KR101342265B1 - Disease resistance-related Mildew resistance Locus O 2 protein gene CaMLO2, plant resistance screening, and transgenic plants using the same - Google Patents

Abstract

The present invention relates to a disease resistance gene CaMLO2 ( C apsicum a nnuum m ildew resistance l ocus O 2 ), the present invention relates to a plant disease resistance screening method and transformed plants. According to the present invention described above, the gene can be usefully used as a genetic material for efficient and effective molecular breeding of the plant's defense response and disease-resistant plants against the attack of pathogens, the search and action mechanism for plant disease resistance There is an advantage that can be used as a tool to identify the.

Description

Disease resistance-related Mildew resistance Locus O 2 protein gene CaMLO2, plant resistance screening, and transgenic plants using the same}

The present invention relates to CaMLO2 , a novel gene related to plant disease resistance. Gene ( CaMLO2 : C apsicum a nnuum M ildew resistance L ocus O 2 ), the present invention relates to a plant disease resistance screening and transformed plants using the same. Specifically, the present invention is a bacterial disease of red pepper bacterial spot pattern ( Xanthomonas campestris pv. When infected with vesicatoria) specific expression is induced CaMLO2 of pepper-derived plant disease resistance genes that are associated with new It provides a base sequence of the gene and an amino acid sequence according to the gene, and relates to a method for detecting the resistance of plants to determine whether the expression of the resistance-related gene CaMLO2 during biotic or abiotic processing using the same . The present invention also inhibit virus induced gene from the pepper plant (VIGS, V irus- I nduced G ene S ilencing) the CaMLO2 using techniques Inhibits gene expression or Arabidopsis thaliana ) The present invention relates to the introduction and overexpression of this gene in plants to provide plant pathogenic effects of the CaMLO2 gene.

Plants have evolved with fast, effective disease resistance mechanisms to protect themselves from invasion of phytopathogens such as bacteria, fungi and viruses. This resistance reaction can be seen as suppressing further progress by using the recognition of pathogen invasion and the defense mechanism of the invading pathogen.

Research into the innate immunity of plants is an interesting topic for identifying disease defenses and signaling pathways in plants and will contribute greatly to understanding resistance responses across eukaryotes. In addition, this disease-resistant gene function research has an important meaning in terms of application, so that the disease-resistant genes transformed by molecular breeding in order to effectively control the disease of endemic plant disease (transgenic resistant) plants can be developed.

Pepper intensively grown and economically important in Korea, Capsicum annuum L., provides an excellent experimental system for studying the signaling pathways resistant to plant diseases. In addition, the interaction of pepper hospital bacteria, viruses, fungi and pepper is a well-known pathosystem and has been a good material for the study of pathogenic reactions. Therefore, the molecular cloning and functional analysis of the major genes involved in resistance in pepper plants can identify various signal transduction pathways that regulate the expression of these genes. Genes can be expressed in pepper plants and other crops to develop disease-resistant transformed plants, and when these resistant plants are commercialized for actual cultivation, they can contribute to environmentally friendly crop cultivation such as pepper, which can be an important gene source.

Plants have developed complex defense mechanisms to combat a variety of microbial pathogens. Strong, broad-spectrum resistance can be conferred by mutation of a single gene in the host plant. In particular, most lace of barley powdery mildew (powdery mildew fungus, Blumeria framinis f.sp. hordei ) wide range resistance to barley m ildew resistance l ocus By mutation of the O ( MLO ) gene. MLO , not present in animals, specific to most plant species The genes encode a protein that binds calmodulin with a 7-transmembrane. Mutations in the MLO gene are known to provide long-term pre-invasive resistance against barley powdery mildew. MLO induced by pathogen invasion Genetic apoptosis signaling is expected to regulate plant cell death and is known to regulate cell-level responses to ongoing pathogen attack by transiently expressed MLO . MLO proteins located in the cell membrane (plasma membrane) by a seven-transmembrane domain (7-transmembrane domains) and interacts with calmodulin-dependent manner with the Ca ^{+ 2.} The calmodulin binding function of MLO plays an important role because the interaction between calmodulin and calcium ions affects plant defense and apoptosis. The interaction of calmodulin with calcium ions attached to the calmodulin binding domain of MLO is a common feature of MLO proteins. Broad and strong plant disease resistance due to mutations in the MLO has been widely reported but, Ca ^{+ 2} -loaded function of MLO to control the calmodulin is not yet clearly understood. Therefore, studying the physiological and biochemical roles of MLO proteins is essential to fully elucidate MLO's ability to control plant defense and apoptosis.

