KR20030068302A - Hypersensitive response elicitor from xantomonas axonopodis and use thereof - Google Patents

Abstract

PURPOSE: Provided is a hypersensitive reaction initiating factor (elicitor) polypeptide or protein which is applied to plants or plant seeds to give disease resistance thereto and prevent it from being damaged by harmful insects while facilitating the growth thereof. CONSTITUTION: A hypersensitive reaction initiating factor protein or polypeptide comprises DNA molecules having the nucleotide sequences of Sequence No. 1, and DNA molecules mixed with the DNA molecules in stringent conditions, and DNA molecules complementary to those DNA molecules. The hypersensitive reaction elicitor has amino acid with the amino acid sequences of Sequence No. 2. The polypeptide or protein is applied to plants or plant seeds in the non-infectious form under the conditions of being effective in acquiring disease resistance. The disease is derived from Xanthomonas axonopodis.

Description

Hypersensitivity reaction inducing factor derived from Xantonamonas axonopodis and its use {HYPERSENSITIVE RESPONSE ELICITOR FROM XANTOMONAS AXONOPODIS AND USE THEREOF}

The present invention relates to a hypersensitivity response inducer derived from Xanthomonas axonopodis and its use, and more particularly to isolated DNA that causes a hypersensitivity response in plants, a plant hypersensitivity response inducing polypeptide The present invention relates to an expression vector transformed by the DNA, a method of imparting disease resistance to plants using the hypersensitivity factor polypeptide, a method of promoting plant growth, and a pest control method.

Plants exhibit sophisticated defense mechanisms caused by germs and environmental stresses, which occur either systemically or locally. Looking at the defense mechanisms of these plants, the plant first starts from the process of recognizing the invasion of pathogens through the receptor. The signal thus recognized passes through an elaborate signal transduction system within the plant cell, and then, through the process of hypersensitive reaction (HR) and systemic acquired resistence (SAR) against a wide range of pathogen invasion, Will defend. The hypersensitivity reaction causes local necrosis in the plant tissue, and when the infected plant cells die, the bacteria can no longer spread. The reaction mechanism of this hypersensitivity reaction is not yet clear, but a signal is transmitted by binding the plant's receptor, the sensitizer, produced by the bacterium, ie an elicitor, so that various defense-related genes (defense-related) It is presumed that hypersensitivity reactions occur because genes are expressed.

The production of the eliminator of the plant's hypersensitivity reactions is regulated by a gene population called the hrp gene that all pathogenic bacteria have in common. To date, the hrp gene has been derived from Erwinia amylovora , Pseudomonas syringae pv.syringae , Ralstonia solanacearum , Xanthomonas campestris pv vesicatoria. Cloned. The hrp gene can be broadly divided into sites that regulate hrp gene expression, sites that express harpin protein, which is a hypersensitive reaction derivative, and sites that are involved in secreting the expressed harpin. There are three known harpin proteins, HrpZ from Pseudomonas syringae pv.syrinage, HrpN from Erwinia amylovora, and Ralstonia solanacearum. PopA.

Understanding the plant's self-defense mechanism can activate the genes associated with it, or isolate and express pathogen-related genes from the plant and microorganisms, thereby making the plant self-protecting from pests. Therefore, the development of biopesticides applying the biodefense mechanism of such plants is being actively made.

Messenger ^TM is manufactured alive the characteristics of a biopesticide began marketed by Eden Bioscience Corporation (Eden · Bioscience) residing in Washington, hapin (Harpin) protein. Harpin proteins are known to exhibit a systematic acquired resistence (SAR) against a wide range of pathogen invasion. Harpin has also been shown to increase early growth and harvest and to confer resistance to pests. Harpin protein is a collection of amino acids produced by bacteria that cause fire blight in apples and pears. When foliar spray is applied, the plant realizes that bacteria have begun to invade and activates the self-protecting functions in the body. However, such messenger should be used after diluting with tap water in actual application. When diluting with tap water, its performance is deteriorated by chlorine component in tap water.

