KR20140041216A - Compositions for enhancing the plant innate immunity and use thereof - Google Patents

Abstract

The present invention relates to a novel composition to enhance the immunity of plants containing a gene carrier including nucleic acid molecules coding an amino acid sequence presented by sequence number 1, particularly, the composition enhances the resistibility to pathogens. If OsMEK2 gene is transformed into Oryza sativa and overexpresses, a lesion by rice blast is not formed and the expression of the genes related to a defense mechanism is induced. Thus, the composition containing the gene carrier inducing the overexpression of the OsMEK2 gene can effectively control the plant′s immune system without effecting the growth of the plant and yield if pathogens invade. In conclusion, the inventors suggest that the OsMEK2 is a protein specifically regulating the resistibility to pathogens, thereby having the potential to be a breeding material to a variety of resistance to plant diseases.

Description

Compositions for enhancing the immunity of plants and uses thereof

The present invention relates to a novel composition for enhancing immunity of plants and a use thereof.

Rice is a very important crop used as a stock of Asian people. It is becoming increasingly important for the development of biotechnology, as well as being used as a model plant to study the functions of genes and the action of genes in monocotyledonous plants. Fungus Magnaporthe oryzae ) or Magnaporthe grisea ) is a threat to rice cultivation around the world and this disease is a problem because it can reduce the probability of infection in the affected area by 10% to 30%. In particular, Pyricularia oryzae ) is an important disease that determines growth and yield. Chemical fungicides (pesticides) and control methods have been developed and used so far. However, it is necessary to overcome the toxicity, safety (residual problem) It has many drawbacks.

In order to solve the above problems, there has been attempted in some plants a molecular biologic effort to introduce a recombinant gene so that the plant has resistance to pests and diseases, and genes with optimal defense ability have been continuously studied. To date, various resistance genes have been identified through gene analysis based on chromosome maps (Proteins, Nucleic Acids, Enzymes, 37 (7), 1353 (1992) Is estimated to be present.

In rice, disease-resistant genes such as Kim et al., Biosci. Biotechnol. Biochem., 58 (6), 1164 (1894), Lipoxigenase (Japanese Patent Publication No. 06-225774) Alexine synthetase [Minami et al., Eur. J. Biochem., 185, 19 (1989)) and peroxidase enzymes (Ito et al., Plant Cell Report, 13, 361 (1994)), but demonstrate any association between these genes and disease resistance There was no report. In this connection, Korean Patent Laid-Open Publication No. 2001-0041277 discloses a novel non-toxic gene AVRI - CO39 from Magnaporthe grisea , a rice blast pathogen, and provides a method for enhancing rice blast resistance using the same , A Korean patent (No. 10-0839027) provides a transgenic plant including OsGlu2, a rice blast resistance gene, which directly protects against diseases of plants (rice) 0682129), a gene consisting of a nucleotide sequence coding for rice-derived OsCK1 and a method for producing the transformant, and a method for producing the transformant, which is resistant to rice blight blight and rice blast disease, 7,345,219) showed resistance to rice blast fungus in transformants overexpressing the MAPK5 gene. In contrast to the present invention, the prior patented invention provides a resistance gene for rice blast pathogens and a transgenic plant using the same, but the genes and resistance-inducing reaction mechanisms involved in rice blast resistance are different from those of the present invention, The way to solve the problem and its composition will be different.

In conclusion, tolerance to plant diseases, including rice blast disease, is highly complicated by pathogenic mechanisms of infection and resistance mechanisms in plants, and no progress has been made between laboratory-level tolerance and field-level tolerance, It is being studied in rice without difficulty.

Therefore, in the present invention, OsMEK2 (OsMAPKK2, Oryza sativa mitogen activated protein kinase kinase), which is a gene related to rice blast resistance, was screened by screening genes associated with the plant immunity response. The present inventors found that OsMEK2 ( OsMAPKK2 ) was overexpressed in rice to induce the expression of various defensive marker genes in rice blast fungus. Thus , the present inventors have found that the expression of OsMEK2 And to provide a novel immunomodulatory gene of rice which exhibits excellent resistance and a transformant using the same.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have tried to develop a composition for enhancing the immunity of plants. As a result, the present inventors have identified OsMEK2 , which is homologous to the amino acid sequence of MAP kinase (MAPKK), which is an immune response modulating protein of tobacco, Arabidopsis thaliana and maize, as a novel plant immunoreactivity modulator Thereby developing a rice blast resistance resistance improving composition. When the OsMEK2 gene was transfected and overexpressed in rice, no lesion was observed for rice blast, and the expression of defense-related genes was induced, thereby completing the present invention.

Accordingly, an object of the present invention is to provide a composition for enhancing the immunity of a plant.

Another object of the present invention is to provide a plant cell transformed with the composition for improving immunity of the present invention.

It is still another object of the present invention to provide a plant transformed with the composition for improving immunity of the present invention.

It is another object of the present invention to provide a method for producing a transgenic plant having improved pathogen resistance.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a composition for immunostaining a plant comprising a gene carrier comprising a nucleic acid molecule encoding an amino acid sequence of the first sequence of the sequence listing.

The inventors of the present invention have found that OsMEK2, which is homologous to the amino acid sequence of mitogen activated protein kinase kinase (MAPKK), which is an immune response modulating protein of tobacco, Arabidopsis thaliana and maize, As a new plant immunoreactivity modulator, we have developed a rice blast resistance resistance improving composition. When the OsMEK2 gene was transfected and transfected into rice, it was confirmed that the expression was induced in the same pattern as that in the case where the overexpression of OsMEK2 gene was shown to be resistant to rice blast disease and showed resistance to defense reaction-related genes.

Conventionally, a chemical disinfectant (pesticide) and a controlling method have been developed and used as a method for preventing the damage caused by rice blast disease, but there are many drawbacks to be overcome such as toxicity, safety (residual problem) Especially after the onset of rice blast disease, the growth and production of rice were greatly influenced by chemical spraying. The present inventors focused their attention on this point and developed a composition for enhancing the immunity of plants by selecting genes that effectively regulate the immune response to plant pathogens without affecting plant growth and production.

The inventors of the present invention for the first time confirmed that OsMEK2 overexpressing mutants using plant immunoconjugates of the present invention are resistant to rice blast and rice blight blight, and the plant immunity control technology of the present invention is OsMEK2 Induced overexpression of the defensive marker gene to rice blast fungus by overexpression of the gene, thereby promptly recognizing the pathogen and activating the defensive mechanism of rice blast.