In particular, MLO's involvement in resistance to barley powdery mildew has been studied, but the function of MLO against phytobacterial pathogens has not been reported yet. Identifying MLO's function against bacterial pathogens in pepper plants is necessary to understand the pathogenic mechanisms of pepper plants and may be used as a major gene source for the production of pathogenic transgenic plants.

As a background technology of the present invention, Korean Patent Publication No. 10-2001-0041943 (2001.05.25) describes a gene encoding MLO protein and confers fungal resistance to plants, but has resistance to phytobacterial pathogens. There is no disclosure of pepper derived MLO gene. In addition, Kim's 2002 Ph.D. dissertation, Department of Biochemistry, Graduate School of Gyeongsang National University, describes the interaction between CaM and Mlo proteins in plant bioprotective mechanisms using OsMlo genes isolated from rice, but their resistance to plant bacterial pathogens. There is no disclosure of the pepper-derived MLO gene. NCBI GenBank Accession No. AY934528 (CaMLO1) and Ralph Panstruga, Plant Molecular Biology (2005) 59: 485-500.

Republic of Korea Patent Publication 10-2001-0041943 (2001.05.25)

Kim Min-cheol, 2002 Ph.D. Thesis, Department of Biochemistry, Graduate School, Gyeongsang National University NCBI GenBank Accession No. AY934528 (2005) Ralph Panstruga, Plant Molecular Biology (2005) 59: 485-500

The present invention is to solve such a problem in the prior art, the purpose of which is pepper bacterial spot pattern disease ( Xanthomonas campestris pv. as involved in the defense response to vesicatoria) Plant disease resistance associated one of the MLO (CaMLO2 of the gene: C apsicum a nnuum m ildew resistance l ocus O 2 ) By separating and deciphering a gene, the nucleotide sequence information and the amino acid sequence information thereof are provided.

In addition, the object of the present invention is the CaMLO2 It is to provide a method for detecting disease resistance of plants that detects the presence or absence of resistance to plant diseases or chemicals using genes.

Furthermore, the present invention provides the CaMLO2 The purpose is to produce a transgenic plant having resistance to plant diseases by introducing the gene into the plant.

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

The above object of the present invention, red pepper bacterial spot disease ( Xanthomonas campestris pv. Vesicatoria strain Bv5-4a) after inoculation, nokgwang pepper varieties caused the overactive cell death (Capsicum annuum cv. Nom Kwang)) identified a new resistance-related gene CaMLO2 isolated from the Nockwang to determine the nucleotide sequence, and to investigate whether the expression of CaMLO2 through biological / chemical treatment, and CaMLO2 gene suppressed via VIGS This was achieved by identifying the plant disease resistance-related function of the CaMLO2 gene in the transgenic Arabidopsis thaliana overexpressing this gene.

Thus, the present invention provides an isolated gene ( CaMLO2 ) encoding a protein having the amino acid sequence of SEQ ID NO: 2.

The present invention also provides an isolated gene ( CaMLO2 ) having a nucleotide sequence of SEQ ID NO: 1.

The present invention further provides an isolated protein (CaMLO2) having the amino acid sequence of SEQ ID NO: 2.

In addition, the present invention provides a recombinant vector comprising the gene ( CaMLO2 ). The recombinant vector is preferably a recombinant plant expression vector. The recombinant vector is, but is not limited to, for example, pBIN35S: CaMLO2 , pBIN35S: CaMLO2: GFP or pTRV2: CaMLO2 . Preferably, the recombinant vector is a gene (CaMLO2) Virus-induced gene silencing (virus-induced gene silencing, VIGS ) the vector of TRV2 (Tobacco Rattle Virus 2) was inserted into a vector a recombinant vector TRV2 prepared for: CaMLO2 to be.

Furthermore, the present invention provides a transformed plant produced using the gene or the recombinant vector. Transformation of a plant means any method of transferring DNA to a plant. Any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable plant cells. The method is based on the calcium / polyethylene glycol method for protoplasts (Krens, FA et al., 1982, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373) (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microinjection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202,179-185 (Klein et al., 1987, Nature 327, 70), the infiltration of plants or the transformation of Agrobacterium tumefaciens Infection by viruses (non-integrative) in virus-mediated gene transfer (EP 0 301 316), and the like. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.