One object of the present invention is to provide novel hypersensitivity response inducers derived from plant pathogens.

Another object of the present invention is to provide a plant sensitive protein or polypeptide.

Still another object of the present invention is an expression vector transformed with the isolated DNA molecule, a host transformed with the isolated DNA molecule, a transformed plant transformed with the isolated DNA molecule, and a transformed DNA molecule. To provide transgenic plant seeds.

Still another object of the present invention is to provide a method for providing disease resistance, a method of promoting plant growth, and a pest control method of the plant using the hypersensitivity response inducer protein or polypeptide.

1 is a genetic map of the hypersensitivity response inducing factor derived from Zantomonas axonopodis according to the present invention,

Figure 2 is an electrophoresis result showing the results of overexpression test for the identification of the hypersensitivity response inducing factor derived from Zantomonas axonopodis according to the present invention,

Figure 3 is a tobacco leaf photograph for showing the results of the hypersensitivity reaction test using the hypersensitivity response inducing factor derived from Xanthomonas axonopodis according to the present invention,

Figure 4 is a tobacco leaf photograph for showing the results of the hypersensitivity reaction test in the non-host host using the hypersensitivity response inducing factor derived from Xanthomonas axonopodis according to the present invention,

Figure 5 is a tobacco leaf photograph to show the results of comparing the stability of the Xanthomonas axonopodis-derived hypersensitivity reaction factor and commercially available Messenger ^TM in tap water according to the present invention.

Hereinafter, with reference to the accompanying drawings will be described in more detail with respect to the present invention.

The inventors have isolated the hypersensitivity response-related genes from the Xanthomonas axonopodis pv.glycines that cause plant hypersensitivity reactions similar to the known hrp genes. Was raised. These genes were named hpaG, which are involved in pathogenic at the host and hypersensitivity reactions at the non-host.

1 is a diagram showing the molecular structure of the genome region of Xanthomonas axonopodis pv. Glycines containing hpaG, a novel hypersensitivity inducer according to the present invention. The boxes and arrows in FIG. 1 represent genes or open reading frames.

The base sequence of the novel hypersensitivity inducing factor according to the present invention is as follows (SEQ ID NO: 1).

ATGAATTCTTTGAACACACAGCTCGGCGCCAACTCGTCCTTCTTTCAGGTTGACCCCGGCCAGAACACGCAATCTAGTCCGAACCAGGGCAACCAGGGCATCTCGGAAAAGCAACTGGACCAGCTGCTGACCCAGCTCATCATGGCCCTGCTTCAGCAGAGCAACAATGCCGAGCAGGGTCAGGGTCAAGGCCAGGGTGGTGACTCTGGCGGTCAGGGCGGCAATCCGCGGCAGGCCGGGCAGTCCAACGGCTCCCCCTCGCAATACACCCAGGCGCTGATGAATATCGTCGGAGACATTCTCCAGGCGCAGAATGGTGGCGGCTTCGGCGGCGGCTTTGGTGGTGGCTTCGGTGGCATCCTCGTCACCAGCCTTGCGAGCGACACCGGATCGATGCAG TAA (402bp)

The novel hypersensitivity triggers of the present invention, in addition to the isolated DNA molecules of SEQ ID NO: 1, DNA molecules that hybridize to DNA molecules comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions and the DNA molecules of SEQ ID NO: 1 Or DNA molecules complementary to DNA molecules hybridized to the DNA molecules of SEQ ID NO: 1. Stringent conditions are, for example, hybridization performed at a hybridization temperature of 60 ° C. or higher or at a buffer concentration of 0.1 × SSC upon washing.