Accordingly, the present inventors have provided a composition that effectively regulates the plant immune system by the presence of OsMEK2 at the time of pathogen invasion, without affecting the plant growth and yield at all using the composition of the present invention, thereby minimizing the influence of pathogenic bacteria In particular, we propose the possibility of minimizing the effect of the OsMEK2 gene on the growth of plants by using the gene related to the immunity regulation, especially the resistance to the pathogen, as a breeding material of a pathogen resistant variety of the plant.

As used herein, the term " immunity " means indicating resistance or resistance to biological or non-biological environmental stresses and is used interchangeably with defense response. Bacterial or pathogen, and the abiotic stress includes stress, such as salt, low temperature, drying or light (light). In the present invention, the immunity is preferably an immunity against biological stress , And more preferably means immunity to a pathogen.

The methods of the present invention can be applied to a variety of known plants and include, for example, food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep, grapes, citrus, persimmon, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; Or feed crops including ragras, red clover, orchardgrass, alpha-alpha, tall fescue and perennialla grass.

The composition for improving immunity of a plant of the present invention comprises a nucleic acid molecule encoding an amino acid sequence of the first sequence of the sequence listing. The first sequence of the sequence listing means the amino acid sequence of MEK2 (Mitogen-activated protein kinase kinase 2, MAPKK2) of rice (Oryza sativa), and the amino acid sequence encoded by the nucleic acid molecule and the MAP kinase The kinase amino acid sequence has at least about 50%, 60% or 70% homology, more preferably at least 75%, 80% or 82% homology, even more preferably at least 90%, 91% , 92%, 93%, 94%, 95%, 96% or 97% homology, even more preferably at least 98%, 99% or more homology. According to a specific embodiment of the present invention, the amino acid sequence is a cigarette (tobacco), Arabidopsis thaliana (Arabidopsis), and the corn MAP (Mitogen-activated protein) 67 % amino acid sequence of the kinase kinase protein and, respectively, 65% and 82% Lt; / RTI >

According to a preferred embodiment of the present invention, the nucleic acid molecule encoding the amino acid sequence of the first sequence of the sequence listing comprises a nucleotide sequence from the 1115th nucleotide to the 4680th nucleotide of the second sequence of the sequence listing, And the nucleotide sequence of the third sequence of the list. The second sequence of the sequence listing includes all the sequences of the OsMEK2 gene promoter, the 5'UTR (untranslated region) and the 3'UTR, and the third sequence of the sequence listing includes the open reading frame (ORF) Lt; / RTI >

As used herein, the term " open reading frame (ORF) " refers to an amino acid sequence or nucleotide sequence between a translation initiation codon and a translation termination codon of a coding sequence of a gene. The initiation codon and termination codon refer to a unit consisting of three adjacent nucleotides in a coding sequence that characterizes initiation and termination of protein synthesis.

In the present invention, the gene carrier carrying the open reading frame sequence may be a variety of gene carriers (vectors) known in the art, including, for example, plasmids and phages, The transporter is a plasmid.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

According to a preferred embodiment of the present invention, the gene carrier of the present invention comprises (i) a nucleic acid molecule encoding the amino acid sequence of the first sequence of the sequence listing; (ii) a promoter that is operatively linked to the nucleic acid molecule and that forms RNA molecules in plant cells; And (iii) a poly A signal sequence that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule.

The recombinant vector of the present invention includes a promoter operable in plant cells. The promoter suitable for the present invention may be any of those conventionally used in the art for transformation of plants. Examples of the promoter include a promoter such as a Cauliflower Mosaic Virus (CaMV) 35S promoter, a nopaline synthase (nos) promoter, A light tolerance promoter of sire KRAWS mosaic virus 35S promoter, a water crane basiliform virus promoter, a combellinella yellow moth virus promoter, a ribulose-1,5-bis-phosphate carboxylase small subituit (ssRUBISCO) (TPI) promoter, adenine phosphoribosyl transferase (APRT) promoter of the Arabidopsis, the octopine synthase promoter and the ubiquitin promoter of corn, and preferably the promoter of the Coliphrauren mosaic virus CaMV) 35S promoter.

As used herein, the term " operably linked " refers to a functional linkage between a nucleic acid expression control sequence (e.g., an array of promoter, signal sequence, or transcription factor binding site) and another nucleic acid sequence, The sequence will control the transcription and / or translation of the different nucleic acid sequences.

In the present invention, the recombinant vector has a poly A signal sequence (3'-non-detoxified region) that causes proper polyadenylation is derived from the nopaline synthase gene of Agrobacterium tumefaciens (nos 3 'end) (Bevan et al., Nucleic Acids Researcher , 11 (2): 369-385 (1983)), derived from the Octopine synthase gene of Agrobacterium tumefaciens, the 3 'end of the protease inhibitor I or II gene of tomato or potato, and CaMV 35S Terminator, preferably a CaMV 35S terminator.

Alternatively, the recombinant vector may further carry a gene encoding a reporter molecule (e.g., luciferase and beta -glucuronidase). In addition, the recombinant vector of the present invention can be used as a selection marker, for example, a gene resistant to antibiotics (e.g., neomycin, carbanicillin, kanamycin, spectinomycin, hygromycin) (e.g., neomycin phosphotransferase ( npt II ) Hygromycin phosphotransferase ( hpt ), etc.).

According to a preferred embodiment of the present invention, the composition of the present invention enhances immunity against a pathogen, thereby imparting pathogen resistance to the plant. The pathogen may be bacteria, fungi, yeast, , Oomycete and virus, more preferably bacteria or fungi.

The bacteria may be selected from the group consisting of Pseudomonas avenae subsp. Avenae, Pseudomonas andropogonis, Bacillus subtilis, Herniparasitic bacteria or Corynebacterium michiganens Corynebacterium michiganense pv. Nebraskense).

The fungus is called Magnaporthe oryzae , Magnaporthe grisea , Glomerella < RTI ID = 0.0 > graminicola Politis), global Melaka Lou Kumar linen System (Glomerella lucumanensis), Aspergillus across spline booth (Aspergillus flavus , Curvularia clavata , C. maculans ), Curvularia ( Curvularia inaequahs , or Curvularia lunata . Preferably, the fungus is selected from the group consisting of Magnaporthe oryzae or Magnaporthe grisea .

The viruses may be selected from the group consisting of the American Wheat Strain Mosaic Virus (AWSMV), the Barley Striped Mosaic Virus (BSMV), the Barrier Sulfur Atrophy Virus (BYDV), the Vein Macaques Mosaic Virus (BYMV), the Eastern Hemorrhoid Stain Virus (CCMV) , Rice black stripe virus (RBSDV), or maize mosaic virus (MMV).

The composition for improving immunity of a plant according to the present invention may be formulated into various forms, and examples thereof include a suspension, a solution, an emulsion, a dusting powder, a dispersible granule But are not limited to, water, wettable powder, emulsifiable concentrate, aerosol, microgranule or paste.