In addition, the present invention is isolated from the plant mRNA and CaMLO2 It provides a method for detecting disease or chemical resistance of a plant comprising the step of identifying differential expression of a gene.

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

First, the present invention is pepper bacterium bacillus bacterium ( Xanthomonas campestris pv. mRNA was isolated from green pepper varieties that showed hypersensitivity resistance to the non-pathogenic strain Bv5-4a inoculation of vesicatoria ), and the cDNA library was prepared. Total mRNA obtained from the leaves of pepper plants inoculated with the non-pathogenic strains and healthy pepper plants not inoculated. Is labeled with digoxygenin to make a probe, and a differential hybridization process using the cDNA and the probe is used to select clones that are expected to be associated with pathogenic resistance. Decoding the nucleotide sequence of, provides the MLO coding gene base sequence (SEQ ID NO: 1) and the corresponding amino acid sequence (SEQ ID NO: 2) of the pepper green mine variety named CaMLO2 (see Fig. 1 and 2).

Analysis of the amino acid sequence of the CaMLO2 protein of the present invention shows that the protein of CaMLO2 has an MLO-related proetin domain, which is typical of plant MLO proteins (FIG. 1), in detail seven transmembrane domains ( t rans m embrane d omain; - ; had a CaMBD) TMD7 TMD 1) and lean binding domain knife module to the C- terminus of the amino acid sequence (ca l m odulin b inding d omain.

The coding region (ORF) of the CaMLO2 cDNA consists of 1587 sequences encoding 528 amino acids, and the calmodulin binding domain of the CaMLO2 protein is conserved lysine (L), tryptophan (W), and alanine (A). And histidine (H) was confirmed. The gene sequences of the identified CaMLO2 and the amino acid sequences encoded by the sequences are shown in FIGS. 1 and 2, and the relationship between the previously reported MLO proteins of other plants is compared with the structure of constructing and preserved motifs. A comparison of other plants previously reported is shown in FIG. 3.

Next, the present invention is shown in Figure 4 to determine the calcium-dependent (Ca ^{2 +} -dependent) calmodulin binding protein CaMLO2. Only the nucleotide sequence encoding the calmodulin binding domain (CaMBD) present at the C-terminus of the CaMLO2 protein was cloned to express the recombinant protein through the GST recombinant E. coli expression system, and then biotin-attached calmodin (Biotin-). confirmed that the CaM), calcium ion (Ca ^{2 +)} is CaMBD and CaM interaction in the presence or absence status GST-CaMBD recombinant protein and Biotin-CaM result confirming the interaction of only the calcium ions (Ca ^{2 +)} is present in using the It was. CaMBD and CaM did not interact with EGTA, a calcium theory remover.

Next, the present invention is a plant expression vector by attaching GFP (green flourescent protein), a fluorescent protein of CaMLO2 protein, to the C-terminus of CaMLO2. Epidermal Cells and Nicotinan of Onion by Recombinant Vector Inserted into pBIN35S Vector Results of confirming the position of the CaMLO2 protein in the plant cells using the epidermal cells of benthamiana are shown in Figures 5a to 5d.

Next, the present invention provides a method for detecting disease or chemical resistance of a plant comprising the step of separating mRNA from a plant subjected to biological / chemical treatment to confirm differential expression of the CaMLO2 gene. More specifically, the biological treatment is exemplified by a pathogenic bacterium such as a pathogenic bacterium of hot pepper, a non-pathogenic bacterium, For example, the chemical treatment may include plant hormone salicylic acid. CaMLO2 In addition to northern blot, real-time RT-PCR and microarray methods, various methods for measuring mRNA expression can be used to confirm gene expression. CaMLO2 according to the present invention Experiments confirmed that genes were differentially induced and expressed by plant pathogens or plant hormone treatment (see FIGS. 6A-6B). Therefore, CaMLO2 according to the present invention Genes can be usefully used for disease detection or chemical resistance of plants.

