WO2003016510A1 - New biopesticide using gene from erwinia pyrifoliaewt#3, novel pathogen that affects asian pear trees - Google Patents
New biopesticide using gene from erwinia pyrifoliaewt#3, novel pathogen that affects asian pear trees Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/18—Erwinia
Definitions
- This invention relates to a novel biopesticide using a gene derived from a novel plant pathogen, Erwinia pyrifoliae WT#3 (KCCM 10283), isolated from Chunchon, Kangwon province, Korea Korea. This pathogen is endemic to Korea.
- This novel biopesticide has more effective properties, such as improved resistance to plant disease, promotion of plant growth, and insect repellency as compared to those of HrpN, a hypersensitive response inducing protein, isolated from Erwinia amylovora (ATCC15580 1 ), which does not exist in Korea.
- it can be utilized as a biopesticide effective to prevent plant diseases caused by pathogens and insects and to enhance plant growth as well as a fertilizer.
- biopesticide used to control pests through a direct application of antagonistic microorganism itself to plants but this is not considered very effective in controlling of the pests. Therefore, recent researches have been progressing to control harmful pests by stimulating the self-defensive system of plants using products of antagonistic microorganisms instead of using microorganisms themselves.
- an essential aim of biological control is to decrease or prevent of pests by activating the self-immunological function of a plant via treatment of microorganism-derived materials to plants.
- the plant disease resistance is primarily conducted by the defense system of a plant by via structural barriers such as cutin in epidermal cells, a wax layer and types of porosity.
- chemicals such as saponin or lectin, secreted by the plant can prevent the increase of pathogens population
- More essential plant disease resistance refers to a hypersensitive response (HR), is a rapid, localized necrosis for preventing spread of pathogens that is associated with the active defense of plants against many pathogens to stimulate their self -defensive system using some microorganism-derived materials. [Richberg, M. H., Aviv, D. H. & Dangl, J. L. 1998. Dead cells do tell tales. Curr. Poin. PlantBiol. 1:480-485].
- the first method for plant disease resistance associated with HR induction is that plants mobilize their early alert system to adjacent cells infected with bacterial pathogens so that these adjacent cells can increase the resistance to pathogens. This defense system is called as 'LAR' (local acquired resistance).
- the second method is that through the activation of defense system in non-infected parts of a plant, more potent defense system is activated against the secondary infection. Consequently, the whole plant may exert a more strongly defense system against pathogens.
- This defense system is called as "SAR" (systemic acquired resistance).
- SAR systemic acquired resistance
- the SAR may be sustained for several weeks or more and the plants exhibit some resistance to a variety of other unrelated pathogens [Hunt, M. D., Neuenschwander, U. H., Delaney, T. P., Weymann, K. B., Friedrich, L. B., Lawton, K. A., Steiner, H. Y. and Ryals, J. A. 1996. Recent advances in systemic acquired resistance research a review. Gene 179:89-95].
- ISR induced systemic resistance
- wound response to harmful insects were reported as the types of another plant resistance [Pieterse, C. M., van Wees, S. C., van Pelt, J. A., Knoester, M., Laan, R., Gerrits, H., Weisbeek, P. J. and van Loon, L. C. 1998.
- SYSTEMIN a polypeptide signal for plant defensive genes. Annu. Rev. Cell Dev. Biol. 14:1-17].
- the mechanism of the plant disease resistance is triggered by elicitors that induce the plant defense system [Kessmann, H, Staub, T., Hofmann, C, Maetzke, T., Herzog, J., Ward, E., Uknes, S. and Ryals, J. 1994. Induction of systemic acquired disease resistance in plants by chemicals. Annu. Rev. Phytopathol. 32:439-459].
- the typical elicitors of SAR include a phenolic signaling compound SA (salicylic acid) produced by plants, elicitin and harpin that are isolated from pathogens [Ponchet, M., Panabieres, F., Milat, M. L., Mikes, V., Montillet, J.
- Harpin is a common name for proteins produced from hrp gene island of plant pathogenic bacteria and one of Harpins, called HrpN, is a protein produced from hrpN gene located at hrp gene island of about 40kb of Erwinia amylovora which is not existing in Korea.
- HrpN When the HrpN is inoculated to host plants, such as an apple, it acts as a pathogenic factor. In contrast, when HrpN is given to non-host plants, it is recognized as a foreign compound in plants and HR is elicited.
- HrpN is an acidic, heat-stable (100 °C) protein with a molecular weight of 44 kDa [The molecular weight was measured in a manner such that after electrophoresis on acrylamide gel, the protein was stained with 0.025% Coomassie Blue R-250 and compared with Molecular Weight Standard of Bio-Rad Co. (Catalog* 161-0305, Bio-Rad Laboratories, 2000 Alfred Nobel Drive Hercules, CA 94547, USA)] with glycine-rich but without cysteine [Zhong-Min, W., Laby, R. J., Zumoff, C. H., Bauer, D. W., He, S. Y., Collmer, A. and Beer, S. V. 1992.
- HrpN a non-chemical protein
- Erwinia amylovora a gram-negative bacteria that cause fire blight of rosaceous plants.
