KR101627462B1 - Method for improving disease resistance using CabZIP2 in plants - Google Patents

Abstract

The present invention relates to a new gene (CabzIP2 annuum bZIP transcription factor 2) derived from pepper associated with plant disease resistance, and more particularly, to a method for enhancing plant pathogen resistance by controlling the expression of CabZIP2 protein.
It has been confirmed that CabZIP2-overexpressed transgenic plant (CabZIP2-OX) of the present invention has an excellent effect in increasing the resistance to bacterial pathogens, so that it can be usefully used for improvement of crops available to mankind do.

Description

[0001] The present invention relates to a method for enhancing plant resistance using CabZIP2,

The present invention relates to a novel capsicum annuum bZIP transcription factor 2 (CabZIP2) derived from pepper and a method for enhancing disease resistance of a plant using the same.

Plants are persistently exposed to environments such as drought, high salt, and pathogens due to their stickiness characteristics. These environmental stresses directly affect plant growth and seed production. Thus, plants have developed mechanisms that protect themselves from a variety of pathogens such as bacteria, fungi, viruses, and nematodes.

The defenses begin to be activated by the recognition of pathogen associated molecular patterns (PAMP) by a small pattern recognition receptor (PRR). Plant pathogen recognition activates a signal transduction pathway that leads to ionic spillage, reactive oxygen species (ROS), and defense genes, and this response allows the plant to be resistant to pathogens . Plant defense responses to pathogens are regulated by a variety of signaling mechanisms.

One of the various defense mechanisms for plants to overcome the attack of pathogens is to increase defense gene expression to produce defensive hormones and antimicrobial proteins.

On the other hand, one of the most abundant transcription factors in plants is the bZIP protein. The bZIP gene is present in all eukaryotes, has a basic region required for DNA binding and a leucine zipper motif that is responsible for dimerization and is homo- or hetero-dimerized before binding to the promoter. dimerization. It also participates in plant defense responses to pathogens. These bZIP transcription factors include OsABF3, CAbZIP1, and the like.

Therefore, there is a study on a method for enhancing resistance to various environmental stresses such as drought, salt, cold, heat, and pathogen using the bZIP transcription factor. The development of crops resistant to environmental stress is expected to contribute greatly to the increase of agricultural production. However, since the technology is mainly focused on drought or dry resistance, there is a need for a method for improving disease resistance using bZIP transcription factors.

Disclosure of the Invention The present invention has been conceived to solve the above-mentioned problems, and it has been confirmed that the resistance of CabZIP2 overexpressed transgenic plant (CabZIP2-OX) to bacterial pathogens is increased.

Accordingly, the present invention provides a recombinant expression vector comprising CabZIP2 comprising the nucleotide sequence of SEQ ID NO: 1 encoding a disease-resistant protein derived from pepper, a peptide of the amino acid sequence of SEQ ID NO: 2 encoded by the gene CabZIP2, Vector. ≪ / RTI >

It is another object of the present invention to provide a method for promoting disease resistance of a plant comprising the step of increasing the expression or activity of a CabZIP2 protein and a transgenic plant having enhanced disease resistance by the method.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

In order to accomplish the object of the present invention, the present invention provides a CabZIP2 (Capsicum annuum bZIP transcription factor 2) gene comprising the nucleotide sequence of SEQ ID NO: 1 encoding a disease-resistant protein derived from pepper.

The present invention provides a peptide of the amino acid sequence of SEQ ID NO: 2 which is encoded by the gene CabZIP2.

The present invention provides recombinant expression vectors containing the CabZIP2 gene described above.

In one embodiment of the present invention, the recombinant expression vector may be pK2GW7, p326GFP, or pGBKT7.

The present invention provides a method for enhancing plant disease resistance comprising increasing the expression or activity of a CabZIP2 protein.

The present invention provides a transgenic plant having enhanced disease resistance by the above method.

The present invention provides a method for screening a plant resistance-enhancing agent, comprising the step of treating the candidate substance and measuring the expression of the CabZIP2 protein.

The present invention relates to a new gene of CabZIP2 derived from pepper associated with resistance to bacterial pathogens of plants, and it was confirmed that CabZIP2-overexpressed transgenic plant (CabZIP2-OX) It is expected that it will be useful for the improvement of available crops.

