bicarbonate (pH 9.3) to a final concentration of 1 mg / ml FITC. Added to protein. After incubation for 4 hours at room temperature under dark conditions, 1 M ethanolamine was added to inactivate the remaining FITC. The solution was placed in the dark for an additional 2 hours and subjected to gel-filtration column chromatography using a 120 cm Sephadex G-50 (Sigma) column to remove unconjugated dye. Separated samples were lyophilized. Conjugation between FITC and purified PepEST was confirmed on SDS-PAGE gels with strong fluorescence of purified PepEST under UV light.

7. Quick Method for Viability of Fungal Spores

Live / Dead BacLight ^™ Bacterial Viability Kit (Molecular Probe) was used according to the manufacturer's instructions to monitor fungal viability. Live / Dead consists of two reagents containing a mixture of fluorescent nucleic acid colorant SYTO9 (green) and propidium iodied (red) in anhydrous DMSO.

8. Observation of Fungal Morphology by SEM (Scanning Electron Microscopy)

Pepper slices were prepared for scanning electron microscopy (SEM) after exogenous PepEST protein treatment. Small segments (5 mm long) were vacuum-infiltrated in sodium phosphate buffer (pH 7.0) containing 2% sucrose, 3% paraformaldehyde and 0.2% glutaraldehyde on ice for 6 hours. After treatment the material was slowly dehydrated in a series of graded ethanol at room temperature. After the last exchange of ethanol the specimens were immediately observed with important point dry, gold coated and scanning electron microscope (Hitachi).

9. Treatment of FITC-labeled Concarbanaline A (ConA)

Fluorescein isothiocyanate-labeled ConA (Sigma) was used to investigate the surface structure of fungi after PepEST treatment. Germinated fungi were treated with 0.1 mg / ml FITC-ConA in PBS and irradiated under fluorescence microscope (Olympus).

(Example)

Example 1 Purification of Recombinant PepEST Protein

An open reading frame of PepEST cDNA (AF122821) was inserted in-frame with glutathione-S-transferase (GST) coding sequence in expression vector pGEX6P-1 between EcoRI and XhoI sites. GST-PepEST fusion protein was digested with GST protein to yield 36.5 KD PepEST protein. Lysis was applied to a 120 cm gel-filtration column (Sephadex G-150, Sigma) to partially separate the PepEST protein. In FIG. 1B, isolated proteins in fractions 9-26 were separated on SDS-PAGE. PepEST protein was detected by western blotting within the same fraction (FIG. 1C).

Example 2 Solubility of a Cutin Monomer

The cuticle is an outer protective layer covering the atmospheric portion of higher plants. To penetrate into the epidermis, fungi use enzymes that hydrolyze plant cuticles. It has been reported that degradation products from cuticles can activate protective responses in host plants. In this experiment, PepEST protein was applied locally to healthy immature pepper fruits to elucidate the possible association of degradation compounds from cuticles. As a result glycerol and 9-octadecenamide were released from the cuticle of the fruit treated with PepEST protein (FIG. 2).

Example 3 Production of Hydrogen Peroxide in Pepper Vertical

To demonstrate the effect of PepEST protein on plant cells, the production of H ₂ O ₂ was investigated as a parameter for the defense and induction of defense related genes in immature fruit treated with PepEST protein for defense. The results in FIG. 3A show that application of distilled water on immature berries as a control did not affect hydrogen peroxide production. However, dark brown precipitate was uniformly detected in epidermal cells of immature berries treated with PepEST protein for 6 hours. . To investigate the time-course of differential accumulation of H ₂ O ₂ by PepEST protein, the amount of hydrogen peroxide was measured for 1 day after treatment. The accumulation pattern of H ₂ O ₂ associated with PepEST treatment could be distinguished quantitatively from the application of distilled water as a control (FIG. 3B). The production of H ₂ O ₂ was induced biphasically 0.5 and 6 hours after treatment. The production of H ₂ O ₂ was significantly higher in PepEST protein treated fruits than in the control.

