Plant disease resistance inducer, method for inducing plant disease resistance, and method for producing plant disease resistance inducer

Abstract

The present disclosure provides a plant disease resistance inducer capable of effectively inducing the resistance of a plant to a disease. The plant disease resistance inducer according to the present disclosure contains secretions of cyanobacteria.

Description

Technical Field

[0001] The present disclosure relates to a plant disease resistance inducer as a natural metabolite that contributes to the induction of disease resistance in plants, a plant disease resistance induction method, and a method for producing the plant disease resistance inducer. Background Art

[0002] With the demand for increased food production accompanying the increase in the world population, there is a need to develop technologies for efficiently producing high-quality crops on limited arable land. Specifically, it is expected that the cultivation time will be shortened by growth promotion, the yield per cultivated area will be increased, the yield rate will be improved by reducing diseases and non-standard products, and the quality of products will be improved by increasing the size of fruits and increasing the sugar content.

[0003] In addition, from the perspective of preventing global warming and reducing environmental load, the use of bio-derived raw materials for fossil-free resources is increasing. Among them, the flexible use of natural-derived materials that consume less fossil energy in the manufacturing process and have less environmental load during use is expected.

[0004] For example, as natural substances that induce plant disease resistance, specific oxo fatty acid derivatives (Patent Document 1) and a polypeptide chain that forms a part of elongation factor (Elongation Factor Tu: EF-Tu) (Patent Document 2) are known. In addition, for example, it is known that plant disease resistance can be induced by inoculating plants with specific microbial species (Patent Document 3).

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent No. 6759448

[0008] Patent Document 2: Japanese Patent Application Publication No. 2015-077116

[0009] Patent Document 3: Japanese Patent No. 4359619 Summary of the invention

[0010] Problems to be solved by the invention

[0011] However, in the above-mentioned prior art, for example, the production of plant disease resistance inducing substances using microorganisms, or the purification or extraction of disease resistance inducing substances, etc., are complicated and time-consuming, and costly. In addition, when the microorganisms themselves are inoculated into plants, the effects vary depending on the combination of the microorganism species used, the target plant species, and the properties of the soil, lacking versatility, and the effect of disease resistance induction is unstable.

[0012] Therefore, the present disclosure provides a plant disease resistance agent and a plant disease resistance induction method that can effectively induce disease resistance in plants. In addition, the present disclosure provides a method for producing a plant disease resistance inducer that can simply and efficiently produce the plant disease resistance inducer.

[0013] Means for solving problems

[0014] One embodiment of the present disclosure relates to a plant disease resistance inducer comprising a secretion of cyanobacteria.

[0015] Effects of the Invention

[0016] The plant disease resistance inducer and the plant disease resistance induction method disclosed in the present invention can effectively induce disease resistance in plants. In addition, the method for producing the plant disease resistance inducer disclosed in the present invention can simply and efficiently produce the plant disease resistance inducer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a flow chart showing an example of a method for producing a plant disease resistance inducer according to an embodiment.

[0018] Figure 2 is a schematic diagram showing the cell surface layers of cyanobacteria.

[0019] Figure 3 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 1.

[0020] Figure 4 yes Figure 3 A magnified image of the dashed area A.

[0021] Figure 5 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2.

[0022] Figure 6 yes Figure 5 A magnified image of the dashed area B.

[0023] Figure 7 This is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1.

[0024] Figure 8 yes Figure 7 A magnified image of the dashed area C.

[0025] Fig. 9 It is a bar graph showing the amount of protein in the culture supernatant of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1 (n=3, error bar=SD).

[0026] Fig.10Displays serial number 01 to serial number 03.

[0027] Fig.11 Displays serial number 04 to serial number 06.

[0028] Fig.12 Displays the serial number 07.

[0029] Fig.13 Displays the serial number 08.

[0030] Fig.14 Displays the serial number 09.

[0031] Fig.15 Serial number 10 to serial number 11 are displayed.

[0032] Fig.16 Serial numbers 12 to 18 are displayed.