Figure 1 compares the amino acid sequence of CabZIP2 (Capsicum annuum bZIP transcription factor 2) cDNA with the bZIP transcription factor amino acid sequence of other plants.
Fig. 2 shows the results of the genealogical analysis of CabZIP2, CabZIP1, and other homologous proteins.
FIG. 3 shows the intracellular location of the CabZIP2 protein through the overexpression of 35S: CabZIP2-GFP in Nicotiana benthamiana.
Fig. 4 shows the functional analysis results of the CabZIP2 protein.
FIG. 5 shows organ-specific expression results of CabZIP2 by RT-PCR in pepper plants.
FIG. 6A shows the expression level of the CabZIP2 gene in the pepper leaves infected with the Xcv strain, and FIGS. 6B to 6D show the expression amounts of the CabZIP2 gene in the pepper leaves treated with salicylic acid (SA), ethylene and methyl jasmonate .
Figure 7 shows the relative expression levels of PR genes in pepper plants inoculated with different strains of Xcv.
Figure 8 shows the increased susceptibility of CabZIP2-silenced pepper plants to Xcv infection. (H: untreated healthy leaves, V: virulent strain Ds1, A: avirulent strain Bv5-4a, CaAMP1: pepper antimicrobial protein 1, CaBPR1: pepper basic PR1)
Fig. 9 shows the results of Pseudomonas syringae pv. tomato (Pst) showed an increased resistance of the CabZIP2-overexpressing Arabidopsis to DC3000 infection, indicating RT-PCR analysis of wild-type and CabZIP2-overexpressing transgenic plants (Fig. 9A), whitening after Pst DC3000 inoculation (Fig. 9B), and bacterial growth (Fig. 9C).
Figure 10 shows the relative expression levels of PR genes in wild-type plants and transgenic plant leaves after inoculation with Pst DC3000 (105 17 cells / ml).

The present inventors have found that increasing the expression of CabZIP2 in plant hormones (salicylic acid (SA), ethylene, and methyl jasmonate) treatment conditions, susceptibility to pathogens in CabZIP2 silenced peppers, and resistance to bacterial pathogens in transgenic Arabidopsis overexpressed CabZIP2 The present invention has been completed on the basis thereof.

Hereinafter, the present invention will be described in detail.

The present invention provides a novel gene, CabZIP2, which encodes a disease-resistant protein derived from pepper, a peptide encoded by the gene CabZIP2, and a recombinant expression vector containing the CabZIP2 gene. The recombinant expression vector may be, but is not limited to, pK2GW7, p326GFP, or pGBKT7.

The gene of the present invention, CabZIP2, was isolated from red pepper by differential hybridization screening and had an N-terminal with an acidic domain. In addition, the CabZIP2 gene of the present invention is preferably composed of the nucleotide sequence of SEQ ID NO: 1, and the peptide encoded by the CabZIP2 gene may preferably be SEQ ID NO: 2, but is not limited thereto.

In one embodiment of the present invention, the CabZIP2 gene was isolated and the homology of the amino acid sequence between the CabZIP2 protein and other bZIP transcription factors was confirmed (see Example 1). In addition, CabZIP2-GFP overexpression was used to confirm that the CabZIP2 protein was located in the cytoplasm and nucleus (see Example 2), and the transcriptional activity was analyzed in yeast (see Example 3). In addition, in order to investigate whether the CabZIP2 gene is involved in the defense reaction, the expression level of CabZIP2 gene was confirmed by using plant hormone, which is a pathogenic infection and non-biologic (see Example 4). Furthermore, in order to investigate the role of CabZIP2 in the defense response of plants, CabZIP2-silenced pepper plants were found to be susceptible to Xcv infection (see Example 5), and CabZIP2-overexpressed (CabZIP2-OX) (See Example 6) to increase the expression or activity of CabZIP2, thereby enhancing disease resistance.

Accordingly, the present invention provides a method of enhancing plant disease resistance comprising increasing the expression or activity of a CabZIP2 protein. The promoting method may include a step of treating a candidate substance that increases the expression or activity of the CabZIP2 protein in a plant, and a step of comparing the expression or activity of the plant with that of the untreated group, but the present invention is not limited thereto.

In another aspect of the present invention, there is provided a transgenic plant in which the expression or activity of the CabZIP2 protein is increased to increase disease resistance.

The plant according to the present invention is a food crop including rice, wheat, barley, corn, soybean, potato, red bean, oats, sorghum; Vegetable crops including Arabidopsis, cabbage, radish, red pepper, strawberry, tomato, watermelon, cucumber, cabbage, melon, squash, onions, onions and carrots; Special crops including ginseng, tobacco, cotton, sesame seed, sugar cane, beet, perilla, peanut, and rapeseed; Fruit trees including apple trees, pears, jujube trees, peaches, sheep grapes, grapes, citrus fruits, persimmons, plums, apricots, and bananas; Roses, gladiolus, gerberas, carnations, chrysanthemums, lilies, tulips; And feed crops including rice, red clover, orchardgrass, alpha-alpha, tall fescue, perennial rice, and the like, preferably Arabidopsis, but are not limited thereto.

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the following examples.

[Preparation example]

Preparation Example 1. Plant and Growth Conditions

(130 μmol photons m ^-2 s ^-1 ) at 24 ° C and 60% under the fluorescent lamp (Capsicum annuum L., cv. Nockwang), Arabidopsis thaliana (ecotype Col-0) and tobacco (Nicotiana benthamiana) Humidity, 16 h light cycle / 8 h dark cycle, and a volume ratio of molybdenum, tin, and vermiculite of 9: 1: 1. Arabidopsis thaliana seeds were sterilized with 70% ethanol for 1 minute before in vitro culture. The seeds were treated with 2% sodium hydroxide for 10 minutes, washed 10 times with sterile distilled water, and seeded on MS medium (Murashige and Skoog agar) supplemented with 1% sucrose. 16h light cycle and 8h dark cycle.