In addition, the present invention is the above CaMLO2 CaMLO2 Specific to Pepper Plants Using Recombinant Vectors That Inhibit Gene Expression Genetically inhibited plant production and disease resistance increased, CaMLO2 By identifying the reduced plant disease resistance of transgenic Arabidopsis plants overexpressed genes can provide new pathogenic transgenic plants and materials for resistant molecular breeding. Therefore, the present invention provides the CaMLO2 Also provided is a method of imparting disease resistance to a plant comprising transforming the plant using a recombinant vector that inhibits gene expression.

More specifically, the present invention provides CaMLO2 by using Tobacco Rattle Virus (TRV), which is used in virus-induced gene silencing (VIGS). The gene was introduced into a TRV vector (Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin. Plant Biol. 2: 109-113) to produce a recombinant vector TRV: CaMLO2 . The recombinant vector was introduced into a plant to prepare a VIGS plant in which CaMLO2 expression was suppressed, thereby increasing resistance and providing a disease-resistant transformed plant (see FIGS. 7A to 8D).

Furthermore, the recombinant vector (pBIN35S: CaMLO2 ) and Agrobacterium tumefaciens ( Agrobacterium ) containing the CaMLO2 gene according to the present invention. tumefaciens strain GV3101) was used to confirm the plant disease resistance reduction function of the CaMLO2 gene through transgenic expression in Arabidopsis plants (see Figures 9a to 12d).

It confirmed that a central role in disease resistance adjustment of CaMLO2 a pepper plant in the present invention as described above, and the resulting reaction and the associated bottle to bottle of CaMLO2 related protein (PR protein) plants are able to accumulate genetic information It can provide an interesting model of signaling for disease, and can be used to develop biotechnological plants that increase disease resistance by understanding the biochemical changes that cause resistance.

As it described above, whereby the invention is completed, pepper plant bacterial etiology pepper bacterial jeommunuibyeong (Xanthomonas campestris pv. Vesicatoria) of plant disease resistance genes as involved in the defense response to CaMLO2 By separating and deciphering the gene, the sequence information and the corresponding amino acid sequence information are provided, and at the same time, it is possible to provide a plant disease resistance search method using the gene.

In addition, according to the present inventionCaMLO2 Increasing resistance through gene suppression and overexpressing the gene in Arabidopsis to confirm the difference in plant disease resistance according to the present inventionCaMLO2Can be provided as a material for the breeding of disease resistant plants. Since the present invention has the effect of promoting the development of resistant varieties is a very useful invention in the plant breeding industry.