The composition for improving immunity of a plant according to the present invention may further contain a surfactant, an inert carrier, a preservative, a humectant, an encapsulating agent, a binder, an emulsifier, a dye, a UV protecting agent, a flow agent, But is not limited thereto.

According to another aspect of the present invention, there is provided a plant cell and a plant transformed with the composition for improving immunity of the present invention. The present inventors have made intensive efforts to develop new transgenic plant cells and plants and have constructed transgenic plant cells and plants resistant to rice blast or rice blight blight disease.

The transformed plant cells and plants of the present invention utilize the above-described composition for improving immunity of the present invention, and the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

To produce the transgenic plant cells and transgenic plants of the present invention, methods generally known in the art ( Methods of Enzymology , Vol. 153, (1987)). Plasmids can be transformed by inserting exogenous polynucleotides into vectors such as plasmids or viruses, and Agrobacterium bacteria can be used as a mediator (Chilton et al. Cell 11: 263: 271 (1977)), directly introducing exogenous polynucleotides into plant cells can be transformed with a plant (Lorz et ai Mol Genet 199: ... 178-182; (1985)). For example, when a vector not containing a T-DNA region is used, electroporation, microparticle bombardment, and polyethylene glycol-mediated uptake may be used. Commonly used in the transformation of plants are the Agrobacterium transformed with exogenous polynucleotides ( Agrobacterium < RTI ID = 0.0 > tumefaciens (see U.S. Patent Nos. 5,004,863, 5,349,124, and 5,416,011).

Transformation of plant cells or plants by Agrobacterium tumefaciens includes methods known in the art. Preferably, the transformation process comprises co-culturing the culture of Agrobacterium tumefaciens with cotton cotyledons. Whereby Agrobacterium tumefaciens is infected into plants. Plant cells or plants transformed by Agrobacterium tumefaciens are regenerated in the regeneration medium, which leads to successful regeneration of shoots. One of ordinary skill in the art can cultivate or plant transformed plant cells or seeds under planted conditions known in the art. Finally, transgenic plants are produced by roots of regenerated shoots in the rooting medium.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system.

The composition of the present invention is applied to various plants, but is preferably applied to rice. In the present invention, leaves suitable for gene introduction include any tissue derived from germinated seeds.

According to another aspect of the present invention, the present invention provides a method for producing a transgenic plant with enhanced pathogen resistance comprising the steps of:

(a) introducing the composition of the present invention into a plant cell; And

(b) obtaining a transgenic plant having enhanced pathogen resistance from the plant cell.

The method for producing transgenic plants with enhanced pathogen resistance can be understood as a method for enhancing pathogen resistance in plants, and the same is used in the present invention. The method for producing a transgenic plant having improved pathogen resistance according to the present invention uses the immunoconjugate composition of the present invention as described above. In order to avoid the excessive complexity of the present invention, It is omitted.

The method for producing the transgenic plant having enhanced pathogen resistance will be described in detail in each step as follows:

Step (a): transformation of plant cells

First, the plant immunity enhancing composition of the present invention is introduced into plant cells. The transformation can be carried out by the above-mentioned transformation method of the plant.

Selection of transformed plant cells can be carried out by exposing the transformed culture to a selection agent such as a metabolic inhibitor, an antibiotic and a herbicide. Plant cells that stably contain a marker gene that is transformed and conferring selectative resistance are grown and divided in the above cultures. Exemplary labels include, but are not limited to, hygromycin phosphotransferase gene, glycophosphate tolerance gene and neomycin phosphotransferase (nptII) system. Methods for the development or regeneration of plants from plant protoplasts or from various expansions are well known in the art. Development or regeneration of plants containing foreign genes introduced by Agrobacterium can be accomplished according to methods known in the art (see US Pat. Nos. 5,004,863, 5,349,124 and 5,416,011).

The plants to which the method of the present invention can be applied are not particularly limited. For example, the plants to which the method of the present invention may be applied include food crops including rice, wheat, barley, corn, soybeans, potatoes, wheat, red beans, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep, grapes, citrus, persimmon, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops including rice grass, red clover, orchardgrass, alpha-alpha, tall fescue and perennial rice.

In the method of the present invention, the term " plant (sieve) " is understood to include not only mature plants but also plant cells, plant tissues, plant seeds and the like which develop into mature plants.

Step (b): obtaining transgenic plants

 Then, a transgenic plant in which pathogen resistance is enhanced from the plant cell is obtained.

The transformed plants according to the present invention can be confirmed whether they are transformed by methods known in the art. For example, PCR using DNA samples from tissues of transformed plants can identify foreign genes inserted into the genome of the transgenic plant. Alternatively, Northern or Southern blotting can be performed to confirm the transformation (Maniatis et al., Molecular Cloning , A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989).)

According to a preferred embodiment of the present invention, the plant is rice, and the pathogen is rice blast fungus or rice blast fungus. In the examples of the present invention, when the above rice plants of the present invention were inoculated with the rice blast fungi or the rice blast fungus, resistance or tolerance to pathogenic bacteria appeared.

According to a preferred embodiment of the present invention, the rice blast fungus is Magnaporthe , more preferably Magnaporthe oryzae , Magnaporthe grisea , Magnaporthe poae , Magnaporthe rhizophila and Magnaporthe salvinii , more preferably the rice blast fungus is Magnaporthe oryzae or Magnaporthe grisea .

According to a preferred embodiment, the rice huinip blight fungus Santo Pseudomonas genus (Xanthomonas) and more preferably Santo Pseudomonas duck chair (Xanthomonas oryzae), and Santo and more preferably Pseudomonas ducks Ducks chair chair (Xanthomonas oryzae pv . oryzae , or Xanthomonas oryzae pv . oryzicola , and even more preferably, Xanthomonas oryzae pv . oryzae .

According to a preferred embodiment of the present invention, the transgenic plant having enhanced pathogen resistance induces expression of a defensive marker gene, and preferably OsPR1b , OsPBZ , OsPAL1, OsAPX1 or OsAPX2 expression is upregulated.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a plant immunity enhancing composition, and more particularly to a novel composition for enhancing resistance to a pathogen.

(b) When OsMEK2 gene is overexpressed by transfection into rice, the lesion does not appear for rice blast, and expression of defense-related genes is induced. Therefore, the composition of the present invention can effectively control the plant immune system by the presence of OsMEK2 upon pathogen invasion without affecting the plant growth and yield.

(d) The present invention therefore presents OsMEK2 as a pathogen resistance-specific regulatory protein, thus suggesting the possibility of a plant-resistant strain as a breeding material.