[0033] Fig.17 Serial number 19 to serial number 22 are displayed. DETAILED DESCRIPTION

[0034] (Insights that form the basis of this disclosure)

[0035] As described in the background art, it is required to use a technology for efficiently producing high-quality crops in limited arable land. In addition, in order to promote crop production, it is required to flexibly utilize natural materials with little environmental load in application. It is particularly desirable to use materials with little fossil energy consumption and little environmental load when manufacturing the material.

[0036] As a technology for promoting crop production, the following prior art is disclosed.

[0037] For example, Patent Document 1 discloses a plant activator that induces systemic resistance in plants and suppresses plant diseases by applying oxy fatty acid derivatives, which are fatty acid metabolites of bacteria, to plants using the salicylic acid pathway. It is also reported that since the oxy fatty acid derivatives are naturally occurring fatty acid oxides, the plant activator has a low environmental load and has little phytotoxicity to the applied plants.

[0038] In addition, for example, Patent Document 2 discloses a polypeptide consisting of a portion of an elongation factor (EF-Tu) widely conserved in plant pathogenic bacteria and non-plant pathogenic bacteria and consisting of a prescribed amino acid sequence, which has the activity of inducing an immune response in Gramineae plants. It is reported that the polypeptide is highly safe for humans, has an extremely low environmental load, and is a relatively low molecular weight polypeptide, so it can be easily and inexpensively manufactured using bacteria. In addition, it is also reported that the peptide and the plant disease resistance inducer containing the polypeptide are not prone to drug resistance mutants and can be applied for a long time.

[0039] In addition, for example, Patent Document 3 discloses the use of a new Paenibacillus bacterium having a plant disease control effect for plant disease control. Specifically, it is reported that the application of Paenibacillus bacterium to plants in various forms, such as spores, vegetative cells, dried cells, or whole cultures or freeze-dried powders thereof, exerts an effect.

[0040] However, in the above-mentioned prior art, the production of plant disease resistance inducing substances using microorganisms, or the purification or extraction of disease resistance inducing substances, etc., are complicated and time-consuming, and costly. In addition, in the above-mentioned prior art, when purifying and extracting plant disease resistance inducing substances, losses such as reduced yield or reduced activity of the plant disease resistance inducing substances occur. On the other hand, when the microorganism itself is inoculated into the plant, the effect is different depending on the combination of the microorganism species used, the plant species that become the object, and the nature of the soil, lacking versatility, and the effect of disease resistance induction is unstable. Therefore, it is expected to develop a material of natural origin that can be produced with cheaper raw materials and simple processes, and has a high plant disease resistance inducing effect.

[0041] The inventors, as the microorganism used in the manufacture of plant disease resistance inducing substances, focus on cyanobacteria. Cyanobacteria (also referred to as cyanobacteria or blue algae) is a group of true bacteria, which decomposes water and produces oxygen through photosynthesis, and fixes CO2 in the air through the energy obtained. Cyanobacteria can also fix nitrogen (N2) in the air according to the species. In this way, cyanobacteria can obtain the required raw materials (that is, nutrients) and most of the energy for the growth and reproduction of thalline from air, water and light, and can cultivate cyanobacteria with cheap raw materials and simple process.

[0042] In addition, as the characteristic of cyanobacteria, it is known that growth and reproduction are fast and light utilization efficiency is high. In addition, genetic manipulation is easy compared with other algae species, so active research and development has been carried out in photosynthetic microorganisms about the material production using cyanobacteria. For example, as an example of material production using cyanobacteria, the production of fuels such as ethanol, isobutanol, alkanes and fatty acids (patent document 4: Japanese Patent Gazette No. 6341676) has been reported. In addition, research and development on the production of materials that become biological nutrient sources is also being carried out. For example, protein is owing to being only synthesized by organisms, and thus requires the development of a technology that produces protein simply and efficiently. As one of the biological species used in this technology, the flexible utilization of cyanobacteria that can utilize light energy and atmospheric CO2 is expected, and active research and development (non-patent document 1: Jie Zhou et al., " Discovery of a super-strong promoter enable efficient production of heterologous proteins in cyanobacteria ", Scientific Reports, Nature Research, 2014, Vol.4, Article No.4500) is being carried out.

