KR100745832B1 - 1 GLIP1 gene and its protein having plant resistance to the fungus - Google Patents

Abstract

A GLIP1(GDSL motif lipase 1) gene and its protein having plant resistance against fungi are provided to confer resistance against fungi through two different mechanisms related to direct destruction of fungi pores and activation of defense signal transfer of plant related to ethylene on a plant. The GLIP1 gene having plant resistance against fungi has the nucleotide sequence of SEQ ID NO:1. The GLIP1 protein encoded by the GLIP1 gene has the amino acid sequence of SEQ ID NO:2. The plant resistance against fungi of the GLIP1 gene or protein is conferred on a plant by treating the plant with the GLIP1 protein of SEQ ID NO:2 or transforming the plant with a vector pBI221-GLIP1 containing the GLIP1 gene of SEQ ID NO:1.

Description

GLIP1 gene and its protein having plant resistance to the fungus

1 is a gene map of pBI221-GLIP1 as an example of a transformation vector of a plant comprising the GLIP1 gene of the present invention.

2 is a gene map of pGEX-GLIP1 as an example of a transformation vector of E. coli comprising the GLIP1 gene of the present invention.

Figure 3 shows the alignment of amino acid sequences of GLIP1 and other GLIPs of the present invention.

Figure 4 is a photograph showing the intracellular location of the GLIP1 protein of the present invention.

5 is a picture of T-DNA insertion mutants of GLIP1 of the present invention.

6 shows disease development of glip1 mutants inoculated with P. syringae and A. brassicicola .

Figure 7 illustrates the lipase activity of the recombinant GLIP1 protein of the present invention.

Figure 8 shows the antimicrobial activity of the recombinant GLIP1 protein of the invention against A. brassicicola .

9 illustrates the function of GLIP1 of the present invention in a cystemic defense response.

The present invention relates to a novel fungal resistance gene derived from Arabidopsis , more specifically to the plant's fungal resistance GLIP1 (GDSL motif lipase 1) gene and its protein and a method for providing fungal resistance of the plant using the same It is about.

The cell wall or extracellular matrix (ECM) is an active area that contains active ingredients that regulate cell-cell interactions. ECM contains secretory proteins that play an important role in various physiological processes such as growth, development and defense response (Showalter, (1993) Plant Cell 5, 9-23; Pennell, (1998) Curr. Opin.Plant Biol. 1, 504-510). The most abundant and most studied cell wall proteins are structural proteins including extensins, Gly-rich proteins, Pro-rich proteins, solanaceous lectins and arabinogalactan proteins (Showalter, 1993). However, ECM also contains nonstructural proteins, including peroxidases, invertases, proteases, mannosidases, galactosidases and glucanases.

ECM peptides or proteins have been reported that participate in conventional plant defense processes. For example, plants have been reported to contain families of antimicrobial peptides (Garcia-Olmedo et al., (1998) Biopolymers 47, 479-491), which are thionins, defensins, lipid transfer proteins, hevein- and knottin-. Similar peptides, maize (Zea mays) basic peptide 1, Impatiens balsamina antimicrobial peptides, snakins and systemins. Of these, Systemins are best defined functionally as the only class of 18-amino acid peptides derived from the precursor polypeptide prosystemin (Ryan et al., (2002) Plant Cell 14 (suppl.), S251-S264). They interact with cytoplasmic receptor kinase SR160 to activate protective signaling. The plant also has a polygalacturonase-inhibiting protein, a ubiquitous cell wall protein that protects the plant from fungal attack (De Lorenzo and Ferrari, (2002) Curr. Opin. Plant Biol. 5, 295-299). The polygalacturonase-inhibiting protein directly inhibits polygalacturonases secreted by pathogenic fungi and enhances the accumulation of oligogalacturonides that activate the defense response.

It has also been reported that conventional fungal plant pathogens secrete hydrolytic enzymes such as cutinases or esterases to break down the components of the plant cell wall during invasion into the host. Moreover, extracellular lipases were considered to be the virulence factor of pathogenic bacteria and fungal species, including A. brassicicola (Berto et al., (1999) FEMS Microbiol. Lett. 180, 183-189; Eddine et al., (2001) Mol. Genet. Genomics 265, 215-224). However, the present invention first revealed that fungal spores can be countermeasured by the plant-derived lipase GLIP1.

In the present invention, we have extensively analyzed the secretome of cultured Arabidopsis cells. We collected proteins secreted into the culture medium and analyzed by 2D gel electrophoresis and MALDI-TOF MS. In order to identify secreted proteins involved in plant pathogen response, salicylic acid (SA) -induced changes in isolated secretions were evaluated. 2D gel analysis showed that 18 protein spots corresponding to 13 different proteins were upregulated or downregulated by SA treatment. Among them, GDSL lipase 1 (GLIP1), a secretory lipase with a GDSL motif, was selected for further characterization. As a result, the present invention was completed by revealing that GLIP1 plays an important role in plant resistance to molds such as Alternaria brassicicola .

Therefore, the main object of the present invention is to provide a novel fungal resistant GLIP1 gene and its protein derived from Arabidopsis.

Another object of the present invention relates to a fungal resistant composition of plants and a method of providing resistance using the GLIP1 gene and its protein.

According to one aspect of the invention, the present invention provides a GLIP1 gene comprising a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 having a fungal resistance of plants.

In the present invention, the GLIP1 gene includes any gDNA or cDNA encoding the amino acid sequence, but preferably has a nucleotide sequence of SEQ ID NO: 1.

According to another aspect of the invention, the invention provides a GLIP1 protein comprising the amino acid sequence of SEQ ID NO: 2 and having fungal resistance of plants.

According to another aspect of the present invention, the present invention provides a fungal resistant composition of a plant containing the GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2 or an expression protein thereof as an active ingredient.