Preparation Example 2. Inoculation of pathogens

Xanthomonas campestris showing toxicity (Ds1) and non-toxicity (Bv5-4a) to red pepper. pv. Vesicatoria (Xcv) strains were used. Bacteria were cultured in yeast-nutrient broth (5 g yeast extract, 8 g nutrient broth, and 1 LH ₂ O) for 28 h at 28 ° C to prepare bacterial suspensions to be inoculated on pepper leaves. The bacterial suspension was adjusted to 10 ⁸ cfu (colony forming units) / ml with sterile tap water before inoculation. Six-leaf stage pepper The bacterial suspension was inoculated on the abaxial side of the plant leaf. The control infiltrated the leaves with sterile tap water. Control and bacterial inoculated pepper plants were cultured in growth room.

In addition, toxic pathogen Pseudomonas syringae pv. tomato DC3000 was used. To prepare a bacterial suspension to be inoculated on Arabidopsis leaf, it was cultured overnight at 28 ° C in King's B medium broth. 5-week-old leaves were inoculated with a 10 mM MgCl ₂ Pst DC3000 (1 × 10 ⁵ cfu / ml) bacterial suspension with a syringe. After inoculation, Arabidopsis plants were maintained at 100% humidity or less for 24 hours and then cultured in growth chambers of the same conditions.

Bacterial density was determined by continuous dilution in King's B medium supplemented with kanamycin (50 μg / mL) and rifampicin (50 μg / mL) at 28 ° C. Data are mean ± standard deviation of three replicate log (cfu cm ^{- 2} ). Bacterial growth in each infected leaf was assessed by Katagiri et al. (2002) method.

Preparation Example 3. Plant hormone treatment

Six-leaf stage The pepper plants were sealed in a capped 500 mL glass bottle and ethylene was injected to obtain a final concentration of 5 μL per liter. Methyl jasmonate (MeJA, 100 μM) and salicylic acid (SA, 5 mM) were sprayed onto the six-leaf stage pepper plants and MeJA-treated red pepper plants were cultured in plastic bags. The control group was sprayed with water. Control and experimental red pepper plants were cultured in a growth chamber at 27 ° C ± 1 ° C for 16 hours per day at approximately 80 μmol photons m ^-2 s ^-1 (white fluorescent lamp).

Preparation 4. RNA isolation, RT-PCR, and real-time qRT-PCR analysis

Total RNA was isolated from red pepper leaf tissues treated with bacterial pathogens or plant hormones using RNeasy Mini kit (Qiagen, Valencia, CA, USA).

To remove DNA (genomic DNA), all RNA samples were treated with RNA-free DNase. After quantification using a spectrophotometer, 1 μg total RNA was used for cDNA synthesis using the Transcript First Strand cDNA synthesis kit (Roche, Indianapolis, Ind., USA). In parallel, cDNA was synthesized without reverse transcriptase, and qRT-PCR was performed to exclude the possibility of contamination of cDNA samples with genomic DNA. The synthesized cDNA was amplified with a CFX96 Touch ^TM Real-Time PCR detection system (Bio-Rad, Hercules, Calif., USA) using iQ ^™ SYBR Green Supermix and specific primers. All reactions were performed in triplicate. PCR was performed as follows: 95 cycles of 5 minutes at 95 C, 45 cycles at 95 C for 20 seconds, 60 C for 20 seconds, and 72 C for 20 seconds. Expression of each gene was calculated by the ΔΔCt method. The relative levels of target gene transcription were normalized using internal control genes (18S rRNA, Actinl, and EFl [alpha]). The primer sequences used were as shown in Table 1.

Gene Direction Sequence SEQ ID NO: CabZIP2 F 5'-CCAGCCGCAGTCATTTGTCCA-3 ' 3 R 5'-TCACAGCCTGCAACACTACCCATT-3 ' 4 18s rRNA F 5'-TATGGTGTGCACCGGTCGTCTCGT-3 ' 5 R 5'-GCAGTTGTTCGTCTTTCATAAATCCAA-3 ' 6 CaActin1 F 5'-GACGTGACCTAACTGATAACCTGAT-3 ' 7 R 5'-CTCTCAGCACCAATGGTAATAACTT-3 ' 8 CaEF1α F 5'-TTAAGGATCTTAAGCGTGGTTATGT-3 ' 9 R 5'-TCTTCAAGAACTTAGGCTCCTTCTC-3 ' 10

[Example]

Example 1. Isolation and Sequence Analysis of CabZIP2 Gene

In order to isolate the new pathogen-induced red pepper genes, hybridization screening of cDNA library constructed with avirulent strain Bv5-4a-infected leaves was performed and the presumed pathogen-induced gene Respectively.

CabZIP2 (Capsicum annuum bZIP transcription factor 2) gene was designed based on the sequence and amino acid sequence of pepper CabZIP2 cDNA. The CabZIP2 clone was 1243 bp, and the deduced amino acid sequence encoded a 344 amino acid protein containing the nuclear localization signal (NLS).

