KR101250168B1 - Pepper pathogenesis-related protein 4b gene and screening method of plant disease resistance using the same - Google Patents

Abstract

The present invention interacts with CaLRR1 (Leucine-rich repeat protein1) of red pepper plants, which are genes specifically induced when infected with pathogenic and non-pathogenic bacteria of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ). The present invention relates to a gene for the pathogenesis-related protein 4b ( CaPR4b ), which is a novel gene, and to the production of a resistant plant and an antifungal agent. According to the present invention described above, it is possible to provide a material for breeding disease resistant plants and a raw material of an antifungal agent, and there is an advantage that can be used as a tool for identifying a mechanism of action against plant disease resistance.

Description

Pepper pathogenesis-related protein 4b gene and screening method of plant disease resistance using the same}

The present invention relates to a pepper pathogenesis-related protein 4b ( CaPR4b ) that interacts with CaLRR1, a Leucine-rich repeat protein (LRR) of pepper plants, a novel gene related to plant disease defense, and pathogenic resistance using the same. The present invention relates to a transformed plant and a disease resistance screening method. More specifically, the present invention relates to the nucleotide sequence and amino acid sequence of the CaPR4b gene, a novel plant-related defense gene, which is strongly expressed in the resistance reaction when infected with pathogenic strains and non-pathogenic strains of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ). To detect whether the expression level of CaPR4b protein in the bio / chemical / physical resistance induction treatment by the differential expression of the resistance-related gene CaPR4b and the immune response using the antibody produced using the peptide of CaPR4b It relates to a method of searching for resistance of plants. In addition, the present invention explored the role of CaPR4b gene resistance in plants through the Agrobacterium-mediated transient expression method, the CaPR4b gene was introduced through transformation into Arabidopsis thaliana , By overexpression to confirm the effect of increasing the plant disease resistance of the CaPR4b gene, and to provide a plant disease resistant transgenic plant through this.

Red pepper is a representative economic crop that has been used as an important condiment in Korean food such as kimchi for a long time in Korea. It is a crop that is cultivated all over the world including many countries of South America. It is a representative economic crop with a large amount of cultivation, boasting the largest scale as a single item among domestic vegetable seeds. Domestic annual consumption is 3.5kg per capita dried pepper and consumption is 200,000 tons, and the market is the second largest crop after rice, which is estimated to reach 1 trillion won. However, in the developed countries, the importance of red pepper is relatively low, so the research is vulnerable and full investment and research have not been made.

In addition, pepper, bacterial, fungal, and viral diseases have been reported variously, the most serious damage to the plague caused by fungi, anthrax caused by fungi and bacterial peak pattern disease caused by bacteria. The damage of pathogens has led to the reduction of the yield and quality of peppers. To solve these problems, stable breeding, reduction of production costs due to pesticides and labor reduction, and breeding of disease-resistant varieties of pepper are considered as important targets. have.

Especially, bacterial spot disease of red pepper is appeared on leaves, petiole, stem, fruit and fruit, and it forms dark brown round or irregular small spot pattern to induce deciduous leaves and reduce yield. Is showing damage. These pepper bacterial spots are difficult to control chemicals using pesticides, so in order to minimize the damage to pepper bacterial spots, it is recommended to control the cultivation environment of pepper plants to control them and to develop resistant pepper varieties and to use biological control appropriately. It is the most efficient and economical control method for farmers. Among these control methods, the development of molecular breeding technology using useful pepper defense genes using biotechnologies for the improvement and breeding of pepper varieties that are resistant to disease can be said to be the most desirable for the production of pepper in the 21st century.

Programs to cultivate disease-resistant varieties of various economic crops, including peppers, have been steadily progressed in order to minimize crop production damage caused by pests, and in the study on the mechanism of resistance in plants for understanding the disease resistance and its practical application. Is used as a model plant.

When a plant is infected, the host plant activates defense mechanisms in a variety of intracellular metabolic pathways, which are determined by the friendly and incompatible reactions in the interaction between the host plant and phytopathogens early in the pathogen infection, thus leading to disease progression. It is determined whether the resistance (defense) reaction can be limited.

In an incompatible resistance response, the infected plant recognizes the invasion of pathogens, and responds to hypersensitive responses, such as local cell death, to the cells at the site of infection, accumulation of callose, and lignin. It shows reactions such as thickening of cell walls due to aging, produces various kinds of antimicrobial substances such as phytoalexin, and produces defense-related proteins called pathogenesis related (PR) proteins. It is known.

These plant defense mechanisms are reported not only when invading pathogens, but also by artificial stimuli such as ethylene or methyl jasmonate, and by environmental stress such as physical injury, salinity, and temperature.

Plants respond to defensive biological / physical / environmental stresses, and in particular, an important role in the plant's ability to defend against pathogens is the expression of plant disease resistance by the interaction of the plant with pathogens. This is the signaling pathway involved. In this regard, the disease defense mechanisms of the plant and how the genes associated with them are recognized as pathogens and as a result of the transmission pathway have been studied in the whole plant, the main concern is biological / biochemical / cell biological methods It has been through the study of the function of disease resistance (defense) gene using.

The study of the function of the disease resistance (defense) gene can have the value of studying the mechanisms related to the physiology and resistance of plants, as well as contribute to the development of resistant varieties through molecular breeding of the crops studied. The recent research on crop molecular breeding technology has been accelerated by the intervention of genetic engineering techniques. Crop molecular breeding through disease-resistant (defense) genes can be used as a material for all species and breeding that has been possible at the existing genome level. By enabling technology at the genetic level, it has the advantage of maximizing the current breeding technology in that it shows more effective breeding effect and can control the effects of such breeding.

