KR101677256B1 - SnRK2.8 Gene Enhancing Pathogen Resistance of Plant and Uses Thereof - Google Patents

Abstract

The present invention provides compositions and transgenic plants that increase plant disease resistance. The present invention relates to a method for inducing SAR to induce a SAR (salicylic acid), which is essential for generating systemic acquired resistance (SAR) in the immune system when an external pathogen enters the plant, It is advantageous in that resistance can be exhibited.
In addition, the present invention is expected to contribute to increasing the production of food, crops, flowers, and fruits by increasing the immunity or resistance of the plant to external pathogens.

Description

The SnRK2.8 gene which increases plant resistance and its use {SnRK2.8 Gene Enhancing Pathogen Resistance of Plant and Uses Thereof}

The present invention relates to a SnRK2.8 gene which increases plant disease resistance and uses thereof.

When bacterial pathogens penetrate plant tissues, oxidative bursts, often called HR (hypersensitive response), are induced in infected plants, which induce apoptosis of the infected area (Fu, ZQ et Al , Annu . Rev. Plant Biol . 64, 839-863 (2013); Torres, MA et al . , Nat . Genet . 37, 1130-1134 (2005)). HR-induced apoptosis inhibits the invasion of pathogens beyond the site of infection (Spoel, S. H et al . , Nat . Rev. Immunol. 12, 89-100 (2012)).

HR induces the biosynthesis of SA in both infected (topical) and terminal (systemic) tissues by activating SAR (systemic acquired resistance) to prevent secondary infection (Fu, ZQ et Al , Annu . Rev. Plant Biol . 64, 839-863 (2013); Alvarez, M. E, et al . , Plant Mol . Biol . 44, 429-442 (2000); Leon, J. et al , Plant Physiol . 108, 1673-1678 (1995)). NPR1 is the highest regulator of SAR. It acts as a coactivator of the TGA transcription factor that regulates the expression of PR (pathogenesis-related) protein genes.

SA regulates NPR1 (nonexpressor PR genes 1) protein stability (Spoel, SH et al ., Cell 137, 860-872 (2009)) mediates the conversion of a dynamic NPR1 oligomer to a monomer, which is a prerequisite for NPR1 nuclear entry (Mou, Z., Cell 113, 935-944 (2003)). Activation of NPR1 requires SA during SAR, while SA levels are only slightly elevated in the distal tissue (Nandi, A. et al , Plant Cell 16, 465-477 (2004)), which implies that additional signals are essential for NPR1 nuclear entry and SAR induction.

On the other hand, the annual loss of crops in Korea is about 2.6 trillion won, 15% of which accounts for about 390 billion won in annual losses, considering the loss due to illness. However, it is a reality that it is very difficult to increase the resistance due to the various kinds of plant pathogenic bacterium and the complex mechanism of action on the plant. Until now, studies on the cultivation of disease resistant cultivars have depended on traditional breeding breeding, and some recently developed resistance cultivars have resistance against resistance due to vertical resistance.

Recently, a method of overexpressing OsWRKY6 gene to increase plant resistance (Korean Patent Registration No. 10-0809671), use of CaSD1 gene derived from pepper (Korean Patent No. 10-1282409) And a method for producing a cyclic depress peptide which is a plant disease resistance inducing substance produced by a microorganism. However, the method for increasing the immunological resistance against external pathogenicity using the immune system is insufficient.

Thus, there is a need for materials and methods that not only have no side effects on plants, but also can significantly increase resistance (immunity) to plant pathogens.

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

SUMMARY OF THE INVENTION The present invention has been made in view of the above-described needs, and it is an object of the present invention to provide an environmentally friendly and powerful immune system capable of suppressing secondary damage caused by the use of an artificial chemical agent such as pesticides, And to develop materials that can As a result, the present inventors completed the present invention by confirming that the genes and proteins associated with SNRK2.8 (SNF1-related protein kinase 2.8) overexpression in plants are associated with systemic acquisition resistance, and thus the resistance to pathogens is remarkable.

The present invention provides a method for producing a transgenic plant having increased disease resistance comprising the steps of: (a) transforming a plant with a vector comprising a gene encoding a SnRK2.8 protein; And (b) regenerating the plant from the transformed plant cell.

The present invention also provides a transgenic plant having enhanced disease resistance and a seed thereof produced by the method for producing a transgenic plant in the present invention.

Also provided is a method for increasing the disease resistance of a plant as compared to a non-transformant comprising the step of over-expressing the SnRK2.8 protein coding gene by transforming the plant with a vector containing the gene encoding the SnRK2.8 protein .

The present invention also provides a composition for increasing plant disease resistance comprising a gene encoding the SnRK2.8 protein having the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

The present invention provides genes and their uses that increase the disease resistance of plants.

The present invention relates to a method for inducing SAR by inducing SAR even when the concentration of SA (salicylic acid), which is essential for generating systemic acquired resistance (SAR) in the immune system, It is advantageous that the resistance can be exhibited.

In addition, the present invention is expected to contribute to increasing the production of food, crops, flowers, and fruits by increasing the immunity or resistance of the plant to external pathogens.