1 is nokgwang pepper cultivars by the present invention (Capsicum annnuum L. cv. Pepper CaMLO2 cloned from Nockwang) A diagram showing the nucleotide sequence and amino acid sequence of the gene,
Figure 2 shows the result of comparing the MLO amino acid sequence of CaMLO2 and other plants according to the present invention,
Figure 3 is a comparison between the amino acid sequence of the CaMLO2 pepper according to the present invention and the Arabidopsis, which is a model plant and MLO proteins of other crops reported previously and the calmodulin binding domain,
4 confirms the interaction between calmodulin binding domain and calmodulin dependent on calcium ions,
5A to 5D show the intracellular location of CaMLO2 protein through the construction of a recombinant vector encoding CaMLO2 and a fluorescent protein,
Figure 6a is red pepper bacterial spot disease ( Xanthomonas campestris pv. vesicatoria ) is a diagram showing the difference in expression of CaMLO2 gene in inoculated and non-inoculated leaves when inoculated with pathogenic and non-pathogenic strains of,
Figure 6b shows the CaMLO2 according to the present invention over time after treatment with salicylic acid (SA) and methyl jasmonate (MJ) A diagram showing a result of gene expression induction,
Figure 7a is a diagram confirming that the expression of the CaMLO2 gene according to the present invention is suppressed using the method of virus-induced gene silencing (VIGS),
Figure 7b is a view showing a reduction in bacterial growth in plants in which the expression of the CaMLO2 gene according to the present invention is suppressed,
When Figure 7c hayeoteul inoculated with the virulent strain Ds1 and pepper jeommunuibyeong bacterial strains of non-pathogenic strains Bv5-4a (Xanthomonas campestris pv. Vesicatoria) in the plant of pepper varieties nokgwang expression is inhibition of gene CaMLO2 according to the present invention, a comparison of the symptoms It is a diagram showing that,
7D and 7E are pepper bacterial bacterial pathogens of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a, respectively, in plants of the red pepper greenery varieties whose expression of the CaMLO2 gene was suppressed according to the present invention ( Xanthomonas campestris pv. vesicatoria ) is a diagram showing the results of expression inhibition of CaMLO2 expression and resistance-related genes when inoculated,
8a and 8b are pepper bacterium spotty bacteria of pathogenic strain Ds1 and non-pathogenic strain Bv5-4a in plants of red pepper green mine varieties in which the expression of CaMLO2 gene according to the present invention is suppressed ( Xanthomonas campestris pv. vesicatoria ) is a diagram showing the change of H ₂ O ₂ in infected pepper leaf tissues through DAB staining (3,3'-diaminobenzidine staining) and quantification, respectively.
8C and 8D are pepper bacterial bacterial pathogens of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in the plant of the red pepper green mine variety in which the expression of the CaMLO2 gene according to the present invention is suppressed ( Xanthomonas campestris pv. vesicatoria ) is a diagram quantifying the apoptosis in infected pepper leaf tissues by trypan staining and measurement of ion conductivity, respectively.
9A shows the recombinant vector pBIN35S: CaMLO2 as Agrobacterium tumefaciens ( Agrobacterium). tumefaciens in transgenic Arabidopsis thaliana plants that overexpressed the gene CaMLO2 using a strain GV3101) strains, showing a result of observing the CaMLO2 which gene is overexpressed,
9b and 9c show bacterial erythema in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention ( Pseudomonas syringae pv. tomato When inoculated with pathogenic strain (DC3000) and non-pathogenic strain (DC3000 avrRpm1) of DC3000, the difference in symptoms and bacterial growth, respectively,
Figure 10a is a bacterial erythema ( Pseudomonas syringae pv. Tomato of pathogenic strain DC3000 and non-pathogenic strain DC 3000 avrRpm1 in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention. When inoculated with pathogenic strain (DC3000) and non-pathogenic strain (DC3000 avrRpm1) of DC3000, it is a diagram showing the quantitative change in H ₂ O ₂ in infected leaf tissue,
Figure 10b is a bacterial erythema ( Pseudomonas syringae pv. Tomato of pathogenic strain DC3000 and non-pathogenic strain DC 3000 avrRpm1 in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention. When inoculated with the pathogenic strain (DC3000) and non-pathogenic strain (DC3000 avrRpm1) of DC3000, the cell death in the infected tissue was quantified by measuring the ion conductivity,
Figure 11 is bacterial pathophysiology of pathogenic strain DC3000 and non-pathogenic strain DC 3000 avrRpm1 in transgenic Arabidopsis plants overexpressing CaMLO2 gene according to the present invention ( Pseudomonas syringae pv. Tomato When the pathogenic strain (DC3000) and the non-pathogenic strain (DC3000 avrRpm1) were inoculated, changes in defense-related genes were shown through real time-RT PCR.
Figure 12a is a fungal disease in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention ( Hyaloperonospora parasitica ) is a picture showing reduced disease resistance to,
Figure 12b is a fungal disease in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention ( Hyaloperonospora parasitica ) is a view showing the results of the examination of mycelial growth after trypan blue staining (trypan blue staining),
Figure 12c and Figure 12d is a fungal pathogen ( Haloperonospora) in transgenic Arabidopsis plants overexpressing the CaMLO2 gene according to the present invention parasitica ) shows the result of confirming the collapse of the disease resistance to spore diameter and spore counting.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. 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  One] CaMLO2 Determination of base / amino acid sequence of gene

MLO ( CaMLO2 : C apsicum a nnuum) , one of the pathogenic proteins associated with red pepper bacterial spot disease m ildew resistance l ocus O 2 ) The following tests were performed to provide protein coding gene sequences and amino acid sequences inferred therefrom.

Capsicum pepper varieties ( Capsicum annuum cv. Nockwang) were sprayed with red pepper bacillus fungi ( Xanthomonas campestris pv. vesicatoria ) were inoculated and treated for 18 hours, and then the plants showing the hypersensitive apoptosis, which were resistant lesions, were taken out of the chamber and leaves were collected.

Next, mRNA was extracted from the leaf sample and reverse transcribed to prepare a cDNA library. Individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed to the nylon membrane (Hybond N +) uniformly. The total mRNA extracted at was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

As a result of the hybridization, the gene showing intensively amplified only for the mRNA probe of the non-pathogenic strain inoculated plant was estimated as a gene related to disease resistance and a clone was obtained.