The plant's hypersensitivity factor polypeptide (or protein) encoded by the isolated DNA causing the hypersensitivity in the plant of the present invention described above has the following amino acid sequence corresponding to SEQ ID NO: 2 in the Sequence Listing. The hypersensitivity inducer polypeptide or protein of the present invention, ie the HpaG protein according to the present invention, has a size of 13.4 kDa, a pi of 3.85, and is rich in glycine among amino acids and has heat stable properties.

MNSLNTQLGANSSFFQVDPGQNTQSSPNQGNQGISEKQLDQLLTQLIMALLQQSNNAEQGQGQGQGGDSGGQGGNPRQAGQSNGSPSQYTQALMNIVGDILQAQNGGGFGGGFGGGFGGILVTSLASDTGSMQ (133 amino acids)

The characteristics of the HpaG protein of the present invention are summarized in Table 1 below in comparison with other hypersensitivity triggers.

HpaG HrpN HrpZ sauce Xanthomonas axonopodis pv.glycines Erwinia amylovora Pseudomonas syringae pv.syringae size 402 bp (133aa) 1212 bp (403aa) 1023 bp (341aa) Molecular Weight 13.4kDa 39.7kDa 34.7kDa pI 3.85 4.44 4.01

The HpaG protein of the present invention is a natural protein produced by Xanthomonas axonopodis pv.glycines , and is believed to be involved in the signaling process that causes plant defense reactions. Since this signaling process is related to plant growth, disease resistance, and pest resistance, when the HpaG protein of the present invention is applied to a plant, it induces the plant to activate its growth and defense mechanisms, thereby promoting plant growth and various Confers resistance to diseases and pests. Suitable plants to which the present invention can be applied include both dicotyledonous plants and monocotyledonous plants. Examples of these plants include dicotyledonous plants such as cucumbers, tobacco, peppers, potatoes, tomatoes, lettuce, cabbages, radishes, sesame leaves, sesame seeds, paprika, bell peppers, eggplants, cotton, beans, and chrysanthemums, as well as rice, barley, wheat, and corn. Includes monocotyledonous plants.

Thus, the novel hypersensitivity reaction inducer according to the present invention can be used to impart disease resistance to plants, to promote plant growth or to control pests.

A method for imparting disease resistance to a plant according to one aspect of the present invention is directed to the hypersensitivity of the present invention from Zantonamonas axonopodis glycines under conditions in which a protein or polypeptide contacts diseased cells of a plant or plant seed to confer disease resistance. Reaction inducing protein, ie, HpaG, in a non-infectious form to a plant or plant seed. In the case of imparting disease resistance to plants using the novel hypersensitivity response factor of the present invention, the disease is not completely immunized against the disease, but the extent of the disease is reduced, the development is slowed, and the area of the lesion is reduced. Examples of plant diseases to which the disease resistance granting method of the present invention can be applied include diseases caused by Rhizoctonia solani , diseases caused by Phytophthora infestans , and pytopes. Disease caused by phytophthora capsici , Disease caused by Leveillula taurica , Disease caused by Gibberela zeae , Puccinia graminis Disease caused by Plasmodiophora brassicae , disease caused by Magnaporthe grisea , disease caused by Fusarium oxysporum , Disease caused by Valsamali , Disease caused by Glomerella cingulata , Botrytis Disease caused by Botrytis cinerea , disease caused by Cladosporium fulvum , disease caused by Ralstonia solanacearum , xanthomonas campestris ( Disease caused by Xanthomonas campestris pv.citri , disease caused by Streptomyces scabies , disease caused by Agrobacterium tumefaciens , Xanthomonas duck oryzae pv. oryzae ), the disease caused by Bukholderia glumae , the disease caused by Erwinia amylovora , Erwinia carotovora subsp . carotovora ), disease caused by Pseudomonas syringae pv.glycinea , disease caused by Xanthomonas axonopodis pv.glycine , Clavibacter michiganense subsp. michiganense ).