Figure 1 shows that SnRK2.8 mediates SAR. Four-week-old plants grown under long-day conditions were used, and transcription levels were examined by RT-PCR. The mean of biological triplicates was calculated and statistically analyzed using Student t - test ( * P <0.01, ** P <0.05). The bars represent the standard error of the mean (SE).
a is the result of the effect of pathogen infection on the expression of SnRK2 . Plants were spray-inoculated with Pst DC3000 cells and cultured for 2 days. Rosetta leaves were analyzed. DPI, days post-infection.
b shows the SnRK2 .8 and PR1 expression dynamics (kinetics) between the terminal blade. The fourth and fifth leaves were pressure-infiltrated with Pst DC3000 / avrRpt2 cells and two top leaves were harvested at each time point.
c is snrk2 .8-1 Mutant and 35S: the results showing the growth of cells in 2.8 Pst DC3000 plant. Plants were spray-inoculated with Pst DC3000 cells and cultured for 6 days. cfu, colony forming unit.
d is snrk2 .8-1 Mutant and 35S: the result showing the expression of the PR1 2.8 plant. The fourth or fifth leaf was analyzed.
e are the results showing the SAR induction of snrk2 .8-1 mutants. The fourth and fifth leaves were pressure-infiltrated with 10 mM MgCl ₂ (-) or Pst DC 3000 / avrRpt2 cells (+) and cultured for 2 days. Two upper leaves were infiltrated with Pst DC3000 cells, cultured for 3 days and photographed (left panel) and bacterial cell counting (right panel).
f is a result showing the local and the expression of the PR1 at the ends of the leaf snrk2 .8 mutants. Pathogenic infections were performed as described in b. Infected leaves (local) and two upper leaves (ends) were analyzed.
Figure 2 shows that the SA is required for the control of the SnRK2 .8 SAR.
a and c were used for gene expression analysis in 2-week-old plants grown on MS-agar plates. Transcript levels were examined by RT-qPCR. The mean of the three biological trials was calculated and statistically analyzed ( t- test, * P <0.01). The bar represents SE.
a illustrates the effects of SA on the SnRK2 .8 expression. Plants were treated with 0.5 mM SA for the indicated period before harvesting whole plants. h, time.
b shows the SnRK2 .8 expression dynamics of the end leaf. The 4th and 5th leaves of 4-week-old plants grown in soil were pressure-infiltrated with Pst DC3000 / avrRpt2 cells and the two top leaves were harvested at the indicated time points for total RNA extraction.
c illustrates a SA induction of PR1 in snrk2 .8-1 mutants. Plants were treated with 0.5 mM SA for the indicated periods.
d shows the effect of over-expression of the SnRK2 .8 PR1 expressed in sid2 and npr1 -2 mutants. Fourth and fifth leaves of 4-week-old plants were harvested for gene expression analysis.
Figure 3 shows that SnRK2.8 phosphorylates NPR1.
a shows the interaction of SnRK2.8 with NPR1 in yeast cells. Yeast cell growth on the Leu, Trp, His and Ade deficient selection media (-LWHA) shows positive interaction.
and b represents BiFC (Bimolecular Fluorescence Complementation). The partial YFP construct fused to either SnRK2.8 or NPR1 and was temporarily co-expressed in Arabidopsis protoplasts. YFP signals were visualized by differential interference contrast (DIC) and fluorescence microscopy.
c is the result of coimmunoprecipitation. MYC-SnRK2.8 and FLAG-NPR1 fused constructs were temporarily co-expressed in tobacco leaves. The input represents 10% protein extract. Control (control), protein expression without transient expression; kDa, kilodalton.
d is the in vitro kinase assay result. The recombinant SnRK2.8 and NPR1 proteins were prepared by MBP fusion in E. coli cells. 1.5 ug of MBP-SnRK2.8 and 5 ug of MBP-NPR1 were used, a black arrow indicates MBP-NPR1, and a white arrow indicates MBP-SnRK2.8.
e represents the protein structure (amino acid sequence basis) of NPR1. M1 to M5 represent mutations in the putative SnRK2.8 target sites of SnRK2.8. BTB, BTB / POZ center motif. ANK, ankyrin - repetitive motifs.
f is the in vitro kinase assay for the NPR1 mutant protein. Preparation of the NPR1 protein and in vitro kinase analysis were performed as described in d. The black and white arrows indicate NPR1 and SnRK2.8, respectively.
g shows the sequence alignment of the NPR1 protein around T373. A SnRK2-mediated phosphorylation motif (K / RXXS / T) is shown. At, baby pole; Nt, Nicotiana tabacum ; Bv, beet ( Beta vulgaris ); Os, rice ( Oryza sativa ); Cp, papaya ( Carica papaya ); Gm, soybeans ( Glycine max ).
Figure 4 shows that SnRK2.8 promotes NPR1 nuclear entry.
a is the result of the effect of pathogen infection on nuclear-cytoplasmic localization of NPR1. MYC operated by the CaMV 35S promoter - NPR1 fusion constructs was overexpressed in Col-0 or snrk2 .8-1 background. Fourth or fifth leaves of 4-week-old plants were pressurized-infiltrated with Pst DC3000 / avrRpt2 cells, and the two upper leaves were harvested 24 hours later before cell fractionation. Nu, nuclear fraction. Cy, cytoplasmic fraction. NPR1 protein was immunologically detected using anti-MYC antibody (left panel). The blot was quantified using the ImageJ software (http://rsbweb.nih.gov/ij/) (right panel). Percentages represent the percentage of nuclei to cytoplasmic distribution.
b is the nucleus of NPR1 from snrk2 .8-1 mutants - a resulting image on the cellular localization. The NPR1 - GFP fusion constructs driven by the CaMV 35S promoter were overexpressed in Col-0 and snrk2.8-1 plants. The plants were treated as described in a. DIC, differential interference contrast microscopy. Scale bar, 10 탆.
c is the result of nuclear-cytoplasmic localization of NPR1 in SA-treated plant cells. Two-week-old plants grown on MS-agar plates were treated with 0.5 mM SA for 24 hours. All plants were cell fractionated and NPR1 detected as described in a.
d relates to the SnRK2.8-mediated feedforward induction of the SAR. The SA signal is integrated into the SnRK2.8 signal to activate NPR1, resulting in the induction of SAR.

