KR20110139032A - Disease resistance-related pepper patatin-like phospholipase gene caplp1, screening of plant disease resistance and transgenic plants using the same - Google Patents

Abstract

PURPOSE: A disease resistance-related Capsicum annuum patatin-like phospholipase gene is provided to obtain genetic material for molecular breeding. CONSTITUTION: A plant disease resistance-related gene is a gene encoding a protein having an amino acid sequence of sequence number 2 or a gene(CaPLP1,Capsicum annuum xyloglucanase inhibitor protein 1) having a base sequence of sequence number 1. The gene is also a gene encoding a protein with an amino acid sequence of sequence number 4, or a gene(CaPLP2, Capsicum annuum xyloglucanase inhibitor protein 2) having a base sequence of sequence number 3.

Description

Disease resistance-related pepper patatin-like phospholipase gene CaPLP1, screening of plant disease resistance and transgenic plants using the same}

The invention peppers (Capsicum annuum L.) plants novel gene pepper patatin similar force porayi phase genes involved in disease resistance isolated from plants (CaPLP1: C apsicum a nnuum P atatin- L ike P hospholipase 1), and using the same. The present invention relates to a method for detecting resistance of plants and to transgenic plants resistant to disease. More specifically, the present invention relates to a nucleotide sequence of CaPLP1 gene, a novel gene related to plant disease resistance, which is induced specifically when infected with red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), which is a bacterial disease of red pepper plants. The present invention provides a sequence, and relates to a method for detecting resistance of a plant to identify whether the resistance-related gene CaPLP1 is differentially expressed in a biological or abiotic process. In addition, the present invention as to the in pepper plants using virus induced gene suppression (VIGS, V irus- I nduced G ene ilencing S) technology inhibiting the expression of the gene or CaPLP1, the gene in the plant Arabidopsis (Arabidopsis thaliana) By introducing and overexpressing (overexpression) to confirm the effect of increasing the plant disease resistance of the CaPLP1 gene, the present invention relates to providing a plant disease resistant transformed plants through this.

Research into the mechanism of disease resistance in plants has a scientific value in determining the in vivo signaling mechanisms that have not been known. In addition, the information on plant disease defense-related genes has the advantage that can be provided as a direct material of molecular genetic breeding for increasing the disease resistance of crops.

Crop breeding using a variety of defense-related genes to increase environmentally-friendly food production can reduce food production costs by minimizing losses due to economically important pathogens, weeds, and environmental stress. In addition to contributing to the recovery of the company's competitiveness, it also contributes to the activation of various industries associated with the development of new varieties.

Plant disease control of red pepper ( Capsicum annuum L. ) as an intensively cultivated and major economic crop in Korea is of great importance and also provides an excellent experimental system for studying the signaling pathway of defense against plant diseases. Separation and functional analysis of genes involved in defensive reactions in the interaction of pepper bacterium pathogens and peppers provides a scientific understanding of plant disease response through overexpression in the pepper plant, as well as the model plant, Arabidopsis. It is the basis of the gene source for the development of disease-resistant transformed plants that promote the expression of resistance-related genes in pepper plants.

When plants are infected with pathogens, host plants activate various intracellular metabolic systems to combat them, activating defense mechanisms. This defense mechanism begins with the recognition of host cells for pathogens, including intracellular Ca ²⁺ , increased free radicals, callose deposition, the production of secondary metabolites, phenol-related antimicrobial agents, and hypersensitivity such as local cell death. Reactions, and to produce a variety of defense-related proteins.

The signal transduction process for this series of resistance reactions delivers lower-level signals through the degradation of phospholipids located on cell membranes and the recognition of degradation products. Phospholipid degradation in this process is a phospholipd bilayer structure in which an enzyme called phospholipase with lipid acyl hydrolase (LAH) activity forms various membranes in the cell. Hydrolysis of the phospholipid acyl bond of the archiconic acid and lysophospholipids. In this process, the degraded product acts as a signaling material, and through low-level signaling, plant hormones enable the production of various secondary metabolites with antibacterial activity as well as jasmonic acid.

Recently, the importance of the role of physiological function and role of phosphorylated phase having individual LAH activity has been highlighted in the growth and defense of plants. In particular, it has been found that the Patatin protein, known as the storage of nutrients in the tubers of the first potatoes, is a phosphorase with LAH activity, and that patatin-like phosphorases preserved in various crops are induced by plant pathogens and There are several reports that play a major role in the hypersensitivity apoptosis. In addition, it has been reported that Mitogen-associated protein kinase (MAP kinase) signaling, which is the backbone of plant immune signaling, is activated through increased stimulation of LAH in model Arabidopsis. Therefore, researches on the enhancement of plant immune response through the modification of the plant's lipid metabolism process by using it are in progress. At present, no functional studies on such patatin-like phospholipases in pepper plants and their role in disease resistance have been reported. Studies on patatin-like phospholipases, which are involved in plant immune responses to external stress as well as plant growth through an important role in lipid metabolism processes within cells, are of great importance, Defining the function is necessary to understand the pathogenic mechanism of pepper plants and may be used as a major gene source for the production of pathogenic transgenic plants.

The present invention is to solve such a prior art problem, the purpose of which is involved in the defensive reaction against red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ), patatin-like phosphoryl which is one of the genes related to plant disease resistance phase: decrypting, by separating the (C CaPLP1 apsicum a nnuum atatin- P L P ike hospholipase 1) gene to provide the sequence information and amino acid sequence information thereof.