After sequencing the obtained clones, the CaMLO2 protein coding gene was identified and named as the pepper MLO ( CaMLO2 ) gene by comparing the sequence with a gene database registered in GenBank using a blast program. Analysis of the amino acid sequence of the CaMLO2 protein was found to belong to the MLO group of plants (see Figures 1 to 3). Gene base sequence (SEQ ID NO: 1) and the amino acid sequence encoded by the base sequence (SEQ ID NO: 2) of the identified CaMLO2 are shown in FIGS. 1 and 2.

[ Example  2] Of CaMLO2 Calmodulin  Binding activity check

In order to assay the calmodulin binding activity of the CaMLO2 protein encoded by the CaMLO2 gene obtained in Example 1 was tested as follows.

[Step 1]

E. coli recombinant protein expression system was used. A nucleotide sequence encoding a Calmodulin binding domain (CaMBD) located at the C-terminus of CaMLO2 was inserted into a recombinant GST vector and transformed into E. coli BL21 strain to express a recombinant protein.

[Step 2]

The calodulin binding ability of the purified protein using the E. coli protein expression system was assayed using the Affinity CB Fusion Protein Detection Kit (Stratagene, La Jolla, Calif.). Biotin-attached calmodulin was used as a primary probe, streptavidin alkaline phosphatase was used as a secondary detection to detect biotin, and biotin attached as a result of the interaction. Silver chemiluminescence substrates were emitted by CD-Star (Sigma-Aldrich, St. Louis, Mo.) and identified by X-ray film. The result confirmed through this is shown in FIG.

[ Example  3] Of CaMLO2  Through fluorescent protein attachment Intracellular  Location verification

The recombinant vector smGFP: CaMLO2 was prepared by introducing the CaMBL1 gene into the 326GFP vector to detect the expression location of CaMLO2 in plants using a recombinant vector labeled with a green fluorescent protein (GFP). (FIG. 5A).

[Step 1]

In order to determine the position where CaMLO2 is expressed in the plant, CaMLO2 labeled with green fluorescence protein was introduced into onion plants by using particle charcoal DNA transfer and observed using confocal microscope. 5B shows the result.

[Step 2]

The recombinant vector thus prepared was Nicotiana using Agrobacterium tumafeciens GV3101 strain. Transient expression in benthamiana was also observed using confocal laser microscope. Proteins were extracted from the expressed plant tissues, and the cytoplasmic portion and membrane layer of the plant cells were separated by ultra centrifugation, and then the location of CaMLO2 could be reconfirmed by western blotting using GFP antibody. 5C and 5D show this result.

[ Example  4] In red pepper plants by pathogen inoculation CaMLO2 Exploration of gene expression

CaMLO2 obtained in Example 1 The following tests were conducted to confirm the use of genes in the search for resistance.

[Step 1]

First, in order to specifically suggested as the method of searching for resistance, pepper bacterial spot patterns pathogen (Xanthomonas campestris pv. Vesicatoria) pathogenic strains Ds1 and the incompatible reaction YN (Yeast- Nutrient) a non-pathogenic strain Bv5-4a representing representing the plant and friendly reaction After incubation in the medium, Capsicum 6-leaf red pepper varieties ( Capsicum) at a concentration of 10 ⁸ cfu / mL annuum cv. Nockwang) was inoculated by injecting the back of the 5 and 6 leaves using a syringe, and after the inoculation was treated with wet conditions at 28 ° C. and 100% relative humidity for 18 hours. After the inoculation, the leaves were sampled at each time point to isolate the RNA, electrophoresed on formaldehyde gel, and transferred to a nylon membrane (Hybond N).

Next, CaMLO2 obtained in Example 1 The gene was labeled with an isotopic α- ³² P [dCTP] using Klenow enzyme, and then the reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% dextran sulfate ( Dextran Sulfate)] was incubated at 65 ° C. for 24 hours to perform hybridization. The isotopically labeled DNA probe is attached to the mRNA attached to the nylon membrane (Hybond N +). When the X-ray film is placed on the nylon membrane (Hybond N +), the isotopically labeled DNA is X-ray film. As a result, the expression and degree of expression were recognized.

Figure 6a were given in the above experiment results of the experimental results CaMLO2 Genes were strongly expressed 20 hours after inoculation of pathogenic pepper bacterial spot disease and 10 hours after inoculation of non-pathogenic pepper bacterial spot disease (FIG. 6A).