Sequence analysis revealed that the CabZIP2 domain contained an N-terminal region, an bZIP region, and a C-terminal region containing an acidic domain. The bZIP domain, which is composed of 59 amino acids and is located in the central region of CabZIP2, is a feature of various plant proteins.

As shown in Figure 1, amino acid sequence alignment and comparison of CabZIP2 with the six bZIP transcription factor families in different plant species revealed that these bZIP proteins have a similar leucine zipper structural signature. The expected CabZIP2 (accession No. AHI85726) was 85.2% with 86.3% potato VIP1-like protein (accession no. XP_006362824), tobacco RSG protein (accession no. BAA97100) and tomato VIP1-like protein (accession No. XP_004237777) The identity was 52.3% with bZIP transcriptional activator (accession no. ACB30357), 53.53% with Arabidopsis VIP1 protein (accession number NP_001237194) and soybean bZIP131 protein (accession no. NP_001237194). This sequence difference reflects the phylogenetic tree, as shown in Figure 2, and CabZIP2 is grouped into six VIP1-like proteins, including CabZIP (accession No. ACB30357; 73% identity and 85% similarity) . In particular, CabZIP1 (accession no. AAX20030; Lee et al., 2006a) isolated from previous studies has 19.45% identity to CabZIP2 and is located in different branches. This indicates that CabZIP2 isolated from pepper is a novel gene.

Example 2. Intracellular location of CabZIP2

To confirm the intracellular location of the CabZIP2 protein, a green fluorescent protein (GFP) sequence was fused to the cDNA of CabZIP2 under the control of 35S promoter.

Without a stop codon was inserted into the gene CabZIP2 encryption area with GFP-fused binary vector p326GFP, the Agrobacterium tumefaciens strain GV3101 carrying the p19 strain structure:; was coupled with (1: 1 concentration ratio of OD ₆₀₀ = 0.5) 5 primary tobacco (N. benthamiana) leaves. Two days after infiltration, the leaf discs were cut and the lower epidermis cells of the leaves were analyzed by confocal laser-scanning microscope LSM 700 (Carl Zeiss, Germany). In order to study the flg22-induced relocalization of the CabZIP2 protein, 10 μM flg22 peptide was infiltrated into the leaves expressing CabZIP2-GFP at least 1 hour before analysis.

As a result, as shown in Fig. 3A, the 35S: CabZIP2-GFP fusion protein in the tobacco epidermal cell produced GFP signal in nucleus and cytoplasm, consistent with DAPI staining results. This suggests that CabZIP2 is a protein located not only in the nucleus but also in the cytoplasm.

Figure 1 shows that CabZIP2 has high homology to VIP1-like proteins in other plant species. Djamei et al. (2007) suggested that VIR1 is rearranged from the cytoplasm to the nucleus by flg22, a bacterial PAMP. In order to determine whether the position of CabZIP2 was affected by bacterial PAMP, the tobacco leaves treated with flg22 and treated with 35S: CabZIP2-GFP were treated.

As a result, as shown in Fig. 3B, the position of CabZIP2 did not change. We confirmed that PAMP did not change the position of CabZIP2 because fluorescent signals were detected in nucleus and cytoplasm. Although the position of CabZIP2 protein was not affected by PAMP, it was found that the amount of CabZIP2 protein expression was sufficient to function in the nucleus.

Example 3. Transactivation assay of CabZIP2 in yeast [

Transcription factors have a proline, glutamine, or acidic amino acid-rich activation domain. However, not all active domains are such.

To confirm that the CabZIP2 protein could function as a transactivator in yeast, a series of CabZIP2 gene deletion constructs were constructed containing the yeast GAL4 DNA binding domain expressed under the control of the Saccharomyces cerevisiae ADH1 promoter, as shown in Figure 4A pGBKT7 vector (Clontech, Palo Alto, Calif.). Fusion proteins were synthesized from Saccharomyces cells carrying integrated copies of the nutritional reporter gene (ADE1 and HIS3) under the control of the GAL4-responsive promoter (BF: 1-344, BN: 1-130, BZ: 131-260, BC: 261-334) S. cerevisiae strain ADH1. Thus, CabZIP2 transcription activation was investigated in Saccharomyces cerevisiae strain AH109 in which Adel and His3 were expressed by the GAL4 promoter. Yeast cells were transformed with an expression vector carrying the GAL4-CabZIP2 fusion gene. Transcriptional activation was confirmed by transferring the transformed yeast cells to the selective medium (SD [-Ade, -His, -Leu, -Trp]) by yeast cells growing in SD medium.

Like other bZIP transcription factors, CabZIP contains an acidic domain at the N-terminal region. The acidic transcriptional activation domain is a common function in eukaryotes. As shown in FIG. 4A, yeast cells carrying the BF or BN-containing construct grew well in the selection medium, indicating that the expression of the ADE1 and HIS3 reporter genes was activated by the CabZIP2 N-terminal domain I could. The CabZIP2 N-terminal region containing the acidic domain functions as a transcriptionally active domain in yeast. In contrast, the structure representing the bZIP region (BZ: 131-260) and the C-terminal region (amino acids 261-334) did not grow yeast in the selective medium. These results indicate that the N-terminal region of CabZIP2 containing the acidic domain can act as a transcription activator in yeast.