- SAR elicitor which controls a variety of a plant diseases, several insects, mites and nematodes and exhibits a plant growth prompting effect by enhancing photosynthesis and nutritional absorption
- HrpN induces disease resistance in Arabidopsis through the systemic acquired resistance pathway mediated by salicylic acid and the NIM1 gene. Plant J. 20:207-215]. Also, HrpN has little toxicity and it does not cause in-any environmental pollution as it is biologically degradable and it is easy to formulate due to heat-resistance even after boiling at 100 °C [Zhong-Min, W., Laby, R. J., Zumoff, C. H, Bauer, D. W., He, S. Y., Collmer, A. and Beer, S. V. 1992. Harpin, elicitor of the hypersensitive response produced by the plant pathogen En ⁇ inia amylovora. Science 257:85-88].
- HrpN derived from En ⁇ inia amylovora was successfully commercialized in 2000 by Eden Bioscience in the trade marker Messenger ® in USA for controlling the diseases of crops, such as cotton, tomato, tobacco, pepper, cucumber, strawberry, and wheat. It has been effectively utilized as fungicide, bactericide, pesticide, and plant growth facilitator [US Pat. Nos. 6,174,717 Bl, 5,849,868, 6,977,060, 5,859,324, and 5,776,889; Korea Examined Pat. Appl. Nos. 1999-022577, 2000-075771, 2000-070495, and 2000-057395].
- the inventor et al. isolated and identified some bacterial pathogens from the infected lesions of a plant showing necrosis in pear growing orchards in Chunchon, Korea and found novel species, Erwinia pyrifoliae WT#3 (KCCM 10283) which is morphologically different from recently reported En ⁇ inia pyrifoliae by German researchers: that is, En ⁇ inia pyrifoliae WT#3 (KCCM 10283) does not have flagella, while En ⁇ inia pyrifoliae, which was reported by a German research team has peritrichous flagella. Also, it was different from the well-known En ⁇ inia amylovora causing fire blight.
- an object of the present invention is to provide a novel species Erwinia pyrifoliae WT#3 (KCCM 10283) and a protein derived from the pathogen as an effective biopesticide, a plant growth activator, a seed-treating agent, an insect repllent, and a fertilizer.
- a further aspect of the present invention is to provide a gene (KCCM 10282) encoding a protein or polypeptide of En ⁇ inia pyrifoliae in a non-infectious form to plants, which induces a hypersensitive reaction to non-hosts or resistance to pathogens in plants when plant cells are in contact with or treated with the gene.
- a further aspect of the present invention is to provide transformants containing a gene (KCCM 10282) encoding a protein or polypeptide in a non-infectious form to plants from Erwinia pyrifoliae WT#3 (KCCM 10283).
- a further aspect of the present invention is to provide a biopesticidal composition containing the protein or polypeptide and a carrier.
- a further aspect of the present invention is to provide the composition which can be applied to plants as a pesticide, a plant growth activator, a seed-treating agent, insect repellent, and a fertilizer.
- a further aspect of the present invention is to provide a method for producing a protein or polypeptide that induces a hypersensitive response or resistance on a mass-scale by isolating and purifying a protein or a polypeptide from a cultures of En ⁇ inia pyrifoliae WT#3 (KCCM 10283) and transformants containing a plant HR-inducing gene of the strain (KCCM 10282) from Erwinia pyrifoliae WT#3 (KCCM 10283).
- the novel En ⁇ inia pyrifoliae WT#3 (KCCM 10283) of the present invention was isolated from the affected stem of a plant showing necrosis of pears orchard in Chunchon (Kangwonzhou, Korea).
- the strain was identified as the genus En ⁇ inia, the same genus of the causal pathogen of fire blight, Erwinia amylovora and necrotic disease, Erwinia pyrifoliae (necrotic disease of Asian pears; Erwinia pyrifoliae reported by a German research team in 1999).
- Erwinia amylovora and Erwinia pyrifoliae are peritrichous flagella, while our pathogen Erwinia pyrifoliae WT#3 (KCCM 10283) is non-flagellated, showing a great morphological difference from En ⁇ inia pyrifoliae which is only found in Korea.
- an isolated gene encoding a protein which induces a hypersensitive response in non-host plants from En ⁇ inia pyrifoliae WT#3 was compared with a hrpN gene encoding a HrpN protein, which was discovered by Cornell University of the US and sold by Eden Bioscience co.
- our novel gene showed less similarity to hrpN gene encoding a HrpN.
- several insertions of nucleotide sequences fragments were found in the gene encoding a protein which induces a hypersensitive response in non-host plants from Erwinia pyrifoliae WT#3 which are not present in hrpN gene from E. amylovora.
- the protein or polypeptide produced by the gene has a different amino acid sequence and molecular weight from those of HrpN peptide.
- a recombinant pKEP3 for higher expression containing the gene derived from the Erwinia pyrifoliae WT#3 was constructed and transformed to Escherichia coli. This transformant was deposited to the Korean Culture Center of Microorganisms on June 11, 2001, and was assigned with Accession No. KCCM 10282.