[0043] For example, in the technology of above-mentioned non-patent literature 1 record, can realize the effective expression of heterologous gene in cyanobacteria.If use this technology, then can produce desired protein in the cell of cyanobacteria (hereinafter, also referred to as in thalline).But the protein that produces in the cell of cyanobacteria is difficult to be secreted into extracellular, thereby needs the cell of broken cyanobacteria to extract the protein that produces in cell.

[0044] So, the inventors find that, by making the outer membrane of the cell wall of coating cyanobacteria partly break away from the cell wall, thereby the protein produced in the thalline of cyanobacteria and the metabolites in the thalline become easily secreted outside the thalline.And then, the inventors also find that the secretion of cyanobacteria has the disease resistance inducing effect of plant.Thus, can not break the thalline of cyanobacteria, efficiently manufacture the plant disease resistance inducing substance that is secreted outside the thalline.In addition, operations such as extraction become unnecessary, thereby the physiological activity of plant disease resistance inducing substance is not easily impaired, thereby by comprising the plant disease resistance inducing agent of this secretion, can effectively induce the disease resistance of plant.

[0045] Therefore, the plant disease resistance inducer and the plant disease resistance induction method disclosed in the present invention can effectively induce disease resistance in plants. In addition, the method for producing the plant disease resistance inducer disclosed in the present invention can simply and efficiently produce the plant disease resistance inducer.

[0046] (Overview of the present disclosure)

[0047] An overview of one aspect of the present disclosure is as follows.

[0048] One embodiment of the present disclosure relates to a plant disease resistance inducer comprising a secretion of cyanobacteria.

[0049] Thus, since the plant disease resistance inducer contains secretions involved in the induction of disease resistance in plants, it can effectively induce the resistance of plants to diseases.

[0050] For example, in the plant disease resistance inducer according to one embodiment of the present disclosure, the cyanobacteria may be a modified cyanobacteria in which the function of a protein involved in the binding of the outer membrane to the cell wall is inhibited or lost.

[0051] Thus, in the transformation of cyanobacteria, due to the combination of cell wall and outer membrane (that is, binding capacity and bonding force) partially reduced, thus outer membrane becomes easy to partially break away from the cell wall.Therefore, in the transformation of cyanobacteria, the protein and metabolites produced in the thalline (that is, the thalline produces material) become easy to leak outside the outer membrane (that is, outside the thalline).Thus, due to the protein and metabolites produced in the thalline by the transformation of cyanobacteria become easy to be secreted outside the thalline, thus for example the extraction process of the thalline producing material such as the broken thalline becomes unnecessary.Therefore, the physiological activity reduction and yield reduction of the material produced in the thalline that produces material in the thalline, the physiological activity reduction and yield reduction of the material (hereinafter, also referred to as the plant disease resistance inducing material) in the thalline of the transformation of cyanobacteria also become difficult to occur.Therefore, the plant disease resistance inducer that a scheme of the present disclosure relates to can effectively induce the resistance of plants to diseases.

[0052] For example, in the plant disease resistance inducer according to one embodiment of the present disclosure, the protein involved in the binding of the outer membrane to the cell wall may be at least one of an SLH (Surface Layer Homology) domain-retaining outer membrane protein and a cell wall-pyruvate modifying enzyme.