In the present invention, preferably, the GLIP1 gene is inserted into a plant transformation vector.

In the present invention, preferably, the GLIP1 protein is a recombinant protein expressed in E. coli.

In the present invention, preferably, the fungal resistance of the plant is achieved by direct destruction of the fungal spores and by signaling of ethylene related cystemic resistance.

According to another aspect of the present invention, the present invention provides a method for imparting fungal resistance to a plant comprising expressing in a plant a GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2.

In the present invention, preferably, the plant is transformed into a vector containing the GLIP1 gene, but is not limited thereto. Expression is expressed by up-regulating transcription of the existing GLIP1 gene of the plant. It includes increasing.

According to another aspect of the present invention, the present invention provides a method for imparting fungal resistance to a plant comprising treating the plant with a GLIP1 protein having the amino acid sequence of SEQ ID NO: 2.

According to another aspect of the invention, the invention provides a transformation vector of a plant comprising the GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2.

In the present invention, the transforming vector of the plant may be a transient expression vector capable of temporarily expressing the GLIP1 gene of the present invention in the introduced plant, but preferably the GLIP1 gene of the present invention. It is preferable to be a binary vector that can be permanently expressed in the introduced plant.

The binary vector that can be used in the present invention can be any binary vector containing BR and BL of T-DNA capable of transforming plants when present with Ti plasmid of A. tumefaciens, eg, pGA series, Although pCG series, pCIT series, pGPTV series, pBECK2000 series, BiBAC series, and pGreen series vectors can be used, it is preferable to use the pBI series (Clontech, USA) which is readily available in the art.

The transient expression vector that can be used in the present invention can be any transient expression vector designed to be transiently expressed in a plant into which a foreign gene has been introduced. For example, pBI221 (Mitsuhara et al., 1996), pMG221 (Maas et al., 1991), pUbiGUS (Christensen and Quail, 1996), and ACT1-D (McElroy et al., 1990) and the like may be used, but preferably pBI221 (Clonetech) readily available in the art. , United States) is good to use.

When the transformed vector of the plant is a binary vector, the plant is transformed according to the Floral dip method (Clough and Bent, 1998, The Plant Journa), and in the case of a transient expression vector, the particle bombardment method (Lacorte et al., Plants can be transformed according to Plant Cell Reports, 1997. The plant transformation vector of the present invention can transform any plant regardless of dicotyledonous or monocotyledonous, sexual or asexual reproduction plants.

In the present invention, preferably, the plant transformation vector, characterized in that pBI221-GLIP1 having a cleavage map of FIG. The parent vector of the vector is the E. coli phagemid vector pBI221, which has a CaMV 35S promoter, which is an expression promoter of a general plant.

According to another aspect of the present invention, the present invention provides a transformation vector of Escherichia coli comprising the GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2.

In the present invention, the E. coli transformation vector includes any conventional transformation vector such as pGEX family vector (Amersham Biosciences), which can be expressed in E. coli, for example, GST gene fusion system. PGEX-GLIP1 having a cleavage map of? The vector has a tac promoter in which expression is induced by IPTG and a GST tag for purification.

In the present invention, secretory proteins involved in various aspects of cell biology were identified by analyzing secretomes of the Arabidopsis using a proteomic method. Next, by examining the change in Arabidopsis screetom for salicylic acid, several proteins involved in the pathogen response were identified. One of them, GDSL lipase 1 (GLIP1), further characterized its function on disease resistance against secretory lipases with GDSL-like motifs. glip1 (mutant) plants were significantly more susceptible to infection by necrotrophic fungus Alternaria brassicicola than parent wild type plants. Recombinant GLIP1 protein has lipase activity as well as antimicrobial activity that directly destroys the fungal spore itself. Furthermore, pretreatment of the glip1 mutant with recombinant GLIP1 protein inhibits A. brassicicola -induced cell death in peripheral and terminal leaves, so that GLIP1 is a systemic resistance signaling plant when infected with A. brassicicola . Started. In addition, the glip1 mutant showed altered expression of defense- and ethylene-related genes. GLIP1 transcription was increased by ethylene releasing agent ethephon, but not by salicylic acid or jasmonic acid. These results suggest that GLIP1 may be an important component in the resistance of plants to A. brassicicola by participating in ethylene signaling. Thus, the GLIP1 of the present invention was found to play an important role in plant resistance to fungi such as Alternaria brassicicola . This disease resistance is regulated by two different mechanisms, which not only directly destroy fungal spores, but also activate cysteine-resistant signaling associated with ethylene.

Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1: Plant Materials, Growth Conditions, and Treatment

Arabidopsis plants were grown in a greenhouse at 23 ° C. under long-term conditions (16 hours light treatment / 8 hours dark treatment cycle). The plants were also grown under single conditions (8-hour light treatment / 16-hour dark treatment cycle) for the inoculation of Pseudomonas syringae , because Arabidopsis is age-related to Pseudomonas syringae . The Arabidopsis Columbia-0 (list number WT-02) ecotype was purchased from Lehle Seeds. Arabidopsis Columbia-6 (list No. CS8155) ecotype and T-DNA inserted mutants glip1-1 (SALK_037272) and glip1-2 (SALK_130146) were obtained from Arabidopsis Biological Resource Center (ABRC).

Callus was generated from the cotyledons of young plants and induced in induction medium (MS medium, pH 5.8, 3% sucrose, 0.8% agar, 2 mg / L 2,4-D). Cell suspension cultures were prepared using 1-2 g of callus tissue in May and Leaver (1993) Plant Physiol. Start was inoculated in 50 ml of MS medium containing 3% sucrose and 2 mg / l 2,4-D as described in 103, 621-627. Suspension cells (10 ml) were incubated in 50 ml of fresh medium each week and gently stirred.