- the high yield of a protein or a polypeptide to elicit hypersensitive response and disease resistance can be produced by mass cultivation of the E. coli transformant containing the expression vector having more effective properties, such as improved resistance to plant disease, promotion of a plant growth, and repellence of insect in compared to that of HrpN from E. amylovora.
- the protein or polypeptide of this invention can be applied to plants as an insect repellent (e.g., aphid) in a conventional procedure to treat stems and leaves of a plant. Further, the seeds of rice treated with a protein or a polypeptide of this invention showed fast growth during the seeding culture period. Thus, the protein or polypeptide of this invention can be applied to plants as an insect repellent and seed-treating agent in a conventional procedure.
- an insect repellent e.g., aphid
- Fig. 1 shows TEM (Transmitted Electro Microscope) photographs of novel En ⁇ inia pyrifoliae WT#3 (KCCM 10283) according to the present invention, En ⁇ inia pyrifoliae Epl6 T and En ⁇ inia amylovora ATCC 15580 ⁇ .
- Fig. 2 shows the growth curves of Erwinia pyrifoliae WT#3 and Erwinia amylovora ATCC15580 ⁇ according to temperature.
- Fig. 3 shows the growth curves of Erwinia pyrifoliae WT#3 and Erwinia amylovora ATCC15580 according to p ' H.
- Fig. 4 shows the numerical analysis of Erwinia pyrifoliae WT#3 (KCCM 10283) according to the Biolog system.
- Fig. 5 shows a phylogenetic analysis based on 16S rRNA gene of Erwinia pyrifoliae WT#3 (KCCM 10283) of this invention.
- Fig. 6 shows the results of phylogenetic analysis of the region encoding tRNA Ala in ITS region of Erwinia pyrifoliae WT#3 (KCCM 10283) of this invention.
- Fig. 7 shows the results of phylogenetic analysis of the region encoding tRNA Glu in ITS region of En ⁇ inia pyrifoliae WT#3 (KCCM 10283) of this invention.
- Fig. 8 shows a plasmid profile analysis of En ⁇ inia pyrifoliae having five plasmids (WT#3, Epl, Epl6) and Erwinia amylovora having one plasmid (ATCC15580 ⁇ , LMG1877, LMG1946)
- WT#3, Epl, Epl6 T the plasmids
- 4 Erwinia pyrifoliae WT#3
- 5 Erwinia amylovora ATCC15580 T
- 6 Erwinia amylovora LMG1877
- 7 Erwinia amylovora LMG1946.
- Fig. 9 shows a hypersensitive response observed 24 hours after inoculation of the genomic library clone constructed from Erwinia pyrifoliae WT#3 (KCCM 10283) on the leaf of tobacco [B: MES buffer, C: a plant HR-inducing protein (HrpN) from
- Erwinia amylovora ATCC 15580 ⁇ 1: a plant HR-inducing protein from clonel
- 2 a plant HR-inducing protein from clone2
- 3 a plant HR-inducing protein from pCEP33
- 4 a plant HR-inducing protein from clone4
- NC protein from pLAFR3 vector
- Fig. 10 shows a physical map encoding the plant HR-inducing gene from the genomic library clone (pCEP33) from En ⁇ inia pyrifoliae WT#3.
- Fig. 11 shows a comparison of genes between a gene (KCCM 10282) encoding the plant HR-inducing protein from Erwinia pyrifoliae WT#3(KCCM 10283) of the present invention and the HR-inducing gene (hrpN) from Erwinia amylovora
- ATCC15580 1 [A: a plant HR-inducing gene from En ⁇ inia pyrifoliae WT#3, B: a plant
- HR-inducing gene from Erwinia amylovora ATCC15580 ⁇ .
- Fig. 12 shows a plant HR-inducing protein (hereinafter referred to as "Pioneer”) from the gene from Erwinia pyrifoliae WT#3 which is expressed in plasmid vector pKEP3 from the gene from [M: protein size marker, 1: Pioneer with 41.1 kD, 2: HrpN with 39.7 kD, 3: pET15b vector].
- Pr plant HR-inducing protein
- Fig. 13 shows a comparison between the amino acid sequences of Pioneer and a plant HR-inducing protein (HrpN) from Erwinia amylovora ATCC15580 T [A: Pioneer, B : HrpN].
- Fig. 14 shows HR on tobacco leaves treated with the Pioneer from Erwinia pyrifoliae WT#3 and HrpN from En ⁇ inia amylovora ATCC15580 T (a control) at different concentrations.
- Fig. 15 shows the disease symptoms on the surface of immature pear fruit 4 days after inoculation of the Pioneer and a buffer (control).
- the analytical results showed that the physiological tests on the strain (e.g., liquefaction of gelatin, motility in 3% agar, and decomposition of pectate) were close to those of type strain Epl6 T of Erwinia pyrifoliae.
- the biochemical tests for the utilization of carbon source revealed different results, especially trehalose and L-arabinose, suggesting that the physiological and biochemical properties of the isolated strain according to the present invention were different from those of type strain Erwinia pyrifoliae Epl6 T and En ⁇ inia amylovora ATCC 15580 ⁇ .