Pathogenesis -related protein 4b ( PR4b ), on the other hand, contains many plants such as potatoes, tobacco, tomatoes, Arabidopsis, barley, rice, rice and corn. 4 (pathogenesis-related protein 4, PR4 ) has been reported to inhibit growth against pathogenic fungi in vitro. It is also known to be induced after the infection of B. aureus and Tobacco mosaic virus. As such, the PR4 family is strongly induced by pathogens and external stimuli. Especially, Arabidopsis plants have been reported to be dependent on the jasminoid acid and ethylene pathways after pathogen infection. In addition, the PR4 family can be divided into forms containing and not containing chitin binding domains. Contrary to these findings, however, research on the function of the PR4 family is still incomplete. Agrobacterium- mediated transient expression of Arabidopsis plants overexpressing the CaPR4b gene and the Agrobacterium- mediated transient expression of CaPR4b genes for the function of pathogenesis-related protein 4b ( CaPR4b ) in red pepper Evaluation of resistance and the response of CaPR4b to bacterial and fungal pathogens of recombinant proteins enabled evaluation of resistance assays. This invention is able to accumulate genetic information of pepper's defense-related gene 4b in response to disease and its associated resistance response, which can provide an interesting model for signaling of plant diseases, and induces biochemical resistance By understanding the change, it may be practically used to develop genetically engineered plants that promote disease resistance or to develop plant protection chemicals that operate to promote the plant's genetic resistance.

The present invention is to solve the above-mentioned problems in the prior art, the purpose of which is involved in the defensive reaction against pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) Leucine- one of the pepper plant related genes related to plant disease defense By separating and decoding the CaPR4b gene encoding the pathogenesis-related protein 4b of red pepper, a new gene that interacts with CaLRR1, a rich repeat protein (LRR), the nucleotide sequence information and the amino acid sequence information thereof are read. To provide.

It is also an object of the present invention to provide a method for detecting resistance of plants using the CaPR4b gene to search for resistance to plant diseases or chemicals.

Furthermore, an object of the present invention is to introduce a CaPR4b gene into a plant to produce a transformed plant having resistance to plant diseases.

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

The object of the present invention is to interact with CaLRR1, which is a Leucine-rich repeat protein (LRR) of red pepper plants isolated from green pepper varieties that exhibits hypersensitivity resistance to inoculation of non-pathogenic strains of the red pepper bacterial bacterium ( Xanthomonas campestris pv.vesicatoria ). Identifying the new pathogenic gene CaPR4b in action to determine its nucleotide sequence, and using it to investigate the expression of CaPR4b through biological / chemical treatment, pepper plant and this gene Agrobacterium-mediated transient expression (Agrobacterium) It was achieved by confirming the induction of plant disease resistance by using the -mediated transient expression method or by overexpression of CaPR4b gene through transformation in Arabidopsis thaliana .

Accordingly, the present invention provides a plant disease defense-related gene (a) a gene encoding pathogenicity-related protein 4b having the amino acid sequence of SEQ ID NO: 2 or (b) an isolated gene ( CaPR4b ) having the nucleotide sequence of SEQ ID NO: 1 do.

The present invention also provides an isolated pathogenic related protein 4b (CaPR4b) having the amino acid sequence of SEQ ID NO: 2.

In another aspect, the present invention 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 :: CaPR4b .

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.

Furthermore, the present invention provides a method of searching for resistance of a plant, comprising separating RNA from a plant and confirming differential expression of a CaPR4b gene or identifying whether CaPR4b protein is expressed using an antibody of the CaPR4b protein as a probe. .

The present invention provides an antifungal agent having antibacterial activity against phytopathogenic fungi comprising the CaPR4b protein as an active ingredient. The fungus is preferably, but not limited to, Alternaria brassicicola .

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

The present invention relates to GAL4, CaLRR1, which is a Leucine-rich repeat protein (LRR) of red pepper plants, a gene specifically induced when infected with pathogenic and non-pathogenic bacteria of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ). PGBKT7: CaLRR1 was prepared by introducing into pGBKT7 comprising a DNA binding domain. In addition, cDNA library extracted from plants infected with non-pathogenic pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) was introduced into pGADT7 vector containing GAL4-activating domain to make recombinant vector. The cDNA library of the red pepper plants introduced into the pGBKT7: CaLRR1 and pGADT7 vectors was transformed by yeast AH109 using a lithium acetate method using a yeast-two-hybrid assay. The transformants transformed in this way were transferred to a selection medium (Adenine-Histidine-Leucine-Leucine-Tryptophan-free medium) to confirm the interaction with each other. Among these identified genes, genes suspected to be involved in disease defense (resistance) are selected to obtain clones, and among these clones, the sequencing of the genes identified by the disease-associated protein 4b (CaPR4b) gene is sequenced. By doing so, CaPR4b protein coding gene nucleotide sequence (SEQ ID NO: 1) and amino acid sequence encoded therefrom (SEQ ID NO: 2) of the red pepper green mine variety named CaPR4b are provided (see FIG. 1).

After sequencing the obtained clones according to the present invention, the 5'-terminal sequence is compared with a gene database registered in GenBank using a Blast program to identify CaPR4b protein coding genes and to relate to pathogenesis. The gene of protein 4b (CaPR4b) ( CaPR4b ) was named. CaPR4b protein showed 100% homology with Capsicum chinense of other cultivars, which showed no homology, and more than 80% homology with PR family of potato and Arabidopsis. This gene has a molecular weight of 22kDa that is resistant to various pathogens as well as pepper bacterial spots. The identified CaPR4b gene nucleotide sequence and amino acid sequence encoded by the nucleotide sequence are shown in FIG. 1. Pathogenicity related protein 4b (CaPR4b) according to the present invention is characterized by having antimicrobial activity against phytopathogenic fungi. Therefore, the antifungal agent can be provided using the protein according to the present invention.

