Patent Application
20180105849
The present invention relates to a method for producing lysophosphatidylethanolamine 18:1 (hereinbelow, also described as LPE18:1) from a microorganism of Pseudomonas sp. More specifically, the present invention relates to a method of producing LPE18:1 by treating phospholipids extracted from bacteria microorganism belonging to Pseudomonas sp. with phospholipase A2 (hereinbelow also described as PLA2).
Lysophosphatidylethanolamine (LPE) is generated according to release of the 2nd acyl chain of phosphatidylethanolamine (PE), which is one of the phospholipids for forming a cell membrane, as a result of PLA2 enzyme activity, and it is present in a small amount in a living body. It has been found that a treatment of LPE can suppress aging of a plant and promote ripening of a fruit (U.S. Pat. No. 5,110,341 and U.S. Pat. No. 5,126,155) and can enhance plant health, protect plants from biotic and abiotic stress-related injuries and enhance the recovery of plant injured as a result of such stresses (U.S. Pat. No. 6,559,099). LPE is mainly produced by treating phosphatidylethanolamine extracted from egg yolk or soy bean with PLA2 enzyme, and the compositional component of LPE is mostly LPE 16:0 and LPE 18:0 in which the 1st acyl chain is 16:0 or 18:0. During the process of studying the biological mechanism related to the suppression of aging caused by LPE, it was found that LPE18:1 is a superior molecule which has much higher biological efficacy than LPE 16:0 or LPE 18:0 (U.S. Pat. No. 6,426,105). At present moment, the technique related to the method for producing LPE uses egg yolk or soy bean as a source material so that the component is mostly LPE 16:0 and LPE 18:0 with almost total absence of LPE18:1 (Korean Patent Registration No. 0331932). Accordingly, efforts have been made to find a source material for producing LPE18:1 but to no avail.
In this regard, it is found according to the present invention that the source material (PE containing the 1st acyl chain of 18:1) for producing LPE18:1 is present in a microorganism like Pseudomonas. The discovery of the source material for producing LPE18:1, and the methods and results of the study are described hereinbelow. Once a plant is infiltrated by a pathogen, a disease-resistant response against part of the pathogen is exhibited by the plant. However, the mechanism related to such response is not clearly known yet. In particular, there is not much information available for the signal transduction mechanism for inducing initial disease resistance. It has been known that, when Pseudomonas syringae pv. tomato DC3000 carrying avrRpm1 (hereinbelow, described as Pst-avrRpm1) invades an extracellular space of Arabidopsis thaliana (Col-0 ecotype) as a host plant, Avr protein (avrRpm1) is introduced by the pathogen to the cytoplasm of a host through the Type III secretion mechanism. It is known that, if the host cell has RPM1, which is a Resistance protein capable of recognizing avrRpm1 protein, the interaction between those proteins causes an increased salicylic acid, activation of NPR1 (NON-EXPRESSER OF PR GENES 1), and an immune response for inducing resistance genes like expression of PR (PATHOGENESIS-RELATED). However, the upper-level molecule for connecting such gene-for gene interaction, genetic basis, and host immune response for inducing plant resistance genes remain mostly unknown.
According to a study regarding the initial signal transduction that is yet to be known, inventors of the present invention found that LPE18:1 is a signaling molecule to induce disease resistance of a plant. It is surprisingly found that LPE18:1 is mainly produced from an invading pathogen (i.e., Pseudomonas). Briefly, the study result indicates that, as Arabidopsis thaliana is attacked by non-pathogenic Pseudomonas (Pst-avrRpm1), intracellular injection of avrRpm1 protein into plant host cells is yielded, and at that time, the protein factor is recognized as a gene-for-gene interaction by the plant and phospholipase A2-alpha (PLA2ª) protein is immediately expressed. PLA2ª protein is secreted to an extracellular region, in which invading pathogens are present, and it decomposes phosphatidylethanolamine present in pathogen membrane to produce LPE18:1 as a main component. This LPE18:1 exhibits an activity of a signaling molecule and, according to propagation to the surrounding, it enters neighboring cells not infected by the pathogen to induce disease resistance.