- the morphological property of the strain WT#3 was observed by TEM (Transmitted Electro Microscope), was different from that of recently reported Erwinia pyrifoliae EP16 T causing necrotic disease in Asian pear trees and Erwinia amylovora ATCC 15580 ⁇ causing fire blight on apples and pears [Fig. 1].
- En ⁇ inia spp. including Erwinia pyrifoliae EP16 T and Erwinia amylovora ATCC 15580 ⁇ were rod shaped with peritrichous flagella whereas the novel strain En ⁇ inia pyrifoliae WT#3 was a slight oval rod shaped "without flagella".
- the temperatures ranging from 12 ° C to 39 ° C were measured at intervals of 3 ° C and doubling time and specific growth rate at different temperatures of Erwinia pyrifoliae WT#3 and En ⁇ inia amylovora ATCC 15580 ⁇ were calculated [Fig. 2].
- En ⁇ inia pyrifoliae WT#3 has higher growth rate (27-30 °C) with short doubling time than that of Erwinia amylovora.
- the optimal temperature was 27 ° C for the strain WT#3.
- Erwinia pyrifoliae WT#3 also showed a better growth compared to Erwinia amylovora ATCC15580 T , suggesting that Erwinia pyrifoliae WT#3 is a cold tolerant because this pathogen is well adapted in the vicinity of Chunchon.
- Chunchon is a relatively cold area in the winter season and has different environmental conditions from that of Erwinia amylovora ATCC15580 T .
- the isolated strain was suspended to the turbidity of 63% in a solution containing 0.4% sodium chloride, 0.03% pluronic F-68, and 0.01% gellan gum and inoculated to the wells containing 96 different carbon and nitrogen sources.
- strain WT#3 was further cultivated in an incubator at 35-37 ° C .
- the violet-turned as utilization of carbon and nitrogen sources were recorded using a reader and their values were numerically divided.
- strains ATCC15580, LMG2068, LMG1877, LMG1946 and ea246 strains (USA), which belong to Erwinia amylovora were showed to be in the same group.
- the strain WT#3 was in the group as Erwinia pyrifoliae (Ep4,
- the nucleotide sequence of its 16S rRNA gene was analyzed considering the fact that it is an essential component to manage a life where its nucleotide sequence is well conserved and can be readily placed for phylogenetic analysis.
- the 16S rRNA gene was amplified by PCR using fDl primer (SEQ. ID. No. 1) and rP2 primer (SEQ. ID. No. 2) and then cloned into pGEM-T vector to analyze its nucleotide sequence.
- Fig. 5 shows a phylogenic tree prepared by a mega program [Kumar, S., Tamura, K. and Nei, M. 1993. MEGA: molecular evolutionary genetics analysis, version 1.0. The Pennsylvania State University, University Park] based on the 16S rRNA nucleotide sequence.
- the strain WT#3 had similarities to En ⁇ inia pyrifoliae Epl6 T and En ⁇ inia amylovora 15580 ⁇ with 98.9% and 97.5% sequence identity, respectively, being more closer to Erwinia pyrifoliae than to En ⁇ inia amylovora.
- Table 2 shows the similarity of 16S rRNA gene of each strain.
- strain WT#3 belongs to the group as En ⁇ inia pyrifolia but was different from that of Erwinia amylovora ATCC 15580 ⁇ , and Enterobacter pyrinus, which was previously reported to infect apples and pears in Korea several years ago. 7) Analysis for 16S-23S ITS (Intergenic Transcribed Spacer) region of strain WT#3
- 16S-23S ITS region was amplified by PCR using R16-1F primer (SEQ. ID. No. 3) and R23-1R primer (SEQ. ID. No. 4), and then cloned into pGEM-T vector to analyze the nucleotide sequence of 16S-23S ITS region.
- 16S-23S ITS region was divided into two groups; Erwinia amylovora had three band patterns with about 1215, 970 and 720 bp in size, whereas domestic pathogen, Erwinia pyrifoliae had two band patterns with 970 and 720 bp in size.
- the band pattern of 970 bp had about 70 bp region of tRNA Ala
- the band pattern of 720bp had a region of tRNA Glu , respectively.
- Fig. 6 shows a phylogenic tree prepared by analyzing the nucleotide sequences of 16S-23S ITS region of both the strain Erwinia pyrifoliae WT#3 and En ⁇ inia amylovora ATCC15580 T .
- Group I Erwinia amylovora (ATCC15580, LMG1877, 10296) (a plasmid > 29 kb)
- Group II Erwinia pyrifoliae (WT#3, Epl, Epl6) (a plasmid > 29 kb, a plasmid 5 kb, and three plasmids 2-4 kb in size)
- necrotic disease pathogens En ⁇ inia pyrifoliae
- Asian pear trees including the strain WT#3 have five plasmids
- fire blight pathogens Erwinia amylovora
- the relatedness in whole genome between the Korea-originated Erwinia pyrifoliae and foreign-originated Erwinia amylovora were was investigated.