It is also an object of the present invention to provide a method for detecting disease resistance of plants using the CaPLP1 gene to search for the presence or absence of resistance to plant diseases or chemicals.

Furthermore, an object of the present invention is to produce a transgenic plant having resistance to plant diseases by introducing the CaPLP1 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, a new resistance-related gene CaPLP1 isolated from Capsicum annuum cv. Nockwang after inoculation of red pepper bacterial bacillus bacterium ( Xanthomonas campestris pv.vesicatoria strain Bv5-4a), which caused hypersensitivity apoptosis To determine the nucleotide sequence, and to determine whether the expression of CaPLP1 through the biological / chemical treatment, using the pepper plant suppressed CaPLP1 gene via VIGS and transgenic Arabidopsis overexpressing this gene ( In Arabidopsis thaliana ), this was achieved by confirming the plant disease resistance induction function of CaPLP1 gene. In addition, the present inventors have confirmed the presence of the gene of the alternative splicing forms Singh (alternative splicing) of CaPLP1 CaPLP2 gene.

Therefore, the present invention provides a plant disease resistance-related gene, (a) a gene encoding a protein having an amino acid sequence of SEQ ID NO: 2, or (b) a gene having a nucleotide sequence of SEQ ID NO: 1. ( CaPLP1 ).

In addition, the present invention has a gene encoding a protein having an amino acid sequence of SEQ ID NO: 4, or (b) having a nucleotide sequence of SEQ ID NO: Gene ( CaPLP2 ).

Furthermore, the present invention provides isolated patatin-like phosphorophase proteins CaPLP1 and CaPLP2 having the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

The present invention also provides a recombinant vector prepared using the gene. The recombinant vector is preferably a recombinant plant expression vector. The recombinant vector is, but is not limited to, for example pBIN35S: CaPLP1 , or pTRV2: CaPLP1 .

Furthermore, the present invention provides a transformed plant prepared 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 progenitor cells. Method is 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), protoplasts Electroporation (Shillito RD et al., 1985 Bio / Technol. 3, 1099-1102), microscopic injection into plant elements (Crossway A. et al., 1986, Mol. Gen. Genet. 202, 179-185 ), (DNA or RNA-coated) particle bombardment of various plant elements (Klein TM et al., 1987, Nature 327, 70), Agrobacterium tumefaci by infiltration of plants or transformation of mature pollen or vesicles And infection with (incomplete) virus (EP 0 301 316) in en mediated gene transfer. A preferred method according to the present invention comprises Agrobacterium mediated DNA delivery.

In addition, the present invention provides a method for detecting disease or chemical resistance of a plant comprising the step of identifying the differential expression of CaPLP1 gene by mRNA separation from the plant.

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

First, the present invention is to prepare a cDNA library by separating the mRNA from the green pepper pepper varieties exhibiting a hypersensitivity resistant response to the non-pathogenic strain Bv5-4a inoculation of the pepper bacterium bacillus bacterium ( Xanthomonas campestris pv.vesicatoria ), peppers inoculated with non-pathogenic strains The total mRNA obtained from the leaves of the plant and healthy red pepper plants not inoculated was labeled with digoxygenin to make a probe, followed by differential hybridization using the cDNA and the probe. Selected clones that are expected to be associated with resistance, decipher the nucleotide sequence of the gene, and sequence the patatin-like phosphorase phase coding gene of the red pepper green mine variety designated as CaPLP1 or CaPLP2 (SEQ ID NO: 1, SEQ ID NO: 3) And the resulting amino acid sequence (SEQ ID NO: 2, SEQ ID NO: 4) (FIG. 1 and 2 TRUE). ).

Analysis of the amino acid sequences of the CaPLP1 and CaPLP2 proteins of the present invention revealed that the Patatin-like phospholipase domain and amino acid sequences typically present in the plant Patatin-like phospholipase in both CaPLP1 and CaPLP2 proteins The signal peptide at the N-terminus of, and the pepper genome was isolated and analyzed by the exon and intron of CaPLP1 , CaPLP2 was found to be part of the exon 1 cut off.

The analysis of the expression induction of the pathogen and of CaPLP1 CaPLP2 of the present invention, expression of CaPLP2 was found to have also a relatively insignificant compared to the CaPLP1. This suggests that CaPLP1 plays a major role in plant defense. The coding region (ORF) of CaPLP1 cDNA consists of 1242 sequences encoding 413 amino acids, and the catalytic dyad, together with the serine hydrolase motif, is present in the patatin-like phosphorylation domain of CaPLP1 protein. Aspartic acid was formed. The identified gene nucleotide sequence of CaPLP1 and the amino acid sequence encoded by the nucleotide sequence are shown in Figs. 1 and 2, and the amino acid sequence difference and expression difference of CaPLP1 and CaPLP2 are shown in Fig. 2. In addition, the relationship between the conserved motif of CaPLP1 protein and patatin-like phosphorylation of other plants previously reported is shown in FIG.