Figure 1 shows the amino acid sequence alignment results between OsMEK2 (rice MAP kinase kinase) and an orthologue.
The amino acid sequence was aligned using the ClustalW2 program. OsMEK2, (Accession No. NP_001056806.1); ZmMEK2, corn (Accession No. ACN31610.1); NbMEK2, Nicotiana tabacum (Accession No. AAF67262.1) and AtMEK2, Arabidopsis thaliana (Accession No. NP - 194710.1).
Fig. 2 schematically shows transcripts of the OsMEK2 overexpressing mutant (OsMEK2 OX). The left panel of Figure 2A shows that the OsMEK2 OX transgenic plant was constructed as a recombinant vector under the CaMV 35S promoter containing a hygromycin selection marker. LB, T-DNA left boundary; RB, T-DNA right border; HPT, hygromycin; NT, Terminator; MCS, multiple cloning site. The right panel of FIG. 2A is a result of real-time PCR analysis of wild type rice (Nipponbare varieties) and transcripts of four OsMEK2 OX lines. Total RNA was extracted from wild-type and OsMEK2OX, cDNA was synthesized and real-time PCR analysis was performed. FIG. 2B shows the results of analyzing transcripts of the OsMEK2 OX line at different time points after inoculation of the pathogen. The OsMEK2 OX transcript was induced within one hour after inoculation of the pathogen.
Figure 3 shows the results of a pathogenicity test on four independent OsMEK2 OX transfection lines inoculated with pathogens. Magnaporthe oryzae ) infection, the severity of the disease in the transformation line was measured. Photographs were taken at 5 dpi (days post inoculation) after infection with KJ201. WT (NB) represents the wild-type Nipponbare varieties, and OX1, OX2, OX3 and OX4 represent four independent transgenic lines (OsMEK2 OX).
Figure 4 quantitatively expresses the expression level of five defense marker genes over time after inoculation with two independent OsMEK2 OX lines (OsMEK2 OX1 and OsMEK2 OX2) and wild type rice (Nipponbare varieties). The top panels of Figures 4A-4E are the results for OsMEK2 OX1 and the bottom panels are for OsMEK2 OX2. FIGS. 4A to 4E show the expression levels of OsPR1B, OsBPZ, OsPAL1, OsPX1 and OsPX2 genes, respectively, in (4a) and (4b), respectively. The measured value was normalized to the expression level of 18s rRNA, and the above test was repeated three times to show an error bar.
Figure 5 shows that OsMPK1 and OsMPK6 expression was increased in the OsMEK2 OX line (OsMEK2 OX1 and OsMEK2 OX2) after inoculation of the pathogen. The top panels of Figures 5A-5B are the results for OsMEK2 OX1 and the bottom panels are for OsMEK2 OX2. In Fig. 5A, OsMK2 OX was induced to express OsMPK1 within 1 hour after inoculation of the pathogen. In Fig. 5b, OsMEK2 OX was induced to express OsMPK6 within 1 hour after inoculation with the pathogen. Total RNA extraction, cDNA synthesis, and real-time PCR were performed according to the experimental materials and experimental methods of the following examples.
FIG. 6 shows a schematic diagram of the correlation between OsMEK2, OsMPK1 and OsMPK6.
Fig. 7 shows the phenotype observed on day 10 after inoculation with PXO099, a rice blast fungus strain, on leaves of OsMEK2 KO transgenic plants. Figure 7a shows the results for OsMEK2 KO5 and Figure 7b shows the results for OsMEK2 KO1. The two OsMEK2 KO lines were shown to be resistant or resistant to PXO099 as compared to the control.
Fig. 8 shows the result of observing the phenotype on day 10 after inoculation of OsMEK2 OX transformant with PXO099, a rice blast fungus. 7A to 7D show the results for OsMEK2 OX1, OsMEK2 OX1, OsMEK2 OX2 and OsMEK2 OX5, respectively.
Fig. 9 is a result of quantifying the lesion area of rice blast fungus on the 10th day after inoculation of OsMEK2 OX transformant with PXO099, a rice blast fungus. OsMEK2 OX (2-8-6-2) It can be seen that three OsMEK2 OX lines except 5 lines have resistance or resistance to PXO099.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Materials and Experiments

Vector construction

To construct an over-expression vector for the OsMEK2 gene, a complete open reading frame (ORF, sequence listing No. 3) of the OsMEK2 cDNA clone (1173 bp) present in the pBluescript SK-vector and the downstream of the cauliflower mosaic virus (CaMV) 35S promoter (SEQ ID No. 4) was inserted into the multicloning site of the pCambia 1300 binary vector (pCambia vector http://www.cambia.org/daisy/cambia/585.html).

Construction of Transgenic-OsMEK2 overexpression

After transforming the wild type rice ( cv. Nipponbare , Japonica type) with Agrobacterium tomefaciens strain LBA4404 containing OsMEK2-overexpressing binary vector (35S: OsMEK2), some modified standard procedures were performed Hiei et al., 1994). Transformed calli were screened by incubating with increasing concentrations of hygromycin (30 mg - 60 mg hygromycin / l). After shooting and rooting in the calli after transformation and selection, the seedling was immersed in water and allowed to acclimate for 3-4 days and then grown in the soil of the greenhouse. The hygromycin resistance of T1 line seeds was analyzed and hygromycin-resistant seedlings were used for functional analysis and T2 homozygote screening.

Pathogenicity Test

For the pathogenicity test, seeds of japonica type wild type rice and OsMEK2 overexpressed rice (OsMEK2 OX) were germinated on a petri dish containing distilled water at 28 DEG C for 3 days. Then, the cells were transplanted into the soil and cultured in a tissue culture incubator for 2 weeks. Tissue culture conditions were as follows: 12 hours light period, 28 ° C, 70% relative humidity. The cultured medium for 2 weeks was inoculated by spraying a spore of Magnaporthe oryzae (Affinity Race KJ201), a rice blast fungus, with an air compressor to 5 x 105 conidia / ml. The conidial spore suspension was prepared as a strain cultured for 14 days with 0.05% (w / v) Tween-20 on 25 ° C oatmeal agar under continuous light conditions. The inoculated plants were cultured at 25 ℃ for 24 hours under dark conditions and then cultured under the normal growth condition at 28 ℃. Rice blast disease occurred within 4 to 6 days after inoculation. Leaves were collected at regular intervals after inoculation, and leaves collected before inoculation were used as control (0 hr). All samples were immediately frozen under liquefied nitrogen and stored at -80 ° C. The severity of rice blast disease was measured at day 5 post-inoculation (dpi, days post inoculation) according to disease index scale at stage 6.