- the purely isolated total DNA was dissolved in 100 ⁇ Jt TE buffer to a concentration lng/ tl, added with ION NaOH, and then denatured by boiling at 80 °C for 10 min. Denatured DNA was applied to Hybond-N + nylon membrane using a slot-blot apparatus.
- DIG 11-dUTP by Dig-High Prime [Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany], prehybridized at 49 ° C for 3 hours in the presence of DNA already fixed to a nylon membrane, and then hybridized for 16 hours at the same temperature. Development of the membranes was conducted by DIG Luminescent Detection Kit [Roche Molecular Biochemicals, Sandhofer Strasse 116, Germany].
- the strain WT#3 was in the group of Erwinia pyrifoliae, which was present only in Korea in 1999, although there were a few other different properties between Erwinia pyrifoliae WT#3 and Epl6 T .
- strain WT#3 does not have flagella in morphology which is first reported in genus Erwinia, it appears to be different from those of pathogen, En ⁇ inia pyrifoliae, which was reported by German researchers and Erwinia amylovora.
- strain WT#3 was designated Erwinia pyrifoliae WT#3 (KCCM 10283) and deposited to the Korean Culture Center of Microorganisms on June 11, 2001.
- the accession number is KCCM 10283.
- a recombinant pKEP3 for higher expression containing the gene derived from the Erwinia pyrifoliae WT#3 was constructed and transformed to Esckerichia coli. This transformant was deposited to the Korean Culture Center of Microorganisms on June 11, 2001, and was assigned with
- Example 2 Properties of specific protein triggering a plant hypersensitive reaction from Erwinia pyrifoliae WT#3 and a gene encoding it
- E.coli strains were cultivated on Luria agar medium containing tetracycline (30 mg/m£) at 37 ° C for 24 hours and 2,000 genomic library clones were generated.
- Fig. 11 compares a gene (KCCM 10282) encoding the plant HR-inducing protein from Erwinia pyrifoliae WT#3 (KCCM 10283) of the present invention with the HR-inducing gene (hrpN)- from Erwinia amylovora ATCC15580 T by analyzing the nucleotide sequences from the selected gene.
- a 1287 bp gene encoding a protein of plant hypersensitive response can be obtained.
- the 1287 bp gene was designated as "a plant HR-inducing gene from WT#3" and its similarity to hrpN gene of Erwinia amylovora ATCC15580 T (1212 bp) was investigated.
- the gene encoding the plant HR-inducing protein of Erwinia pyrifoliae WT#3 has a different nucleotide sequences from hrpN gene of En ⁇ inia amylovora and also has the insertions of novel nucleotide sequences fragments, thus showing that the plant HR-inducing gene from WT#3 has a novel gene structure which is not found in hrpN gene of En ⁇ inia amylovora.
- the nucleotide sequence of the gene encoding the plant HR-inducing protein of Erwinia pyrifoliae WT#3 was denoted as SEQ. ID. No. 5.
- an expression vector pKEP3 containing the gene was constructed as follows:
- Recombinant protein expression system in E.coli (Novagen, Inc. Madison, WI53711 USA) was employed so as to construct the expression vector pKEP3.
- This system was derived from pBR322 plasmid, where T7 promoter and operator can bind to lac repressor before the insertion site of a foreign gene.
- This structure can easily facilitate the expression of an inserted gene in a larger volume by T7 RNA polymerase produced in a host E.coli genome.
- a substrate IPTG 3 hours after incubation is added, the combination of both lac repressor produced from lacl gene and IPTG does not repress the expression of T7 RNA polymerase and thus, a larger amount of protein is synthesized.
- pKEP3 was constructed to contain an ampicillin-resistance gene and ampicillin can be used to the medium as a selective marker.
- the gene encoding a plant HR-inducing protein from Erwinia pyrifoliae WT#3 and the plasmid of recombinant protein expression system were digested at 37 ° C for 12 hrs in the presence of the restriction enzymes Ndel and Bam ⁇ I to generate the same 5'- and 3'-end. They were then ligated into the restriction sites using DNA ligase at 14 °C for 16 hrs and transferred into E.coli via CaCl 2 transformation.
- pKEP3 has many advantages that (1) ampicillin-resistant gene can be used as a selected marker, (2) its possession with His tag may make an easier purification, and (3) its possession of a strong T7 lac promoter may ensures a larger volume of protein production from ligated insertion DNA.
- the E. coli transformant containing the expression vector pKEP3 was deposited to the Korean Culture Center of Microorganisms on June 11, 2001 and was assigned with the Accession No. KCCM 10282.
- hrpN gene of Erwinia amylovora ATCC15580 T was cloned by using the same recombinant protein expression system (Novagen, Inc., Madison, WI 53711, USA) and used as a control for the biological test of pKEP3, which contains novel plant HR-inducing gene from Erwinia pyrifoliae WT#3. 3) Expression of a plant HR-inducing protein
- bacterial cells were inoculated to a LB broth by adding ampicillin (50 as a selective marker and chloramphenicol (33 ⁇ g/i ⁇ l) for inhibiting the synthesis of other proteins produced from E.coli genome, and subcultured at 37°C for 12 hours. Then, the bacterial transformant (KCCM 10282) of the present invention was cultured at 30 °C for 7 hours using the same medium. When the O.D. of the transformant (KCCM 10282) reached 0.6 about 3 hours after the cultivation, the culture was added with 0.4 mM IPTG and cooled down to 30 ° C and then cultured again for 4 hours.