Next, the present invention provides a method for detecting disease resistance of a plant, comprising the step of identifying the differential expression of the CaPLP1 gene by RNA separation from the plant subjected to biological / chemical treatment. More specifically, the biological treatment may be pathogens such as pathogenic or non-pathogenic bacteria of pepper bacterial spot pattern disease. For example, the chemical treatment may include plant hormone salicylic acid. The CaPLP1 gene expression check may be used in addition to the Northern blot, real-time RT-PCR, microarray method, a variety of methods for measuring the expression of mRNA can be used. Experiments confirmed that CaPLP1 gene according to the present invention is differentially induced expression by plant pathogens or plant hormone treatment (see FIGS. 4b to 4d). Therefore, CaPLP1 gene according to the present invention can be usefully used for the disease or chemical resistance screening method of plants.

In addition, the present invention using the recombinant vector containing the above CaPLP1 gene to produce a plant specifically inhibited CaPLP1 gene in pepper plants and confirmed that the disease resistance is reduced, the transgenic Arabidopsis overexpressed CaPLP1 gene By identifying whether the plant produces phytopathogenic resistance, it is possible to provide materials for new pathogenic transgenic plants and resistant molecular breeding. Therefore, the present invention also provides a method of imparting disease resistance to a plant, comprising the step of transforming the plant using a recombinant vector comprising the CaPLP1 gene.

More specifically, the present invention relates to a CaPLP1 gene using a TRV2 vector (Baulcombe DC (1999) using a Tobacco Rattle Virus (TRV), which is used for virus-induced gene silencing (VIGS). Fast forward genetics based on virus-induced gene silencing.Curr.Opin.Plant Biol. 2: 109-113) to produce a recombinant vector TRV2 :: CaPLP1 . The recombinant vector was introduced into the plant to prepare a VIGS plant in which the expression of CaPLP1 was suppressed, thereby reducing the resistance (see FIGS. 5A to 7).

Furthermore, by using the recombinant vector (pBIN35S :: CaPLP1 ) and the Agrobacterium tumefaciens strain GV3101 strain containing the CaPLP1 gene in accordance with the present disease, transgenic expression in Arabidopsis plants was obtained. Confirming the plant disease resistance activation function of the CaPLP1 gene (see FIGS. 8 to 9c), thereby providing a disease resistant transgenic plant.

As described above, in the present invention, it was confirmed that CaPLP1 is essential for the pathogenic expression of pepper plants, and it is possible to accumulate genetic information of the response to CaPLP1 disease and related disease-associated protein (PR protein). It can provide an interesting model of signaling for, and can be used to develop biotechnological plants that increase disease resistance by understanding the biochemical changes that cause resistance.

As described above, by completing the present invention, it is involved in the defensive reaction against pepper plant bacterial disease ( Xanthomonas campestris pv.vesicatoria ) and isolates the Patatin- like phosphorus phase CaPLP1 gene, a gene related to plant disease resistance. By decoding, the sequence information and amino acid sequence information can be provided, and at the same time, the gene can be used not only to provide a plant disease resistance search method, but also to contribute to the production of resistant plant breeding and transgenic plants.