In addition, the present invention provides a method of searching for resistance of a plant, including checking whether CaPR4b protein is expressed using an antibody of the CaPR4b protein as a probe. The method for preparing the antibody of the CaPR4b protein and the method for confirming the expression of the CaPR4b protein may use a variety of techniques known in the art to which the present invention belongs.

In addition, the present invention, a non-pathogenic induction method, such as salicylic acid, or methyl jasmonate plant hormonal chemicals to the plant is treated with a non-biological induction method, and using the resistance screening method to check whether the expression of CaPR4b protein This provides a new resistance induction method that is easier and saves time.

Next, the present invention provides a method for searching for resistance of a plant, comprising the step of identifying RNA and differential expression of the CaPR4b gene by separating RNA 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. Examples of the chemical derivatives include salicylic acid and methyl jasmonate. The CaPR4b gene expression can be determined by various methods for measuring mRNA expression in addition to Northern blot, real-time RT-PCR, microarray method. It was confirmed through experiment that CaPR4b gene according to the present invention is differentially induced expression by phytopathogens , plant hormones or chemical derivatives. Therefore, the CaPR4b gene according to the present invention can be usefully used for the resistance detection method of plants.

The present invention also confirms the effect of the gene on plants by using the Agrobacterium-mediated transient expression method, which causes the CaPR4b gene to overexpress temporarily in tobacco plants. It is possible to provide new disease-resistant transgenic plants and resistant molecular breeding materials by confirming whether or not phytopathogenic plants have been produced.

Furthermore, by introducing a recombinant vector (pBIN35S :: CaPR4b ) containing the CaPR4b gene caused by the onset into a plant, a transgenic Arabidopsis plant overexpressed CaPR4b according to the present invention was confirmed to increase plant disease resistance.

Through the Arabidopsis plants overexpressing the pathogenic gene 4b according to the present invention, the resistance assay against phytopathogens was performed to determine whether disease resistance occurred. The present invention can accumulate relevant information through studies such as the response to the disease of the pathogenic gene 4b and the change in the resistance response through interaction with CaLRR1, Leucine-rich repeat protein (LRR) of the pepper plant. This could provide an interesting model for signaling plant diseases, and by understanding the biochemical changes that cause resistance, they could be used to develop biotech plants that promote disease resistance.

As described above, by completing the present invention, by separating and deciphering the CaPR4b gene, which encodes a novel pathogenic related protein 4b, which is a plant disease defense related gene, which is involved in the defense against pepper bacterial bacterial spot pattern disease, In addition to providing sequence information and corresponding amino acid sequence information, it is possible not only to provide a method for detecting resistance of plants using antibodies of this gene and protein, but also to contribute to the development of resistant plant breeding and transgenic plants. .

Therefore, the present invention has an effect that can promote the development of resistant varieties is a very useful invention in the plant breeding industry.