[0053] Thus, in the modified cyanobacteria, for example, the function of at least one of (i) the SLH domain-retaining outer membrane protein combined with the cell wall and the enzyme (i.e., cell wall-pyruvate modifying enzyme) that catalyzes the reaction of the binding sugar chain on the cell wall surface being modified by pyruvate is suppressed or lost, or (ii) the expression of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate modifying enzyme is suppressed. Therefore, the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the binding (i.e., binding amount and binding force) of the covalently bonded sugar chain on the cell wall surface are reduced. Thus, the outer membrane becomes easy to detach from the cell wall in the weak part of the outer membrane and the cell wall. As a result, in the modified cyanobacteria, the combination of the outer membrane and the cell wall is reduced, so that the outer membrane becomes easy to partially detach from the cell wall, and thus the proteins and metabolites produced in the thalline as described above become easy to leak out of the thalline. Thus, the secretion productivity of the plant disease resistance inducing substance produced in the thalline to the outside of the thalline is improved by the modified cyanobacteria. Therefore, the plant disease resistance inducing agent according to one embodiment of the present disclosure contains a plant disease resistance inducing substance efficiently secreted by the modified cyanobacterium, and thus can effectively induce plant resistance to diseases.

[0054] For example, in a plant disease resistance inducer involved in one embodiment of the present invention, the SLH domain-retaining outer membrane protein can be Slr1841 composed of the amino acid sequence shown in sequence number 1, NIES970_09470 composed of the amino acid sequence shown in sequence number 2, Anacy_3458 composed of the amino acid sequence shown in sequence number 3, or a protein whose amino acid sequence is more than 50% identical to any of the above SLH domain-retaining outer membrane proteins.

[0055] Thus, in the modified cyanobacteria, for example, (i) the function of any SLH domain-maintained outer membrane protein shown in the above sequence numbers 1 to 3 or a protein whose amino acid sequence is 50% or more identical to any of the above SLH domain-maintained outer membrane proteins is suppressed or lost, or (ii) the expression of any SLH domain-maintained outer membrane protein shown in the above sequence numbers 1 to 3 or a protein whose amino acid sequence is 50% or more identical to any of the above SLH domain-maintained outer membrane proteins is suppressed. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-maintained outer membrane protein in the outer membrane or a protein having the same function as the SLH domain-maintained outer membrane protein is suppressed or lost, or (ii) the expression level of the SLH domain-maintained outer membrane protein in the outer membrane or a protein having the same function as the SLH domain-maintained outer membrane protein is reduced. As a result, in the modified cyanobacteria, the binding amount and binding force of the binding domain (e.g., SLH domain) of the outer membrane for binding to the cell wall are reduced, so that the outer membrane becomes easy to partially detach from the cell wall. As a result, substances produced in the bacteria become easily leaked out of the bacteria, and thus plant disease resistance inducing substances produced in the bacteria also become easily leaked out of the bacteria. Therefore, the plant disease resistance inducing agent involved in one embodiment of the present disclosure can effectively induce plant disease resistance because it contains plant disease resistance inducing substances effectively secreted by the modified cyanobacteria.

[0056] For example, in a plant disease resistance inducer involved in one embodiment of the present invention, the cell wall-pyruvate modification enzyme can be Slr0688 composed of the amino acid sequence shown in sequence number 4, Synpcc7942_1529 composed of the amino acid sequence shown in sequence number 5, Anacy_1623 composed of the amino acid sequence shown in sequence number 6, or a protein whose amino acid sequence is more than 50% identical to any of the above cell wall-pyruvate modification enzymes.

[0057] Thus, in the modified cyanobacteria, for example, (i) the function of any of the cell wall-pyruvate modification enzymes shown in the above sequence numbers 4 to 6 or the protein whose amino acid sequence is 50% or more identical to any of the above cell wall-pyruvate modification enzymes is inhibited or lost, or (ii) the expression of any of the cell wall-pyruvate modification enzymes shown in the above sequence numbers 4 to 6 or the protein whose amino acid sequence is 50% or more identical to any of the above cell wall-pyruvate modification enzymes is inhibited. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modification enzyme or the protein having the same function as the enzyme is inhibited or lost, or (ii) the expression level of the cell wall-pyruvate modification enzyme or the protein having the same function as the enzyme is reduced. Thus, the covalently bonded sugar chains on the cell wall surface become less susceptible to modification by pyruvate, and thus the binding amount and binding force of the sugar chains of the cell wall to the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane are reduced. As a result, in the modified cyanobacteria, the sugar chain of the covalent bond type on the cell wall surface becomes difficult to be modified by pyruvic acid, so the binding force between the cell wall and the outer membrane is weak, and the outer membrane becomes easy to partially detach from the cell wall. Thus, the substance produced in the thalline becomes easy to be leaked out of the thalline, and the plant disease resistance inducing substance produced in the thalline also becomes easy to be leaked out of the thalline. Therefore, the plant disease resistance inducing agent that one scheme of the present disclosure is related to can effectively induce the disease resistance of plants because it comprises the plant disease resistance inducing substance effectively secreted by the modified cyanobacteria.