In addition, by confirming the resistance reduction through the inhibition of the CaPLP1 gene according to the present invention, by overexpressing the gene in the Arabidopsis to confirm plant disease resistance, CaPLP1 according to the present invention can be provided as a material for breeding 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 a view showing the nucleotide sequence of the pepper CaPLP1 gene cloned from the red pepper greenery varieties ( Capsicum annnuum L. cv. Nockwang) according to the present invention,
Figure 2a shows the results of the exon and intron analysis of CaPLP1 and CaPLP 2 according to the present invention,
Figure 2b shows the result of comparing the amino acid sequence of CaPLP1 and CaPLP2 according to the present invention,
Figure 2c shows the results of RT-PCR analysis for comparing the expression of CaPLP1 and CaPLP2 according to the present invention after inoculating pathogenic, non-pathogenic pathogens,
Figure 3 is a schematic diagram showing the relationship between the amino acid sequence of CaPLP1 and CaPLP2 of the present invention and the Arabidopsis, a model plant, and patatin-like phosphorylation of other crops previously reported, a conserved catalytic dyad motif important for LAH activity. Shows an analysis of
Figure 4a shows the expression results of CaPLP1 gene according to the present invention in each organ of the red pepper cultivation varieties,
Figure 4b and Figure 4c shows the difference between CaPLP1 gene expression in the inoculated and non-vaccinated leaves and CaPLP1 protein expression after pathogens when inoculated with pathogenic and non-pathogenic strains of pepper bactericidal spot ( Xanthomonas campestris pv.vesicatoria ) The figure shown,
Figure 4d is a diagram showing the results of induction of the expression of CaPLP1 gene according to the present invention after treatment with salicylic acid (Salicylic acid),
Figure 5a is a diagram confirming that the expression of the CaPLP1 gene according to the present invention is suppressed using the method of virus-induced gene silencing (VIGS),
Figure 5b is the disease resistance of the red pepper when the inoculation of pepper bactericidal strains ( Xanthomonas campestris pv. Vesicatoria ) of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a inoculated to the plant of the red pepper greenery varieties suppressed CaPLP1 gene expression according to the present invention The symptom is enlarged and the apoptosis which is a resistance reaction is reduced.
Figure 5c is a diagram showing the increase in bacterial growth in plants in which the expression of the CaPLP1 gene according to the present invention is suppressed,
Figure 5d is a red pepper bacterium of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a ( Xanthomonas campestris pv. Vesicatoria ) in plants in which expression of the CaPLP1 gene is suppressed by Western blotting using an antibody of CaPLP1 protein. Is a diagram showing the results of comparing the CaPLP1 protein induction results with the control when inoculated,
Figure 6a is when the inoculation of the pepper bacteriophage bacterium ( Xanthomonas campestris pv. Vesicatoria ) of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a in the pepper green cultivars suppressed the expression of CaPLP1 gene according to the present invention in This is a diagram showing the change of H ₂ O _{2 through} Dab (DAB) staining (3,3'-diaminobenzidine staining) and quantitative,
Figure 6b is when the inoculation of pepper bacteriophage bacteria ( Xanthomonas campestris pv. Vesicatoria ) of the pathogenic strain Ds1 and non-pathogenic strain Bv5-4a inoculated to the plant of the pepper greenery varieties inhibited the expression of CaPLP1 gene according to the present invention Cell death of quantified by trypan staining and measurement of ion conductivity,
Figure 7 when inoculated with the virulent strain Ds1 and pepper jeommunuibyeong bacterial strains of non-pathogenic strains Bv5-4a (Xanthomonas campestris pv. Vesicatoria) in plants of the expressed the inhibition of pepper varieties nokgwang CaPLP1 gene according to the present invention, inhibition of expression CaPLP1 Is a diagram showing the results of expression differences between the gene and resistance,
Figure 8 is a recombinant vector pBIN35S: CaPLP1 and Agrobacterium tumefaciens (Agrobacterium tumefaciens strain GV3101) with the strain, and the CaPLP1 gene expression over in transgenic Arabidopsis thaliana plants that overexpressed the CaPLP1 gene, which results in Arabidopsis thaliana disease resistance The figure shows the result of observing that the expression of the related gene AtPDF1 gene is increased,
Figure 9a is a photograph showing the pathogenic resistance to Hyaloperonospora parasitica in transgenic Arabidopsis plants overexpressed CaPLP1 gene according to the present invention,
Figure 9b is a diagram showing the results of the analysis through the trypan blue staining (trypan blue staining) after the inoculated apoptosis ( hyaloperonospora parasitica ) inoculated in transgenic Arabidopsis plants overexpressing the CaPLP1 gene according to the present invention ,
Figure 9c is a view showing the results confirmed by measuring the spore hard water and spore count increased the pathogenic resistance to Hyaloperonospora parasitica in transgenic Arabidopsis plants overexpressed CaPLP1 gene according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 CaPLP1 Base / Amino Acid Sequencing of Genes

Pepper, one of pathogen resistance related protein for bacterial jeommunuibyeong patatin similar force porayi phase (CaPLP1: C apsicum a nnuum atatin- P L P ike hospholipase 1) to to provide the protein coding gene sequence and amino acid sequence deduced therefrom The same test was performed.

Capsicum annuum cv.Nockwang was inoculated with red pepper bacterium bacterium ( Xanthomonas campestris pv.vesicatoria ), and after 18 hours of wet treatment, the plants showing resistance to apoptosis, which were resistant lesions, were taken out of the room and harvested leaves. .

Next, mRNA was extracted from this leaf sample and reverse transcribed to prepare cDNA libraries. The individual cDNA clones were prepared from the cDNA library thus prepared and adsorbed to the nylon membrane (Hybond N +) uniformly, and then the leaves of the red pepper plants inoculated with the non-pathogenic strains of the red pepper bacterial spot pattern bacteria and the leaves of whole red pepper plants not inoculated. The total mRNA extracted at was labeled with dioxygenin (digoxygenin) to make a probe, and then differential hybridization was performed.

As a result of hybridization, the genes amplified intensively for mRNA probes of non-pathogenic strain inoculated plants were assumed to be genes related to pathogenic resistance, and clones were obtained.

After sequencing the obtained clones, the CaPLP1 protein coding gene was identified by using a blast program and compared with the gene database registered in GenBank and named as pepper patatin-like phosphorase ( CaPLP1 ) gene. In the construction of CaPLP1 cDNA clones from the cDNA library, CaPLP2 was found to have less than 132 sequences compared to CaPLP1 , and the genome analysis of pepper revealed that CaPLP2 was an alternative splicing form of CaPLP1 . As a result of analyzing the amino acid sequence of CaPLP1 protein, it was found that it belongs to the patatin-like phosphorylated group having LAH activity of plants (see FIG. 3). Gene base sequences (SEQ ID NO: 1 and SEQ ID NO: 3) of the identified CaPLP1 and CaPLP2 and amino acid sequences encoded by the base sequences (SEQ ID NO: 2 and SEQ ID NO: 4) are shown in FIGS. 1 and 2.

Example 2 In Red Pepper Plant by Pathogen Inoculation CaPLP1 Exploration of Gene Expression

In order to confirm the method of using the CaPLP1 gene obtained in Example 1 for the resistance search was tested as follows.

[Step 1]

First of all, in order to present a specific resistance screening method, the non-pathogenic strain Bv5-4a exhibiting an incompatible reaction with the pathogenic strain Ds1 of Xanthomonas campestris pv.vesicatoria , which has a friendly reaction with plants, was identified as YN (Yeast- Nutrient). After incubation in the medium, inoculated by infiltrating the back of the five and six leaves of Capsicum annuum cv. Nockwang 6 capillary green pepper varieties ( Capsicum annuum cv. Nockwang) at a concentration of 10 ⁸ cfu / ㎖, inoculated by 28 ℃, Wet was treated for 18 hours at 100% relative humidity. After inoculation, the leaves were sampled at different time intervals to separate RNA and protein, and electrophoresed on formaldehyde gel and SDS-PAGE gel, respectively, and then transferred to a nylon membrane (Hybond N AND P).