As hormonal treatment, salicylic acid (1 μM), methyl jasmonate (50 μM) and ethephon (1.5 mM) were dissolved in water and sprayed on 4 week old plants. Treated plants were stored under 100% relative humidity and harvested at labeled time.

Inoculation of Pseudomonas syringae was performed as described below (Alvarez et al., (1998) Cell 92, 773-784). Pst DC3000 (avrRpt2) and Pst DC3000 were grown overnight in King's B medium with or without 100 μg / ml rifampicin and 50 μg / ml kanamycin respectively, washed twice and then mixed again with 10 mM MgCl 2. Four-week-old plants absorbed 20 μl of 10 mM MgCl 2 or Pst DC3000 (avrRpt2) (106 colony-forming units / ml) in two of the two first leaves, followed by 20 μl of Pst DC3000 (avrRpt2) (106 colony) in the second leaves. -forming units / ml) were absorbed the next day.

Inoculation of Alternaria brassicicola was performed by dropping 10 μg of spore suspension drops onto the leaves of each of the 4 week plants. By pretreatment of GLIP1 protein, the plants absorbed 10 μl of PBS (control) or GLIP1 protein (1 μg) in one of the axial sides of each leaf and incubated for 24 hours prior to pathogen inoculation. Inoculated plants were stored under 100% relative humidity of the growing room for the labeled time. To count the spores, 10 leaves were collected and shaken vigorously in 5 ml of 0.1% tween20 in the test tube. The leaves were removed and the suspension containing spores was centrifuged at 5000 g for 15 minutes. Spores were mixed again in 200 μl of 0.1% tween20, diluted in sequence, and numbered under a microscope.

Example 2: Preparation of Secretory Proteins

After subculture, the suspension culture sample was treated with 0.5 mM salicylic acid for 4 days, and the untreated sample was passed through a 0.2 μm membrane filter under vacuum at the same time to remove cells from the liquid medium of the sample. The collected medium (secretion sorting step) is dialyzed with 10 mM MES, pH 5.8 buffer and lyophilized.

Example 3 2D Electrophoresis

Electrophoresis of 2D is described by Lee et al. 2004 Arabidopsis. Plant Cell 16, 1378-1391. Secretory protein isoelectric focusing sample buffer (7 M urea, 2 M thiurea, 4% 3-[(3-cholamidopropyl) -dimethyl ammonio]-1-propane sulfate, 1% carrier ampholytes, 20 mM DTT, 0.001% bromophenol blue ) And hydrate with a strip (240 mm, pH 4 to 7; Amersham Bioscience) to dissolve the protein at pH. Isoelectric focusing is performed with an IPGphor system at 320,000 to 350,000 voltage hr. 12% SDS polyacrylamide gel was used to electrophores the gel in the second direction. After electrophoresis, the gel was stained with silver nitrate according to the manufacturer's instructions (Amersham Bioscience), and analyzed by ImageMaster 2D Elite software (Amersham Bioscience) by scanning the gel to make a protein map. Each condition was analyzed with three to five gels from three different protein extracts. The amount of silver staining was normalized according to internal reference amount. Salicylic acid-induced changes were analyzed statistically by student's t test, and each protein was identified by MALDI-TOF MS with P <0.05.

Example 4 Data Search with MALDI-TOF MS

Peptide samples were prepared by the method described in Lee et al., (2004) Plant Cell 16, 1378-1391. Peptide mass was measured with a MALDI-TOF MS system (Voyager-DE STR; Applied Biosystems) and the fingerprint data was matched in a National Center for Biotechnology Information non-redundant database using the MS-Fit program. The assay was set to 50 ppm mass tolerance and one incomplete cleavage. The acetylation of N-terminus, alkylation of Cys by carbamidomethylation, oxidation of Met, and modification of N-terminus Gln to pyroGlu were all possible modifications. The molecular weight and pi were set at 10 to 200 kD and 4 to 7, respectively. It was confirmed that the protein was confirmed to be identical when the five peptides matched, more than 15% sequence match, and molecular weight search number 103 or more.

Example 5: Protein Analysis

For immunoblotting, proteins were isolated on 12% SDS polyacrylamide gel and electrophoresed on nitrocellulose membrane. The membranes are anti-GST, anti-AnnAt1 (Lee et al., 2004), anti-OEE2 (Yang et al., 2003), anti-cyclin D (Cho et al., 2004), and anti-myrosinase (Husebye et al. , 2002). Anti-myrosinase antibodies are described by Dr. Provided by Atle Bones, other antibodies were prepared at the Kumho Life Science Institute. Antibody-binding proteins were detected by an enhanced chemiluminescence system (Amersham Biosciences) using horseradish peroxidase linked to secondary antibodies.

Example 6: DNA and RNA Analysis

Genomic DNA was digested, isolated and blotted on Hybond N + nylon membranes. For DNA analysis, T-DNA-specific probes containing partial sequences of the left border of T-DNA were made by PCR using 5'-GCTGCCTGTATCGAGTGGTGATTTTGT-3 'and 5'-TCCTTTCGCTTTCTTCCCTTCCTTTCTC-3' primers. . Hybridization was performed at 60 ° C for 16 to 24 hours. Membranes were washed with 1 x SSC with 0.1% SDS (0.15 M NaCl, 0.015 M sodium citrate), then with 0.5 x SSC with 0.1% SDS, and finally with 0.1 x SSC with 0.1% SDS. The band was visualized by radiographic photography.

RT-PCR was performed with 0.1 μg total RNA using the Access RT-PCR system (Promega). Primers used in RT-PCR are shown in Table 1 below.