Example 4 Expression of Defense-Related Genes in Pepper Cells

RNA blot analysis was performed on immature berries after treatment with PepEST protein to test whether PepEST protein induces PR gene expression. In addition, since H ₂ O ₂ is known to induce resistance responses in plants, the levels of PR gene transcripts in immature berries were investigated after treatment with hydrogen peroxide. PR genes such as PR3, PR5, PR10 and PepThi were derived from the fruit treated with hydrogen peroxide (FIG. 4). PR3 and PR5 were strongly induced at the early stage of induction and gradually decreased. In contrast, the PR 10 transcript increased significantly with time. The expression of the PR gene after PepEST protein treatment showed the same pattern as that induced by hydrogen peroxide, but the expression level was slightly reduced except for PepThi where transcripts accumulated 6 hours after treatment.

Treatment of immature pepper with PepEST protein led to the induction of defense-related genes in the absence of fungal pathogens, indicating that PepEST protein-induced resistance protects pepper fruit from fungal pathogens. To investigate whether the induction of the PR gene occurs in infected fruit with anthrax fungi, the expression pattern of the PR gene was monitored in the fruit treated after inoculation of the fungal spores. 5 shows that transcripts corresponding to PR 5 and 10 genes accumulated only in the treated fruit 72 hours after inoculation.

Example 5 Inhibition of Fungal Development by PepEST Treatment

The conidia of C. gloeosporioides started germination 1 hour after precipitation on the surface or cover glass of immature pepper fruit. One conidia produced a germination tube with an inflated tip forming an apressorium. The first apresorium developed in 6 hours. However, the normal development of fungi was severely impaired by the application of PepEST recombinant protein. 7A shows abnormal development of fungi treated with PepEST protein on cover glass at 24 hours. At low concentrations of PepEST protein, the growth tips of the mycelium tended to elongate and often produced new spores without developing apressorium. Fungal growth was completely blocked at 0.5 mg / ml PepEST.

The viability of fungi exhibiting abnormal growth patterns was examined by staining with Live / Dead BacLight ^™ Bacterial Viability Kit based on 2-color fluorescence assay of cell viability. Staining has different spectral properties and different ability to penetrate fungal cell membranes. Cells with complete membranes were labeled with green fluorescence while cells with damaged membranes were stained with red fluorescence. The results show that the viability of fungi is severely affected even at low concentrations of PepEST protein (FIG. 6).

Moreover, fungal morphology on immature pepper fruit was observed using a scanning electron microscope (Hitachi). Spores of C. gloeosporioides were incubated for 24 hours after inoculation on immature pepper berries with PepEST protein. As a control, fungal growth was routinely progressed with distilled water and germination tubes with apresorum were observed (FIG. 7A). Apresorium did not develop fungi by treatment with 1 μg PepEST protein and the fungus showed severe abnormalities in 5 μg protein.

Example 6 Surface Damage of Fungi After Exogenous Treatment with PepEST Protein

To observe the cellular localization of PepEST protein, PepEST protein was labeled with FITC as described above (FIG. 8A). GST protein was used as a control. GST-FITC protein accumulated inside fungal cells 24 hours after incubation. Spore growth and differentiation were not affected at all. However, PepEST-FITC protein was attached to the outer surface of the hyphae (Figure 8B).

Since the PepEST protein is a hydrolytic enzyme with esterase activity, the surface of the protein-treated fungi was investigated by both fluorescence and scanning electron microscopy. Labeling of the cell wall with ConA-FITC resulted in even fluorescence on the outer cell wall of the fungi germinated in distilled water as a control. However, the labeled walls of fungi treated with PepEST protein were found to be severely altered. PepEST protein induced changes in cell wall morphology indicate that PepEST protein dissolves material precipitated out of the wall (FIG. 9A). To determine whether PepEST protein hydrolyzes the cell wall, an ultrafine structure on the affected fungal surface was observed with a Field-Emission Scanning Electron Microscope (Hitachi). The surface of the fungal wall was covered with undefined mucus material in distilled water but the framework of the wall structure was exposed after treatment with PepEST protein (FIG. 9B). Decomposed material from the treated fruit was analyzed using GC-mass spectrometry. Two compounds, glycerol and 1,2-dithiane-4,5-diol, were detected in the digested solution (FIG. 10).