Next, α- ³² P [dCTP] labeled with an isotope was labeled with CaPLP1 gene obtained in Example 1 using Klenow enzyme, and then the reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% 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.

In addition, specific antibodies of CaPLP1 were prepared using rabbits, and protein expression of CaPLP1 was also examined using the same. After the extracted protein hybridized with 5% milky containing CaPLP1 antibody to the transferred nylon membrane, immunoglobulin mouse (IgG) antibody conjugated with Horseradish peroxidase was used. CaPLP1 protein expression was confirmed (FIG. 4C).

4A to 4C, the CaPLP1 gene was strongly expressed 10 hours after inoculation with non-pathogenic pepper bacterial spot disease (FIG. 4B).

Example 3 Plant Hormone CaPLP1 Induced Expression of Genes

In order to find out whether the CaPLP1 gene of the present invention can be induced by salicylic acid, which is a plant hormone commonly used to induce resistance to bacterial pathogens, the following experiment was performed.

[Step 1]

First, in order to search for derivatives useful for inducing expression of CaPLP1 gene, resistance induction test was performed using salicylic acid among chemicals known to be involved in the induction of plant disease resistance expression. Six-leaf peppers were used for all experiments.

Salicylic acid was treated by spraying pepper leaves at a concentration of 5 mM, and each plant collected leaves 1, 5, 10, 15, 20 and 25 hours after treatment, extracted RNA from each sample and extracted RNA. Was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +), followed by northern blotting. The test method is the same as described in Example 2.

As a result of the experiment, the CaPLP1 gene was treated with salicylic acid, as shown in FIG. 4D, and the expression was induced from 10 hours, and the expression was maintained strongly until 25 hours.

Example 4 CaPLP1  Changes in Disease Resistance by Inhibition of Gene Expression

When in the pepper plant is suppressed to a specific expression of the CaPLP1 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 CaPLP1 gene obtained from of ( The recombinant vector TRV2: CaPLP1 was prepared by introducing CaPLP1 gene into TRV2 vector using TRV (Tobacco Rattle Virus), which is used in VIGS, virus-induced gene silencing (Bulcombe DC (1999) Fast forward genetics based on virus-induced gene silencing.Curr. Opin.Plant Biol. 2: 109-113).

The recombinant vector TRV2 thus prepared: CaPLP1 was introduced into Agrobacterium tumefaciense strain GV 3101 through electrophoresis, and the GV3101 strain containing the recombinant vector was added to the seedlings of pepper seedlings. Inhibition of CaPLP1 gene expression was induced by inoculation in pepper plants by infiltration using syringes. The effects of gene expression inhibition were reverse transcriptase PCR (RT-PCR) and real-time RT-PCR (real). -time RT-PCR), and Western blot confirmed that the expression was reduced at the protein level (see FIG. 5A).

[Step 1]

In order to investigate the change in the resistance to bacterial diseases in plants with inhibition of CaPLP1 expression, the pepper bacterial spot pattern bacterium was applied to the pepper plants that were inhibited with the expression of CaPLP1 gene and pepper green mine varieties inoculated with TRV2: 00 vector as a control. after inoculation of the pathogenic strain Ds1 and non-pathogenic strains of Bv5-4a (Xanthomonas campestris pv. vesicatoria) to 10 ⁷ and 10 ⁸ cfu ml ^-1 concentration of the symptoms observed in the inoculated leaf in 5 x 10 ⁴ cfu ml ^-1 Bacterial growth was measured in infected plant tissues at 0 and 3 days after inoculation (FIG. 5B and FIG. 5C).

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. 6a and 6b).

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 CaPLP1 through real-time RT-PCR. In comparison with the control plants at the time of inoculation with the effect, the difference in expression of the gene associated with disease resistance was observed (FIG. 7).

As shown in FIG. 5B, the experimental results were observed after the inoculation of the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a in the plants inhibited by the expression of CaPLP1 by VIGS. Sulfation and necrosis were observed, and when the non-pathogenic strains were inoculated, local apoptosis was observed in the control plants on day 2, but in the plants inhibited by the expression of CaPLP1 , the local apoptosis was slowed.

As shown in FIG. 5C, bacterial growth of pathogenic bacteria and non-pathogenic bacteria in infected tissues was measured, and more bacterial growth was observed in plants in which CaPLP1 expression was suppressed compared to the control.

As shown in FIGS. 6A and 6B, the accumulation of H ₂ O ₂ in CaPLP1 -inhibited plants was reduced in both pathogenic and non-pathogenic strains compared to the control plants, and in particular, less ionic conductivity was observed in non-pathogenic strains. there was.

In addition, as a result of the real-time Alti- pishial test, as shown in Figure 7, it was observed that the inhibition of the CaPLP1 gene effectively in the plants inhibited CaPLP1 , CaBPR1 gene ( Coi et al. (2007) Hydrogen peroxide generation by the pepper extracellular peroxidase CaPO2 activates local and systemic cell death and defense response to bacterial pathogens.