TABLE 1

gene Forward primer Reverse primer GLIP1 5'-CGATTGTGCACCAGCCTCATTGGTT-3 ' 5'-CAGCGCTTTGAGATTATAGGGTCC-3 ' PR-1 5'-TCGTCTTTGTAGCTCTTGTAGGTG-3 ' 5'-TAGATTCTCGTAATCTCAGCTCT-3 PR-2 5'-CGTTGTGGCTCTTTACAAACAACAAAAC-3 ' 5'-GAAATTAACTTCATACTTAGACTGTCGAT-3 ' PR-3 5'-CGGTGGTACTCCTCCTGGACCCACCGGC-3 ' 5'-CGGCGGCACGGTCGGCGTCTGAAGGCTG-3 PR-4 5'-GACAACAATGCGGTCGTCAAGG-3 ' 5'-AGCATGTTTCTGGAATCAGGCTGCC-3 ' PR-5 5'-ATGGCAAATATCTCCAGTATTCACA-3 ' 5'-ATGTCGGGGCAAGCCGCGTTGAGG-3 ' PDF1.2 5'-GCTAAGTTTGCTTCCATCATCACCCTT-3 ' 5'-AACATGGGACGTAACAGATACACTTGTG-3 ' EIN2 5'-TGCAGCTCGCATAAGCGTTGTGACTGGTA-3 ' 5'-CGCTCTCTCCATTTAACCGAGTTAACAC-3 ' EIN3 5'-GATGTTGATGAATTGGAGAGGAGGATG-3 ' 5'-ACGTCTCTGAGGAGGATCACAGTGT-3 ' Erf1 5'-CGGCTTCTCACCGGAATATTCTATCG-3 ' 5'-TCTCCGAAAGCGACTCTTGAACTCTCT-3 ' Actin1 5'-GGCGATGAAGCTCAATCCAAACG-3 ' 5'-GGTCACGACCAGCAAGATCAAGACG-3 '

Example 7: Analysis of Endogenous Enzyme Activity

Endogenous ADH and G6PDH activity is described by Ranieri et al., (1996) Physiol. Plant. 97, 381-387; Chung and Ferl, (1999) Plant Physiol. It was measured according to the method described in 121, 429-436. Protein was extracted with extraction buffer (50 mM Tris, pH 8.0, and 15 mM DTT) and centrifuged at 12,000 g for 15 minutes at 4 ° C. The reaction mixture for ADH included 50 mM Tris (pH 9.0), 0.867 mM NAD +, 20% ethanol and separated protein extracts. The reaction for G6PDH was performed with protein extracts separated with 86.3 mM triethanolamine (pH 7.6), 6.7 mM MgCl 2, 0.37 M NADP +, 12 mM glucose-6-phosphate. The increase in absorbance was monitored every 15 seconds for 60 seconds at 340 nm. Activity is expressed as μmole with mg protein reduced to NAD + or NADP + per minute.

Example 8: Location of GLIP1 in Cells

GLIP1, GLIP1ΔSP (GLIP1 lacking a signal peptide) and SP (signal peptide alone) were generated by PCR using the following primers.

GLIP1 is 5'-GATCTCTAGAATGGAAAACTCTCAATTAG-3 '

5'-GATCCTCGAGATTAAGTTCAAACAGCGC-3 '

GLIP1ΔSP and 5'-GATCTCTAGAATGGACAACAATAATCTTGTA-3 '

5'-GATCCTCGAGATTAAGTTCAAACAGCGC-3 '

SP is 5'-GATCTCTAGAATGGAAAACTCTCAATTAG-3 '

5'-CCGCTCGAGGACACTAGAGAGAGTATC-3 '

The amplified DNA was cloned into the XbaI and XhoI sites of the pBI221 vector (Clontech) having the 35S promoter of CaMV to fuse smGFP to the distal end of the DNA product to prepare pBI221-GLIP1 (FIG. 1). The result is Shieh et al. (1993) Plant Physiol. 101, 353-361 was introduced into onions via a helium bioparticle transmitter. After incubation at 12 ° C. for 48 hours at 23 ° C., intracellular distribution of GFP fusion protein was confirmed by fluorescence microscopy (Olympus BX-51TRF).

Example 9 Purification and Lipase Activity Analysis of Recombinant GLIP1

In Example 6, the GLIP1 cDNA amplified by RT-PCR was cloned into BamHI and XhoI of a pGEX 6P-1 vector (Amersham Biosciences) having a tac promoter and GST tag in which expression was induced by IPTG and recombinant vector pGEX-GLIP1 (Fig. 2) was prepared. The GLIP1 (S44A) mutant was generated by mutant PCR and the primers were as follows.

5'-TCGTGTTTGGAGATgCTGTGTTCGATGCTGGA-3 ',

5'-TCCAGCATCGAACACAGcATCTCCAAACACGA-3 '.

These products were introduced into E. coli cells and cultured, and the recombinant protein was induced by the addition of 0.1 mM isopropyl ß-D-thiogalactopyranoside. This GST-GLIP1 fusion protein was purified, dialyzed with PBS and stored at 70 ° C. Lipase activity was reacted by adding 2 to 4 μg of recombinant protein in 0.5 M HEPES, pH 6.5, 1 mM substrate (p-nitrophenyl acetate or p-nitrophenyl butyrate) for enzymatic reaction. After 1 hour of reaction at 30 ° C., the absorbance was measured at 405 nm every 5 minutes for 1 hour.

Example 10 Microscopic Analysis

After dropping the fungus on the leaves 4 days old leaves were dipped in lactophenol-aniline blue. Lactophenol was first mixed with phenol, acetic acid, sterile water, and glycerol in a ratio of 1: 1: 1: 2, and then aniline blue (0.1%) was mixed with this lactophenol in a 1: 3 ratio. Apoptosis was detected by lactophenol and trypan blue staining, and bleaching was done by chloral hydrate. The stained samples were fixed under 60% glycerol and observed under a microscope.