According to another aspect of the present invention, a method for promoting plant growth may induce a hypersensitivity reaction of the present invention derived from Zanthonamonas axonopodis glycines under conditions in which a protein or polypeptide contacts a cell of a plant or plant seed to promote plant growth. And applying the factor protein, ie, HpaG, to the plant or plant seed in non-infectious form. Plant growth promotion according to the present invention appears in various aspects, for example, effects such as singularity improvement and early maturation can be obtained.

A pest control method for a plant according to another aspect of the present invention is to induce a hypersensitivity reaction of the present invention derived from Zantomonas axonopodidis glycines under conditions in which a protein or polypeptide contacts a cell of a plant or plant seed to control a pest. And applying the factor protein, ie, HpaG, to the plant or plant seed in non-infectious form. The plants to which the hypersensitivity inducing factor according to the present invention are applied prevent the contact of the plants with the pests and kill the pests close to the plants.

The hypersensitivity-inducing factor polypeptides or proteins according to the invention can be used alone or directly applied to plants in the form of compositions comprising other additives. The composition may further include components such as fertilizers, insecticides, fungicides, buffers, wetting agents, coatings, and the like, in addition to the hypersensitivity reaction factor protein according to the present invention, HpaG as an active ingredient. Such a hypersensitivity response triggering polypeptide or protein composition may be applied to a plant or seed of a plant using known methods such as spraying, injecting, applying, dipping and the like.

Another method of imparting disease resistance to plants, promoting plant growth, or controlling pests using the novel hypersensitivity triggers according to the present invention is that the plant or plant seeds are separated into the DNA molecules of the present invention. To transform.

Isolated DNA molecules of the present invention can be introduced into cells using conventional DNA recombination techniques. In plants, reproductive plants can be re-differentiated from the transformed single cells, and thus the genes introduced into the plant cells can finally be passed down through the seed to generations.

Another aspect of the invention is an expression vector transformed with the isolated DNA molecule of the invention. Expression vectors usable in the present invention include various viral vectors or plasmid vectors. Preferred expression vectors include pUC18, pBluescript.

Host vector systems for expressing novel hypersensitivity response inducing polypeptides or proteins of the invention may include transformed bacteria, microorganisms such as yeasts, viruses, insect cell systems infected by viruses, plant cells infected by bacteria. have. Specifically, the E Coli-pBluescript host-vector system may be mentioned.

Recombinant molecules according to the present invention can be introduced into plant cells by a variety of methods. One of the methods of introducing the isolated DNA of the present invention into cells is a method using Ti plasmid. The soil bacterium Agrobacterium tumefaciens has a huge (about 200,000 base-pair) plasmid called Ti plasmid. When the bacteria come into contact with the plant cells, fragments called T DNA (about 23,000 base pairs) are transferred from the Ti plasmid to the nucleus of the plant cells and integrated at any one of the chromosomes of the plant during transformation. Similarly, Ri plasmids of Agrobacterium rhizogenes can be used.

Another method of introducing isolated DNA into plant cells is biolithics. Biological ballistics, also called particle bombardment, is a method of introducing small genes covered with or contained in foreign genes into plants to introduce the desired genes into plant cells, resulting in plant It is a way to achieve transformation. Biolithographic methods can be used to transform many plants, especially monocotyledonous plants, in which the Agrobacterium transformation method does not function.

Another method of introducing DNA is microinjection. The microinjection method is a method of injecting a small amount of material into a specific portion of the plant cell in the form of a protoplast from which the cell wall is removed through a very thin glass capillary tube using a micromanipulator attached to a microscope.

Another method is using liposomes. When the lipid solution is dropped into an aqueous solution of DNA or RNA, a lipid vesicle (liposome) containing a small amount of the DNA or RNA solution is formed by hydrophobic interaction between lipids. Liposomes containing DNA or RNA are fused with protoplasts because their chemical properties are similar to those of the plasma membrane or can be injected into specific internal organs (eg vacuoles) of plant cells by microinjection.