[ Example  5] caused by plant hormones CaMLO2 Induced Expression of Genes

CaMLO2 of the invention In order to find out whether the gene can be induced by salicylic acid and methylzamonate, which are plant hormones commonly used to induce resistance to bacterial pathogens, the following experiments were performed.

[Step 1]

First, CaMLO2 In order to search for derivatives useful for inducing gene expression, resistance induction tests were conducted using salicylic acid and methyl jasmonate among chemicals known to be involved in the induction of plant disease resistance expression. Six-leaved pepper leaves were used in all experiments.

Salicylic acid was treated by spraying pepper leaves at a concentration of 5 mM, and methylzasmonate was treated at a concentration of 100 μM. The plants were harvested after 1, 5, 10, 15, 20 and 25 hours of treatment, RNA was extracted from each sample and the extracted RNA was electrophoresed on a gel containing formaldehyde followed by a nylon membrane (Hybond N +). ) And then Northern blotting was performed as follows. The test method is the same as described in Example 2.

As a result of the experiment, the CaMLO2 gene was observed to be expressed from 5 hours after salicylic acid treatment as shown in FIG. 6b, and the expression was maintained strongly until 25 hours, and weakly expressed compared to salicylic acid when methylzamonate was treated. It can be observed that it is maintained when induced (FIG. 6B).

[ Example  6] CaMLO2  Changes in disease resistance due to inhibition of gene expression

When in the pepper plant is suppressed to a specific expression of the CaMLO2 genetic disease occurrence and other related genes induced and whether to evaluate the collapse if the resistance to the disease, in Example 1 inhibit virus induced gene a CaMLO2 gene obtained from of ( The recombinant vector TRV: CaMLO2 was prepared by introducing CaMLO2 gene into TRV vector using TRV (Tobacco Rattle Virus), which is used in VIGS, virus-induced gene silencing (Baulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin.Plant Biol. 2: 109-113).

Thus prepared recombinant vector TRV: CaMLO2 to Agrobacterium tumefaciens ( Agrobacterium Tumefaciense strain GV 3101) was introduced through electrophoresis, and the GV3101 strain containing the recombinant vector was inoculated in pepper seedlings by infiltration using a syringe on the cotyledons of pepper seedlings. Thereby inducing the inhibition of the expression of the CaMLO2 gene, and the effect of gene expression inhibition was confirmed through reverse transcriptase PCR (RT-PCR) and real-time RT-PCR (see FIG. 7A).

[Step 1]

In order to investigate the change in the resistance to bacterial diseases in plants inhibited by the expression of CaMLO2 , the pepper bacterium in the pepper green mine varieties inoculated with TRV2: 00 vector as a control and the pepper plants suppressed by the expression of CaMLO2 gene ( Xanthomonas campestris pv. vesicatoria ) were inoculated with the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a at concentrations of 10 ⁷ and 10 ⁸ cfu ml ^{- 1} , and then the leaves were inoculated with 5 x 10 ⁴ cfu ml ^{- 1} . On day 3, bacterial growth in infected plant tissues was measured (FIGS. 7B and 7C).

In addition, RNA was extracted from infected leaves of experimental and control groups 18 and 36 hours after inoculation with 10 ⁷ cfu ml ^-1 concentration of pathogens to inhibit specific genes of CaMLO2 through real-time RT-PCR. With the effect, the expression difference of the disease resistance related genes were observed in comparison with the control plants at the time of inoculation (Figs. 7D and 7E).

As a result, as shown in FIG. 7B, bacterial growth of pathogenic bacteria and non-pathogenic bacteria in infected tissues was measured. As a result, less bacterial growth was observed in plants in which CaMLO2 expression was suppressed only in plants inoculated with pathogenic bacteria.

As shown in FIG. 7C, after the inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in the plants inhibited by the expression of CaMLO2 by VIGS, the yellowing was observed even after 6 days after the inoculation of the pathogenic strain. Delayed and necrotic phenomena were observed, and there was no difference between control plants and inoculated non-pathogenic strains.

In addition, real-time Alti-fish egg test results Figure 7d, and as shown in Figure 7e, has been effectively observed that inhibition of CaMLO2 gene found in the plant a CaMLO2 is suppressed, pathogenic strain-dependent typical resistance-related gene CaPR1 gene inoculation when salicylic acid (Choi et al. (2007) Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens.