The antimicrobial activity of GLIP1 was determined by treating 10 μl spore suspension (5 × 10 5 spores / ml) with 0.1 to 5 μg recombinant protein. The sample was placed on a glass slide and reacted at 23 ° C. for 24 hours, followed by optical microscopy. Mold samples were fixed in a fixed solution (0.1 M sodium phosphate, pH 7.0, 1% sucrose, 3% paraformaldahyde, 0.2% glutaraldehyde) under vacuum for 6 hours, followed by dehydration and drying, followed by scanning electron microscopy (S-2500). scanning electron microscope, Hitachi).

Experimental Result 1: Isolation of Secretory Protein from Arabidopsis Cell Culture

Cell cultures were established from Arabidopsis ecological Columbia (Col-0) to isolate proteins secreted into the medium. The culture medium was harvested on day 4 when cells actively reproduced in the mid-log phase. The secreted proteins were isolated from cell wall protein myrosinase myrosinase (Husebye et al., 2002), cytoplasmic protein glutathione S-transferase (GST) and cyclin D, cytoplasmic and membrane protein annexin1 (AnnAt1), and chloroplast protein oxygen-evolving enhancer protein2 (OEE2). Antibodies were tested for contamination by protein gel blot analysis using antibodies specific to). Extracellular protein myrosinase was found mostly in the secreted fractions, while others were exclusively detected in cultured cells (ie, non-secreted fractions). To confirm contamination exclusion of the culture medium, we measured the cytoplasmic marker enzyme activity, ie, alcohol dehydrogenase (ADH) and glucose-6-phosphate dehydrogenase (G6PDH), in the isolated medium and callus fraction. The enzyme activity was high in the cells but not detected in the medium. These data indicate that the secretome prepared in the culture medium was not essentially contaminated with nonsecretory proteins.

Experimental Result 2: Identification of Salicylic Acid (SA) -Responsive Secretion Proteins

To identify secretory proteins involved in pathogen response, Arabidopsis cryptome responds to SA (Durner et al., (1997) Trends Plant Sci. 2, 266-274), a plant hormone involved in defense signaling. The change of was analyzed. Arabidopsis cell cultures were either untreated or treated with 0.5 mM SA for 2 hours, then the culture medium was collected and analyzed by comparative 2D gel electrophoresis. Protein spots were detected on silver-stained 2D gels. Protein spots with varying intensity in SA-treated media compared to untreated control media were quantitatively analyzed. Three independent experiments were performed and only spots with more than two times the change were reproduced. MALDI-TOF MS analysis was performed with 18 protein spots selected to identify proteins (Table 2).

Table 2 Identification of SA-responsive Proteins Secreted in Arabidopsis Cell Culture

The identified proteins corresponded to 13 different proteins, some with multiple spots, suggesting that they undergo posttranslational modifications. Of the 13 proteins, three had a protein name or known function, but ten appeared new or unknown proteins in Arabidopsis genome databases. These unknown proteins were then scanned in Prosite (http://ca.expasy.org/tools/#pattern), a protein family and domain database, to assign additional functional motifs (ie, the jacalin-associated protein, Leu-Rich). Repeat, and active site of esterase / lipase / thioesterase; Table 2).

Experimental Result 3: Sequence Comparison of GLIP1 with Other GLIPs

Further analysis was performed by selecting the GDSL motif lipase / hydrolase-like protein (spot 119) named in Table 2, since no plant-derived secretory lipase has been reported to be involved in the pathogen response yet. In the BLAST search of Arabidopsis genome database, six additional GLIP genes were identified and named GLIP2 through GLIP7 (FIG. 3). 3 shows the alignment of amino acid sequences of GLIP1 with other GLIPs. The sequence was aligned using ClustalW. The signal sequence is shown in bold at the N terminus, and residues of the catalytic triad (Ser, Asp and His) are shown in arrows and bold. Lipase signature sequences are indicated by solid lines and GDSL-like motifs are indicated by boxes. The common symbols in the sequence are as follows: Asterisks are identical residues in all sequences, and colon and period indicate conserved and semiconserved substitutions, respectively. Comparison of amino acid sequences showed that GLIP1 was 68-78% identical to GLIP2, GLIP3, and GLIP4, while having low homology with other GLIPs (32-52% identical with GLIP5, GLIP6, and GLIP7). The GLIPs include GDSL-like motif (Brick et al., (1995) FEBS Lett. 377, 475-480) and signal peptide as predicted by Signal P (Nielsen et al., (1997) Protein Eng. 10, 1-6). A novel lipase gene family with All seven GLIPs have catalytic triads of Ser, Asp and His preserved in lipase and esterase families. Another feature of the GLIPs is that the traditional lipase signature sequence GxSxG is replaced by GxSxxxxG and it overlaps with the GDSL motif (Brick et al., 1995).