Another method of introducing the isolated DNA of the present invention into plant cells is by electroporation and the use of polyethylene glycol. The electroshock method introduces a foreign gene into a plant chromosome by inserting a plant protoplast into an aqueous buffer in which foreign DNA to be introduced is dissolved, and applying a strong and short current to instantaneously introduce the DNA into the protoplast. Polyethylene glycol (PEG) has a very strong absorption capacity, and when cells are placed in a high concentration PEG solution, the cell membrane is denatured, and the DNA added to the PEG aqueous solution is transferred into the cell.

Selective markers, such as antibiotics, are added to the in-flight regeneration medium to select transformed cells after introduction of foreign genes, and the transformed protoplasts are regenerated into plants. This regeneration method can be employed differently depending on the plant species, Evans et al., Handbook of Plant Cell, Vol. 1: known methods described in the literature (MacMillan Publishing Co., New York, 1983) and the like can be used.

Example

Hereinafter, the present invention will be described in more detail with reference to examples. However, the following examples are merely for illustrative purposes, and these examples should not be construed as limiting the protection scope of the present invention.

hpaG Gene overexpression

The clone that put hpaG into pT7-7 as the expression vector is pTJ1. E. coli BL21 (DE3), and then transformed into E. coli . Overexpress in large quantities in E. coli. First of all, E. bred on LB plate. A single colony of E. coli BL21 (DE3) pTJ1 is inoculated in 10 ml LB broth. Inoculated cells are incubated overnight (about 12 hours to about 16 hours) at about 230 rpm in a 37 ° C. shaking incubator. Secondary culture was carried out in 10 ml of 1 L LB broth cultured overnight and incubated in a shaking incubator at 37 ° C. The culture was centrifuged for 20 minutes at 6,000 rpm at 4 ℃. After centrifugation, the supernatant is discarded and the cell pellet is resuspended in 20 ml 50 mM Tris-HCl, pH 8.0. HpaG protein is present in this suspension, and the suspension is subjected to SDS-PAGE and hypersensitivity reaction (HR) tests to confirm the presence of HpaG. The results of these experiments are shown in FIG. 2.

2 is this. SDS-polyacrylamide gel electrophoresis of the overexpressed HpaG of the present invention expressed by E. coli BL21 (DE3) (pTJ1).

Referring to Figure 2, lane 1 is E. E. coli is a protein from BL21 (DE3) (pTJ1) and lane 2 is IPTG-derived E. coli . E. coli is a protein obtained from BL21 (DE3) (pTJ1). Lane 3 is IPTG-derived E. coli. After sonication obtained from E. coli BL21 (DE3) (pTJ1), the protein was heated at 100 ° C. for 10 minutes.

-SDS-PAGE test method

After assembling the BIO-RAD mini-gel unit, 15% separation gel (SDW 2.35 ml, 1.5M Tris-HCl, pH 8.8, 2.5 ml, 10% SDS 0.1 ml, 30% acrylamide solution 5 ml, 10% APS) 0.05 ml, TEMEI 0.005 ml) is poured to solidify. 4% stacking gel (SDW 2.44 ml, 0.5M Tris-HCl, pH 6.8, 1 ml, 10% SDS 0.04 ml, 3032 acrylamide solution 0.532 ml, 10% APS 0.02 ml, TEMED 0.004 ml) Pour in and insert the comb to harden for 1 hour. The prepared protein sample was mixed in the same amount with 2 × loading buffer (0.5M Tris-HCl pH 6.8, 100% glycerol, 10% SDS, 2-mercaptoethanol, 1% bromophenol blue, SDW), and then wells Load in After running the gel for 1 hour and 30 minutes at 50V, run the gel at 150V when the dye passes the concentrated gel and reaches the separation gel. When the dye has reached the end of the gel, it is stopped and the gel is shaken in a rocker for one hour in a staining solution (1.0 g of Coomassie Blue R-250, 450 ml of methanol, 100 ml of glacial acetic acid, 450 ml of SDW). While dyeing, destaining solution (methanol 100ml, glacial acetic acid 100ml, SDW 800ml) and desalted by shaking in a rocker. Rinse the gel four times, replacing the desalting solution every 30 minutes. When the desalination process is completed, exchange with distilled water and check the band by comparing it with a size marker.