As it is shown by the study result described above, it was possible to recognize that the source material (PE containing the 1st acyl chain of 18:1) for producing LPE18:1 is present in a microorganism like Pseudomonas. Thus, the inventors of the present invention carried out large-scale culture of Pseudomonas bacteria, and according to extraction of lipids therefrom and treatment with PLA2 enzyme, confirmed production of a large amount of LPE18:1. The LPE18:1 produced by the inventors was applied to a plant, and the biological effect is compared with a known mixture liquid of LPE 16:0/LPE 18:0 and also with commercially available LPE18:1 of high purity. LPE produced by the inventors using the microorganism Pseudomonas is found to be mostly consisting of LPE18:1 and LPE 16:0 with extremely small amount of LPE 16:1. In terms of the effect, crude LPE18:1 mixture with LPE16:0 produced by the inventors is superior to LPE 16:0/LPE 18:0 mixture. However, it was inferior to pure LPE18:1. Investigation is also made to see whether or not a source material for producing LPE18:1 can be obtained from a microorganism other than Pseudomonas. In this regard, as a result of analyzing phospholipids after separating them from Escherichia coli as Gram negative bacteria, Bacillus subtilis and Arthrobacter citres as Gram positive bacteria, Saccharomyces cerevisiae as yeast, and Chlorella vulgaris as algae, it is found that, according to the treatment with PLA2, LPE18:1 is produced hardly from other microorganisms but only from Pseudomonas.
Meanwhile, “methods of enhancing plant health, protecting plants from biotic and abiotic stress related injuries and enhancing the recovery of plants injured as a result of such stresses” is described in Korean Patent Application Publication No. 2002-0086604, and “a composition and a method for immunizing plants against diseases” is disclosed in Korean Patent Application Publication No. 1997-0001484. However, the method for producing LPE18:1 from a microorganism of Pseudomonas sp. as described in the present invention has not been disclosed before.
The present invention is devised under the circumstances described above. Specifically, the inventors of the present invention confirmed that lysophosphatidylethanolamine 18:1 for inducing plant immunity is produced by phospholipase A2ª (PLA2ª) from cell membrane phospholipids of Pseudomonas syringae as a pathogen which invades a plant. On the basis of this result, large-scale culture of Pseudomonas (Pst) is carried out followed by extraction of lipids and treatment with PLA2 enzyme, and production of a large amount of LPE18:1 is confirmed.
Furthermore, in order to see whether or not a source material (PE containing the 1st acyl chain of 18:1) for producing LPE18:1 can be obtained from a microorganism other than Pseudomonas, phospholipids are isolated from Escherichia coli as Gram negative bacteria, Bacillus subtilis and Arthrobacter citres as Gram positive bacteria, Saccharomyces cerevisiae as yeast, and Chlorella vulgaris as algae, and then treated with PLA2. As a result, it is confirmed that not much amount of LPE including LPE18:1 is produced from other microorganisms except Pseudomonas in which significant production of LPE18:1 is shown. Furthermore, in order to see whether or not LPE18:1 can be produced in a large amount from other types belonging to Pseudomonas sp., phospholipids are extracted from Pseudomonas caeni, Pseudomonas fluorescens, and Pseudomonas putida. As a result, it is found that content of PE containing the 1st acyl chain of 18:1 as an original source material of LPE18:1 is quite high in all types of Pseudomonas, and LPE18:1 is produced in a large amount according to a treatment with PLA2 enzyme.
According to the present invention, the Pseudomonas microorganism is cultured at large scale, and phospholipids are extracted and treated with PLA2 obtained from an animal. As a result, it is confirmed that LPE18:1 can be produced in a large amount, and the present invention is completed accordingly.
In order to solve the problems described above, the present invention provides a method for producing LPE18:1 according to treatment of phospholipids extracted from a microorganism of Pseudomonas sp. with phospholipase A2.