Experimental Result 4: Intracellular Location of GLIP1

To investigate the intracellular location of GLIP1, soluble modified green fluorescent protein (smGFP) was fused to GLIP1 expressed under the control of the cauliflower mosaic virus (CaMV) 35S promoter. The smGFP fusion construct of full length GLIP1, GLIP1 lacking signal peptide (GLIP1DSP) and signal peptide alone (SP) was introduced into epithelial cells of onion (Allium cepa). After incubation for 24 hours, fluorescence from smGFP was examined by fluorescence microscopy (FIG. 4). Figure 4 is a photograph showing the intracellular location of the GLIP1 protein. GFP fusion constructs were introduced into onion epithelial cells by particle transfer. Expression of the fusion protein was induced with the CaMV 35 promoter. The bright field (left) and the fluorescence image (right) are shown. The schematic structure of the fusion structure is shown at the bottom of each panel. (A) expression of GFP control, (B) expression of full-length GLIP1-GFP fusion protein, (C) expression of GLIP1ΔSP-GFP fusion protein, (D) expression of SP-GFP fusion protein, (E) in the presence of 1M mannitol Expression of SP-GFP (Plasmolysis Induced). smGFP control and GLIP1DSP were expressed in the cytoplasm. However, GLIP1 and SP fluorescence signals were clearly located in the intercellular space, indicating that signal sequences lead to the secretion of GLIP1. Whereas the fluorescence of GLIP1 was localized in the cell gap, SP showed a diffusion pattern. The intercellular location was evident by the presence of mannitol, which induces plasmolysis that distinguishes each other by dropping the cytoplasm from the cell wall (FIG. 4E). These results are consistent with proteomic data showing that GLIP1 is secreted into the cell wall or extracellular space.

Experimental Result 5: Isolation of GLIP1 T-DNA Insertion Mutant

In order to analyze the in vivo function of GLIP1, the Salk Institute insertion sequence database was searched for T-DNA insertion mutations so that the T-DNA inserts were found in the two alleles, glip1-1 and glip1- , in the fifth exon and the second intron, respectively. 2 was obtained (FIG. 5A). Mutant seeds were originally heterozygous for single T-DNA insertion at a 3: 1 isolation ratio. Homozygous lines were separated in the next generation. To determine the exact location of T-DNA insertion, genomic DNA fragments of the GLIP1 gene and the T-DNA junction were amplified and sequenced (FIG. 5A). DNA gel spot analysis determined that these lines contained a single T-DNA insert (FIG. 5B). It was also demonstrated that homozygous mutants do not express GLIP1 transcripts (FIG. 5C). Under normal growth conditions, glip1 plants showed similar growth as Col-0 (data not shown). 5 is a picture of T-DNA insertion mutants of GLIP1. (A) The genomic structure of GLIP1 and the location of T-DNA insertion in glip1 mutants. The arrow points to the location (triangle) of the T-DNA insertion. Genomic GLIP1 DNA is represented by exons (black) and introns (white). The T-DNA order is indicated by the left boundary (LB) and the right boundary (RB). Numbers refer to nucleotides in genomic GLIP1 DNA. (B) DNA gel blot analysis of T-DNA insertion in Col-0 and glip1 plants. Genomic DNA was digested with EcoRI (E) and HindIII (H) and blots were probed with T-DNA left border-specific DNA. (C) an RNA analysis of pictures Col-0 and GLIP1 glip1 gene expression in plants. GLIP1 expression was analyzed by RT-PCR. Total RNA (0.1 μg) was used to detect GLIP1 and Actin1 (loading control) expression.

Experimental Results 6: GLIP1 Function in Disease Resistance

Since GLIP1 was isolated from SA-responsive secretion proteins, it was first examined for infection by the glip1 mutant, Pseudomonas syringae (Crute et al., (1994) Arabidopsis, pp 705-747), a bacterial pathogen closely related to SA signaling. It was analyzed whether it showed a changed response. Bacterial growth and disease development after inoculation with the toxic Pseudomonas syringae pv tomato DC3000 (Pst DC3000) were not significantly different between Col-0 and glip1 plants (FIG. 6D). Treatment of primary leaves with toxic avrRpt2 with Pst DC3000 [Pst DC3000 (avrRpt2)] prior to inoculation of secondary leaves with Pst DC3000 showed that the development of cystemic acquisition resistance (SAR) in both Col-0 and glip1 plants was improved. Was observed (FIG. 6D). These results suggest that GLIP1 is not related to resistance to P. syringae in Arabidopsis.

We also examined the resistant phenotypes of glip1-1, glip1-2, Col-0, and another Colombian ecotype, Col-6 after inoculation with necrotrophic fungus A. brassicicola . In the infection cycle, the germ tube on the leaf surface expands after spore germination. At the apex of the hyphae, the fungus develops appressoria and penetrates directly into the host, eventually colonizing tissue (McRoberts and Lennard, 1996). Col-0 and Col-6 were used as A. brassicicola (Kagan and Hammerschmidt, (2002) J. Chem. Ecol. 28, 2121-2140), respectively, as wild-type ecotypes that are resistant and sensitive. Inoculation of Col-0 wild-type plants with A. brassicola has been shown to induce hypersensitivity responses resulting in small necrotic lesions localized at the site of infection (Thomma et al., (1999) Plant J. 19, 163-171). We also observed that Col-0 inoculated with A. brassicicola forms small brown necrotic lesions (FIG. 6A). In contrast, glip1-1 , glip1-2 , and Col-6 plants developed widespread lesions upon inoculation (FIGS. 6A and 6C). When visualized by lactophenol-aniline blue staining, the unfolded lesions were severely colonized with fungal hyphae (FIG. 6B). Moreover, when determining the number of newly generated spores at the site of inoculation, it was observed that new spores were abundantly produced in glip1 and Col-6 leaves, unlike Col-0, where little new spores were formed (FIG. 6C). No difference was observed between glip1-1 and glip1-2 in disease resistance, indicating that the observed phenotype is the result of the insertion of T-DNA into the GLIP1 gene.