1 is a view showing the nucleotide sequence and amino acid sequence of the pepper disease-related protein 4b (CaPR4b) gene cloned from Capsicum annuum L. cv. Nockwang according to the present invention.
Figure 2 is an alignment diagram comparing the amino acid sequence of the pepper disease-related protein 4b (CaPR4b) gene cloned from Capsicum annuum L. cv. Nockwang according to the present invention with the amino acid sequence of other plants.
3 is mutually associated with CaLRR1, a Leucine-rich repeat protein (LRR) of red pepper plants, a gene specifically induced when infected with pathogenic and non-pathogenic bacteria of red pepper bacterial spot disease ( Xanthomonas campestris pv.vesicatoria ) It is a diagram showing the results of the yeast-two-hybrid experiment to confirm the interaction of the pathogenic-related protein 4b gene ( CaPR4b ), a novel gene acting.
4 is a view showing the results of performing a Bimolecular fluorescence complementation (BiFC) assay to confirm the interaction of CaLRR1 and CaPR4b in plants.
5 is a diagram of co-immunoprecipitation for confirming the interaction of CaLRR1 and CaPR4b in plants.
Figure 6 is showing a pepper bacterial strains induced jeommunuibyeong CaPR4b genes in pepper leaves inoculated pepper leaves non-inoculated when inoculated with the virulent strain Ds1 and non-pathogenic strains of Bv5-4a (Xanthomonas campestris pv. Vesicatoria) results in nokgwang Pepper Varieties Drawing.
Figure 7 shows the results of CaPR4b gene expression induction over time after the chemical / physical stimulation treatment for red pepper cultivation varieties.
Figure 8 shows the results of CaPR4b protein induction and salicylic acid in red pepper leaves and inoculated red pepper leaves when inoculated with the pathogenic strain Ds1 and the non-pathogenic strain Bv5-4a of the red pepper bacteriophage ( Xanthomonas campestris pv.vesicatoria ) And the result of inducing the expression of CaPR4b protein over time after treatment with methyl jasmonate .
9 is a diagram confirming CaPR4b purified protein after CaPR4b protein purification.
Fig. 10 shows the results of the antimicrobial activity of CaPR4b protein against plant pathogens.
Figure 11 is a photograph showing the results of the antimicrobial activity of CaPR4b protein against Alternaria brassicicola .
Figure 12 is a photograph showing the results of measuring the germination rate of the pathogen to confirm the antimicrobial activity of Alternaria brassicicola of CaPR4b protein.
13 is a view showing the results of measuring the mycelial growth of pathogens to confirm the antibacterial activity of Alternaria brassicicola CaPR4b protein.
Figure 14 shows Botrytis cinerea , Colletotrichum orbiculare Fusarium oxysporumf of CaPR4b protein. sp. Photograph showing the results of antimicrobial activity against matthiolae .
FIG. 15 is a photograph showing apoptosis result after transient expression of CaPR4b in tobacco plants.
FIG. 16 is a photograph showing the apoptosis result after performing a transient assay by inoculating CaPR4b, CaLRR1 and two genes in tobacco plants.
FIG. 17 shows the rate of cell death after transient expression of CaPR4b in tobacco plants.
FIG. 18 shows ionic conductivity after transient expression of CaPR4b in tobacco plants.
FIG. 19 shows the results after different concentrations of CaLRR1 and transient expression of CaPR4b in order to determine whether CaLRR1 inhibits apoptosis induced by overexpression of CaPR4b in tobacco plants. .
FIG. 20 shows the apoptosis rate after different concentrations of CaLRR1 and transient expression of CaPR4b in order to determine whether CaLRR1 inhibits apoptosis induced by overexpression of CaPR4b in tobacco plants. It is a figure which shows one result.
21 is a measure of the ionic conductivity after performing the respective transient expression of different levels and CaPR4b of CaLRR1 (transient assay) In order to examine whether CaLRR1 of inhibiting cell death induced by overexpression of CaPR4b in tobacco plants is dependent on the concentration The figure which showed the result.
22 is a view showing a result of the result and expression of resistance genes in the Arabidopsis Transgenic for which over-the pepper CaPR4b gene expression in Arabidopsis thaliana plants over-expression of the gene CaPR4b.
FIG. 23 is a diagram showing the results of overexpression of CaPR4b protein in transgenic Arabidopsis plants overexpressed red pepper CaPR4b gene. FIG.
Figure 24 shows the Arabidopsis leaf which is resistant to the disease when inoculated with bacterial pathogen Pseudomonas syringae pv. Tomato DC3000, a transgenic Arabidopsis plant overexpressing CaPR4b gene. It is a photograph.
25 is a bacterial pathogen Pseudomonas syringae pv. Tomato DC3000 and a non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-A-RPM) in a transgenic Arabidopsis plant overexpressing the CaPR4b gene. ) Shows the results of pathogenic expression due to reduced bacterial growth after inoculation of Pseudomonas syringe Pasova tomato DC3000.
FIG. 26 is a diagram showing changes in ion conductivity and active oxygen after inoculation of bacterial pathogen Pseudomonas syringe Pasova tomato DC3000 to transgenic Arabidopsis plants overexpressed CaPR4b gene.
FIG. 27 is a diagram showing changes in NO (nitric oxide) after inoculation of the bacterial pathogen Pseudomonas syringe Pasova tomato DC3000 to the transgenic Arabidopsis plants overexpressed CaPR4b gene.
28 is a high-allo FER North Fora Noco2 (Hyaloperonospora arbidopsidis Noco 2) the growth of symptom change in the inoculated after cotyledon and symptoms enlarged, overactive cell death and mycelia (trypan causing downy mildew on Arabidopsis in Arabidopsis the CaPR4b overexpression Blue dye).
29 is a view showing the results for bunsaengjagyeong formation after inoculation of high egg FER North Fora Noco2 (Hyaloperonospora arbidopsidis Noco 2) causing downy mildew in Arabidopsis thaliana Arabidopsis thaliana in the CaPR4b overexpression.

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 1 CaPR4b  Determination of base / amino acid sequence of gene

The following tests were performed to provide the pathogenicity related protein 4b (CaPR4b) coding gene sequence, which is one of the pathogenicity related proteins (PR protein) for red pepper bacillus, and the amino acid sequence inferred therefrom.

Capsicum annuum cv. Nockwang was inoculated with red pepper bacterial spot bacterium ( Xanthomonas campestris pv.vesicatoria ), and after 18 hours of wet treatment, the plants which showed hypersensitivity reactions, which were resistant lesions, were taken out of the room and leaves were collected. Next, cDNA libraries were prepared by extracting mRNA from the leaf samples, reverse transcripting, and amplifying the polymerase chain reaction (PCR). Then, individual cDNA clones were prepared from the thus prepared cDNA library and introduced into a pGADT7 vector containing a GAL4-activating domain to make a recombinant vector. In addition, CaLRR1 , a Leucine-rich repeat protein (LRR) of red pepper plants, is a gene specifically induced when infected with pathogenic and non-pathogenic bacteria of red pepper bacterial bacterium ( Xanthomonas campestris pv.vesicatoria ). PGBKT7: CaLRR1 was prepared by introduction into pGBKT7 comprising the domain.

The transformants transformed in this way are transferred to a selection medium (adenine-Histidine-Leucine-Leucine-Tryptophan) which can confirm the interaction with each other and then interact with each other. Among these identified genes, genes suspected to be involved in disease defense (resistance) were selected to obtain clones, and among these clones, the sequencing of genes identified as genes of the disease-associated protein 4b (CaPR4b) was sequencing. ) provides by, peppers CaPR4b protein coding gene sequence of nokgwang varieties named CaPR4b (SEQ ID NO: 1) and amino acid sequence encoded therefrom (SEQ ID NO: 2) (see Fig. 1).