According to the present invention, it is confirmed that the crude LPE18:1 mixture is produced by phospholipase A2ª from cell membrane phospholipids of a pathogen which invades a plant, and a local immune response of a plant is induced as a result. As a result, the crude LPE18:1 mixture produced by the treatment with phospholipase A2ª of the present invention is a material originating from natural products. LPE18:1 can be used as a plant vaccine material for preventing the plants from injuries caused by pathogen infections and/or environmental stresses and accelerating the recovery of plants injured by pathogen infections and/or environmental stresses. LPE18:1 can also be used as a composition for enhancing fruit ripening (color and sweetness) and storage properties, and as it can be used for an application in plant tissues, food products, pharmaceuticals, cosmetics, and agricultural use, it would be very advantageously used in related industries.
In order to achieve the purpose of the present invention, the present invention provides a method for large scale production of lysophosphatidylethanolamine comprising:
For the method according to one embodiment of the present invention, the lysophosphatidylethanolamine (hereinbelow, described as LPE) is produced by phospholipase A2 protein from phospholipids of a cell membrane of pathogen which invades a plant, and LPE can be LPE 16:0, LPE 16:1, LPE 18:0, LPE18:1, LPE 18:2, or LPE 18:3. LPE can be preferably LPE 16:0, LPE 16:1, or LPE18:1, and more preferably LPE18:1, but not limited thereto.
Furthermore, for the method of the present invention, the microorganism may be a microorganism of Pseudomonas sp. It is preferably Pseudomonas caeni, Pseudomonas fluorescens, Pseudomonas putida, or Pseudomonas syringae. It is more preferably Pseudomonas caeni or Pseudomonas fluorescens, but not limited thereto.
Furthermore, for the method of the present invention, the phospholipids may be phosphatidylcholine, phosphatidylserine, or phosphatidylethanolamine, preferably phosphatidylethanolamine, and more preferably phosphatidylethanolamine in which the acyl chain at the first position is 18:1, but not limited thereto.
For the method according to one embodiment of the present invention, the phospholipase A2 is an enzyme which hydrolyzes the ester bond at sn-2 position of glycerol phospholipid as a main component of a biological membrane to release lysophospholipids and fatty acids. Phospholipase A2 may be an enzyme originating from an animal like snake toxin, honey bee toxin, or pig pancreatic juice, or from a microorganism or a plant. It can be also a recombinant protein, but not limited thereto.
Furthermore, for the method of the present invention, any method well known in the pertinent art can be used as a method for extracting phospholipids from harvested bacteria and a method for separating and purifying lysophosphatidylethanolamine.
The present invention further provides lysophosphatidylethanolamine produced by the aforementioned method. Lysophosphatidylethanolamine produced by the method of the present invention is preferably lysophosphatidylethanolamine 18:1, but not limited thereto.
Hereinbelow, the present invention is explained in greater detail in view of the Examples. However, it is evident that the following Examples are given only for exemplification of the present invention and by no means the present invention is limited to the following Examples.
Arabidopsis plant was cultivated at temperature of 22° C. and relative humidity of 60% with conditions including 16 hours of light period and 120 μmoles m−2s−1 of photon flux density. Because the plant defense response is age-dependent, fully mature leaves of the 4 week old plant were used for various analyses. Arabidopsis (Arabidopsis thaliana) mutants including pla2ª, pla2ª-II, sid2, npr1-5, rps2 and ein2 (Salk_099415, CS857021, Salk_042603, CS3724, Salk_087581, CS3071 and CS8072, respectively) were purchased from Arabidopsis Biological Resource Center, USA. Arabidopsis mutants eds1 and pad4-5 were obtained from S. A. Whitham (Iowa State University, USA) and J. E. Parker (Max Planck Institute for Plant Breeding Research, Germany), respectively. The transgenic event expressing NPR1-eGFP and NahG were obtained from X. Dong (Duke University, USA) and O. M. Park (Korea University, South Korea), respectively.