-Hypersensitivity reaction test

The HpaG prepared by the above procedure is quantitatively diluted and injected into the leaves of the plant to be tested for hypersensitivity. At the time of injection, needle leaves are punctured on the back of the leaf, and then HpaG is injected into the leaf cell gap through the needle mark using a syringe. Hypersensitivity reactions can be determined to occur if necrosis occurs only in the injected part of the leaves within 24 hours after the injection. The cultured bacteria are washed twice with sterilized water, diluted with 0.5 to 0.5, and injected into the leaves.

Figure 3 is a photograph of tobacco leaves (cv. Samsun) showing the reaction in the state after 24 hours infiltrated by the following experimental procedure. Each number indicated on the leaf of Figure 3 was treated as follows.

1. IPTG-induced tooth. Cell extracts heat treated from E. coli BL21 (DE3) (pTJ1).

2. IPTG-induced teeth. Cell extract from E. coli BL21 (DE3) (pTJ1).

3. Surviving IPTG-induced teeth. E. coli BL21 (DE3) (pTJ1).

4. This. Cell extract from E. coli BL21 (DE3) (pTJ1).

5. Surviving Teeth. E. coli BL21 (DE3) (pTJ1) cells.

6. 50 mM Tris-HCl, pH 8.0

7. IPTG-induced tooth. Cell extract from E. coli BL21 (DE3) (pT7-7).

8. Surviving IPTG-Induced Tooth. E. coli BL21 (DE3) (pT7-7).

9. This. Cell extract from E. coli BL21 (DE3) (pT7-7).

10. To survive. E. coli BL21 (DE3) (pT7-7).

This experiment indicates that the polypeptide produced in the HpaG gene is a factor that triggers hypersensitivity. Results from panels 6-10 show that E. coli BL21 (DE3) (pT7-7) strains used in this experiment did not cause any hypersensitivity reactions to tobacco leaves. The E. coli BL21 (DE3) (pTJ1) strain, in which the HpaG gene was inserted, shows that the polypeptide is produced by IPTG and causes hypersensitivity. Panel 1 also shows that the polypeptide thus produced is highly stable to heat. Panel 5 indicates that the HpaG gene does not produce polypeptide in the absence of IPTG.

In conclusion, the polypeptide synthesized by HpaG gene is a factor that induces hypersensitivity reaction, and this gene is synthesized by IPTG, and the synthesized hypersensitivity factor is very stable to heat.

Sensitization test in non-host

4 shows the results of hypersensitivity test in non-host. Figure 4a is a photograph showing the results of hypersensitivity test in red pepper (cv. Chokwang), Figure 4b is a photograph showing the results of hypersensitivity test in tobacco (cv. Samsun), Figure 4c is a hypersensitivity in tomato (cv. Seokwang) A photograph showing the test results.

Panel 1 shows IPTG induced tooth. Treated with heat-treated sonicates from E. coli BL21 (DE3); Panel 2 is treated with sterile distilled water; Panel 3 shows the Xanthomonas axonopodis pv. Glycines 8ra ( X. axonopodis pv.glycines 8ra), panel 4 was inserted into the transposon Tn3 gus A to induce mutations in Xanthomonas axonopodis pv. Glycines 8ra 4-9 ( X. axonopodis pv. Glycines 8ra). Zantonas axonopodis pv. Glycines 8ra ( X. axonopodis pv. Glycines 8ra) induced hypersensitivity in pepper and tomato plants, but not in tobacco. HpaG protein also induced hypersensitivity in pepper and tobacco, but not in tomato. The test results of this hypersensitivity reaction are summarized in Table 2 below.

pepper tobacco tomato HpaG + + - Xag 8ra + _ + Xag 8ra hpaG :: Tn3gusA 70-1 + _ +

In the table, + indicates that the hypersensitivity reaction is induced, and-indicates that it did not cause the hypersensitivity reaction.