After sequencing the obtained clones according to the present invention, the 5'-terminal sequence is compared with a gene database registered in GenBank using a blast program to identify CaPR4b protein coding genes and the defense related genes. 4b ( CaPR4b ) gene. CaPR4b protein showed 100% homology with Capsicum chinense of other cultivars, which showed no homology, and more than 80% homology with PR family of potato and Arabidopsis. In addition, the result of comparing the amino acid sequence of CaPR4b is a form containing a chitin binding domain among the PR4 family. This gene is a protein having a molecular weight of 22 kDa that has resistance to various pathogens in addition to pepper bacterial spot disease (see FIG. 2).

[Example 2] CaPR4b  Confirmation of Gene's Interaction with CaLRR1

The CaPR4b gene obtained in Example 1 was tested as follows to confirm the interaction with CaLRR1.

[Step 1]

First of all, in order to present the resistance search method, the pGBKT7 including the GAL4 DNA binding domain prepared during the screening process was introduced to confirm the interaction between pGBKT7: CaLRR1 and pGADT7: CaPR4b including the GAL4-activating domain. The genes were recombined into different vectors (pGAD: CaPR4b / pGBKT7: CaLRR1 ) and transformed into yeast AH109 using the lithium acetate method. In addition, the interaction with CaLRR1 was confirmed by using a recombinant vector containing only each domain in order to determine whether these interactions bind to two domains of the CaPR4b, a chitin binding domain and a BARWIN domain. As a result, the interaction between CaPR4b and CaLRR1 was found to be the strongest in the chitin binding domain of CaPR4b. (See Figure 3)

[Step 2]

Next, Bimolecular fluorescence complementation (BiFC) assay was used to confirm whether CaPR4b and CaLRR1 interaction is actually performed in plants. Recombinant vectors were introduced by introducing the two genes into the PUC-pSPYNE vector and the PUC-pSPYCE vector, and then introduced into the Agrobacterium tumefaciense strain GV 3101 by electroporation and the recombinant vector enters again. The inoculated GV3101 strain was inoculated into tobacco plants and then confirmed the interaction using a confocal laser scanning microscope (CLSM). When the interaction occurs in plants, the C-terminal and N-terminal of each vector combine to emit yellow fluorescence and can be observed under a high focus microscope. Walter et al., 2004. Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation. Plant J. 40: 428438.) These results confirm that CaPR4b and CaLRR1 interact strongly with each other. In particular, the chitin binding domain of CaPR4b plays a role in CaLRR1 interaction. (See Fig. 4)

[Step 3]

In addition, co-immunoprecipitation was used to confirm whether CaPR4b and CaLRR1 interaction occurs at the plant protein level. Recombinant vectors were prepared after introduction into pSPYCE and pSPYNE, which are vectors that can confirm the interaction between the two proteins. The recombinant vector has a form of HApSPYCE: HA: CaLRR1 containing HA and pSPYNE: c-Myc: CaPR4b containing c-Myc. Each recombinant vector is introduced into Agrobacterium tumefaciense strain GV 3101 via electroporation and used for inoculation of tobacco plants. In particular, the total protein is extracted by collecting tobacco plants inoculated with each recombinant vector alone and tobacco plants inoculated with the two recombinant vectors. Each extracted protein was subjected to immunoprecipitation using anti-HA agarose affinity gel and anti-c-Myc agarose affinity gel at 4 ° C. The protein thus obtained was electrophoresed using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to the nylon membrane (Hybond P +). After transfer, expression was confirmed using an antibody (anti-c-Myc) at a dilution ratio of 1: 5,000. As a result, the interaction between the two proteins was confirmed (see FIG. 5).

Example 3 In Pepper Plants by Pathogen Inoculation CaPR4b  Gene Expression Detection Method

In order to confirm the method of using the CaPR4b gene obtained in Example 1 for the resistance search was tested as follows.

[Step 1]

First, in order to specifically presented in the resistive search method, and then culturing the non-pathogenic strain Bv5-4a pepper bacterial spot patterns indicating the pathogen (Xanthomonas campestris pv. Vesicatoria) of the pathogenic strain Ds1 and the incompatible reaction indicating the plants and friendly reaction, 10 ⁸ Inoculate by infiltration using a syringe on the back of the 1st and 2nd leaves of Capsicum annuum cv.Nockwang at the concentration of cfu / ml, and inoculate 28 ℃, 100% relative after inoculation. It was wet-treated for 18 hours under humidity conditions. After 1, 5, 10, 15, 20, and 25 hours of inoculation, RNA was isolated by sampling 1 and 2 leaves, respectively, and the transferred RNA was electrophoresed on a gel containing formaldehyde and then transferred to a nylon membrane (Hybond N +). I was.

Next, the CaPR4b gene obtained in Example 1 was labeled using 14-dCTP-biotin and then amplified by PCR (Polymerase Chain Reaction), and then a reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% Dextran Sulfate] was incubated at 65 ° C. for 16-24 hours to perform hybridization. The biotin-labeled DNA probe is attached to the mRNA attached to the nylon membrane. When the X-ray film is placed on the nylon membrane, the biotin-labeled DNA photosensitizes the X-ray film. This made it possible to recognize the expression.

By monitoring the amount of ribosomal RNA during the Northern blotting it was confirmed that the same amount of RNA sample was loaded (loading). The results of the above experiments are shown in FIG. 6. As a result of the experiment, CaPR4b gene was expressed in both peppery and non-pathogenic strains. More specifically, in the pathogenic strain, that is, the friendly response, expression of the CaPR4b gene increased after 5 hours, and expression was observed up to 25 hours. In the non-pathogenic strain, which showed an incompatible reaction, the strongest expression was observed at 15 hours, and the increased expression level was maintained until 25 hours, and CaPR4b gene expression was much stronger than the friendly response. (See Fig. 6)

Example 4 By Chemical / Physical Derivatives CaPR4b  Induced Expression of Genes

In order to find out whether the CaPR4b gene of the present invention can be induced by chemical derivatives commonly used for resistance induction and to provide a specific induction method, the following test was performed.