Many studies have reported that bZIP transcription factors prefer dimerization in different species including Homo sapiens, Drosophila melanogaster, Saccharomyces cerevisiae, and Arabidopsis thaliana. Unlike most other bZIP transcription factors, subfamily I members were expected to form homodimers rather than heterodimers. Since CabZIP2 contains the subfamily I of bZIP, homozygosity of CabZIP2 was confirmed by BiFC (bimolecular fluorescence complementation) analysis. Agrobacterium-mediated overexpression analysis was performed on tobacco (N. benthamiana) leaves to confirm the homodimerization of CabZIP2. Detailed methods of BiFC analysis are as follows.

To construct a BiFC construct, the full length cDNA of CabZIP2 was cloned into the 35S-SPYNE (R) 173 and 35S-SPYCE (M) vectors without a stop codon. For overexpression, the constructs harboring Agrobacterium tumefaciens strain GV3101 were mixed with the p19 strain to prevent gene silencing and cultured on the abaxial side of a 5-week-old tobacco (N. benthamiana) leaf using a 1 ml needless syringe. ≪ / RTI > For microscopic analysis, leaf discs were cut 4 days after infiltration. The lower epidermis cells of the leaves were analyzed by confocal microscopy (model Zeiss 510 UV / Vis Meta).

As a result, as shown in FIG. 4B, the coexpression of CabZIP2: SPYCE and CabZIP2: SPYNE in the cytoplasm and nucleus appeared as yellow fluorescence, indicating that CabZIP2 functions as a homodimer.

In this transcription factor family, the protein dimerization of bZIP involves a leucine zipper motif that requires recognition of cis-acting elements. In contrast, some bZIP transcription factors lose DNA-binding activity during heterodimerization.

Example 4. Induction of CabZIP2 gene expression by bacterial pathogen infection and abiotic elicitor in pepper leaves

RT-PCR analysis was performed to determine whether CabZIP2 was always expressed in red pepper tissues such as leaves, stems, roots, and flowers (see Preparation Example 4).

As a result, as shown in Fig. 5, CabZIP2 was always expressed in all of the above red pepper tissues, and in particular, CabZIP2 expression was found to be high in stem and root.

To determine whether CabZIP2 was induced by the compatible and incompatible interactions of pepper and Xcv, the expression of CabZIP2 was analyzed in pepper leaves infected with toxic and non-toxic Xcv strains.

As a result, as shown in FIG. 6A, the pepper leaves infected with the toxic strain CabZIP2 had no visible symptoms for 24 hours after inoculation, but white matter and gangrene symptoms were observed 6 days after the inoculation. In contrast, pepper leaves infected with non-toxic strain Bv5-4a showed a hypersensitive response 18 hours after inoculation. These various disease responses are due to differential induction of CabZIP2.

In a compatible interaction, CabZIP2 transcription was first detected at 2 hours post-inoculation and at 6 hours post-transcription. In incompatible interactions, CabZIP2 transcription accumulated faster and more potently than observed during the affinity interaction. The CabZIP2 gene could be detected within 0.5 hours of inoculation and the expression gradually decreased gradually. Defensive signals in the early stages of plant and microbial interaction are needed for an effective defense response against pathogens and can bind to one of the terminal pathways leading to the transcriptional activity of the PR gene. In a friendly and unfriendly interaction, strong CabZIP2 expression occurred at 0.5 and 2 hours after inoculation, respectively.

In this example, the expression of these genes was compared with CabZIP2 since the pepper PR gene transcription, including CaBPR1 and CaAMP1, was induced by bacterial pathogens.

As a result, as shown in Fig. 7, expression of CaBPR1 and CaAMP1 occurred after CabZIP2 induction, and CabZIP2 transcription was induced faster and more strongly in unfriendly interaction than affinity interaction. This suggests that the CabZIP2 gene is involved in the defense reaction.

The plant hormones SA (salicylic acid), ethylene, and MeJA (methyl jasmonate) are defense-related hormones that accumulate during pathogen infection and are involved in the signal transduction pathway. These defense-related hormones have disease resistance in plants. To evaluate the effect of these hormones on the expression of CabZIP2, SA, ethylene, or MeJA was applied to pepper leaves. SA is known to be a key factor in signal transduction pathways to resist biotrophic pathogens. SA treatment significantly promoted CabZIP2 after 0.5-12 hours of treatment, as shown in Figure 6B. Ethylene treatment, as shown in Figure 6C, detected CabZIP2 transcription first after 0.5 hours and rapidly increased between 6-12 hours after treatment. In MeJA-treated pepper leaves, CabZIP2 transcription began to accumulate at 0.5 hour after treatment and reached a peak level in 2-12 hours, as shown in Figure 6D. Expression of CabZIP2 was induced faster and more strongly in unfriendly interactions, followed by treatment with defense hormones. Therefore, it was found that the CabZIP2 gene was involved in the defense reaction.