6 shows disease development of glip1 mutants inoculated with P. syringae and A. brassicicola . Four-week-old plants were inoculated with 20 mL of P. syringae bacterial suspension (10 ⁶ colony forming units [cfu] / mL) for the first and second inoculations (D) or 10 mL of fungal spore suspension (5 X). 10 ⁵ spores / mL) ([A] to [C]). Inoculated leaves were removed after 4 days and analyzed. The experiment was repeated three times, yielding similar results. (A) Necrotic lesion phenotype. (B) Necrotic lesions stained with lactophenol-aniline blue. The formation of fungalmycelium on glip1 and Col-6 leaves was visualized microscopically. (C) Measurement of lesion diameter and number of newly formed spores. Bars and error bars represent the mean and SD of three independent experiments of 10 leaves, respectively. (D) Bacterial growth determined on secondary leaves infected with Pst DC3000 1 day after seeding of primary leaves with 10 mM MgCl 2 or Pst DC3000 (avrRpt2). Primary and secondary inoculations with M + V, MgCl 2 and Pst DC3000, respectively; Primary and secondary inoculations with A + V, Pst DC3000 (avrRpt2) and Pst DC3000, respectively. Bars and error bars represent the mean and SD of three independent experiments, respectively.

Experimental result 7: antimicrobial activity of recombinant GLIP1 protein

To determine if GLIP1 has the lipase activity required for resistance to A. brassicicola , recombinant GLIP1 protein was expressed in Escherichia coli (FIG. 7A). Along with the wild-type protein, a (S44A) GLIP1 mutant was prepared in which Ser-44 of the catalytic triad was substituted with Ala. For these proteins, lipase activity was measured using p-nitrophenyl acetate and p-nitrophenyl butyrate as substrates (FIG. 7B). Wild type GLIP1 showed high activity, but GLIP1 (S44A) mutant did not, indicating that GLIP1 is a lipase and Ser-44 of the catalytic triad participates in hydrolytic activity. 7 depicts lipase activity of recombinant GLIP1 protein. (A) Coomassie blue-stained gel photograph of purified recombinant GST-GLIP1 protein. (B) Lipase activity of recombinant GLIP1 and GLIP1 (S44A) proteins and GST control. In the measurements, 2 mg (open symbol) and 4 mg (closed symbol) proteins were incubated with two substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate. Each data point represents the mean and SD of five independent measurements.

Since GLIP1 is a secreted protein, we hypothesized that GLIP1 could directly inhibit the pathogenic activity of A. brassicicola . Therefore, the antimicrobial activity of GLIP1 against A. brassicicola was measured. When fungal spores were incubated with recombinant wild type and mutant proteins, spore germination was inhibited by the active form of GLIP1 (FIGS. 8A and 8B). GLIP1 (S44A) had little effect on mold growth. Furthermore, scanning electron microscopy showed that GLIP1 caused severe morphological changes of fungal spores (FIG. 8C). These results suggest that GLIP1 directly destroys the structure of the fungal cell wall and / or cell membrane in a specific way. Figure 8 depicts the antimicrobial activity of recombinant GLIP1 protein against A. brassicicola . Recombinant protein was added to 10 mL of spore suspension (5 × 10 ⁵ spores / mL). Samples were incubated at 23 ° C. for 24 h and observed under a microscope. PBS was used as a mock control. (A) The inhibitory effect of recombinant protein (5 mg) on the germination of fungal spores was observed by light microscopy. (B) The germination rate was quantitatively analyzed. Optical microscopy determined the percentage of germinated spores shown in (A). Bars and error bars represent the mean and SD of three independent experiments, respectively. (C) Scanning electron microscopy images of fungal spores incubated with GLIP1 protein (5 mg). In each condition, five spores were observed with similar results. PBS was used as a mock control.

Experimental Result 8: GLIP1 Function in Systemic Defense Response

When inoculated with A. brassicicola , Col-0 activates SAR responses leading to the induction of defense-related genes such as PDF1.2 , PR-3 and PR-4 (Penninckx et al., (1996) Plant Cell 8 , 2309-2323; Thomma et al., (1998) PNAS USA 95, 15107-15111). Therefore, we investigated whether GLIP1 is involved in local and cystemic defense responses induced in plants by pathogen inoculation. When the leaves of A. brassicicola -inoculated plants were stained with trypan blue, extensive cell death associated with cystemic diffusion of this fungus was observed in the peripheral and terminal leaves of glip1-1 but not in Col-0 (FIG. 9A). ). In SAR-induced Col-0, cell death was localized at the site of inoculation. Thus, expression of SAR can be determined by monitoring the presence of trypan blue-stained cells. glip1-1 plants were pretreated with recombinant GLIP1 protein and then inoculated with topical A. brassicicola (1 ° p) (FIG. 9B). After topical inoculation (1 ° p) on the pretreated leaves, the other terminal leaves were inoculated with A. brassicicola (1 °). Apoptosis of both topical (1 ° p) and cystemic (1 ° and 2 °) was severely reduced, indicating that SAR was induced even in leaves far from infection (FIG. 9B). It is noteworthy that pretreatment of glip1-1 with GLIP1 protein in topical leaves (1 ° p) cystically inhibits apoptosis in distant leaves (1 °) inoculated with A. brassicicola which would otherwise strongly induce apoptosis. . In contrast, pretreatment (p) of GLIP1 without topical A. brassicicola inoculation did not inhibit apoptosis of both 1 ° and 2 ° leaves (FIG. 9B, right). Here, primary (1 °) and secondary (2 °) refer to inoculated and uninoculated leaves, respectively. These data suggest that GLIP1 activates cystemic resistance signaling only when infected with A. brassicicola .