[Step 1]

First, in order to search for derivatives useful for inducing expression of CaPR4b gene, resistance induction test was conducted using salicylic acid and methyljasmonate among chemicals known to be involved in the induction of plant disease resistant expression. Six-leaf peppers were used for all experiments. 5 mM salicylic acid, 100 μM of methyl jasmonate was sprayed on the front and back of pepper leaves using a sprayer.

Each plant was harvested after 1,5,10,15,20,25 hours, RNA was extracted from each sample, and Northern blotting was performed using the extracted RNA as follows.

Next, the CaPR4b gene obtained in Example 1 was labeled with 14-dCTP-biotin (14-dCTP-biotin) and amplified using PCR (Polymerase Chain Reaction), followed by reaction solution [0.25 M phosphate buffer, 7% SDS, 1 mM EDTA, 5% Dextran Sulfate] was incubated at 65 ° C. for 16-24 hours to perform hybridization. The biotin-labeled DNA probe is attached to the mRNA attached to the nylon membrane. When the X-ray film is placed on the nylon membrane, the biotin-labeled DNA photosensitizes the X-ray film. This made it possible to recognize the expression.

By monitoring the amount of ribosomal RNA during the Northern blotting it was confirmed that the same amount of RNA sample was loaded. The results of the above experiments are shown in FIG. 7 and the CaPR4b gene was strongly accumulated after 5 hours of treatment with salicylic acid. (FIG. 7) Also, after the treatment with methyljasmonate, it was observed to accumulate weakly compared to salicylic acid.

[Step 2]

In this way, CaPR4b gene response to chemical / physical stress was observed. Ethylene was treated at a concentration of 10 μl / L by injection into a closed glass container with plants in a gaseous state. Sodium chloride was treated by making an aqueous solution at a concentration of 200 mM to hold the root of the plant. 100 μM of methyl viologen and absic acid were treated by dipping the roots of the plants in 100 μM of aqueous solution of absic acid and spraying the same aqueous solution onto the pepper leaves. Each plant was harvested after 1,5,10,15,20,25 hours, RNA was extracted from each sample, and Northern blotting was performed using the extracted RNA as follows. Expression patterns for dryness and wound stress were also confirmed through northern blotting. (See Figure 7)

[Example 5] Detection of CaPR4b protein expression in red pepper plants by pathogen inoculation and chemical derivatives

Experiments were performed by sampling proteins that were treated in the same manner as in Example 4 to separate proteins. Next, the CaPR4b gene obtained in Example 1 was used to produce antibodies in rabbits using peptide synthesis to confirm the protein expression of CaPR4b. The antibody was prepared by requesting an antibody manufacturer (AbFRONTIER, Korea), and the experiment was performed using the antibody.

First, in order to specifically presented in the resistive search method, and then culturing the non-pathogenic strain Bv5-4a pepper bacterial spot patterns indicating the pathogen (Xanthomonas campestris pv. Vesicatoria) of the pathogenic strain Ds1 and the incompatible reaction indicating the plants and friendly reaction, 10 ⁸ Inoculate by infiltration using a syringe on the back of the 1st and 2nd leaves of Capsicum annuum cv.Nockwang at the concentration of cfu / ml, and inoculate 28 ℃, 100% relative after inoculation. It was wet-treated for 18 hours under humidity conditions. Proteins were extracted by sampling 1 and 2 leaves after 5, 15, 25, and 35 hours of inoculation, respectively. The extracted proteins were electrophoresed using 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (HDS). P +). After transfer, expression was confirmed using an antibody (α-CaPR4b) at a dilution ratio of 1: 5,000. When performing the experiment of protein expression it was confirmed that the same amount of protein was loaded by quantifying the amount of protein. The experimental results are shown in FIG. 8.

As shown in FIG. 8, the CaPR4b gene was expressed in both the pathogenic and non-pathogenic strains, but the CaPR4b gene was weakly expressed in the pathogenic and non-pathogenic strains. It was identified and most strongly expressed at 25 hours in non-pathogenic strains showing incompatible reactions. As shown in FIG. 8, salicylic acid increased expression at 12 hours and was most strongly expressed at 24 hours, and expression of protein level of methyl jasmonate was also observed.

Example 6 Confirmation of Antimicrobial Activity of CaPR4b Protein

After overexpressing the CaPR4b gene in E. coli, the recombinant vector was prepared by introducing the CaPR4b gene obtained in Example 1 into a pMAL vector containing a maltose binding protein. The recombinant vector was transformed into Escherichia coli strain BL21 and the protein obtained by culturing Escherichia coli containing the recombinant vector was used for the next experiment. (Fig. 9)

[Step 1]

In order to confirm the antibacterial activity of CaPR4b protein, Alternaria brassicicola , Botrytis cinerea , Colletotrichum orbiculare Fusarium oxysporumf.sp. matthiolae, Xanthomonas campestris pv. vesicatoria, Pseudomonas syringae pv. Antimicrobial activity was confirmed by measuring the minimum inhibitory concentration of CaPR4b protein of pathogens such as tomato (see Figure 11). Each pathogen was inoculated in a medium containing CaPR4b protein with different concentrations, and then compared with the growth pathogens inoculated in a medium containing a protein that only purified maltose binding protein. As a result, CaPR4b protein was not significantly affected in pathogenic bacteria, but strong antibacterial activity was observed in pathogenic fungi. (See Figures 11 and 14)

[Step 2]

In particular, spore germination and mycelial growth of pathogens were measured to determine the antibacterial activity of CaPR4b protein against Alternaria brassicicola . (See FIGS. 12 and 13) The germination rate and mycelial growth of Alternaria brassicicola inoculated in a medium containing CaPR4b protein with different concentrations and then inoculated with a medium containing only purified protein of maltose binding protein were compared. As shown in Figure 12, 13 CaPR4b protein strongly inhibited germination rate and mycelial growth of Alternaria brassicicola .