Example 5. Identification of increased susceptibility to Xcv infection in CabZIP2-silenced pepper plants

As shown in Example 4 above, since the expression of CabZIP2 gene in pepper leaves is activated by abiotic elicitor treatment to induce Xcv infection and defense signals, virus-induced gene silencing (VIGS, virus- induced gene silencing technique was used to investigate the role of CabZIP2 in the defense of pepper to bacterial pathogens. Since the production of stable transgenic peppers by overexpression or knock-out of genes is very difficult, VIGS analysis provides a fast and effective means of knock-down expression and helps to understand the gene function of interest in the pepper.

The TRV (tobacco rattle virus) -based VIGS analysis used the CabZIP2 gene knocked down in pepper plants. To silence the CabZIP2 gene, a 254 bp fragment of CabZIP2 cDNA was injected into the pTRV2 vector. Agrobacterium tumefaciens strain GV3101 harboring pTRV1 was combined with pTRV2: 00 or pTRV2: CabZIP2 (1: 1 concentration ratio) and infiltrated together with a fully extended cotyledon (OD ₆₀₀ = 0.2 of each structure) of pepper plants. The plants were placed in a growth room under the same conditions as in Preparation Example 3 above.

The pepper seedlings were inoculated with recombinant TRV silencing constructs and phenotypic differences were not observed during growth and development in plants containing the empty vector control or CabZIP2-silenced (TRV: CabZIP2) pepper. VIGS efficiency was assessed by qRT-PCR using CabZIP2-silenced pepper leaves and CabZIP2 co-vector control after 12 hours of inoculation with Xcv toxin (Ds1) and non-toxic (Bv5-4a) strains. CabZIP2 expression decreased in CabZIP2-silenced pepper leaves regardless of Xcv inoculation. Since CabZIP2 functions as transcriptional activator (see FIG. 4) and VIP1 transcription factor regulates PR1 expression, RT-PCR was performed to determine whether CabZIP2 silencing affects the induction of defense-related genes. The primer sequences used were as shown in Table 2. < tb > < TABLE >

Gene Direction Sequence SEQ ID NO: CaBPR1 F 5'-CAGGATGCAACACTCTGGTGG -3 ' 11 R 5'-ATCAAAGGCCGGTTGGTC -3 ' 12 CaAMP1 F 5'-GAATTCATGATGAATGCTAATGGATT -3 ' 13 R 5'-GAATTCAGTCTGTGATCCC CGC -3 ' 14

As a result, as shown in Fig. 8A, CaBPR1 and CaAMP1 expression levels were significantly reduced in CabZIP2-silenced pepper leaves as compared to co-vector control plants.

Bacterial growth of the blank vector and CabZIP2-silenced red pepper was measured at 0, 3, and 5 days post inoculation (dpi) to determine if the expression of the defense-related genes including CabZIP2 reflected plant bacterial growth.

As a result, as shown in Fig. 8B, in a friendly interaction, CabZIP2 silencing not only damaged the expression of some defense-related genes, but also increased the susceptibility of plants to Xcv toxin strain infection. CabZIP2-silenced plants had significantly higher bacterial growth at 3 or 5 dpi than the control plants. Based on the induction of CabZIP2 expression by Xcv infection and non-biologic treatment, these results indicate that CabZIP2 is required for the resistance of pepper to toxic bacterial pathogens.

In addition, as shown in Fig. 8C, in the unfavorable smoothing action, the bacterial growth was not noticeable in the CabZIP2-silenced pepper leaves as compared to the ball vector control pepper leaves. CabZIP2 expression was rapidly and potently induced in non-toxic Xcv infected pepper leaves, but CabZIP2 silencing did not significantly change the susceptibility of pepper to non-toxic Xcv infection.

Thus, it was found that CabZIP2 plays a key role in basal resistance rather than R gene-mediated resistance. In addition, we could confirm that CabZIP2 plays an important role in the defense of the pepper to bacterial pathogens, influencing other defense gene expression.

Example 6. Confirmation of increased resistance to bacterial pathogens in CabZIP2-overexpressed (CabZIP2-OX) transgenic plants

To further investigate the effect of CabZIP2 in in vivo resistance response, we generated a CabZIP2 Arabidopsis transgenic plant overexpressing the entire precursor under control of the 35S promoter. Detailed methods for the development of the transgenic plant are described below.

The CabZIP2 full-length cDNA sequence was integrated into the pK2GW7 binary vector to induce constant expression (constitutive expression) of the CabZIP2 gene under the CaMV (cauliflower mosaic virus) 35S promoter in Arabidopsis. Next, the CabZIP2 binary vector was introduced into the Agrobacterium tumefaciens strain GV3101 by electroporation and the Agrobacterium-mediated transformation of Arabidopsis thaliana with the CabZIP2 gene was performed using the floral dip method . To select the transgenic lines, seeds harvested from the transgenic plant were seeded on MS agar plates containing 50 μg / mL kanamycin. RT-PCR analysis was also performed using genomic mRNA from wild-type plants and CabZIP2-OX plants in order to confirm the expression of the CabZIP2-OX transgene in independent homozygous T3 lines (see Preparation Example 4).