After inoculation with pathogen 10 ⁷ cfu ml ^-1 , H ₂ O ₂ , a plant-resistance signaling-related substance, was quantified for each time period after inoculation, and cell death of plant tissue, which is an expression of the resistance response, was quantified by measuring ion conductivity (FIG. 8a-8d).

As shown in FIGS. 8a to 8d, H ₂ O ₂ accumulation and ion conductivity were increased only when inoculated with pathogenic plants in plants inhibited by CaMLO2 compared to control plants.

[ Example  7] CaMLO2  Production of transgenic plants using genes CaMLO2  Variation of disease resistance of Arabidopsis plants by overexpression of genes

Model to investigate with respect to increased if the resistance to the induced and whether the bottle of genes associated with different disease occurs when over expression of CaMLO2 gene in Arabidopsis thaliana plants used in the plant, the CaMLO2 gene obtained in Example 1 in pBIN35S vector PBIN35S: CaMLO2 was prepared by introduction, and the recombinant vector pBIN35S: CaMLO2 thus prepared was introduced into Agrobacterium tumefaciense strain GV 3101 through electroporation and GV3101 containing the recombinant vector. By introducing the strain into Arabidopsis plants through floral dipping method, a transgenic Arabidopsis induced the overexpression of CaMLO2 gene was prepared and the following test was performed.

[Step 1]

As described above, the plants overexpressed CaMLO2 were inoculated with Pseudomonas syringe-Pasova tomato DC3000 pathogenic and non-pathogenic strains at a concentration of 5 x 10 ⁴ cfu / ml, and the change of the plants was observed. As shown in Figure 9b, as compared to wild-type plants in the CaMLO2 the over-expression has been shown that the sensitivity observed for the bottle of pathogenic bacterial, as shown in Figure 9c, the observed results of the bacterial growth in the infected tissue CaMLO2 Pathogenic bacterial growth in overexpressed genes was higher than that in wild-type plant tissues. As shown in Figure 10a and 10b, the accumulation of H ₂ O ₂ was lower than the control at the beginning of pathogenic bacterial inoculation, the ion conductivity was confirmed to be higher in the late time zone after the pathogenic bacterial inoculation. This suggests that the defensive response of the pathogenic bacteria at the initial time was more disintegrated than the control, but the ionic conductivity was increased due to the increase of necrotic reaction caused by the growth of the late pathogens. There was no change in non-pathogenic bacterial inoculation compared to the control.

[Step 2]

24 and 48 hours after inoculation with pathogenic and non-pathogenic bacteria at 10 ⁷ cfu ml ^-1 concentration, RNA was extracted from infected leaves of experimental and control groups, and control plants were inoculated via real-time RT-PCR. Compared with the expression difference of the genes related to disease resistance was observed, the results are shown in FIG.

[Step 3]

Hylooperonospora parasitica Noco2 ( hyaloperonospora) parasitica Noco 2) was inoculated and the change of disease resistance was observed. Hyeloferonospora parasitica Noco2 was sprayed at a concentration of 5 × 10 ⁴ spores ml ⁻¹ at the cotyledon stage of CaMLO2 and non-overexpressed wild type control plants. After inoculation, the cells were subjected to wet treatment at a temperature of 17 캜.

As a result, in the transgenic Arabidopsis plants overexpressing CaMLO2 , the mycelial growth of the Bacillus pathogens was increased, and the results are shown in FIGS. 12A to 12D.

Although specific portions of the present invention have been described in detail, those skilled in the art will appreciate that these specific embodiments are merely examples of preferred embodiments and that the scope of the present invention is not limited thereto. It is therefore intended that the scope of the present invention be defined by the appended claims and their equivalents.

Attach an electronic file to a sequence list

Claims (6)

An isolated gene ( CaMLO2 ) encoding a protein having the amino acid sequence of SEQ ID NO: 2 . An isolated gene having the nucleotide sequence of SEQ ID NO: 1 ( CaMLO2 ). An isolated protein having the amino acid sequence of SEQ ID NO: 2 (CaMLO2). A recombinant vector comprising the gene of claim 2 ( CaMLO2 ). Arabidopsis or pepper plants transformed with the recombinant vector according to claim 4. delete

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