According to one aspect of the present invention, the present invention provides a method for producing a transgenic plant having increased disease resistance comprising the steps of:

(a) transforming a plant with a vector comprising a gene encoding a SnRK2.8 protein; And

(b) regenerating the plant from the transformed plant cell.

The present inventors have found that the SnRK2.8 protein plays an important role in the immune system when invading foreign pathogens in plants. More specifically, the present invention relates to a method for phosphorylating the NPR1 protein when overexpression of a nucleotide encoding a SnRK2.8 protein in a plant, inducing pathogenesis-related protein gene expression by transferring the phosphorylated NPR1 protein into the nucleus And ultimately induces an immune system, especially SAR (systemic acquired resistance), to induce an independent signal to SA (salicylic acid).

The expression "amino acid sequence of SEQ ID NO: 2" in the present specification means "SnRK2.8 protein amino acid sequence &quot;, preferably Arabidopsis lt; / RTI &gt; protein amino acid sequence "derived from S. thaliana .

The SnRK2 .8 gene of the present invention may preferably be formed of a base sequence of SEQ ID NO: 1. In addition, homologues of the nucleotide sequences are included within the scope of the present invention. Specifically, the gene has a nucleotide sequence having a sequence homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more, with the nucleotide sequence of SEQ ID NO: 1 .

"% Of sequence homology to base" is determined by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence (addition or deletion) of the optimal alignment of the two sequences (I. E., Gap), as compared to &lt; / RTI &gt;

The range of the SnRK2.8 protein according to the present invention includes a protein having the amino acid sequence represented by SEQ ID NO: 2 and a functional equivalent of the protein. Is at least 70% or more, preferably 80% or more, more preferably 90% or more, still more preferably 90% or more, more preferably 90% or more, Quot; refers to a protein having a homology of at least 95% with a physiological activity substantially equivalent to that of the protein represented by SEQ ID NO: 2. "Substantially homogenous physiological activity" means an activity that increases plant disease resistance compared to non-transformant.

According to a preferred embodiment of the present invention, the gene sequence encoding the amino acid sequence of the SnRK2.8 protein in the present invention includes the nucleotide sequence of SEQ ID NO: 1.

The term "recombinant" refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

In the present invention, a gene carrier known in the art may be used for carrying the nucleotide sequence of SEQ ID NO: 1, and preferably a vector system.

When the vector system of the present invention is used, it includes a plasmid, a phage, and the like, preferably a plasmid.

The vector system of the present invention can be constructed through various methods known in the art, and specific methods for this can be found in Sambrook et &lt; RTI ID = 0.0 &gt; al ., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

The recombinant vectors of the present invention include promoters operable in plant cells. The promoter suitable for the present invention may be any of those conventionally used in the art for transformation of plants. Examples of the promoter include a promoter such as a Cauliflower Mosaic Virus (CaMV) 35S promoter, a nopaline synthase (nos) promoter, Wort mosaic virus 35S promoter, and the like, preferably the promoter of Collifura mosaic virus (CaMV) 35S.

In the recombinant vector of the present invention, conventional terminators can be used. Examples thereof include nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, phaseoline terminator, Agrobacterium tumefaciens (Agrobacterium tumefaciens ) Octopine gene terminator, but the present invention is not limited thereto. Regarding the need for terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of a terminator is highly desirable in the context of the present invention.

As used herein, the term " resistant "means showing resistance to a pathogen and is used in the same sense as a defense response.

According to a preferred embodiment of the present invention, the gene of the present invention increases immunity to pathogens, thereby imparting pathogen resistance to the plant. The pathogens include bacteria, fungi, yeast, , Oomycete, and viruses, and more preferably bacteria.

The method of the invention comprises the step of transforming a plant cell with a recombinant vector according to the present invention, the transformant is, for example, Agrobacterium tyumeo Pacific Enschede may be mediated by (Agrobacterium tumefiaciens). In addition, the method of the present invention comprises regenerating a transgenic plant from the transformed plant cell. Any of the methods known in the art can be used for regeneration of transgenic plants from transgenic plant cells.

Transformed plant cells must be regenerated into whole plants. Techniques for the regeneration of mature plants from callus or protoplast cultures are well known in the art for a number of different species.

In one embodiment of the present invention, the plant is selected from the group consisting of Arabidopsis, potato, eggplant, cigarette, pepper, tomato, burdock, ciliaceae, lettuce, bellflower, spinach, modern sweet potato, celery, carrot, parsley, parsley, cabbage, Rice, barley, wheat, rye, corn, sorghum, oats, onion, etc., such as rice bran, watermelon, melon, cucumber, amber, pak, strawberry, soybean, mung bean, kidney bean and pea , Preferably Arabidopsis or rice plants, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a transgenic plant having enhanced disease resistance and seeds thereof compared to the non-transformant produced by the above method. The present inventors have made intensive efforts to develop new transgenic plants and plant cells, and as a result, have found that transgenic plant cells and plants having resistance to diseases caused by external pathogens can be obtained by inducing SAR (systemic acquired resistance) Respectively.

The transformed plants and plant seeds of the present invention are produced using the method of the present invention for increasing the disease tolerance of plants of the present invention described above. In order to avoid the excessive complexity of the present specification, It is omitted.

According to still another aspect of the present invention, there is provided a method for producing a plant, comprising the step of transfecting a plant with a vector containing a gene encoding a SnRK2.8 protein to overexpress the SnRK2.8 protein- Lt; RTI ID = 0.0 &gt; resistance. &Lt; / RTI &gt;

The present invention relates to a method for increasing disease resistance using the same method as the method for producing the plant having increased disease resistance. Accordingly, the description common to both is omitted in order to avoid the excessive complexity of the present specification.