As a result, as shown in Fig. 9A, the PCR product was not detected for wild-type plants, but the CabZIP2 gene was amplified from three CabZIP2-OX plants, which was used for phenotypic analysis.

The phenotype of the CabZIP2-OX plant was difficult to distinguish from wild-type plants under normal growth conditions. To determine if overexpression of CabZIP2 could improve disease resistance, wild-type plants and CabZIP2-OX plants were inoculated with Pst DC3000, which causes similar symptoms to bacterial speck disease in tomatoes and Arabidopsis. As a result, as shown in Fig. 9B, after 7 days of inoculation, the wild-type plants exhibited typical bleaching phenomenon in the inoculated leaves, whereas none of the leaves of the CabZIP2-OX lines had prominent bleeding symptoms.

Bacterial growth was measured at 0, 1, 3, and 5 dpi of leaf tissue to determine whether macroscopic disease symptoms, such as whitening, reflected bacterial growth in plants. As a result, as shown in Fig. 9C, bacterial growth did not differ between wild type and CabZIP2-OX plants at 0 or 1 dpi. Bacterial growth of CabZIP2-OX plants decreased 15-30 fold after 3 days of inoculation. Five days after inoculation, the number of bacteria in wild-type plants decreased slightly and the growth of Pst DC3000 bacteria in CabZIP2-OX plants was 18-20 times lower than wild-type plants.

From the results shown in Fig. 9, it was found that CabZIP2 overexpression increased resistance to bacterial pathogens. Increased resistance to the virulent bacterial pathogen Pst DC3000 in the CabZIP2 transgenic line resulted from overexpression of the capsicum CabZIP2 gene. In previous studies, the CAbZIP1-OX plants exhibited resistance to the Pst DC3000 phenotype. However, CAbZIP1 is a bZIP transcription factor different from CabZIP2.

(Pathogenesis-related) 1 and NPR1 (Nonexpressor of Pathogenesis) in wild-type and CabZIP2-OX plants in order to investigate whether the increased resistance to bacterial pathogens in the CabZIP2-OX plant is associated with SA (salicylic acid) -Related protein 1) expression levels were compared.

As a result, as shown in Fig. 10, expression of PR1 and NPR1 genes was more active in CabZIP2-OX leaves than in wild type plants. However, the expression of PDF1.2 and LOX genes associated with JA was not significantly different between wild-type and CabZIP2-OX plants.

Thus, it was found that the SA-dependent defense mechanism contributes to the increased resistance of CabZIP2-OX to bacterial pathogen infection.

From the above preparation examples and examples, it was confirmed that the CabZIP2 protein is located in the cytoplasm and the nucleus, and functions as a transcription activator there. In addition, the susceptibility of pepper plants to bacterial pathogens was increased by CabZIP2 silencing, and the resistance to Pst DC3000 was increased by ectopic expression of the CabZIP2 transgenic Arabidopsis thaliana. These results indicate that CabZIP2 can function as a transcription activator of the defense response against bacterial pathogens in pepper plants.

It will be understood by those skilled in the art that the foregoing description of the present invention is for illustrative purposes only and that those of ordinary skill in the art can readily understand that various changes and modifications may be made without departing from the spirit or essential characteristics of the present invention. will be. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive.