Complementarity test of pla2ª mutant carrying native-promoter:PLA2ª and generation of PLA2ª overexpressing transgenic event
For complementation, pla2ª mutant plant was transfected by a floral dip method by using pCAMBIA1300 clone, which is a binary vector for plant transfection including PLA2ª genomic DNA having native promoter part (i.e., from −1175 to +922) at BamHI site. In order to have an artificial deformation for intracellular translocation of PLA2ª (i.e., not allowing secretion to apoplast), pCAMBIA1300 carrying pPLA2ª::PLA2ª with no signal peptide was prepared, and introduced to pla2ª plant by using Agrobacterium. To generate PLA2ª-overexpressing transgenic event, pBIG binary vector carrying p35S::PLA2ª was introduced to the wild type plant.
Pseudomonas syringae pv. tomato DC3000 (hereinbelow, Pst) and Pst-avrRpm1, which is avrRpm1-containing bacterial strain with no virulency, were obtained from Y. J. Kim (Korea University, South Korea). Pst-avrRpt2, which is avrRpt2-containing bacterial strain with no virulency, was obtained from J. M. Park (KRIBB, South Korea) and Pst-avrRps4, which is avrRps4-containing bacterial strain with no virulency, was obtained from R. Innes (Indiana University, USA). The bacterial strains were cultured and treated according to the method of Katagiri, et al. (Arabidopsis Book, 2002, 1:e0039).
Total RNA was extracted by using RNA isolator (Gibco, USA) from a sample which has been frozen with liquid nitrogen (i.e., 2 leaves per sample). Reverse transcription polymerase chain reaction (RT-PCR) and real-time quantitative polymerase chain reaction (real-time qPCR) were carried out by using gene-specific primers. For the analysis of a real-time quantitative polymerase chain reaction, Applied Biosystems 7900 Real-Time PCR SYSTEM™ was used. 2X SYBR® Green qPCR master mix was used for PCT amplification. The analyzed data were normalized first against the amount of ACTIN1 gene, and the fold increase of gene expression according to treatment was determined in comparison with a mock or non-treatment plant (1.0 fold). Reference gene as an alternative (AT1G13320) was obtained from the previous study (Hong et al., 2010, Plant Cell Physiol. 51:1694-1706). The gene-specific primers that are used in the present invention are as described in Table 1 and Table 2 below.
To have histochemical localization of GUS activity, transgenic Arabidopsis plant carrying PLA2ª-promoter::GUS structure was generated (Jung et al., 2012, Front Plant Sci. 3:126). To have clear expression of PLA2ª in an infected area of a leaf tissue, 3 hours after switching to night condition by turning off the light, the pathogen was syringe-infiltrated to an abaxial surface of the PLA2ª-promoter:GUS transgenic Arabidopsis leaf. 1.5 Hours later, histochemical analysis of GUS was carried out according to the method by Jefferson (EMBO J. 1987, 6:3901-3907).
According to the method described before (Ryu et al., 1996, Biochem Biophys Acta. 1303:243-250), total lipid was extracted from leaf and bacterial samples. Each phospholipid and free fatty acid (FFA) were quantified by Kansas Lipidomics Research Center based on ESI-MS/MS analysis.
Every phospholipid was purchased from Avanti Polar Lipids Inc. (USA), and free fatty acids (16:0, 18:0 and 18:1) were purchased from Sigma Co. (USA). The solvent was dried under nitrogen gas stream, and the lipids were suspended therein according to ultrasonication so as to have final concentration of 100 nmol ml−1 in 0.018% silwet L-77 (or 200 nmol ml−1 in H2O). Among the LPE (lysophosphatidylethanolamine) species, LPE18:1 showed the biggest increase in Pst-avrRpm1 inoculation, and thus it is used for the present invention.