According to still another aspect of the present invention, there is provided a composition for increasing plant disease resistance comprising a gene encoding the SnRK2.8 protein comprising the amino acid sequence of SEQ ID NO: 2 as an active ingredient. The composition of the present invention contains, as an active ingredient, a gene encoding the SnRK2.8 protein consisting of the amino acid sequence of SEQ ID NO: 2, and can be used to increase the disease resistance of a plant by transforming the gene into a plant.

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

Materials and methods

Plant material and growth conditions

All used Arabidopsis ( Arabidopsis thaliana line was in the background of Col-0 (Columbia). Arabidopsis was grown on MS-agar plates at 23 ° C under long-day conditions (LDs, 16 h light and 8 h dark conditions). White light (120 μmol photons m ^-2 s ^-1 ) was provided by fluorescence FLR40D / A tube (Osram, Seoul, Korea). SnRK2 .8 - deficient snrk2 .8-1 .8-2 snrk2 and mutants (SALK-073395 and SALK-053423) is stored in the T-DNA (The Arabidopsis Information Resource, Ohio State University, OH) TAIR insertion mutant It was separated from the pool. The NPR1 -deficient npr1 -2 mutant described in the previous study (Cao, H. et al. , Cell 88, 57-63 (1997)) was obtained from Xinnian Dong. Homozygous mutants were obtained by selection and segregation analysis of three or more generations, and the lack of gene expression was confirmed by RT-PCR of the mutants. A full-length SnRK2.8 cDNA driven by the CaMV (Cauliflower Mosaic Virus) 35S promoter was overexpressed in the Col-0 plant resulting in a 35S: 2.8 plant. A number of independent transfection lines were obtained and two representative lines were analyzed simultaneously (in parallel). Agrobacterium tumefaciens-mediated Arabidopsis transformation was carried out by the modified floral dip method as described in previous studies (Zhang, X. et al . , Nat . Protoc . 1, 641-646 (2006)). Overexpression of foreign genes in transgenic plants was confirmed by quantitative real-time RT-PCR (RT-qPCR).

Gene transcription analysis

RT-qPCR was performed to measure the relative levels of gene transcripts. Total RNA sample preparation, reverse transcription and quantitative PCR were performed according to the proposed rules for reproducible measurement (Udvardi et al , Plant Cell 20, 1736-1737 (2008)). RT-qPCR reactions were performed in 96-well blocks of an Applied Biosystems 7500 Real-Time PCR System (Foster City, Calif.) Using a 20 [mu] l volume of SYBR Green I master mix. PCR primers are listed in Table 1 below. The thermal cycling profile of the second step was a 15 second reaction at 95 ° C for denaturation and a 1 minute reaction at 60 ° C for annealing and polymerization. The eIF4A gene (At3g13920) was included as an internal control to normalize the amount of cDNA used in the reaction.

Biological triplicates were performed using total RNA samples extracted from three independent plant materials grown under the same conditions. The relative ΔΔC _T method was performed to determine the relative amount of each amplified product from the sample. The threshold cycle (C _T ) was automatically measured for each reaction using the default parameters of the system.

Preparation of recombinant proteins

The full length SnRK2.8 and NPR1 cDNA sequences were subcloned into pMAL-c2X E. coli expression vector (NEB, Ipswich, Mass.) Containing a maltose-binding protein (MBP) Recombinant MBP-SnRK2.8 and MBP-NPR1 proteins were synthesized in E. coli Rosetta2 (DE3) pLysS strain (Novagen, Madison, Wis.). Cells were harvested and resuspended in MBP buffer (20 mM Tris-HCl, pH 7.4, 200 mM NaCl, 1 mM EDTA) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO; Cat No. P9599) , 10 mM 2-mercaptoethanol and 1 mM PMSF). Cell lysates were prepared by centrifugation after 3 cycles of freezing and thawing. The fusion protein was purified by affinity chromatography.

Yeast two-hybrid assay

Yeast double-hybridization assays were performed using the BD Matchmaker system (Clontech, Mountain View, Calif.) To investigate whether SnRK2.8 interacts with NPR1. The pGADT7 vector was used for GAL4 activation domain (AD) fusion and the pGBKT7 vector was used for GAL4 DNA-binding domain (BD) fusion. The PCR products of the full length SnRK2.8 and NPR1 cDNAs were restricted with Nde I and Eco RI and subcloned into pGBKT7 and pGADT7 vectors. Yeast strain AH109, containing the lacZ encoding the beta -galactosidase integrated into the chromosome under the control of the GAL1 promoter and the HIS reporter gene mediating histidine biosynthesis, was used for transformation of the expression construct. Transformation of AH109 cells was performed according to the manufacturer's instructions. The obtained colonies were streaked on a medium free of histidine (His), adenine (Ade), leucine (Leu) and tryptophan (Trp).

BiFC (Bimolecular Fluorescence Complementation ) analysis

BiFC assays were performed using the Arabidopsis mesophyll protoplasts as described in the prior art (Seo, PJ et &lt; RTI ID = 0.0 &gt; al . , Nat . Commun . 2, 303 (2011)). Full-length cDNA sequence is SnRK2 .8 pSATN-cEYFP-C1 vector N- terminal half (nYFP) of the 3 'end of the DNA sequence encoding the EYFP of (E3082) - was fused to the frame (in-frame). The full length NPR1 cDNA sequence was fused in-frame to the 3 'end of the DNA sequence encoding the C-terminal half (cYFP) of the EYFP of the pSATN-nEYFP-C1 vector (E3081). Co-transfection of the nYFP-SnRK2.8 and cYFP-NPR1 constructs into Arabidopsis protoplasts was performed by the PEG (polyethylene glycol) -calcium transfection method (Yoo, SD et al , Nat . Protoc . 2 , 1565-1572 (2007)). After 16 hours of co-transfection, the intracellular distribution of the fusion construct was visualized by differential interference contrast (DIC) microscopy and fluorescence microscopy using a Zeiss LSM510 confocal microscope (Carl Zeiss, Yena, Germany) . Reconstitution of YFP fluorescence was observed using confocal microscopy with the following YFP filter setup: Excitation 515 nm, 458/514 dichroic and emission 560-615 nm BP filter.