[0058] For example, in the plant disease resistance inducer involved in one embodiment of the present disclosure, the cyanobacteria may be a modified cyanobacteria in which the gene expressing the protein involved in the binding of the outer membrane to the cell wall is deleted or inactivated.

[0059] Thus, in the transformation of cyanobacteria, because the expression of the protein participating in the combination of cell wall and outer membrane is suppressed, or because the function of this protein is suppressed or lost, thus the combination of cell wall and outer membrane (that is, binding amount and bonding force) is partially reduced. As a result, in the transformation of cyanobacteria, outer membrane becomes easy to partially break away from the cell wall, thus the protein produced in the thalline and metabolites and other substances produced in the thalline become easy to leak outside the outer membrane, i.e. outside the thalline. Therefore, the secretion productivity of the plant disease resistance induction substance produced in the thalline of the transformation of cyanobacteria improves. Thus, the extraction process of the substance produced in the thalline such as the broken thalline becomes unnecessary, thus the physiological activity reduction and yield reduction of the substance produced in the thalline become difficult to occur, and the physiological activity reduction and yield reduction of the plant disease resistance induction substance produced in the thalline also become difficult to occur. Therefore, the plant disease resistance induction agent that a scheme of the present disclosure relates to can effectively induce the disease resistance of plants.

[0060] For example, in the plant disease resistance inducer according to one embodiment of the present disclosure, the gene expressing a protein involved in the binding of the outer membrane to the cell wall may be at least one of a gene encoding an SLH domain-retaining outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme.

[0061] Thus, in the modified cyanobacteria, at least one gene of the gene encoding the SLH domain-retaining outer membrane protein and the gene encoding the cell wall-pyruvic acid modifying enzyme is deleted or inactivated. Therefore, in the modified cyanobacteria, for example, the expression of at least one of (i) the SLH domain-retaining outer membrane protein and the cell wall-pyruvic acid modifying enzyme is suppressed, or the function of at least one of (ii) the SLH domain-retaining outer membrane protein and the cell wall-pyruvic acid modifying enzyme is suppressed or lost. Therefore, the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the combination (that is, binding amount and binding force) of the covalently bonded sugar chain on the cell wall surface are reduced. Thus, the outer membrane becomes easy to detach from the cell wall at the weak part of the outer membrane combined with the cell wall. As a result, in the modified cyanobacteria, the outer membrane becomes easy to partially detach from the cell wall by reducing the combination of the outer membrane and the cell wall, so that the protein and metabolites produced in the bacteria become easy to leak out of the bacteria, and the plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria. Therefore, the plant disease resistance inducing agent related to one embodiment of the present invention can effectively induce the disease resistance of plants because it contains the plant disease resistance inducing substances efficiently secreted by the modified cyanobacteria.

[0062] For example, in a plant disease resistance inducer involved in one embodiment of the present invention, the gene encoding the SLH domain-retaining outer membrane protein can be slr1841 composed of the base sequence shown in sequence number 7, nies970_09470 composed of the base sequence shown in sequence number 8, anacy_3458 composed of the base sequence shown in sequence number 9, or a gene whose base sequence is more than 50% identical to any of the above genes.