Transcription analysis by quantitative real-time polymerase chain reaction

As a pathogenic test, a real-time PCR (Stratagene, Mx3000P) was performed to confirm expression of the resistance marker gene. Total RNA was isolated from rice tissue using a triazole according to the Invitrogen protocol and quantitated by absorption spectroscopy. Before real-time polymerase chain reaction, total RNA samples were treated with DNAase (DNase) without RNAse. The first strand of the cDNA was synthesized in a 20 μl reaction mixture according to the manufacturer's protocol using a cDNA synthesis kit (Invitrogen). In the synthetic reaction, 2 μg of RNA isolated from rice leaves collected after a certain period of time in rice plants inoculated with rice blast fungus Magnaporthe grisea KJ201 was used. Real-time PCR was performed using 1 cDNA of cDNA as a template. Stratagin master mix (Brilliant III Ultra-Fast SYBR Green QPCR Master Mix, Stratagene) was used as a reaction reagent. 18S rRNA was used as a control.

Experiment result

Discovery of OsMEK2 as a positive regulator of positive response through phylogenetic analysis

We performed a phylogenetic analysis to find the closest orthologue to OsMEK2 from other plant species providing functional clues to OsMEK2. To this end, the present inventors have carried out an amino acid sequence analyzes among tobacco (tobacco), Arabidopsis thaliana (Arabidopsis) and MAP (Mitogen-activated protein) kinase kinase protein and of corn protein OsMEK2 (Fig. 1). As a result, OsMEK2 showed 67%, 65% and 82% homology with Tobacco, Arabidopsis and corn MAP kinase kinase proteins, respectively. Previous studies on Tobacco MAP kinase kinase (NbMEK) and Arabidopsis MAP kinase kinase (AtMEK2) as the closest homologous gene to OsMEK2 have been shown to actively involve these MAP kinase kinases in the defense response (Jin et al , 2003, Brader et al., 2007). These studies demonstrate that OsMEK2 can function as a positive regulator in the disease resistance pathway of rice.

OsMEK2 expression induced by pathogen inoculation

To demonstrate the function of OsMEK2 in defense mechanisms, the present inventors conducted transgenic transgenic tests in rice. To this end, a transgenic plant comprising the CaMV35S: OsMEK2 construct was constructed and named OsMEK2 OX (Fig. 2A). In general, the phenotype of OsMEK2 OX was very similar to wild-type individuals. We selected four independent OsMEK2 OX lines (OX1, OX2, OX3 and OX4) based on the OsMEK2 transcription pattern for functional analysis (Figure 2A). After inoculation of the virulent strain (KJ201), OsMEK2 OX lines induced OsMEK2 transcription rapidly (within 1 hour) compared to wild-type and MOCK-treated controls (FIG. 2B). These results demonstrate that OsMEK2 is a pathogen-induced MAP kinase kinase that is involved in positive regulation of the defense mechanism.

OsMEK2 Overexpression of Magnaporte In Oriza (Magnaporthe oryzae, KJ201)  Increased resistance to

To analyze the OsMEK2 function in the defense mechanism, four independent OsMEK2 OX lines (OX1, OX2, OX3 and OX4) and wild type (Nipponbare varieties) rice were inoculated with rice blast fungus strain KJ201. The phenotype of the disease was observed at 5 dpi (days post inoculation) (Fig. 3). Wild-type individuals showed severe disease symptoms with lesions from 3 dpi, but OsMEK2-overexpressed individuals did not show any symptoms within 5 dpi (Fig. 3) and showed almost no disease symptoms after 5 dpi. Thus, the present inventors have demonstrated that the OsMEK2 OX line improves the resistance to Magnaporthe oryzae early.

Pathogen-inoculated OsMEK2 OX  Defense from objects Marker  Up-regulation of genes

Five different defense marker genes ( OsPR1b , OsPBZ , OsPAL1, OsAPX1) in pathogenic-inoculated wild-type and OsMEK2 OX individuals And OsAPX2 ) were analyzed by time-based quantitative expression analysis. As a result, a rhythmic pattern was observed. OsPR1b transcription was induced within 1-3 hours after inoculation at very early time, whereas transcription of OsPBZ and OsPAL1 was induced within 1-24 hours after inoculation (Figures 4a, 4b and 4c). The other defense marker genes, OsAPX1 and OsAPX2, were introduced relatively late, i.e. within 48 hours after inoculation (Figures 4d and 4e). These results show that OsMEK2 actually functions as a positive regulator of defense mechanisms by inducing the activity of various defensive marker genes and thus the presence of OsMEK2 is important for rapid cognitive and defense against plant diseases have.

OsMEK2 OX  By pathogen inoculation of the individual OsMPK1  And OsMPK6  Induction increase

The present inventors have previously confirmed that OsMPK1 and OsMPK6 act as OsMEK2 interactors by yeast two hybrid assay (Singh et al., 2012). In the present invention, by confirming that OsMPK1 and OsMPK6 were induced within 1 hour of infection, OsMEK2 OX line showing high resistance to pathogenic bacteria was constructed (FIGS. 5A and 5B). These results show that OsMPK1 and OsMPK6 are involved in defense signaling and should be confirmed in future studies.

rice plant Blight of white blight  Phenotype change by inoculation

Xanthomonas oryzae pv . oryzae) suspension of the strain PXO099 (OD ₆₀₀ = 0.4) using, over-expression or knock the OsMEK2 - out (Scissors-dip method in the knock-out) Germination (booting) step in which transformed object (Song et al., 1995) Lt ; RTI ID = 0.0 > PXO099 . ≪ / RTI > PXO099 The strain is a strain showing strong virulence in wild type rice (cv. Nipponbare, japonica type), OsMEK2 KO is a gene deletion mutant, OsMEK2 OX is a gene knock-out mutant. The control (OsMEK2 overexpressing individual) was inoculated only with water, and the lesion length was measured using a method known in the art (Chern et al., 2005).

 As a result, as shown in FIG. 7 and FIG. 8, there was almost no lesion of the rice blast fungus in the control group, but it was confirmed that the rice leaf blotting occurred in the OsMEK2 KO (FIG. 7) and OsMEK2 OX (FIG. 8) mutants.