NPR1 Intracellular Localization  Cells for Fractionation )

The MYC - NPR1 fusion construct fused in-frame to the 5 'end of the full length NPR1 cDNA was overexpressed in the background of Col-0 or snrk2 .8-1, the MYC - coding sequence driven by the CaMV 35S promoter. The 4th and 5th leaves of 4-week-old plants grown in soil under LD conditions were pressure-infiltrated for 2 days using Pst DC3000 / avrRpt2 cells. Two upper leaves were harvested prior to cell fractionation. The production of nuclear and cytoplasmic fractions was carried out as described in the prior art (Wang, W. et al ., Plant Cell 23, 3565-3576 (2011)). Fractions were analyzed on 10% SDS-PAGE and blotted onto a Millipore Immobilon-P membrane (Billerica, MA). NPR1 protein was immunologically detected using anti-MYC antibody. The α-tubulin protein, a marker for cellular localization, was detected using an anti-tubulin antibody (Sigma-Aldrich). The blot was quantified using ImageJ software (http://rsbweb.nih.gov/ij/).

Analysis of protein infection and salicylic acid (SA) treatment

Four - week - old Arabidopsis plants grown in soil at 23 ℃ under long - day conditions were used for pathogen infection and gene expression analysis. Pathogenic Pst DC3000 ( Pseudomonas syringae pv. tomato strain DC3000) was cultured in LB (Luria-Bertani) supplemented with rifampicin (50 mg / l) for 2 days at 28 ° C. The bacterial cell suspension was prepared with 10 ⁷ cfu (colony forming unit) / ml in 10 mM MgCl ₂ with 250 ppm of TWEEN 80 and sprayed directly onto the rosette leaf surface. After 16 hours of incubation at 25 ° C and 100% relative humidity in complete darkness, the inoculated plants were transferred to a growth chamber set at 23 ° C and 80% relative humidity and grown for 2 days under LD conditions. Inoculated leaves were used for total RNA extraction. To measure bacterial cell growth, spray-inoculated plants were cultured for 3 to 6 days under LD conditions and bacterial cells were counted as described in previous studies (Park, JE et al ., J. Biol . Chem . 282, 10036-10046 (2007)).

At the end leaves in order to investigate the induction of systemic acquired resistance (systemic acquired resistance, SAR), 4 second and fifth Rosetta leaf is 10 mM MgCl pressurized with a non-pathogenic Pst DC3000 / avrRpt2 cells suspended in the _second solution-was infiltration, LD conditions For 2 days. The two top leaves were infiltrated with Pst DC3000 cells and then further cultured for 3 days as described above before photographing and bacterial counting. For SA treatment, 0.5 mM SA was sprayed on 2-week old plants grown on MS-agar plates and whole plants were cultured for an appropriate time before harvesting.

Co - IP (Coimmunoprecipitation ) analysis

Co-IP analysis was performed using Nicotiana benthamiana cells as described in previous studies (Song, YH et al al. , Science 336, 1045-1049 (2012)). CaMV 35S and pBA002 pEarleyGate202 vector that is activated by the promoter (ABRC, Columbus, Ohio) each subcloned in the MYC - SnRK2 .8 and FLAG - NPR1 sequence and fusion expression constructs were temporarily co-expressed in tobacco leaves. The plant material was pulverized in liquid nitrogen and the protein was dissolved in Co-IP buffer (50 mM Tris-Cl pH 7.4, 500 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100 and 1% Nonidet P-40). 10% of the extract was used as an input control. Anti-FLAG antibody (Sigma-Aldrich) was added to the extract and incubated for 2 hours. After the incubation, protein G agarose beads were added and further incubated for 2 hours. The beads were then washed 5 times with Co-IP buffer without protease inhibitor cocktail. To elute bound proteins, 50 μl of 2X SDS loading buffer (100 mM Tris-Cl pH 6.8, 4% SDS (sodium dodecyl sulfate), 0.2% bromophenol blue, 20% glycerol and 200 mM DTT) was added and heated for 5 minutes. Twenty percent of the eluted protein was used as an IP control. Anti-FLAG and anti-MYC (Millipore, Billerica, MA) antibodies were used for immunological detection of FLAG-NPR1 and MYC-SnRK2.8 fusion proteins, respectively.

In vitro phosphorylation assay

In vitro phosphorylation assays were performed as described in the prior art. 5 ㎍ of the recombinant protein and 1.5 ㎍ NPR1 protein purification SnRK2.8 of 1 μCi [γ- ³² P] reaction buffer containing the ATP [20 mM Hepes (pH 7.4 ), 10 mM MgCl 2, 1 mM Na 3 VO ₄ , 2 mM DTT, 0.5 mM PMSF, 2 mM EDTA and 200 nM dATP]. The reaction mixture was incubated at 30 ° C for 30 minutes and the reaction was terminated by addition of 4 μl of 6 × SDS loading buffer. The mixture was then heated to 100 &lt; 0 &gt; C for 5 minutes and loaded on a 10% SDS-PAGE gel. The gel was stained with Cooassie Blue and dried under vacuum for detection of the radiation signal.