- the mixture was centrifuged at 6,000 rpm for 15 min. Then, the supernatant was discarded and a pellet was suspended in a solution containing 5 mM MES buffer and 0.1 mM PMSF.
- the suspension of transformant (KCCM 10282) was lysed by sonication until the suspension became transparent and then boiled for 10 min at 100 ° C. Then, the mixture was centrifuged at 15,000 rpm for 10 min and the supernatant was discarded. After adding a protein inhibitory cocktail at the ratio of 1/1,000, the mixture was filtrated by using a 0.45 ⁇ m filter and weighed the extract amount of protein.
- the plant HR-inducing protein producing from transformant encoding the plant HR-inducing gene (KCCM 10282) of En ⁇ inia pyrifoliae WT#3 was named as 'Pioneer'.
- hrpN gene of Erwinia amylovora ATCC15580 T was cloned by the same recombinant protein expression system (Novagen, Inc. Madison, WI53711 USA) and used as a control for the biological test of Pioneer. As shown in Fig. 12, it was demonstrated that both genes of En ⁇ inia pyrifoliae
- WT#3 and Erwinia amylovora ATCC15580 T encoding plant HR-inducing protein were successfully expressed by the recombinant protein expression system and were synthesized a large volume of a plant HR-inducing proteins.
- novel protein domains of Pioneer were produced from the N-terminal at the sites of 76-79 (Thr-Gly-Leu-Leu), 88 - 92 ( L e u - G l y - G l y - G l y - S e r ) , 102 - 113 (Gly-Leu-Gly-Gly-Leu-Gly-Gly-Asp-Leu-Gly-Ser-Thr), and 131-137 ( G l y - A l a - T h r - N a l - G l y - T h r - S e r ) .
- the Pioneer had a low amino acid sequence identity of 85.9% homology to that of Hrp ⁇ .
- the molecular weight of Pioneer was 41.1 kD as compared with 39.7 kD of Hrp ⁇ .
- the molecular weight of both Pioneer and Hrp ⁇ was not compared with the molecular weight standards on an acrylamide gel but a molecular weight of each amino acid deduced from, the nucleotide sequence of gene using Winstar program. Therefore, Pioneer has a novel type of a protein by the insertion of novel peptide domains and such difference is expected to bring about much improved biological activity, which cannot be found in that of Hrp ⁇ .
- ATCC15580 T were inoculated onto tobacco (non-host plants) leaves using a syringe, together with MES buffer (protein lysis buffer) as a control [Fig.14].
- MES buffer protein lysis buffer
- FIG.14 it was noted that Pioneer and Hrp ⁇ exhibited a clear HR in the front tobacco leaf at the dose of 10 ⁇ g/ml and 20 ⁇ g/ml, respectively; in the same reverse tobacco leaf, Pioneer and Hrp ⁇ exhibited a clear HR at the dose of 5 ⁇ g/ml and 10 ⁇ g/ml, respectively. This reflected that Pioneer induces HR at a relatively lower concentration than that of Hrp ⁇ .
- Purified Pioneer at a dose of 500 ⁇ g/ml was inoculated to the surface of an immature fruit by punching it a hole (0.5 mm in diameter and 10 mm in depth).
- the surface of the immature fruit turned black as a progressive symptom 4 days after treatment, compared with that of a control.
- Example 3 The study of biological activity using plant HR-inducing protein (Pioneer)
- Cucumbers were cultivated by the conventional "rain-protecting" method prevailing in agricultural farms. The test materials were applied based on the 3-time repeated randomized complete block design. The stems and leaves of cucumbers were treated with the HR-inducing protein, Pioneer, at a dose of 20 ⁇ g/ml according to the recommended dose by EDEN Bioscience corporation.
- EDEN Bioscience's Messenger® was used as a control at the same dose above, together with locally produced Fenarimol (chemical pesticide) based on the instruction in use.
- the treatment methods are as follows: ⁇ Treatment by three times: Pioneer, Messenger® and Fenarimol were sprayed to the stems and leaves of cucumber three times each at intervals of 10 days after early stage of powdery mildew (Sphaerotheca fuliginea).
- the disease severity of powdery mildew- (Sphaerotheca fuliginea) from the upper 8 leaves to the bottom 3 leaves of cucumbers was measured 7 days after treatment based on the following criteria (0: no disease, 1: 1-5%, 2: 5.1-20%, 3: 20.1-40%, 4: more than 40%).
- Table 5 The disease severity of powdery mildew- (Sphaerotheca fuliginea) from the upper 8 leaves to the bottom 3 leaves of cucumbers was measured 7 days after treatment based on the following criteria (0: no disease, 1: 1-5%, 2: 5.1-20%, 3: 20.1-40%, 4: more than 40%).