FIG. 9 is a graph showing the lesion site lengths of the OsMEK2 KO and OsMEK2 OX mutants measured. The KO 1 and KO 2 lines showed a larger lesion area than the control group, and the OX 1, OX 2 and OX 4 lines also showed a larger lesion area than the control group. The OX 3 line showed less lesion sites than the control, suggesting that the OX 3 line is specifically resistant to PXO099 , a blight of rice blight.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Sejong University Industry Academy Cooperation Foundation <120> Compositions for Enhancing the Plant Innate Immunity and Use          the <130> PN120402 <160> 4 <170> Kopatentin 2.0 <210> 1 <211> 353 <212> PRT <213> Artificial Sequence <220> <223> Amino Acid Sequence of OsMEK2 <400> 1 Met Gly Lys Pro Gly Lys Leu Ala Leu Pro Ser His Glu Ser Thr Ile   1 5 10 15 Gly Lys Phe Leu Thr Gln Ser Gly Thr Phe Lys Asp Gly Asp Leu Leu              20 25 30 Val Asn Lys Asp Gly Leu Arg Ile Val Ser Gln Ser Glu Glu Gly Glu          35 40 45 Ala Pro Pro Ile Glu Pro Leu Asp His Asn Gln Leu Ser Leu Asp Asp      50 55 60 Leu Asp Ala Ile Lys Val Ile Gly Lys Gly Ser Ser Gly Ile Val Gln  65 70 75 80 Leu Val Arg His Lys Trp Thr Gly Gln Phe Phe Ala Leu Lys Val Ile                  85 90 95 Gln Leu Asn Ile Gln Glu Asn Ile Arg Arg Gln Ile Ala Gln Glu Leu             100 105 110 Lys Ile Ser Leu Ser Thr Gln Cys Gln Tyr Val Val Ala Cys Cys Gln         115 120 125 Cys Phe Tyr Val Asn Gly Val Ile Ser Ile Val Leu Glu Tyr Met Asp     130 135 140 Ser Gly Ser Leu Ser Asp Phe Leu Lys Thr Val Lys Thr Ile Pro Glu 145 150 155 160 Pro Tyr Leu Ala Ala Ile Cys Lys Gln Val Leu Lys Gly Leu Met Tyr                 165 170 175 Leu His His Glu Lys His Ile Ile His Arg Asp Leu Lys Pro Ser Asn             180 185 190 Ile Leu Ile Asn His Met Gly Glu Val Lys Ile Ser Asp Phe Gly Val         195 200 205 Ser Ala Ile Ile Ala Ser Ser Ser Ala Gln Arg Asp Thr Phe Thr Gly     210 215 220 Thr Tyr Asn Tyr Met Ala Pro Glu Arg Ile Ser Gly Gln Lys His Gly 225 230 235 240 Tyr Met Ser Asp Ile Trp Ser Leu Gly Leu Val Met Leu Glu Leu Ala                 245 250 255 Thr Gly Glu Phe Pro Tyr Pro Pro Arg Glu Ser Phe Tyr Glu Leu Leu             260 265 270 Glu Ala Val Val Asp His Pro Pro Pro Ser Ala Pro Ser Asp Gln Phe         275 280 285 Ser Glu Glu Phe Cys Ser Phe Val Ser Ala Cys Ile Gln Lys Asn Ala     290 295 300 Ser Asp Arg Ser Ser Ala Gln Ile Leu Leu Asn His Pro Phe Leu Ser 305 310 315 320 Met Tyr Asp Asp Leu Asn Ile Asp Leu Ala Ser Tyr Phe Thr Thr Asp                 325 330 335 Gly Ser Pro Leu Ala Thr Phe Asn Thr Ser Asn Arg Tyr Asp Asp Arg             340 345 350 ***     <210> 2 <211> 5028 <212> DNA <213> Artificial Sequence <220> <223> OsMEK2 total DNA sequence including promoters, intron and 3'UTR <400> 2 aatagtatgt agaatggtga ttcaaactaa caagtaaggt ggtatgactt tatggaagaa 60 gaaaaataga gagatagttt ggaccgttga ttaatcatct aaaggctaaa aacaattgat 120 gtgatataac ttaattagag gaaaaaaagg agaaatataa tttggatcat agattaatca 180 tttaagcact aactaagcga ggatgaacta tataagtata taggatatca taaaacataa 240 taaagatata cattgaaaca ttatgtgaat tatgttggat gataatttaa catgtttgta 300 tttgaaaagc tcttaaatta taaaaactat aaagatatag catgtttgca cgatgtttaa 360 atgtgatgat tgttagtgga taatgatgtg gcatcttgtt aattagtgga tgatgatgtg 420 gcatcttgtt agtagataat gatgttgcat ctttgtatat taagcttaaa gagttagtgg 480 aggataattt tatagtaaga taaaattcca tctgtaacaa ctaacggtct taacctgcct 540 atgtaaataa tacccgccat atttatccgt attttatttg acaaaatact tcagtacaat 600 tttagattat ttcaccccca atattgggtg tacaactgta catcgcaata gtgatggccc 660 tagatagtgt tgcactatat gtgtgtcttt caaggcattg accaaaacac acacgtgccc 720 aagctgcaat ttatctcacc atttcagatt atgagaatct tcaattctcc tagacagcaa 780 agaaaaagca tatacaattt tacatcacca ttttcagatt aggagaatct tcaatagaca 840 tcaaagaaaa agcatatgca aattttacct cacaatttca gattaggaga atattcaaaa 900 gacagctaat tagaaaaaaa aagagagtta aaaaaaccca taataaaaaa agcaaaagga 960 gactagtact agcaaggcta gccagcccat ggtgttcgac ttcgtctcca tttcgcgtct 1020 cctcctcctc ctcctccggc catctcaccc tcccggcgag cagcgagcgg gaggcctccg 1080 ccaccgccgc cgccgccgcc gccgtcgccg cccgatgggg aagccgggga agctagcgct 1140 gccctcgcac gagtcaacca tcggcaaatt cctgtgatct cccgttcccc ccatctctct 1200 ctctcccgct tttccgtgtc ctctgctgct cgcatcacgc ttgctgttgt tgttggatgt 1260 gtgttgcttg tgctgattcc ggtggtgttt cgggggtttg ggttttcggg ctgcaggacg 1320 cagagcggga cgttcaagga cggcgatctg ctcgtcaaca aggacggcct ccgcatcgtc 1380 tcgcagagcg aggaagggga ggtaaagtca ccagcggaaa tgatatagtt tcacctgttt 1440 ggtattccgt ttggcaatcg taggagtgaa ctcgccatgg ttggatcttc tcttgggttt 1500 tgttgaggag atgaaggtgt gaattggatg atgggcgggt cgctagcggc gttcctggat 1560 tcactctgga gtgaattgtt gttgagctct gattatattc tggattggaa tgtcatgcgt 1620 ttggcaatca gggtgctgtt tgcctgtttg gtaatagggc agatgcatac gctaattttc 1680 tacattaaaa tgtgccaata atttgcaacc atttttatat ggtagtgaag aaatcctgaa 1740 atagtttaat tcacattttc taatccacag cgaactagga taggaaacag catgcctttt 1800 tatcttcatt gtttcgatga gcgtcatact tatatagtgg tttctcatgt ggagtgtcta 1860 gacactaact agtggtatct gcatatgcaa taaattactg aaagcatata tgctgcatct 1920 ggtagctctg ttcctggtgc tcacttatag tgtgagaatg ttctttaata ctatcattac 1980 tttttgtact ctccattcat ttaatatgtt cttttatttt gtgtactcac cttctctcta 2040 ttggcatgct aaaattgttg caggcccctc ctatagaacc tttagatcat aatcagttga 2100 gtctagatga tctagacgca atcaaagtta tcggtaaagg tagtagtgga atcgtgcagt 2160 tggttcgcca caaatggact ggccagtttt ttgctctgaa ggtattatat gaaagtacat 2220 tctttgcttt ttatgatttc ttccactctc cccttaccag ttttaactac aatcttttta 2280 gtgttcccaa tagttatagc ttcatgtgag tccagtgttt tttcatgtgg tcttttgcct 2340 aacacttgca aaggatcttt agttctttac ctccattgat atattattgc gtttacaggt 2400 aatacaactc aatatccagg agaatatacg cagacagatt gcgcaggaat tgaaaatcag 2460 cttgtcaaca cagtgccaat atgttgttgc atgctgtcag tgtttttacg ttaatggtgt 2520 catttcaatt gttttggagt atatggacag tggctctctc tcagatttcc tgaagacagt 2580 taaaaccatt ccagagccct accttgctgc aatctgtaag caggccagtg acatgtggta 2640 gaagctatca cacaatttct ttgctttgca aatgttactt aagcaccaca tggactttat 2700 aataaaaata aataaatctt tcctgcgcag gtgctaaaag