Example 5 CaPLP1  Preparation of Transgenic Plants Using Genes CaPLP1  Disease Resistance Changes in 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 CaPLP1 gene in Arabidopsis thaliana plants used in the plant, the CaPLP1 gene obtained in Example 1 in pBIN35S vector PBIN35S: CaPLP1 was prepared by introduction, and the recombinant vector pBIN35S: CaPLP1 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 inducing overexpression of CaPLP1 gene was prepared and the following tests were performed.

[Step 1]

CaPLP1 gene order hwalmul in overexpressed plant pathogens (biotrophic pathogen), to investigate whether the resistance is increased on the CaPLP1 gene overexpression of Arabidopsis and the wild-type Arabidopsis CaPLP1 and the Arabidopsis plants in the pole plant as control AtPR1 ( Huffaker and Ryan .. (2007) Endogenous peptide defense signals in Arabidopsis differentially amplify signaling for the innate immune response) and AtPDF1 (Huffaker and Ryan .. (2007) Endogenous peptide defense signals in Arabidopsis differentially Expression of the amplify signaling for the innate immune response) gene was observed and the results are shown in FIG. 8.

Figure # 1, # 2, # 3 from 8 denotes a system code of the transformed using the plant, the experimental results CaPLP1 and gene CaPLP1 gene-overexpressed effectively in the over-expression transgenic Arabidopsis plants, the AtPDF1 gene compared with the control A large amount of expression was confirmed.

[Step 2]

CaPLP1 overexpressed plants were inoculated with hyaloperonospora parasitica Noco2 ( hyaloperonospora parasitica Noco 2), a scavenger- producing parasitic parasite, in the Arabidopsis. Hyeloferonospora parasitica Noco2 was sprayed at a concentration of 5 × 10 ⁴ spores ml ⁻¹ at the cotyledon stage of CaPLP1 overexpressed and non-overexpressed wild type control plants. After inoculation, it was wet-treated at the temperature of 17 degreeC.

As a result, the resistance was increased in the transgenic Arabidopsis plants overexpressing CaPLP1 , it was observed that mycelial growth of Bacillus pathogens is suppressed and the results are shown in Figures 9a to 9d.

Although specific portions of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus 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.