- the stems and leaves of cucumbers were treated with Pioneer at a dose of 20 ⁇ g/ml 5 times each at intervals of 14 days from day 7 before implantation.
- HrpN a control, was also given to the stems and leaves of cucumbers at the same dose as aforementioned.
- the cucumbers for this experiment were harvested at the implantation of day 34, 42 and 56 and investigated using a portable photosynthesis device (LCA-4 system, ADC BioScientific Ltd., UNK; light source: 1,500 ⁇ mole) and a chlorophyll device (Chlorophyll meter SPAD-502, Minolta, Japan).
- a portable photosynthesis device (LCA-4 system, ADC BioScientific Ltd., UNK; light source: 1,500 ⁇ mole)
- a chlorophyll device Chlorophyll meter SPAD-502, Minolta, Japan
- Pioneer can be effectively used as a pesticide at a lower dose than HrpN protein.
- the peppers for this experiment were harvested at the implantation of day 34, 42 and 56 and investigated using a portable photosynthesis device (LCA-4 system, ADC BioScientific Ltd., UNK; light source: 1,500 ⁇ mole) and a chlorophyll device (Chlorophyll meter SPAD-502, Minolta, Japan).
- a portable photosynthesis device LCD-4 system, ADC BioScientific Ltd., UNK; light source: 1,500 ⁇ mole
- a chlorophyll device Chlorophyll meter SPAD-502, Minolta, Japan
- the treatment was performed as follows: ⁇ Immersion plus spray method: Oriental melon seeds were immerged in both Pioneer and HrpN protein at a dose of 10 ⁇ g/ml for 24 hours. Then, these seeds were sowed in a pot. At the implementation of day 17 and 28, the stems and leaves of melons were treated with both Pioneer and HrpN. ⁇ Spray method: At the implementation of day 17 and 28, the stems and leaves of oriental melons were treated with both Pioneer and HrpN. The disease severity of downy mildew was measured 55 days after treatment.
- HrpN a control
- the treatment was performed as follows: ⁇ 8 days after implantation, the stems and leaves of sweet peppers were treated with both Pioneer and HrpN.
- Phytophthora capsici (2 X10 6 cells/ ml) was inoculated to sweet peppers, so treated, and disease severity was measured 45 days after treatment.
- Sweet peppers were implanted to a 25 cm port and cultivated at a glass house. Sweet peppers, so treated, were investigated at the implantation of day 41 and 45. Table 14
- Rice was cultivated by the conventional "open field culture” method prevailing in agricultural farms.
- the stems and leaves of rice was treated with Pioneer at the doses of 10 ⁇ g/ml, and immerged at 28 ° C for 24 hours.
- Rice seeds were sowed to a seedbed, grown for 16 days and implanted to a test field.
- Cucumbers were selected as host plants of aphis.
- Cucumbers were cultivated by the conventional "rain-protecting" method prevailing in agricultural farms.
- the stems and leaves of cucumbers were treated with Pioneer at a dose of 20 ⁇ g/ml.
- HrpN a control, was also given to the stems and leaves of cucumbers at the same dose.
- a higher dose (40 ⁇ g/ml) of Pioneer was required for better enhancement of rice's growth.
- Pioneer can be more effectively used as a seed-treating agent which can promote the growth of rice seeds than HrpN.
- KCCM 10283 a novel Erwinia pyrifoliae WT#3 (KCCM 10283) according to the present invention is isolated and identified, and a novel protein (Pioneer) or a polypeptide translated from a plant HR-inducing gene (KCCM 10282) of this strain shows that Pioneer has improved properties in inducing plant resistance, plant growth promotion, insect repellent effect, increase of photosynthesis and chlorophyll, and seed treatment effect than a HrpN from Erwinia amylovora ATCC15580 1 .
- En ⁇ inia pyrifoliae WT#3 (KCCM 10283) is quite suitable for the development of a novel and better plant hypersensitive reaction eliciting biopesticide.
- this invention is advantageous in developing a novel and improved biopesticide and a fertilizer.