gactgatgta cttacatcat 2760 gagaagcaca tcatacaccg agatctgaag ccgtcaaata tattaataaa tcatatgggt 2820 gaagtaaaaa tatccgactt tggtgttagt gccatcattg ctagttcctc ggcacaacga 2880 gatacattta ctgggacata taactacatg gcggtgcgta aaagtttctg tgtcatgaaa 2940 tgtatctctg attattgcat gaaatgctat ctattttgtt tgtcttgttg aaccaaaggg 3000 aaattctgtt cttgacagcc agaaagaatc agtgggcaaa aacatggtta tatgagtgat 3060 atctggagct tgggcctcgt tatgctagaa ttggccactg gtgaatttcc atatcctcct 3120 cgtgaaagct tttatgaact ccttgaagct gttgtcgacc acccaccacc ttctgctcca 3180 tcagaccagt tttcagagga attctgttca ttcgtctctg catggtaatt gtacttcttt 3240 gatgatttct cttatccact tacatttgca gtactagcaa tttgttttaa tttttagttt 3300 acaatcacgt gcttctaata tcccaagttg gtgcctgttt tatacatttt acatgcaaaa 3360 tttaagtgaa aaggtgacta ccttcagagt gtgtttggta ccctgtatgt cattcagcct 3420 ggcttgcatc agcagagctt gcccaaccag tcctgtgtag atgctgaaaa ttgacaagtt 3480 tagttacatg cttttgctta gctaggccat gtagggagtt tgtttggttt acagcatgta 3540 tacgtgcaaa gcccaaaata tatctttgga gccagtgcat gccagcaatt tcgtgctctc 3600 accagcaggc cagcaggaaa cggattctga atacggaaaa cgtttctatg gggccatgcc 3660 catacctcac tttggaatgc gcagggattg gcccatccaa tcacacagaa aaccaaacat 3720 tggaactgtt ccagggaaca aatgccccct tactgccttg gtatgcagat tgtagggacc 3780 caattgatgc atggcgggat tacacccaac taaatcattt attgtgtggc gtgttccata 3840 ctccctccat acttataaag gaagtcgttt aggacaatat ttaagtcaaa ccttgagaat 3900 ataaatcgtg aataactctc aagttgttga gtttgaaaat ataaaaatta tatgtataga 3960 tttgtcttga aaaatacttt cataaaagta tgcatatatc actttttaat aaatattttt 4020 atagaaataa gaagtcaaat ttgtgttttg gagaccgtgt tgctttccta aacgactttc 4080 tttacgagta tggagggagt acttcttttc ttgagatatt tttgttactc ttacatgtga 4140 accaaatagg tctaatgtct ttcaactctt catattcata tggactttca atgttaagaa 4200 ggctgctaag ttttataaag ggaaataaat aaataaacag ggctaataca gttcctctgt 4260 ctgttctgtg ggttgcagta tccaaaagaa tgcttcggat aggtcatctg cacaaatctt 4320 attagtaagt gaaaattctt atccacgctg ttctttttaa ggcatctttt gcacaagtct 4380 agctcactga tcttgttgcg tcctccctct gctctttgat acccagaatc atccattcct 4440 gagcatgtat gacgacctga atatagatct tgcttcgtac ttcacaaccg atggatctcc 4500 gcttgccacc ttcaatacaa gtaatgatga gcaacttgtc ttttttcttt gtttttgcca 4560 tccattttgt catagacaca tgttagtgcc attctttatg attccagtgg atgaaccatc 4620 aagggaacct tacattcttt tttttcgtag gcaaccgata tgatgacaga tagtctgatg 4680 caacactgcg tcatcatcta gctcgaagga tgatcctttg gctgtccatc tgtagaatct 4740 accatctacc taactggccc caaatgtttc gtgtattttt tgggagaaaa ctttgccctg 4800 tgtactgagc tgggattgat gtctttgata tgtaaatttc gcgtaatttc atatgtagaa 4860 caattctagg atcattgggc attgaagttt tgtttggccg tgcaccagga tgatctgttc 4920 ttgatcgctt tacctttgtt tctcacttct gtaactaaaa cctatattta ccatttaccg 4980 gattgttcat ggcctcttct gggcaagtcg gtgtcgactg aaagtctc 5028 <210> 3 <211> 1059 <212> DNA <213> Artificial Sequence <220> <223> Open Reading Frame DNA sequence of OsMEK2 <400> 3 atggggaagc cggggaagct agcgctgccc tcgcacgagt caaccatcgg caaattcctg 60 acgcagagcg ggacgttcaa ggacggcgat ctgctcgtca acaaggacgg cctccgcatc 120 gtctcgcaga gcgaggaagg ggaggcccct cctatagaac ctttagatca taatcagttg 180 agtctagatg atctagacgc aatcaaagtt atcggtaaag gtagtagtgg aatcgtgcag 240 ttggttcgcc acaaatggac tggccagttt tttgctctga aggtaataca actcaatatc 300 caggagaata tacgcagaca gattgcgcag gaattgaaaa tcagcttgtc aacacagtgc 360 caatatgttg ttgcatgctg tcagtgtttt tacgttaatg gtgtcatttc aattgttttg 420 gagtatatgg acagtggctc tctctcagat ttcctgaaga cagttaaaac cattccagag 480 ccctaccttg ctgcaatctg taagcaggtg ctaaaaggac tgatgtactt acatcatgag 540 aagcacatca tacaccgaga tctgaagccg tcaaatatat taataaatca tatgggtgaa 600 gtaaaaatat ccgactttgg tgttagtgcc atcattgcta gttcctcggc acaacgagat 660 acatttactg ggacatataa ctacatggcg ccagaaagaa tcagtgggca aaaacatggt 720 tatatgagtg atatctggag cttgggcctc gttatgctag aattggccac tggtgaattt 780 ccatatcctc ctcgtgaaag cttttatgaa ctccttgaag ctgttgtcga ccacccacca 840 ccttctgctc catcagacca gttttcagag gaattctgtt cattcgtctc tgcatgtatc 900 caaaagaatg cttcggatag gtcatctgca caaatcttat taaatcatcc attcctgagc 960 atgtatgacg acctgaatat agatcttgct tcgtacttca caaccgatgg atctccgctt 1020 gccaccttca atacaagcaa ccgatatgat gacagatag 1059 <210> 4 <211> 1528 <212> DNA <213> Artificial Sequence <220> <223> Downstream Sequence of CMV promotor <400> 4 tcggtctcca tttcgcgtct cctcctcctc ctcctccggc catctcaccc tcccggcgag 60 cagcgagcgg gaggcctccg ccaccgccgc cgccgccgcc gccgtcgccg cccgatgggg 120 aagccgggga agctagcgct gccctcgcac gagtcaacca tcggcaaatt cctgacgcag 180 agcgggacgt tcaaggacgg cgatctgctc gtcaacaagg acggcctccg catcgtctcg 240 cagagcgagg aaggggaggc ccctcctata gaacctttag atcataatca gttgagtcta 300 gatgatctag acgcaatcaa agttatcggt aaaggtagta gtggaatcgt gcagttggtt 360 cgccacaaat ggactggcca gttttttgct ctgaaggtaa tacaactcaa tatccaggag 420 aatatacgca gacagattgc gcaggaattg aaaatcagct tgtcaacaca gtgccaatat 480 gttgttgcat gctgtcagtg tttttacgtt aatggtgtca tttcaattgt tttggagtat 540 atggacagtg gctctctctc agatttcctg aagacagtta aaaccattcc agagccctac 600 cttgctgcaa tctgtaagca ggtgctaaaa ggactgatgt acttacatca tgagaagcac 660 atcatacacc gagatctgaa gccgtcaaat atattaataa atcatatggg tgaagtaaaa 720 atatccgact ttggtgttag tgccatcatt gctagttcct cggcacaacg agatacattt 780 actgggacat ataactacat ggcgccagaa agaatcagtg ggcaaaaaca tggttatatg 840 agtgatatct ggagcttggg cctcgttatg ctagaattgg ccactggtga atttccatat 900 cctcctcgtg aaagctttta tgaactcctt gaagctgttg tcgaccaccc accaccttct 960 gctccatcag accagttttc agaggaattc tgttcattcg tctctgcatg tatccaaaag 1020 aatgcttcgg ataggtcatc tgcacaaatc ttattaaatc atccattcct gagcatgtat 1080 gacgacctga atatagatct tgcttcgtac ttcacaaccg atggatctcc gcttgccacc 1140 ttcaatacaa gcaaccgata tgatgacaga tagtctgatg caacactgcg tcatcatcta 1200 gctcgaagga tgatcctttg gctgtccatc tgtagaatct accatctacc taactggccc 1260 caaatgtttc gtgtattttt tgggagaaaa ctttgccctg tgtactgagc tgggattgat 1320 gtctttgata tgtaaatttc gcgtaatttc atatgtagaa caattctagg atcattgggc 1380 attgaagttt tgtttggccg tgcaccagga tgatctgttc ttgatcgctt tacctttgtt 1440 tctcacttct gtaactaaaa cctatattta ccatttaccg gattgttcat ggcctcttct 1500 gggcaagtcg gtgtcgactg aaagtctc 1528