<110> Korea University Industrial and Academic Collaboration Foundation <120> Disease resistance-related pepper patatin-like phospholipase gene          CaPLP1, screening of plant disease resistance and transgenic          plants using the same <130> P11-100518-01 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 1491 <212> DNA 213 Capsicum annuum L. cv. Nockwang <400> 1 cttttaacat attaacatac gccattctcc ctctctttca aaaaatcaga tagataaatt 60 tttttttgtg tattggataa ctcaacatgg ggaggatggt tcttatagct gcagcaatga 120 ttctgtttgt gacccttcaa gttttacaac ctccattaat ggtttctgct gctaccaaag 180 gaaagatggt aactgttttg agtatcgatg gaggtggtat tagaggcatt attcctggca 240 cccttctagc tttccttgaa tccaaacttc aggaacttga tggaccaaat gcaagagttg 300 cagattattt tgatgtagta gcaggaacaa gcacaggtgg attagtaaca actatgctta 360 ctgctcctaa caaggataat cgccctttat atcaagcaaa agaaatttcc agtttctaca 420 tgcagcatgg ccctcaaatt tttcctcaaa gcaggcgtaa cagcttcttg agaagagtca 480 caaatttgtt tgggggacca aagtatgatg gcaagtactt gagaacaatt attacttcaa 540 tattaggcaa tcttactatg aagcaaacgt tgactaatac agtcatacca acttttgata 600 tcaaacgcct tcaaccaatt atcttcagta ctgctgatgc aaaagcaaat atgtctaaga 660 atgctcaatt gtcagacgtt tgcctcagta cctcggccgc acctacttat ttcccagtgc 720 actattttga gactaaggat gctcaaggga aaacacgtac atttaatctg gtcgatggag 780 gcgtagctgc aaataatcca accttaatgg caataacaca cgtgtcaaaa caaatcatga 840 ggggcaactt tcagtatgag gatatggaga atgtggactg caagaaaatg ttggttttgt 900 cattgggaac gggtataggc aagcacgaag agaagtacaa cgctacagtg gcttccaggt 960 gggggatgct aggctgggtg ttcaacaatg gtgccacccc tttgatagat atttttggtg 1020 atgctagtgc tgatatggta gatatacatg tttccaccat gtttcagacc tttggcagtg 1080 agaagaatta cgtcagaatt caggacgaca atttggtcgg ggaggctgca tctatggata 1140 ttgcaaccag agaaaacatg gagacacttg tacaaattgg taatggtcta ttgaagaagc 1200 caatttcaag ggtcaactta gaaacagggc gatacgaacc agttgttggg gaaggcacca 1260 acgaagctgc tatggtccgt tttgcccagt tgctttcaga ggaaaggaag ctccgaatag 1320 ccaactaaca agtgcacttc acagatcacc ctaactggtg atatatacta caagaattta 1380 tcacacaatt ttttggatga ttagaagatt aattttcatt ctttgacttt caatagtaaa 1440 ataaacatcc ccacgaggaa aataaaattt aaaaaaaaaa aaaaaaaaaa a 1491 <210> 2 <211> 413 <212> PRT 213 Capsicum annuum L. cv. Nockwang <400> 2 Met Gly Arg Met Val Leu Ile Ala Ala Ala Met Ile Leu Phe Val Thr   1 5 10 15 Leu Gln Val Leu Gln Pro Pro Leu Met Val Ser Ala Ala Thr Lys Gly              20 25 30 Lys Met Val Thr Val Leu Ser Ile Asp Gly Gly Gly Ile Arg Gly Ile          35 40 45 Ile Pro Gly Thr Leu Leu Ala Phe Leu Glu Ser Lys Leu Gln Glu Leu      50 55 60 Asp Gly Pro Asn Ala Arg Val Ala Asp Tyr Phe Asp Val Val Ala Gly  65 70 75 80 Thr Ser Thr Gly Gly Leu Val Thr Thr Met Leu Thr Ala Pro Asn Lys                  85 90 95 Asp Asn Arg Pro Leu Tyr Gln Ala Lys Glu Ile Ser Ser Phe Tyr Met             100 105 110 Gln His Gly Pro Gln Ile Phe Pro Gln Ser Arg Arg Asn Ser Phe Leu         115 120 125 Arg Arg Val Thr Asn Leu Phe Gly Gly Pro Lys Tyr Asp Gly Lys Tyr     130 135 140 Leu Arg Thr Ile Ile Thr Ser Ile Leu Gly Asn Leu Thr Met Lys Gln 145 150 155 160 Thr Leu Thr Asn Thr Val Ile Pro Thr Phe Asp Ile Lys Arg Leu Gln                 165 170 175 Pro Ile Ile Phe Ser Thr Ala Asp Ala Lys Ala Asn Met Ser Lys Asn             180 185 190 Ala Gln Leu Ser Asp Val Cys Leu Ser Thr Ser Ala Ala Pro Thr Tyr         195 200 205 Phe Pro Val His Tyr Phe Glu Thr Lys Asp Ala Gln Gly Lys Thr Arg     210 215 220 Thr Phe Asn Leu Val Asp Gly Gly Val Ala Ala Asn Asn Pro Thr Leu 225 230 235 240 Met Ala Ile Thr His Val Ser Lys Gln Ile Met Arg Gly Asn Phe Gln                 245 250 255 Tyr Glu Asp Met Glu Asn Val Asp Cys Lys Lys Met Leu Val Leu Ser             260 265 270 Leu Gly Thr Gly Ile Gly Lys His Glu Glu Lys Tyr Asn Ala Thr Val         275 280 285 Ala Ser Arg Trp Gly Met Leu Gly Trp Val Phe Asn Asn Gly Ala Thr     290 295 300 Pro Leu Ile Asp Ile Phe Gly Asp Ala Ser Ala Asp Met Val Asp Ile 305 310 315 320 His Val Ser Thr Met Phe Gln Thr Phe Gly Ser Glu Lys Asn Tyr Val                 325 330 335 Arg Ile Gln Asp Asp Asn Leu Val Gly Glu Ala Ala Ser Met Asp Ile             340 345 350 Ala Thr Arg Glu Asn Met Glu Thr Leu Val Gln Ile Gly Asn Gly Leu         355 360 365 Leu Lys Lys Pro Ile Ser Arg Val Asn Leu Glu Thr Gly Arg Tyr Glu     370 375 380 Pro Val Val Gly Glu Gly Thr Asn Glu Ala Ala Met Val Arg Phe Ala 385 390 395 400 Gln Leu Leu Ser Glu Glu Arg Lys Leu Arg Ile Ala Asn                 405 410 <210> 3 <211> 1359 <212> DNA 213 Capsicum annuum L. cv. Nockwang <400> 3 cttttaacat attaacatac gccattctcc ctctctttca aaaaatcaga tagataaatt 60 tttttttgtg tattggataa ctcaacatgg ggaggatggt tcttatagct gcagcaatga 120 ttctgtttgt gacccttcag gaacttgatg gaccaaatgc aagagttgca