Example 7 Protection of Immature-Green Pepper Fruit Cells

By applying PepEST protein in a dose-dependent form, fungal apresorum formation was significantly inhibited in infected fruit with anthrax fungi. Blocking the development of apresorum resulted in the protection of immature fruit against fungi (FIG. 11). The epidermal cells of the red pepper fruit remained healthy in the treated fruit 72 hours after inoculation of the fungus, while the fungus penetrated into the epidermal cells of the untreated fruit.

Example 8 Formula Consisting of PepEST Protein

Soluble protein, including PepEST protein, was isolated by size fractionation. The fractions 9-26 containing the protein were mixed together. PepEST protein was considered 1% isolated protein fraction. The protein mixture obtained was lyophilized and dissolved at 250 μg per milliliter of the final concentration. DTT was then added to the protein solution at a final concentration of 1 mM and named Formula KH1.

Spore germination was completely inhibited as shown in FIG. 12 when spores of anthrax fungi were treated with Formula KH1.

The effect of the present invention is to provide pepper esterase (PepEST) recombinant protein as a biological control substance, and more particularly to block fungal penetration into plant cells and biological control substances that can elicit protective responses in plant cells. To provide a recombinant protein as a direct fungal inhibitor.

<110> KOREA KUMHO PETROCHEMICAL CO., LTD. <120> Pepper esterase as a biocontrol agent <160> 10 <170> KopatentIn 1.71 <210> 1 <211> 328 <212> PRT <213> Capsicum annuum <400> 1 Met Ala Ser Gln Ser Phe Val Pro Pro Ile Phe Glu Asn Pro Phe Leu   1 5 10 15 Asn Ile Glu Glu Leu Ala Gly Asp Thr Ile Val Arg Lys Pro Glu Pro              20 25 30 Leu Thr Gln Ala Asn Ser Asp Pro Asn Gly Thr Ser Leu Val Val Ser          35 40 45 Lys Asp Val Asp Leu Asp Ile Asn Lys Lys Thr Trp Leu Arg Ile Tyr      50 55 60 Val Pro Gln Arg Ile Ile Thr Asn His Asn Asp Asp Glu Lys Leu Pro  65 70 75 80 Val Ile Phe Tyr Tyr His Gly Gly Gly Phe Val Phe Phe His Ala Asn                  85 90 95 Ser Phe Ala Trp Asp Leu Phe Cys Gln Gly Leu Ala Gly Asn Leu Gly             100 105 110 Ala Met Val Ile Ser Leu Glu Phe Arg Leu Ala Pro Glu Asn Arg Leu         115 120 125 Pro Ala Ala Tyr Asp Ala Met Asp Asp Gly Leu Tyr Trp Ile Lys Ser     130 135 140 Thr Gln Asp Glu Trp Val Arg Lys Tyr Ser Asp Leu Ser Asn Val Tyr 145 150 155 160 Leu Phe Gly Ser Ser Cys Gly Gly Asn Ile Ala Tyr His Ala Gly Leu                 165 170 175 Arg Val Ala Ala Gly Ala Tyr Lys Glu Leu Glu Phe Val Lys Ile Lys             180 185 190 Gly Leu Ile Leu His Gln Pro Tyr Phe Ser Gly Lys Asn Arg Thr Glu         195 200 205 Ser Glu Glu Lys Leu Lys Asp Asp Gln Leu Leu Pro Leu His Ala Ile     210 215 220 Asp Lys Met Phe Asp Leu Ser Leu Pro Lys Gly Thr Leu Asp His Asp 225 230 235 240 His Glu Tyr Ser Asn Pro Phe Leu Asn Gly Gly Ser Lys His Leu Asp                 245 250 255 Asp Val Ile Ala Gln Gly Trp Lys Ile Leu Val Thr Gly Val Ser Gly             260 265 270 Asp Pro Leu Val Asp Asn Ala Arg Asn Phe Ala Asn Phe Met Glu Glu         275 280 285 Lys Gly Ile Lys Thr Phe Lys Leu Phe Gly Asp Gly Tyr His Ala Ile     290 295 300 Glu Gly Phe Glu Pro Ser Lys Ala Ala Ala Leu Ile Gly Ala Thr Lys 305 310 315 320 Asp Phe Ile Cys Ala Thr Thr Asn                 325 <210> 2 <211> 1217 <212> DNA <213> Capsicum annuum <400> 2 ttatctgtgt gatcaattat tatggctagc caaagttttg ttcctccaat ttttgaaaat 60 ccctttctta acattgaaga attagcaggt gacacaattg tacgtaaacc tgaacccctc 120 acacaagcca attctgatcc caatggcacg tccttagttg tatctaaaga cgtagacctt 180 gacatcaaca aaaagacatg gctgcgaata tacgtcccac aacgaataat cacaaatcat 240 aatgatgatg aaaaattgcc tgtcattttc tactaccatg gtggaggctt tgttttcttc 300 catgccaata gttttgcctg ggatttgttt tgtcaaggac ttgctggaaa ccttggggca 360 atggttatct cccttgaatt tcgtctggcc cctgaaaatc gccttcctgc agcttacgac 420 gatgccatgg atgggttata ttggattaaa tcaactcaag atgaatgggt ccgaaaatat 480 tcagatttga gtaacgttta tctttttgga tctagttgcg gtggaaacat agcttaccat 540 gcagggttac gggtagcagc tggggcatat aaagaactag agccagtgaa gatcaaaggg 600 ctaattttgc atcaaccata tttcagtgga aaaaacagga cagaatctga agagaagcta 660 aaggatgatc aacttttgcc attacatgca attgacaaaa tgttcgactt gtccttgcca 720 aaagggacac ttgatcatga tcatgaatat tccaatccat ttcttaatgg agggtccaag 780 catttagatg atgtgatcgc acaaggctgg aagattcttg taactggtgt ctctggagat 840 cctctggttg ataatgcgcg caactttgca aattttatgg aagaaaaagg cataaaaact 900 ttcaagctct ttggagatgg ttatcatgca attgaggggt ttgaaccatc aaaggcagca 960 gctttaattg gcgccaccaa agatttcata tgtgctacta caaattaaaa atatgtaacg 1020 tagcatcctg ctagcgttgt gtttgtttca tttccttcaa ataaatcaag tgagcttctt 1080 tgtgcaaata agaggggttt acaccctcct tcctgttaga gattacttta aaatattata 1140 tttctcttga agatcaaagt tttagagatg agttattgct gaaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa aaaaaaa 1217 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 atggagttct ctgtatcacc agtgg 25 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 tccgaatgtc taaagtggta caag 24 <210> 5 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 atgggctatt tgagatcatc t 21 <210> 6 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 tcacctctct gcaatcaata t 21 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 ctgacaagtc cacagcctca g 21 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 gttctttcca tgacaaccaa ttg 23 <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gggggatcca aaatggctcg ttcc 24 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctcggtaccc tttatttaat tttgtgtgac act 33