Example  One

The present inventors SnRK2 .8 transfer the Pst DC3000 (Pseudomonas syringae pv. tomato DC3000) (Fig. 1 a), which supports the notion that SnRK 2.8 is involved in pathogen resistance. Some other SnRK2 genes, such as SnRK2 .3 , 2.6 , 2.7 and 2.9 , are also slightly induced after pathogen infection.

Then the inventors examined whether affected by a systemic signal (systemic signal) produced by the SnRK2 .8 transfer primary infection. SnRK2 .8 transfer was rapidly induced in the end leaf within 6 hours after topical infection by Pst DC3000 / avrRpt2 cells (15) (b in FIG. 1). On the other hand, systemic expression of PR1 was initiated 6 hours after local infection. This sequential expression pattern suggests that SnRK2.8 acts as a SAR in the upstream of PR1 regulation by TGA.

Consistent with SnRK2 .8 induced by pathogen infection, SnRK2 .8 - overexpressing plants (35S: 2.8) will appear Despite the improved resistance to Pst DC3000 infection, SnRK2 .8 - defective mutants (snrk2 .8-1) is Lt; RTI ID = 0.0 &gt; Col-0 &lt; / RTI &gt; Thus, the 35S transcription PR1: but a significant increase in plant 2.8, .8-1 snrk2 mutants in not change significantly (Fig. 1 d).

The present inventors investigated the potential role of SnRK2.8 in SAR through bacterial cell counting of terminal leaves, which was induced by Pst DC3000 / avrRpt2 infection. In particular, and snrk2 snrk2 .8-1 .8-2 mutants compared to the Col-0 plants, did not show this increased resistance (e in FIG. 1). snrk2 .8 - reduced SAR phenotype of the mutant is defective SA- deficient mutants sisd2 and NPR1 - was similar to the phenotype of the mutants npr1 -2 defects, which are SnRK2.8 as reported in previous studies for the SA and NPR Supporting the concept that it plays a role in SAR induction. Transferring irradiation of PR1 in SAR- active plant is only slightly adversely affected, the leaves of the local induction PR1, said to be substantially reduced, the end leaf (f in Fig. 1). In contrast, the transcriptional induction of PR1 was markedly reduced as previously reported in both the local and terminal leaves of the nprl- 2 mutant. These observations demonstrate that SnRK2.8 plays a predominant role in the endothelial tissue than the focal tissue.

Example  2

SA is essential for induction of SAR (Fu, ZQ et Al , Annu . Rev. Plant Biol. 64, 839-863 (2013); Delaney, TP et al ., Science 18, 1247-1250 (1994)). Therefore, the present inventors investigated whether SnRK2.8 is associated with SA. The present inventors made investigation into the effect on the SA SnRK2 .8 transfer and discovered that SnRK2 .8 transfer is not affected by the SA (a in Fig. 2). Furthermore sid2 mutation did not affect the induction SAR- SnRK2 .8 expressed in the terminal leaves (b in Fig. 2). At the same time, the induction of PR1 and SA PR5 generally snrk2 .8-1 one generated in the mutants, (c in FIG. 2), which SA- mediated induction of PR npr1-2 did the observed mutants requiring NPR1 However, it does not depend on SnRK2.8. However, high levels of PR1 induced by over-expression was not observed in SnRK2 .8 sid2 npr1 -2 and the background (background) (according (in FIG. 2 FIG. 3, but this observation was passed SnRK2.8 and SA signals are independent, PR Lt; / RTI &gt; in the expression phase, perhaps through NPR1.

Example  3

NPR1 acts as a coactivator with TGA transcription factors to regulate PR gene expression (Despres, C. et al , Plant Cell 12, 279-290 (2000); Despres, C. et al , Plant Cell 15, 2181-2191 (2003)). Thus, the present inventors have investigated whether SnRK2.8 interacts with upstream modulators. The yeast two-hybrid assay showed that SnRK2.8 interacted only with NPR1 among the upstream modulators examined (Fig. 3a). The SnRK2.8-NPR1 interaction occurred in both nuclear and cytoplasm, as investigated by BiFc (bimolecular fluorescence complementation) analysis (Fig. 3b). The in planta interaction of SnRK2.8 and NPR1 was confirmed by a coimmunoprecipitation assay using tobacco cells that co-express FLAG-NPR1 and MYC-SnRK2.8 fusion proteins 3c).

The SNRK2 family constituent proteins are serine / threonine (S / T) protein kinases that mediate plant development and environmental stress resistance (Kulik, A. et al , OMICS . 15, 859-872 (2011)). Therefore, the present inventor investigated whether or not SnRK2.8 phosphorylates NPR1. Recombinant protein was prepared by MBP (maltose-binding protein) fusion of Escherichia coli and in vitro kinase analysis was performed. These results indicate that SnRK2.8 can phosphorylate NPR1 (Fig. 3d). In addition, SnRK2.8 was autophosphorylated. Conversely, the near homologue of SnRK2.8, SnRK2.6, was autophosphorylated (Boudsocq, M. et al, J. Biol. Chem. 279, 41758-41766 (2004)). Basophilic kinases such as the SnRK2 constitutive protein preferentially phosphorylate S and T residues in the sequence of K / RXXS / T (where X represents any amino acid) (Kim, MJ et al ., Biochem. J. 448, 353-363 (2012)).