<110> Chung-Ang University Industry-Academy Cooperation Foundation <120> Method for improving disease resistance using CabZIP2 in plants <130> P20150068_PB15-12550 <160> 14 <170> KoPatentin 3.0 <210> 1 <211> 1035 <212> DNA <213> CabZIP2      <400> 1 atggacccga agttcgccgg aaagcccata cccgctccat ttctagcggg tcgaactgac 60 cttgaccaga tgcccgatac tcctacccgt attgcccgtc accgtcgagc tcaatcggag 120 actttcttcc ggttcccgga ctttgacgat gatattctac tggacgacgt cgttgctgac 180 ttcaacatcg acatttctgc gccgtcggct gatacacata tgcagcagcc ggctaattcg 240 gctgactcgt catcaaccgg accccttttt gctggaccgt ctggtggtga tcaaaaccct 300 agaccgttga atcatttccg gagtctgtca gttgacgctg acttttttga tgggttggag 360 tttggtgatg ttggagcagc gacaacaccg gcggcggcga aggaagaaga gaagtgggtg 420 atgggttcgg gttcgggttc gggttcgcgg cataggcata gtaattcgat ggatggttct 480 tttagtacgg cgtcgtttga agctgagagt tctgttaaga aagctatggc gcctgatcgc 540 cttgctgaat tggctttaat tgatcccaag agagctaaaa ggattcttgc aaacagacaa 600 tctgctgcac gttctaagga gcggaaaatc cgttatacta atgaactgga gaggaaggtg 660 cagactctgc agactgaagc tactaatcta tctgcacaaa tcacggttct gcagagagac 720 aatagtggat tgactactga gaacaaagaa ctcaagctac ggttgcaagc tttggaacaa 780 caagcccatc ttagagatgc cctgaatgaa gctttgaggg aagaattgca gcggctaaag 840 atagcagctg gtcaaatgac agctgcaaat ggaaccaggg ggccgcgtcc acatttccct 900 ccccagccgc agtcatttgt ccaatgtggt aatctccatt cacaaguca gcagcagcct 960 caatctaaca caagttccca aaatattggt ggtcagaccc aacctagctt gatgaacttc 1020 agtaacaggg gttga 1035 <210> 2 <211> 344 <212> PRT <213> CabZIP2 <400> 2 Met Asp Pro Lys Phe Ala Gly Lys Pro Ile Pro Ala Pro Phe Leu Ala   1 5 10 15 Gly Arg Thr Asp Leu Asp Gln Met Pro Asp Thr Pro Thr Arg Ile Ala              20 25 30 Arg His Arg Arg Ala Gln Ser Glu Thr Phe Phe Arg Phe Pro Asp Phe          35 40 45 Asp Asp Asp Ile Leu Leu Asp Asp Val Val Ala Asp Phe Asn Ile Asp      50 55 60 Ile Ser Ala Pro Ser Ala Asp Thr His Met Gln Gln Pro Ala Asn Ser  65 70 75 80 Ala Asp Ser Ser Ser Thr Gly Pro Leu Phe Ala Gly Pro Ser Gly Gly                  85 90 95 Asp Gln Asn Pro Arg Pro Leu Asn His Phe Arg Ser Leu Ser Val Asp             100 105 110 Ala Asp Phe Phe Asp Gly Leu Glu Phe Gly Asp Val Gly Ala Ala Thr         115 120 125 Thr Pro Ala Ala Ala Lys Glu Glu Glu Lys Trp Val Met Gly Ser Gly     130 135 140 Ser Gly Ser Gly Ser Arg His Arg His Ser Asn Ser Met Asp Gly Ser 145 150 155 160 Phe Ser Thr Ala Ser Phe Glu Ala Glu Ser Ser Val Lys Lys Ala Met                 165 170 175 Ala Pro Asp Arg Leu Ala Glu Leu Ala Leu Ile Asp Pro Lys Arg Ala             180 185 190 Lys Arg Ile Leu Ala Asn Arg Gln Ser Ala Ala Arg Ser Ser Lys Glu Arg         195 200 205 Lys Ile Arg Tyr Thr Asn Glu Leu Glu Arg Lys Val Gln Thr Leu Gln     210 215 220 Thr Glu Ala Thr Asn Leu Ser Ala Gln Ile Thr Val Leu Gln Arg Asp 225 230 235 240 Asn Ser Gly Leu Thr Thr Glu Asn Lys Glu Leu Lys Leu Arg Leu Gln                 245 250 255 Ala Leu Glu Gln Gln Ala His Leu Arg Asp Ala Leu Asn Glu Ala Leu             260 265 270 Arg Glu Glu Leu Gln Arg Leu Lys Ile Ala Ala Gly Gln Met Thr Ala         275 280 285 Ala Asn Gly Thr Arg Gly Pro Arg Pro His Phe Pro Pro Gln Pro Gln     290 295 300 Ser Phe Val Gln Cys Gly Asn Leu His Ser Gln Gln Gln Gln Gln Pro 305 310 315 320 Gln Ser Asn Thr Ser Ser Gln Asn Ile Gly Gly Gln Thr Gln Pro Ser                 325 330 335 Leu Met Asn Phe Ser Asn Arg Gly             340 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CabZIP2 Forward <400> 3 ccagccgcag tcatttgtcc a 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> CabZIP2 Reverse <400> 4 tcacagcctg caacactacc catt 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA Forward <400> 5 tatggtgtgc accggtcgtc tcgt 24 <210> 6 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> 18s rRNA Reverse <400> 6 gcagttgttc gtctttcata aatccaa 27 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaActin1 Forward <400> 7 gacgtgacct aactgataac ctgat 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaActin1 Reverse <400> 8 ctctcagcac caatggtaat aactt 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaEF1-alpha Forward <400> 9 ttaaggatct taagcgtggt tatgt 25 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CaEF1-alpha Reverse <400> 10 tcttcaagaa cttaggctcc ttctc 25 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> CaBPR1 Forward <400> 11 caggatgcaa cactctggtg g 21 <210> 12 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> CaBPR1 Reverse <400> 12 atcaaaggcc ggttggtc 18 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> CaAMP1 Forward <400> 13 gaattcatga tgaatgctaa tggatt 26 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> CaAMP1 Reverse <400> 14 gaattcagtc tgtgatcccc gc 22

Claims (6)

delete delete delete delete Comprising increasing the expression or activity of the CabZIP2 protein consisting of the amino acid sequence of SEQ ID NO: 2.
A transgenic plant having enhanced disease resistance by the method of claim 5.

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