[Example 7] Agrobacterium-mediated transient expression method CaPR4b  Effect of Gene Expression on Plants

Pepper Agrobacterium the CaPR4b gene in plants tumefaciens-mediated transient expression (Agrobacterium-mediated transient expression) as through the method sikyeoteul the over-expression In order to examine the change in the pepper plant, carried pBIN3S vector available from Stratagene the CaPR4b gene obtained in Example 1 PBIN35S :: CaPR4b was prepared by introducing into the recombinant vector pBIN35S :: CaPR4b thus introduced into the Agrobacterium tumefaciense strain GV 3101 through electroporation and the recombinant vector was added. Overexpression of the CaPR4b gene was induced by introducing the GV3101 strain into the pepper plants using an infiltration method using a syringe for each concentration.

[Step 1]

15 and in the plant CaPR4b is expressed in excess, such as from 16 became a plant cell death observed compared to control CaPR4b is not introduced into the rate and ion conductivity of the cell death In order to examine the results of the resistance reaction in these plants Was performed. As shown in FIG. 17, apoptosis was increased in CaPR4b -overexpressed plants as compared to the control group, and as shown in FIG. 18, ionic conductivity was also increased in CaPR4b -overexpressed plants.

[Step 2]

To determine the effect of apoptosis caused by overexpression of CaPR4b by CaLRR1 interacting with this protein, the two genes were mixed and inoculated into plants. Results Apoptosis did not occur at the site of overexpression of CaLRR1 . However, over-expression of CaLRR1 is inhibited cell death occurs by the over-expression of CaPR4b. These results were also confirmed by the cell death rate and ion conductivity. (See Figures 16, 17 and 18)

[Step 3]

In order to confirm whether apoptosis due to overexpression of CaPR4b inhibited by overexpression of CaLRR1 was dependent on CaLRR1 concentration, CaLRR1 was mixed with CaPR4b, and then inoculated to plants. Inoculation result, cell death caused by over-expression of the CaPR4b was observed doeeojim dependent inhibition with a concentration of aLRR1. (See Fig. 19) performs a result the ratio of the ionic conductivity of the cell death was also confirmed cell death is suppressed most strongly by the over-expression of CaPR4b when the CaLRR1 CaPR4b and inoculated at the same rate. (See Figures 20, 21)

[Example 8] CaPR4b  Production of transgenic plants using genes CaPR4b  Increased disease resistance due to overexpression of genes

By introducing the CaPR4b gene obtained in Example 1 in pBIN3S vector available from Stratagene to investigate as to whether the increase in resistance to induction, and whether the bottle of genes associated with different disease occurs when over expression of CaPR4b gene in a plant pBIN35S : PBIN35S :: Recombinant vector thus prepared after CaPR4b: CaPR4b was introduced into Agrobacterium tumefaciense strain GV 3101 by electroporation and GV3101 strain containing the recombinant vector Was transformed by introducing into the Arabidopsis plants through floral dipping method to induce overexpression of CaPR4b gene, and the following tests were performed.

[Step 1]

In order from the CaPR4b gene is overexpressed plants Learn whether the resistance to bacterial disease increases, first the CaPR4b expression in wild-type Arabidopsis thaliana plants as the CaPR4b gene is overexpressed Arabidopsis thaliana and control at the gene and protein levels Observations were made and the results are shown in FIGS. 22 and 23. In FIG. 25, # 6, # 8, and # 18 indicate the line numbers of the transgenic plants used, and as a result, CaPR4b was overexpressed in Arabidopsis plants.

[Step 2]

As above, inoculated with Pseudomonas syringe Pasova tomato DC3000 and non-pathogenic strain Pseudomonas syringe Pasova tomato DC3000 (ABR-Al-MW1) at a concentration of 10 ⁵ cfu / ml and observed changes of plants (FIG. 26). As a result, as shown in FIGS. 24 and 25, it was observed that CaPR4b showed an increased resistance to bacterial diseases in plants overexpressed compared to wild-type, and the leaves were collected on days 0 and 3 after inoculation to 1 cm ² of plant leaves. After measuring the number of bacteria is shown in Figure 25 the results. In particular, in the plants overexpressed CaPR4b gene, the growth of pathogens was significantly reduced compared to wild-type plants after inoculation of Pseudomonas syringe-Pasova tomato DC3000, and disease resistance was observed.

[Step 3]

In order to determine the resistance CaPR4b the reactions in the overexpressed plants after inoculation of Pseudomonas when ringge lost bar tomato DC3000 after inoculation was measured and ion conductivity of the active oxygen and NO (Nitric oxide). As a result of these measurements, the CaPR4b overexpressed plants contributed to resistance through Pseudomonas syringe Pasova tomato DC3000 inoculation after ionic conductivity, free radicals and NO content. Was observed.

Example 9 After Inoculation of Hyelofernospora Noco2 CaPR4b  Changes in Disease Resistance in Overexpressed Plants of Genes

Tests were performed after inoculation of Hyaloperonospora arabidopsidis Noco 2, a causal plant, overexpressed with CaPR4b , to cause Downy mildew. Hyalofernospora Noco2 was sprayed at a concentration of 5 × 10 ⁴ mL ⁻¹ by spraying the cotyledon of CaPR4b overexpressed cephalococci. After inoculation, the cells were subjected to wet treatment at a temperature of 17 캜.