The present inventors obtained the M1-M5 protein by replacing the S / T residue with alanine (A) in NPR1 (Fig. 3e). In vitro phosphorylation analysis showed that SnRK2.8-mediated phosphorylation was reduced by about 70% in M4 (Fig. 3f), indicating that T373 is the primary target residue of SnRK2.8. Sequence analysis showed that T373 is entirely conserved in the NPR1 protein of various plant species (g in FIG. 3). On the other hand, SnRK2.8 did not phosphorylate NPR1 S11 / 15, which is known to be phosphorylated in SA-treated plants (Spoel, SH et al ., Cell 137, 860-872 (2009)).

Example  4

SA causes its monomerization required for nuclear localization of NPR1 (Mou, Z., Fan et al. , Cell 113, 935-944 (2003)). It has been reported that low concentrations of SA still induce the conversion of the NPR1 oligomer to the monomer, but are insufficient for its nuclear flux (Kinkema et al , Plant Cell 12, 2339-2350 (2000)). NPR1 is effectively introduced into the nuclei of distal tissues with slightly elevated SA levels after pathogen infection (Nandi, A. et al ,, Plant Cell 16, 465-477 (2004)). Therefore, the present inventors evaluated whether SnRK2.8 is required for nuclear inflow of NPR1 during SAR.

After primary infection with Pst DC3000 / avrRpt2 cells, the nuclear and cytoplasmic fractions of the terminal leaves were isolated and the relative distribution of NPR1 was examined in each fraction. In the mock-treated Col-0 plants, 47% of NPR1 was located in the nucleus (Fig. 4a) and the proportion of nuclear NPR1 after SAR induction increased to 66%. In contrast to the screen, mock- treated snrk2 .8-1 was nuclei of the mutants, the 16% of the NPR1 position, eopeotneunde a significant effect of the amount of SAR induction of the nuclear NPR1, which SnRK2.8 the NPR1 nuclear location for SAR Lt; / RTI &gt;

The present inventors also investigated whether or not SnRK2.8 affects the conversion of the NPR1 oligomer to the monomer by comparing the snrk2.8-1 mutant overexpressing the MYC-NPR1 fusion protein driven by the CaMV (Cauliflower Mosaic Virus) 35S promoter Respectively. It was equally elevated levels of monomers in both Col-0 and snrk2 .8-1 background, indicating that this does not affect the SnRK2.8 NPR1 monomer Tuesday at the plant pathogen infection.

In snrk2 .8-1 mutants compromised (impaired) NPR1 nuclear 35S influx of snrk2 .8-1 background: NPR1 - it was investigated further using a GFP-terminal leaves of transgenic plants. After SAR induction, a GFP (green fluorescence protein) signal was predominantly detected in the nucleus of the Col-0 background (Fig. 4b). In contrast, the GFP signal in the background of snrk2 -8-1 was strongly expressed in the cytoplasm, but relatively weak in the nucleus, as compared to the GFP signal detected in the Col-0 background. Conversely, NPR1 nuclear localization aspects of the local organization was shown similarly in both Col-0 and snrk2 .8 the background, which supports the role of nuclear NPR1 SnRK2.8 in the position of the terminal leaves angry.

The present inventors ultimately examined whether the SNRK2.8-mediated NPR1 nuclear localization is associated with SA. In the mock-treated Col-0 plants, 6.4% of NPR1 was located in the nucleus, and the amount of NPR1 in the nucleus was significantly increased up to 39% after SA treatment (Fig. Notably, the mutant snrk2 .8-1 similar nuclear-cytoplasmic distribution pattern was observed, indicating that the SA does not directly related to the parameters SnRK2.8- NPR1 nuclear localization.

SA is essential for SAR development but should be kept low at plant sites other than the HR site to avoid toxic effects such as growth retardation, DNA damage and physiological disturbance. In this regard, it is likely that SA is not required for a high level of SA because it is used in the initiation signal that links the induction of SAR and local infection in the endothelial tissue. When NPR1 is enhanced by SA through the conversion of oligomers to monomers, the SnRK2.8-mediated feedforward pathway activates NPR1 through protein phosphorylation, leading to the induction of downstream PR genes.

We believe that SA signaling, which provides an adaptation strategy under biological stress conditions, and integration of the SnRK2.8-mediated activation system in NPR1, while maintaining normal plant growth in the endothelial tissue, (Fig. 4 (d)). The results of this study can contribute to the identification of long-range signaling molecules and methods during the future of SAR.