The mature form of the recombinant PLA2ª protein was produced in Escherichia coli which is fused with Dsbc protein. According to the method described before (Ryu et al., 2005, Biochem Biophys Acta. 1736:144-151), it was purified by affinity chromatography. The protein mixture in which PLA2ª and Dsbc are separated from each other or Dsbc:PLA2ª fusion protein (10 μgml−1) was treated on the leaf of pla2ª mutant by syringe infiltration, each in an amount of 0.5 μg. The enzyme activity of PLA2ª was determined by in vitro analysis using substrate PE for a reaction mixture containing 10 mM Ca2+ and 0.05% Triton X-100 in 50 mM Tris·HCl (pH 8.5). The mock-treated plant was infiltrated with a buffer (50 mM Tris·HCl, pH 8.0) or Dsbc protein. For inactivation of the PLA2ª activity, recombinant PLA2ª was pre-treated for 30 minutes at 30° C. with 2 μM manoalide as an irreversible inhibitor. Inactivation of the PLA2ª activity caused by manoalide was confirmed by an in vitro analysis of PLA2 activity.
In order to determine the antibacterial activity of PLA2ª, 3 μg of recombinant PLA2ª in free and mature form, which has been prepared according to the method by Ryu et al. (Biochem Biophys Acta. 2005, 1736:144-151), was added to 100 μl of a solution (Tris·HCl (50 mM, pH 8.0), 10 mM calcium chloride (CaCl2)) in which virulent Pst (5×105 CFU ml−1) is suspended. The bacteria sample was cultured in a weak shaking state at 28° C. for 6 hours. Thereafter, only the survived bacteria were titrated. As a mock control group, a suspension in which the bacteria are suspended in a solution having no PLA2ª was used.
According to the method described before (Bowling et al., 1994, Plant Cell 6:1845-1857; Heck et al., 2003, Plant 1 36:342-352), salicylic acid, ethylene, and jasmonic acid were quantified from 0.5 g of a Arabidopsis leaf sample which has been sprayed with Pst-avrRpm1 suspension (1×108 CFU ml−1 in 0.018% Silwet L-77) or LPE (100 nmol ml−1 in 0.018% Silwet L-77 or 200 nmol ml−1 in H2O).
NPR1 transition was determined by spraying LPE (100 nmol ml−1 in 0.018% Silwet L-77), salicylic acid (0.3 mM in 0.018% Silwet L-77), or mock solution (0.018% Silwet L-77) to a transgenic plant carrying 35S::NPR1-eGFP, and observing, 6 hours later, the plant leaf with a laser scanning confocal microscope (Zeiss, Germany).
Leaf tissue (i.e., 4 leaves for each sample) was collected, and after vacuum infiltration for 5 minutes with sterilized water, they were cultured at room temperature for 2 hours under shaking. Level of electrolyte leakage from the sample was measured by using a conductivity meter (Mettler Toledo, Switzerland). Data appeared to be the percentage of total electrolyte leakage was obtained by, after freezing, carrying out melting at room temperature under shaking for 6 hours.
Student's t-test was employed to determine the statistical significance among groups. The data of bacterial growth analysis and ion leakage analysis were expressed in terms of mean ±standard deviation, and the significant value was as follows: *P<0.05; **P<0.01. All data other than those were expressed in terms of mean±standard error in which *P<0.05; **P<0.01.
Pseudomonas cell culture was freeze-dried, and total lipids were extracted from the bacteria according to the method described before (Ryu et al., 1996, Biochem Biophys Acta. 1303:243-250). Thereafter, commercially available phospholipase A2 (powder form) was dissolved in water and 20 μl was collected. A mixture of the Pseudomonas lipid extract (20 μl), phospholipase A2 enzyme (20 μl), and the reaction buffer (50 mM Tris-HCl, 10 mM CaCl2, 0.05% Triton X-100) (160 μl) was allowed to react for 30 minutes at 30° C. Thereafter, 750 μl of Chl:MeOH (1:2) was added thereto to terminate the reaction. By adding 200 μl of chloroform and 200 μl of KCl (2 M) thereto followed by centrifugation and removal of supernatant, LPE18:1 was obtained.