Signaling molecules such as SA, jasmonic acid (JA) and ethylene accumulate accumulate during defense to initiate coordinated expression of protective genes. A. Resistance to brassicicola involves the coordination of JA and ethylene (Dong, (1998) Curr. Opin. Plant Biol. 1, 316-323). PDF1.2, PR-3 and PR-4 require JA and ethylene for activation, while PR-1, PR-2 and PR-5 are induced by SA (Kunkel and Brooks, (2002) Curr Opin, Plant Biol. 5, 325-331). In addition, many genes (eg, ERF1, EIN2 and EIN3) have been shown to function in ethylene signaling (Wang et al., (2002) Plant Cell 14 (suppl.), S131-S151). Therefore, we analyzed the expression levels of these genes, including GLIP1 in Col-0 and glip1-1 upon A. brassicicola treatment. GLIP1 transcription was strongly induced at Col-0 (Figure 9C). On the other hand, PR gene expression was not different in glip1-1 and PDF1.2 was less induced. Moreover, the basal RNA levels of the SA dependent PR genes, PR-1, PR-2 and PR-5 were much higher in glip1-1 than Col-0. Among these ethylene-related genes, EIN2 and EIN3 also showed altered expression in glip1-1 . EIN2 expression was inhibited in the Col-0, the glip1-1 A. was slightly induced in response to brassicicola. Increasing EIN3 in Col-0 was reversed by decreased expression in glip1-1 . These results suggest that glip1 is defective in ethylene signaling. In addition, the high basal expression levels of SA related PR genes in glip1-1 are consistent with previous reports that the SA and ethylene pathways interact in an antagonistic manner (Wang et al., 2002). Although A. brassicicola infection induces PR-1, it has been shown that resistance to this pathogen is dependent on JA / ethylene signaling pathways (Penninckx et al., (1996) Plant Cell 8, 2309-2323). Surprisingly, GLIP1 was strongly induced by ethephon over 24 hours (FIG. 9D). However, only a slight increase in GLIP1 expression was observed upon treatment with SA and JA. These results demonstrate that the increase in GLIP1 protein in the secretome is regulated posttranslationally (ie by secretion) and / or transcriptionally. These results also suggest that GLIP1 is an important component in the resistance associated with ethylene signaling.

9 shows the function of GLIP1 in the cystemic defense response. Four week plants were inoculated with 10-mL drops of A. brassicicola spore suspension (5 × 10 ⁵ spores / mL). In (A) and (B), primary (1 °) and secondary (2 °) refer to inoculated and uninoculated leaves, respectively. The term 1 ° p refers to leaves inoculated with A. brassicicola pretreated with PBS or GLIP1 protein. A large magnified (X400) bottom photo shows cell death at the site of inoculation of primary leaves (1 ° p and 1 °). These experiments ([A] to [D]) showed the result of repeating three times. (A) Cell death shown by lactophenol-trypan blue staining. The primary and secondary leaves were stained 4 days after the primary leaves were treated with A. brassicicola . (B) Inhibition of A. brassicicola -induced local and cystemic cell death in glip-1 mutants by pretreatment with GLIP1 protein. For pretreatment, PBS (control) or GLIP1 protein (1 μg) was absorbed and incubated for 1 day in one of the hypocotyls of each leaf of the plant and then inoculated with A. brassicicola . One day after topical inoculation (1 ° p) of the pretreated leaves, the other terminal leaves were inoculated with A. brassicicola . As a control (right), only GLIP1 protein was pretreated without topical inoculation of A. brassicicola (p). Cell death was monitored by trypan blue staining over 4 days. (C) Induction of pathogenesis- and ethylene-related genes in Col-0 and glip1-1 plants infected with A. brassicicola . Four-week-old plants were inoculated with 10-mL drops of spore suspension (5 × 10 ⁵ spores / mL) and total RNA was extracted using harvested one day after treatment. RNA levels were determined by RT-PCR analysis. Actin1 was used as a control. (D) RNA analysis of GLIP1 induction in response to hormonal treatment. SA (1 mM), methyl jasmonate (MJ; 50 mM), and ethylene releasing agent ethephon (ET; 1.5 mM) were dissolved in water and applied to 4 main Col-0 plants. Total RNAs were isolated at the indicated time post-treatment and RNA levels were determined by RT-PCR analysis. Actin1 was used as a control.

As described above, in the present invention, a comprehensive analysis of the secretotom was carried out in the cultured cell medium of Arabidopsis. In addition, in order to identify secreted proteins that participate in plant defense, protein accumulation was increased or decreased in secretos in response to SA treatment. Among them, the function of defense against the A. brassicicola of secreted lipase GLIP1 was analyzed by GLIP1 in two different ways: by directly destroying the fungal spores themselves and activating the plant's defense signaling . It has been found to protect plants from brassicicola attacks. Therefore, the GLIP1 of the present invention can impart resistance to molds such as Alternaria brassicicola to desired plants by not only directly destroying mold spores but also by activating cystemic resistance signaling associated with ethylene.

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Claims (12)

GLIP1 (GDSL motif lipase 1) gene having fungal resistance of a plant consisting of a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2. The GLIP1 gene according to claim 1, which has a nucleotide sequence of SEQ ID NO: 1. GLIP1 protein consisting of the amino acid sequence of SEQ ID NO: 2 and having fungal resistance of plants. Mold resistance composition of plants containing the GLIP1 gene or its expression protein encoding the amino acid sequence of SEQ ID NO: 2 as an active ingredient. 5. The composition of claim 4 wherein the fungal resistance of the plant is achieved by direct destruction of the fungal spores and by signaling of ethylene related systemic resistance. A method for imparting fungal resistance to a plant comprising expressing in a plant a GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2. The method of claim 6, wherein the plant is transformed with the vector comprising the GLIP1 gene. A method for imparting fungal resistance to a plant comprising treating a plant with a GLIP1 protein having the amino acid sequence of SEQ ID NO: 2. Transformation vector of the plant comprising the GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2. The vector according to claim 9, which is pBI221-GLIP1 having a cleavage map of FIG. E. coli transformation vector comprising a GLIP1 gene encoding the amino acid sequence of SEQ ID NO: 2. The vector according to claim 11, which is pGEX-GLIP1 having a cleavage map of FIG.

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