gattattttg 180 atgtagtagc aggaacaagc acaggtggat tagtaacaac tatgcttact gctcctaaca 240 aggataatcg ccctttatat caagcaaaag aaatttccag tttctacatg cagcatggcc 300 ctcaaatttt tcctcaaagc aggcgtaaca gcttcttgag aagagtcaca aatttgtttg 360 ggggaccaaa gtatgatggc aagtacttga gaacaattat tacttcaata ttaggcaatc 420 ttactatgaa gcaaacgttg actaatacag tcataccaac ttttgatatc aaacgccttc 480 aaccaattat cttcagtact gctgatgcaa aagcaaatat gtctaagaat gctcaattgt 540 cagacgtttg cctcagtacc tcggccgcac ctacttattt cccagtgcac tattttgaga 600 ctaaggatgc tcaagggaaa acacgtacat ttaatctggt cgatggaggc gtagctgcaa 660 ataatccaac cttaatggca ataacacacg tgtcaaaaca aatcatgagg ggcaactttc 720 agtatgagga tatggagaat gtggactgca agaaaatgtt ggttttgtca ttgggaacgg 780 gtataggcaa gcacgaagag aagtacaacg ctacagtggc ttccaggtgg gggatgctag 840 gctgggtgtt caacaatggt gccacccctt tgatagatat ttttggtgat gctagtgctg 900 atatggtaga tatacatgtt tccaccatgt ttcagacctt tggcagtgag aagaattacg 960 tcagaattca ggacgacaat ttggtcgggg aggctgcatc tatggatatt gcaaccagag 1020 aaaacatgga gacacttgta caaattggta atggtctatt gaagaagcca atttcaaggg 1080 tcaacttaga aacagggcga tacgaaccag ttgttgggga aggcaccaac gaagctgcta 1140 tggtccgttt tgcccagttg ctttcagagg aaaggaagct ccgaatagcc aactaacaag 1200 tgcacttcac agatcaccct aactggtgat atatactaca agaatttatc acacaatttt 1260 ttggatgatt agaagattaa ttttcattct ttgactttca atagtaaaat aaacatcccc 1320 acgaggaaaa taaaatttaa aaaaaaaaaa aaaaaaaaa 1359 <210> 4 <211> 369 <212> PRT 213 Capsicum annuum L. cv. Nockwang <400> 4 Met Gly Arg Met Val Leu Ile Ala Ala Ala Met Ile Leu Phe Val Thr   1 5 10 15 Leu Gln Glu Leu Asp Gly Pro Asn Ala Arg Val Ala Asp Tyr Phe Asp              20 25 30 Val Val Ala Gly Thr Ser Thr Gly Gly Leu Val Thr Thr Met Leu Thr          35 40 45 Ala Pro Asn Lys Asp Asn Arg Pro Leu Tyr Gln Ala Lys Glu Ile Ser      50 55 60 Ser Phe Tyr Met Gln His Gly Pro Gln Ile Phe Pro Gln Ser Arg Arg  65 70 75 80 Asn Ser Phe Leu Arg Arg Val Thr Asn Leu Phe Gly Gly Pro Lys Tyr                  85 90 95 Asp Gly Lys Tyr Leu Arg Thr Ile Ile Thr Ser Ile Leu Gly Asn Leu             100 105 110 Thr Met Lys Gln Thr Leu Thr Asn Thr Val Ile Pro Thr Phe Asp Ile         115 120 125 Lys Arg Leu Gln Pro Ile Ile Phe Ser Thr Ala Asp Ala Lys Ala Asn     130 135 140 Met Ser Lys Asn Ala Gln Leu Ser Asp Val Cys Leu Ser Thr Ser Ala 145 150 155 160 Ala Pro Thr Tyr Phe Pro Val His Tyr Phe Glu Thr Lys Asp Ala Gln                 165 170 175 Gly Lys Thr Arg Thr Phe Asn Leu Val Asp Gly Gly Val Ala Ala Asn             180 185 190 Asn Pro Thr Leu Met Ala Ile Thr His Val Ser Lys Gln Ile Met Arg         195 200 205 Gly Asn Phe Gln Tyr Glu Asp Met Glu Asn Val Asp Cys Lys Lys Met     210 215 220 Leu Val Leu Ser Leu Gly Thr Gly Ile Gly Lys His Glu Glu Lys Tyr 225 230 235 240 Asn Ala Thr Val Ala Ser Arg Trp Gly Met Leu Gly Trp Val Phe Asn                 245 250 255 Asn Gly Ala Thr Pro Leu Ile Asp Ile Phe Gly Asp Ala Ser Ala Asp             260 265 270 Met Val Asp Ile His Val Ser Thr Met Phe Gln Thr Phe Gly Ser Glu         275 280 285 Lys Asn Tyr Val Arg Ile Gln Asp Asp Asn Leu Val Gly Glu Ala Ala     290 295 300 Ser Met Asp Ile Ala Thr Arg Glu Asn Met Glu Thr Leu Val Gln Ile 305 310 315 320 Gly Asn Gly Leu Leu Lys Lys Pro Ile Ser Arg Val Asn Leu Glu Thr                 325 330 335 Gly Arg Tyr Glu Pro Val Val Gly Glu Gly Thr Asn Glu Ala Ala Met             340 345 350 Val Arg Phe Ala Gln Leu Leu Ser Glu Glu Arg Lys Leu Arg Ile Ala         355 360 365 Asn    

Claims (9)

An isolated gene ( CaPLP1) of (a) or (b) which codes for pepper-derived patatin-like phosphorus phase as a plant disease resistance related gene:
(a) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 2;
(b) a gene having the nucleotide sequence of SEQ ID NO: 1. An isolated protein having the amino acid sequence of SEQ ID NO: 2 (CaPLP1). An isolated gene ( CaPLP2) of (a) or (b), which codes for pepper-derived patatin-like phosphorus phase as a plant disease resistance related gene:
(a) a gene encoding a protein having the amino acid sequence of SEQ ID NO: 4;
(b) a gene having the nucleotide sequence of SEQ ID NO: 3. An isolated protein having the amino acid sequence of SEQ ID NO: 4 (CaPLP2). A recombinant vector comprising the gene of claim 1. A plant transformed with the recombinant vector according to claim 5. Separation of mRNA from the plant to determine the differential expression of the gene ( CaPLP1, CaPL2 ) described in claim 1 or claim 3. 8. The method of claim 7, wherein the plant is a plant inoculated with pathogenic or non-pathogenic bacteria of red pepper bacterial spot pattern disease. The method of claim 7, wherein the plant is a salicylic acid-treated plant.

Images