In order to identify the cellular function(s) of secretory PLA2ª in plant defense responses, the pla2ª mutants, pla2ª (knockout) and pla2ª-II (knockdown), were obtained from TAIR. Under normal growth conditions, the pla2ª mutants did not differ phenotypically from wild-type plants. However, the local immune responses of the pla2ª mutants to Pst-avrRpm1 were impaired (
In Col-0 wild type plants, PLA2ª is expressed at low levels. PLA2ª expression is rapidly and moderately induced upon inoculation with Pst-avrRpm1, but weakly in response to virulent Pst inoculation (
Inoculation of wild type leaves with Pst-avrRpm1 increased LPE level by about 35% over the amounts present in mock-treated leaves (
Based on the analysis of pla2ª mutant plants compared to wild type, we hypothesized that the defect in immune responses of the pla2ª mutant may be due to the failure to generate the lipid products that mediate downstream immune responses. To test this hypothesis, we supplemented pla2ª mutant with LPE18:1 1.5 h after inoculation of Pst-avrRpm1. Indeed, LPE restored the ability of the pla2ª mutant to induce local immune responses and restricted the propagation of disease symptoms beyond the HR region (
ICS1 plays a role in host immune responses as a key enzyme in SA biosynthesis. When pla2ª mutant plants were inoculated with Pst-avrRpm1, they reduced ICS1 expression by 59% compared to wild type plants (
NPR1 is a key regulator of SA-mediated immune responses leading to PR1 gene expression. NPR1 activation requires its translocation from the cytoplasm to the nucleus due to SA-induced redox changes. Since NPR1 activation is mediated by SA, it is conceivable that the LPE-induced increase in SA may activate NPR1. Application of LPE to transgenic plants carrying 35S::NPR1-eGFP indeed activated NPR1 by inducing its translocation from the cytoplasm to the nucleus, as did SA treatment (
The pla2ª mutant is also defective in PDF1.2 expression in response to inoculation with Pst-avrRpm1 (
The aforementioned results consistently support the hypothesis that the defects of pla2ª mutant plants in local host immune responses under long-day conditions are due to the failure of PLA2ª to generate lipid products such as LPE, which mediate R gene-induced immune response. In support of this hypothesis, exogenous treatment of rPLA2ª proteins to pla2ª mutant leaves generated endogenous lipid products including LPE (
Based on our results, we propose that PLA2ª-derived lipid-based signaling operates under long-day conditions as follows. Upon inoculation with avirulent Pst, PLA2ª is rapidly, moderately, and transiently expressed in an R gene-mediated manner. PLA2ª is secreted into the extracellular spaces, where it generates its lipid products including LPE from the membranes of invading bacteria and host cells. The LPE then triggers R gene-induced downstream immune responses through both the ICS1/NPR1-dependent SA signaling leading to PR1 expression and the ACS-dependent ethylene signaling leading to PDF1.2 expression (
Pseudomonas syringae was cultured in a large amount, and, after harvesting, lipids were extracted therefrom. The lipid extract was treated with phospholipase A2, and by using HPLC-ELSD analysis, decomposition of PE (phosphatidylethanolamine) and production of LPE (lysophosphatidylethanolamine) were determined (
In order to see whether or not LPE18:1 can be produced from microorganisms other than Pseudomonas, phospholipids were isolated from Escherichia coli as Gram negative bacteria, Bacillus subtilis and Arthrobacter citres as Gram positive bacteria, Saccharomyces cerevisiae as yeast, and Chlorella vulgaris as algae. As a result of the analysis of phospholipids, it was found that PE as a raw material source of LPE is hardly detected from the microorganisms other than Pseudomonas (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2015-0089016 | Jun 2015 | KR | national |
The present application is a continuation in part application to International Application No. PCT/KR2015/013227, with an International Filing Date of Dec. 4, 2015, which claims the benefit of Korean Patent Application No. 10-2015-0089016, filed in the Korean Intellectual Property Office on Jun. 23, 2015, the entire contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/KR2015/013227 | Dec 2015 | US |
| Child | 15852864 | US |