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
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JP2003521819A JP3976731B2 (en) | 2001-08-14 | 2002-08-14 | A new biochemical pesticide that uses a gene derived from a black wilt fungus WT # 3 (KCCM10283), a novel phytopathogenic fungus that affects pear trees |
CA 2457060 CA2457060A1 (en) | 2001-08-14 | 2002-08-14 | New biopesticide using gene from erwinia pyrifoliaewt#3, novel pathogen that affects asian pear trees |
EP02758919A EP1417299A4 (en) | 2001-08-14 | 2002-08-14 | New biopesticide using gene from erwinia pyrifoliaewt 3, novel pathogen that affects asian pear trees |
BR0210787A BR0210787A (en) | 2001-08-14 | 2002-08-14 | Biopesticide using erwinia pyrifoliae wt # 3 gene, new pathogen affecting Asian pear |
HU0402172A HU225133B1 (en) | 2001-08-14 | 2002-08-14 | New biopesticide using gene from erwinia pyrifoliaewt#3, novel pathogen that affects asian pear trees |
AU2002324348A AU2002324348B2 (en) | 2001-08-14 | 2002-08-14 | New biopesticide using gene from Erwinia pyrifoliaeWT#3, novel pathogen that affects Asian pear trees |
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KR2001/49047 | 2001-08-14 | ||
KR1020010049047A KR20030015010A (en) | 2001-08-14 | 2001-08-14 | New biopesticide using WT#3-1 gene from Erwinia pyrifoliae WT#3, novel pathogen that affects Asian pear trees |
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JP (1) | JP3976731B2 (en) |
KR (2) | KR20030015010A (en) |
CN (1) | CN100494344C (en) |
AU (1) | AU2002324348B2 (en) |
BR (1) | BR0210787A (en) |
CA (1) | CA2457060A1 (en) |
HU (1) | HU225133B1 (en) |
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Cited By (4)
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US9181592B2 (en) * | 2007-03-30 | 2015-11-10 | Case Western Reserve University | Method for detecting a bacterial pathogen |
WO2021152096A1 (en) * | 2020-01-30 | 2021-08-05 | Ocean Dx | Multiplex pcr method for detecting microorganisms and use thereof |
WO2023288294A1 (en) | 2021-07-16 | 2023-01-19 | Novozymes A/S | Compositions and methods for improving the rainfastness of proteins on plant surfaces |
WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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KR100718549B1 (en) | 2006-04-25 | 2007-06-21 | 대한민국 | Vector comprising hrp gene from erwinia pyrifoliae transgenic agrobacterium tumefaciens and pathogenic resistant transgenic plant using the same |
KR101249854B1 (en) | 2011-01-11 | 2013-04-03 | 재단법인 제주테크노파크 | Composition and Method for Controlling Plant Disease Occurred by Magnaporthe grisea Using a Fruit Extract of Pittosporum tobira Ait or Saponin ⅢA3 Isolated from the Fruit Extract |
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US5859324A (en) * | 1995-06-07 | 1999-01-12 | Cornell Research Foundation, Inc. | Hypersensitive response induced resistance in plants |
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EP0648266B1 (en) * | 1992-07-01 | 2006-02-08 | Cornell Research Foundation, Inc. | Elicitor of the hypersensitive response in plants |
US5850015A (en) * | 1995-06-07 | 1998-12-15 | Cornell Research Foundation, Inc. | Hypersensitive response elicitor from Erwinia chrysanthemi |
CN1126817C (en) * | 2000-12-15 | 2003-11-05 | 南京农业大学 | Gene for coding plant growth regulator and its expression product and application |
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Non-Patent Citations (5)
Title |
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BAUER D.W. ET AL.: "Erwinia chrysanthemi harpinEch: an elicitor of the hypersensitive response that contributes to soft-rot pathogenesis", MOL. PLANT MICROBE INTERACT, vol. 8, no. 4, July 1995 (1995-07-01) - August 1995 (1995-08-01), pages 484 - 491, XP002085275 * |
DATABASE GENBANK [online] March 1999 (1999-03-01), XP002977937, Database accession no. (M92994) * |
MUKHERJEE A. ET AL.: "Molecular characterization and expression of the Erwinia carotovora htpNEcc gene, which encodes an elicitor of the hypersensitive reaction", MOL. PLANT MICROBE INTERACT, vol. 10, no. 4, May 1997 (1997-05-01), pages 462 - 471, XP002977309 * |
See also references of EP1417299A4 * |
WEI Z.M. ET AL.: "Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora", SCIENCE, vol. 3, no. 257(5066), July 1992 (1992-07-01), pages 85 - 88, XP002912474 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US9181592B2 (en) * | 2007-03-30 | 2015-11-10 | Case Western Reserve University | Method for detecting a bacterial pathogen |
WO2021152096A1 (en) * | 2020-01-30 | 2021-08-05 | Ocean Dx | Multiplex pcr method for detecting microorganisms and use thereof |
WO2023288294A1 (en) | 2021-07-16 | 2023-01-19 | Novozymes A/S | Compositions and methods for improving the rainfastness of proteins on plant surfaces |
WO2023225459A2 (en) | 2022-05-14 | 2023-11-23 | Novozymes A/S | Compositions and methods for preventing, treating, supressing and/or eliminating phytopathogenic infestations and infections |
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CN100494344C (en) | 2009-06-03 |
CN1582329A (en) | 2005-02-16 |
BR0210787A (en) | 2004-08-17 |
HU225133B1 (en) | 2006-06-28 |
HUP0402172A3 (en) | 2005-07-28 |
HUP0402172A2 (en) | 2005-02-28 |
KR20030015010A (en) | 2003-02-20 |
EP1417299A1 (en) | 2004-05-12 |
AU2002324348B2 (en) | 2005-09-15 |
EP1417299A4 (en) | 2005-02-16 |
CA2457060A1 (en) | 2003-02-27 |
KR20030015163A (en) | 2003-02-20 |
KR100389143B1 (en) | 2003-06-25 |
JP3976731B2 (en) | 2007-09-19 |
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ZA200400008B (en) | 2005-01-05 |
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