In this experiment, it was shown that purified HpaG according to the present invention acts as an eliminator in pepper and tobacco but not as an eliminator in tomato. On the other hand, Xanthomonas axunopodis, which possesses the biosynthetic gene HpaG, acts as an eliminator in peppers and tomatoes, and in tobacco, it can be seen that the produced polypeptides are not traced or excreted extracellularly, causing no hypersensitivity reactions. . And 70-1, a mutant with transposon [Tn3 gus A] inserted into the open reading frame (see Fig. 1), lacks the HpaG gene and loses pathogenicity, but it is still an eliminator that induces hypersensitivity in pepper or tomato. Tobacco did not trigger a response.

Through the results of Table 2, it can be confirmed that the polypeptide produced by HpaG acts as a hypersensitivity inducing factor to pepper and tobacco, and various substances can act as a hypersensitivity inducing factor by plant.

Comparative test of stability in tap water

Comparison of the hypersensitivity response inducer (HpaG protein) derived from Xanthomonas axonopodis according to the present invention with HrpN (Comparative Example 1) and HrpZ (Comparative Example 2) proteins contained in a commercial biopesticide (Messenger ^TM ) It was. The proteins used in this experiment were all overexpressed separately and used purified proteins, and stability and concentration comparison experiments were carried out in tap water, and the results are shown in Table 3 below.

Concentration test results in tap water of HpaG, HrpN and HrpZ Example ( HpaG ) Comparative Example 1 ( HrpN ) Comparative Example 2 ( HrpZ ) Concentration (㎍ / ml) time 100 10 One 0.1 100 10 One 0.1 100 10 One 0.1 4 hours + + - - - - - - - - - - 2 hours + (3) + (6) -(9) - -(3) -(6) -(9) - - - - - 1 hours + (2) + (5) -(8) - + (2) -(5) -(8) - - - - - 30 minutes + (1) + (4) -(7) - + (1) -(4) -(7) - - - - - 0 hours + + - - + - - - - - - -

In the above table, "+" means that the hypersensitivity reaction was induced in the non-host plants used, and "-" means not causing the hypersensitivity reaction. In addition, the number in parentheses indicates the panel number in FIG.

In Table 3, the time represents the elapsed time after dissolving in tap water before starting the hypersensitivity reaction test. In this experiment, it can be seen that the hypersensitivity inducer according to the present invention is 10 times more effective in concentration than the conventional hypersensitivity inducer and has very stable characteristics against tap water.

In this experiment, a commercial messenger (Messenger ^™ ) was used as a comparative example, and the HpaG protein lyophilized powder of the present invention was used as an example. The reagents prepared in this way were adjusted to the same concentration and injected into the leaves of tobacco (cv. Samsun) to observe whether hypersensitivity was induced for 24 hours. As a result, a photograph is shown in FIG. 5. In Figure 5, A is the result using the HpaG protein of the present invention, B is the result using the messenger.

As confirmed through the results of FIG. 5, the conventional Messenger ^TM (B) hardly induces hypersensitivity reactions after 1 hour when diluted in tap water, whereas the HpaG protein (A) of the present invention was 10-fold in tap water. Dilution did not deteriorate the performance due to the chlorine component in the tap water and showed a hypersensitivity reaction. Therefore, the HpaG protein of the present invention has the advantage that its efficacy can be maintained without deterioration even when diluted with tap water when used as a biopesticide for imparting disease resistance, promoting plant growth or controlling pests.