<110> SNU R & DB FOUNDATION <120> SnRK2.8 Gene Enhancing Pathogen Resistance of Plant and Uses          Thereof <130> PN15020 <160> 12 <170> KoPatentin <210> 1 <211> 1032 <212> DNA <213> Arabidopsis thaliana <400> 1 atggagaggt acgaaatagt gaaggatatt gggtctggta acttcggagt agcaaagctt 60 gttcgtgaca aattttccaa agagcttttc gctgttaagt tcatcgagcg aggccaaaag 120 attgatgaac atgtacaaag agaaatcatg aaccataggt cgctgatcca tcccaatata 180 ataagattca aggaggtttt attgacggca acacatttgg cgttagtaat ggaatacgcc 240 gccggaggag aactgttcgg aagaatctgc agcgccggaa gattcagtga agacgaggca 300 aggtttttct ttcagcagct tatatcagga gttaattact gtcacagtct tcaaatatgc 360 catagagatt taaagctaga gaacacgtta cttgatggaa gcgaagcgcc acgtgtaaag 420 atttgcgact ttggatattc aaaatcagga gttcttcatt cgcaaccaaa gacaacagta 480 ggaacacctg cttacattgc acctgaagtg ctctccacga aagagtatga cggcaaaatc 540 gctgatgttt ggtcttgtgg agtcactttg tatgttatgc ttgttggtgc ttatcctttt 600 gaagatcctt ctgatcctaa agattttcgg aagacgatcg gtcggattct caaagctcag 660 tatgctattc ctgattatgt tcgagtttcg gatgaatgca gacatcttct ctctcggata 720 ttcgttgcca accctgaaaa gagaataaca atagaggaga taaagaatca ttcttggttt 780 ctcaagaact tgccggtaga gatgtatgaa ggatcattga tgatgaatgg tccatcgact 840 cagacagtag aagagatagt gtggatcatt gaagaagctc ggaaacctat caccgtagct 900 actggactcg caggtgctgg tggctctggt ggaagcagta atggtgccat tggaagtagc 960 agtatggatc tcgatgactt ggacacagat ttcgacgaca tcgataccgc tgatctcctt 1020 tcccctttgt ga 1032 <210> 2 <211> 343 <212> PRT <213> Arabidopsis thaliana <400> 2 Met Glu Arg Tyr Glu Ile Val Lys Asp Ile Gly Ser Gly Asn Phe Gly   1 5 10 15 Val Ala Lys Leu Val Arg Asp Lys Phe Ser Lys Glu Leu Phe Ala Val              20 25 30 Lys Phe Ile Glu Arg Gly Gln Lys Ile Asp Glu His Val Gln Arg Glu          35 40 45 Ile Met Asn His Arg Ser Leu Ile His Pro Asn Ile Ile Arg Phe Lys      50 55 60 Glu Val Leu Leu Thr Ala Thr His Leu Ala Leu Val Met Glu Tyr Ala  65 70 75 80 Ala Gly Gly Glu Leu Phe Gly Arg Ile Cys Ser Ala Gly Arg Phe Ser                  85 90 95 Glu Asp Glu Ala Arg Phe Phe Phe Gln Gln Leu Ile Ser Gly Val Asn             100 105 110 Tyr Cys His Ser Leu Gln Ile Cys His Arg Asp Leu Lys Leu Glu Asn         115 120 125 Thr Leu Leu Asp Gly Ser Glu Ala Pro Arg Val Lys Ile Cys Asp Phe     130 135 140 Gly Tyr Ser Lys Ser Gly Val Leu His Ser Gln Pro Lys Thr Thr Val 145 150 155 160 Gly Thr Pro Ala Tyr Ile Ala Pro Glu Val Leu Ser Thr Lys Glu Tyr                 165 170 175 Asp Gly Lys Ile Ala Asp Val Trp Ser Cys Gly Val Thr Leu Tyr Val             180 185 190 Met Leu Val Gly Ala Tyr Pro Phe Glu Asp Pro Ser Asp Pro Lys Asp         195 200 205 Phe Arg Lys Thr Ile Gly Arg Ile Leu Lys Ala Gln Tyr Ala Ile Pro     210 215 220 Asp Tyr Val Arg Val Val Ser Asp Glu Cys Arg His Leu Leu Ser Arg Ile 225 230 235 240 Phe Val Ala Asn Pro Glu Lys Arg Ile Thr Ile Glu Glu Ile Lys Asn                 245 250 255 His Ser Trp Phe Leu Lys Asn Leu Pro Val Glu Met Tyr Glu Gly Ser             260 265 270 Leu Met Met Asn Gly Pro Ser Thr Gln Thr Val Glu Glu Ile Val Trp         275 280 285 Ile Ile Glu Glu Ala Arg Lys Pro Ile Thr Val Ala Thr Gly Leu Ala     290 295 300 Gly Ala Gly Gly Ser Gly Gly Ser Ser Asn Gly Ala Ile Gly Ser Ser 305 310 315 320 Ser Met Asp Leu Asp Asp Leu Asp Thr Asp Phe Asp Asp Ile Asp Thr                 325 330 335 Ala Asp Leu Leu             340 <210> 3 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 3 tgaccacaca gtctctgcaa 20 <210> 4 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 4 accagggaga cttgttggac 20 <210> 5 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 5 cctgaagtgc tctccacgaa 20 <210> 6 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 6 gcattcatcc gaaactcgaa 20 <210> 7 <211> 21 <212> DNA <213> Unknown <220> <223> primer <400> 7 tgatcctcgt gggaattatg t 21 <210> 8 <211> 24 <212> DNA <213> Unknown <220> <223> primer <400> 8 tgcatgatca catcattact tcat 24 <210> 9 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 9 agttcctccc gtcactctgg 20 <210> 10 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 10 tcctccggat ggtcttatcc 20 <210> 11 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 11 tctgcagtga agctttggct 20 <210> 12 <211> 20 <212> DNA <213> Unknown <220> <223> primer <400> 12 ggtcgtcttt cggactggtt 20

Claims (8)

A method for producing a transgenic plant having increased disease resistance comprising the steps of:
(a) transforming a plant with a vector comprising a gene encoding the SNRK1.8-related protein kinase 2.8 protein consisting of the amino acid sequence of SEQ ID NO: 2; And
(b) regenerating the plant from the transformed plant cell. delete A transgenic plant having enhanced disease resistance of the plant compared to the non-transformant produced by the method of claim 1. 4. The transgenic plant according to claim 3, wherein the plant is a dicot or a monocotyledon. A transformed seed of a plant according to claim 3. A method for screening a non-transformed host cell comprising the step of transfecting a plant with a vector comprising a gene encoding a SNRK2.8 (SNF1-related protein kinase 2.8) protein consisting of the amino acid sequence of SEQ ID NO: 2 to overexpress the SnRK2.8 protein- A method of increasing plant disease resistance compared to sieve. delete A composition for increasing plant disease resistance comprising a gene encoding SNRK2.8 (SNF1-related protein kinase 2.8) protein consisting of the amino acid sequence of SEQ ID NO: 2 as an active ingredient.

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