Claims (14)

Immunity enhancement composition of a plant comprising a gene carrier comprising a nucleic acid molecule encoding the amino acid sequence of SEQ ID NO: 1 sequence.
The composition of claim 1, wherein the composition enhances immunity to a pathogen to confer pathogen resistance to the plant.
3. The composition of claim 2, wherein said pathogen is selected from the group consisting of bacteria, fungi, yeast, oromycetes and viruses.
2. The composition of claim 1, wherein said nucleic acid molecule comprises a nucleotide sequence from nucleotide 1115 to nucleotide 4680 of SEQ ID NO: 2.
2. The composition of claim 1, wherein the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 3.
2. The method of claim 1, wherein the gene delivery vehicle comprises: (i) a nucleic acid molecule encoding an amino acid sequence of the first sequence of the sequence listing; (ii) a promoter that is operatively linked to the nucleic acid molecule and that forms RNA molecules in plant cells; And (iii) a poly A signal sequence that acts in plant cells to cause polyadenylation of the 3'-end of the RNA molecule.
Plant cells transformed with the composition of any one of claims 1 to 6.
Plant transformed with the composition of any one of the preceding claims.
A method for producing a transgenic plant with enhanced pathogen resistance, comprising the following steps:
(a) introducing the composition of any one of claims 1 to 6 into a plant cell; And
(b) obtaining a transgenic plant having enhanced pathogen resistance from the plant cell.
10. The method according to claim 9, wherein the plant is selected from the group consisting of food crops including rice, wheat, barley, corn, soybean, potato, wheat, red bean, oats and millet; Vegetable crops including Arabidopsis, cabbage, radish, pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame, sugar cane, beet, perilla, peanut and rapeseed; Apple trees, pears, jujubes, peaches, sheep, grapes, citrus, persimmon, plums, apricots and banana; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies and tulips; And feed crops comprising raglass, red clover, orchardgrass, alpha alpha, tall fescue and ferrier alla grass.
10. The method of claim 9, wherein the plant is rice, and the pathogen is rice blast or rice leaf blight.

12. The method of claim 11, wherein the rice blast is genus Magnaporthe .
The method of claim 11, wherein the transgenic plant with enhanced pathogen resistance is a defense marker gene OsPR1b , OsPBZ , OsPAL1 , OsAPX1 or Up-regulating expression of OsAPX2 .
The method of claim 11, wherein the rice leaf blight is a genus of Xanthomonas ( Xanthomonas ).

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