The novel plant hypersensitivity response factor polypeptide or protein according to the present invention, when applied to a plant or plant seed, can impart disease resistance to the plant, promote growth, and provide an effect of controlling pests. Moreover, this effect provides the advantage of not being degraded by tap water.

On the other hand, plants grown from plants or plant seeds transformed with the novel hypersensitivity inducing factor DNA according to the present invention have excellent disease resistance, pest resistance, and growth.

Claims (34)

(a) a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 1; (b) a DNA molecule that hybridizes to a DNA molecule comprising the nucleotide sequence of SEQ ID NO: 1 under stringent conditions; And (c) An isolated DNA encoding a factor protein or polypeptide that causes a hypersensitivity reaction of a plant selected from the group consisting of DNA molecules complementary to the DNA molecules of (a) or (b). A polypeptide causing a hypersensitivity reaction of a plant having an amino acid comprising the amino acid sequence of SEQ ID NO: 2. Expression vector transformed with the isolated DNA molecule of claim 1 A host transformed with the isolated DNA molecule of claim 1. A transformed plant transformed with the isolated DNA molecule of claim 1. A transformed plant seed transformed with the isolated DNA molecule of claim 1. A method of imparting disease resistance to a plant comprising applying the polypeptide or protein of claim 2 to a plant or plant seed in a non-infectious condition under conditions effective to confer disease resistance. 8. The method of claim 7, wherein said disease is a disease caused by Phytophthora infestans . 8. The method of claim 7, wherein said disease is a disease caused by Phytophthora capsici . 8. The method of claim 7, wherein said disease is a disease caused by Leveillula taurica . 8. The method of claim 7, wherein said disease is a disease caused by Gibberela zeae . 8. The method of claim 7, wherein said disease is a disease caused by Puccinia graminis . 8. The method of claim 7, wherein said disease is a disease caused by Plasmodiophora brassicae . 8. The method of claim 7, wherein said disease is a disease caused by Magnaporthe grisea . 8. The method of claim 7, wherein said disease is a disease caused by Fusarium oxysporum . 8. The method of claim 7, wherein said disease is a disease caused by Valsa mali . 8. The method of claim 7, wherein said disease is a disease caused by Glomerella cingulata . 8. The method of claim 7, wherein said disease is a disease caused by Botrytis cinerea . 8. The method of claim 7, wherein the disease is a disease caused by Cladosporium fulvum . 8. The method of claim 7, wherein said disease is a disease caused by Ralstonia solanacearum . 8. The method of claim 7, wherein said disease is a disease caused by Xanthomonas campestris pv. Citri . 8. The method of claim 7, wherein the disease is a disease caused by Streptomyces scabies . 8. The method of claim 7, wherein said disease is a disease caused by Agrobacterium tumefaciens . 8. The method of claim 7, wherein said disease is a disease caused by Xanthomonas oryzae pv. Oryzae . 8. The method of claim 7, wherein said disease is a disease caused by Bukholderia glumae . 8. The method of claim 7, wherein said disease is a disease caused by Erwinia amylovora . 8. The method of claim 7, wherein said disease is a disease caused by Erwinia carotovora subsp. Carotovora . 8. The method of claim 7, wherein said disease is a disease caused by Pseudomonas syringae pv. Glycinea . 8. The method of claim 7, wherein said disease is a disease caused by Xanthomonas axonopodis pv. Glycines . 8. The method of claim 7, wherein said disease is a disease caused by Clavibacter michiganense subsp. Michiganense . 8. The method of claim 7, wherein said disease is a disease caused by Rhizoctonia solani . A method of promoting plant growth, comprising applying a polypeptide or protein of claim 2 to a plant or plant seed in a non-infectious form under conditions effective to promote plant growth. A method of controlling pests of a plant comprising applying the polypeptide or protein of claim 2 to a plant or plant seed in a non-infectious condition under conditions for effectively controlling the pest. A composition comprising the polypeptide of claim 2 as an active ingredient.