[Step 1]

Each plant was observed on day 6 after inoculation to observe resistance to hyaluronic perospora, and trypan blue staining was performed on day 7. As shown in FIG. 28, in the CaPR4b overexpressed plant, the formation of sporangiophores was reduced compared to the wild type, and the resistance reaction was observed, and the mycelial growth was less developed than the wild type through trypan blue staining. there was.

[Step 2]

Count the number of conidia formed after inoculation of hyalofernospores for the resistance test of hyalurofernospores in each plant, and then group them to 0, 1-5, 6-10, 11-15, 15 or more. After making the average value and expressed as a percentage (%). As shown in FIG. 29, the CaPR4b -overexpressed plant exhibited higher resistance than the wild-type in the mean value and ratio compared to the healthy plant.

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

<110> KOREAN UNIVERSITY RESEARCH AND BUSINESS FOUNDATION <120> Pepper pathogenesis-related protein 4b gene and screening method          of plant disease resistance using the same <130> P11-101209-02-CaPR4b <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 627 <212> DNA 213 Capsicum annuum L. cv. Nockwang <400> 1 atggagaaac gaactagtac tactattctt ctagccctag tcctcttcat cgtatccgga 60 gtggccaacg cacagcagtg cggaaggcaa aggggcggag ccttatgcgg cggaaactta 120 tgttgcagcc aatttggatg gtgtggatcg acacccgaat actgttcacc tagccaaggt 180 tgccagagcc aatgcagtgg aagtggtccg actccagaag gaagcgcgca aaatgtacgt 240 gcaacgtatc atttgtataa cccgcagaat gttggttggg acttgaatgc tgttagtgct 300 tattgctcga catgggatgc taataagcct ttggcttgga ggagcaagta tggttggact 360 gctttctgtg gtcctgttgg acctcgtggt caagcctcct gtggcaagtg cttaagggtc 420 acaaacagac gaaccagagc tcaaacaacg gtgagaatcg tggatcaatg cagcaacggt 480 ggactagact tggacattaa cgttttccgg caaatcgaca cagacggagt gggaaatcaa 540 caaggtcacc ttatggtgga ctaccaattt gttaattgcg gtgacaatgt gaatgttcct 600 ctcttgtctg tagttgacac acaatga 627 <210> 2 <211> 208 <212> PRT 213 Capsicum annuum L. cv. Nockwang <400> 2 Met Glu Lys Arg Thr Ser Thr Thr Ile Leu Leu Ala Leu Val Leu Phe   1 5 10 15 Ile Val Ser Gly Val Ala Asn Ala Gln Gln Cys Gly Arg Gln Arg Gly              20 25 30 Gly Ala Leu Cys Gly Gly Asn Leu Cys Cys Ser Gln Phe Gly Trp Cys          35 40 45 Gly Ser Thr Pro Glu Tyr Cys Ser Pro Ser Gln Gly Cys Gln Ser Gln      50 55 60 Cys Ser Gly Ser Gly Pro Thr Pro Glu Gly Ser Ala Gln Asn Val Arg  65 70 75 80 Ala Thr Tyr His Leu Tyr Asn Pro Gln Asn Val Gly Trp Asp Leu Asn                  85 90 95 Ala Val Ser Ala Tyr Cys Ser Thr Trp Asp Ala Asn Lys Pro Leu Ala             100 105 110 Trp Arg Ser Lys Tyr Gly Trp Thr Ala Phe Cys Gly Pro Val Gly Pro         115 120 125 Arg Gly Gln Ala Ser Cys Gly Lys Cys Leu Arg Val Thr Asn Arg Arg     130 135 140 Thr Arg Ala Gln Thr Thr Val Arg Ile Val Asp Gln Cys Ser Asn Gly 145 150 155 160 Gly Leu Asp Leu Asp Ile Asn Val Phe Arg Gln Ile Asp Thr Asp Gly                 165 170 175 Val Gly Asn Gln Gln Gly His Leu Met Val Asp Tyr Gln Phe Val Asn             180 185 190 Cys Gly Asp Asn Val Asn Val Pro Leu Leu Ser Val Val Asp Thr Gln         195 200 205

Claims (7)

An isolated gene ( CaPR4b) of (a) or (b) which encodes pepper-derived pathogenic protein 4b as a plant disease defense 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 (CaPR4b). A recombinant vector comprising the gene of claim 1. Pepper or Arabidopsis plant transformed by the gene of claim 1 or the recombinant vector of claim 3. Bacterial spot bacteria ( Xanthomonas campestri pv. Vesicatoria ), black pattern bacteria ( Alterriaria brassicicola ) or Bacillus fungi of peppers or Arabidopsis plants comprising the step of identifying the differential expression of the CaPR4b gene according to claim 1 by separating mRNA from the plant Method of searching for resistance to Hyaloperonospora arabidopsidis Noco 2). The bacterium Streptococcus ( Xanthomonas campestri pv. Vesicatoria ), Black Streptococcus ( Alternaria brassicicola ) of peppers or Arabidopsis plants comprising the step of confirming the expression of CaPR4b protein using the antibody of the CaPR4b protein of claim 2 as a probe Screening method for resistance against Hyaloperonospora arabidopsidis Noco 2 An antifungal agent having antimicrobial activity against phytopathogenic fungi comprising the protein of claim 2 as an active ingredient.

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