Claims (9)

In a pepper ester biological control substance having the amino acid sequence of SEQ ID NO: 1, the effective amount of the pepper esterase is a biological control substance, characterized in that 0.05 to 0.2 mg per milliliter The method according to claim 1, wherein the pepper esterase has a function of protecting the plant by releasing the cutin monomer produced by hydrolyzing the cuticle from the immature pepper fruit and inducing the production of hydrogen peroxide and expression of the PR gene. Biological control substances The plant protection function against infiltrating fungal pathogens such as anthrax and blast fungus of the pepper esterase biological control material of claim 1 is achieved by applying the esterase to the plant externally. The biological control material according to claim 2, wherein the cutin monomer is 9-octadecenamide. 4. The biological control substance according to claim 3, wherein the protective function against anthrax is based on 1,2-dithiane-4,5-diol, which is a dithiane compound produced by hydrolysis of anthrax cell wall. The method of claim 1, wherein the pepper esterase ⅰ) inhibits germination of fungal spores at a low concentration depending on the concentration of the esterase; Ii) biologically controlled substances characterized by their high anti-fungal function of inhibiting fungal penetration into the pepper fruit at high concentrations; The biological control substance according to claim 2, wherein the plant is a vegetable crop including red pepper. The red pepper esterase of claim 1 is produced by the expression of a microorganism comprising a recombinant plasmid containing a cDNA fragment of SEQ ID NO: 2, the genetic engineering method of biological control material The method according to claim 8, wherein the pepper esterase is partially purified by size classification of total protein from the microorganism and mixed with a formula KH1 comprising dithiothreitol (DTT). Genetic engineering method