[0063] Thus, in the modified cyanobacteria, any gene encoding the SLH domain-maintaining outer membrane protein shown in the above sequence numbers 7 to 9 or a gene with a base sequence more than 50% identical to any of the above genes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above SLH domain-maintaining outer membrane proteins or a protein having the same function as any of the above proteins is inhibited, or (ii) the function of any of the above SLH domain-maintaining outer membrane proteins or a protein having the same function as any of the above proteins is inhibited or lost. As a result, in the modified cyanobacteria, the binding amount and binding force of the binding domain (such as the SLH domain) used by the outer membrane to bind to the cell wall are reduced, so that the outer membrane becomes easy to partially detach from the cell wall. As a result, the proteins and metabolites produced in the bacteria become easy to leak out of the bacteria, and thus the plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria. Therefore, the plant disease resistance inducing agent according to one embodiment of the present disclosure contains a plant disease resistance inducing substance efficiently secreted by the modified cyanobacteria, and thus can effectively induce disease resistance in plants.

[0064] For example, in a plant disease resistance inducer involved in one embodiment of the present invention, the gene encoding the cell wall-pyruvate modification enzyme can be slr0688 composed of the base sequence shown in sequence number 10, synpcc7942_1529 composed of the base sequence shown in sequence number 11, anacy_1623 composed of the base sequence shown in sequence number 12, or a gene whose base sequence is more than 50% identical to any of the above genes.

[0065] Thus, in the modified cyanobacteria, any gene encoding a cell wall-pyruvate modification enzyme shown in the above sequence numbers 10 to 12 or a gene with a base sequence more than 50% identical to that of the gene encoding any of the above enzymes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modification enzymes or proteins having the same function as any of the above enzymes is inhibited, or (ii) the function of any of the above cell wall-pyruvate modification enzymes or proteins having the same function as any of the above enzymes is inhibited or lost. Thus, the covalently bonded sugar chains on the cell wall surface become less susceptible to modification by pyruvic acid, thereby reducing the amount and binding force of the sugar chains of the cell wall binding to the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane. As a result, in the modified cyanobacteria, the amount of sugar chains used by the cell wall to bind to the outer membrane is reduced by pyruvic acid, thereby the binding force between the cell wall and the outer membrane is weak, and the outer membrane becomes easy to partially detach from the cell wall. As a result, proteins and metabolites produced in the bacteria become easy to leak out of the bacteria, and thus the plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria. Therefore, the plant disease resistance inducing agent involved in one embodiment of the present disclosure can effectively induce plant disease resistance because it contains the plant disease resistance inducing substances effectively secreted by the modified cyanobacteria.

[0066] Furthermore, one embodiment of the present disclosure relates to a method for inducing plant disease resistance using a plant disease resistance inducer comprising a secretion of cyanobacteria.

[0067] According to a method for inducing plant disease resistance according to one embodiment of the present disclosure, since a plant disease resistance inducer containing a secretion involved in inducing plant disease resistance is used, disease resistance of a plant can be effectively induced.

[0068] Furthermore, one embodiment of the present disclosure relates to a method for producing a plant disease resistance inducer, comprising the steps of preparing cyanobacteria and causing the cyanobacteria to secrete a secretion that participates in the induction of plant disease resistance.

[0069] Thus, since the secretion produced in the cyanobacteria and involved in the induction of plant disease resistance can be secreted simply by culturing the cyanobacteria, a plant disease resistance inducer containing the secretion can be easily and efficiently produced.

[0070] Hereinafter, the embodiments will be described in detail with reference to the drawings.

[0071] In addition, the embodiments described below all represent comprehensive or specific examples. The numerical values, materials, steps, and the order of the steps shown in the following embodiments are examples and do not limit the subject matter of the present disclosure. In addition, among the constituent elements in the following embodiments, constituent elements not recorded in the independent claims representing the highest concept are described as arbitrary constituent elements.

[0072] In addition, each figure does not necessarily illustrate strictly. In each figure, the same code|symbol is attached|subjected to the substantially same structure, and the repeated description may be abbreviated or simplified.

[0073] In addition, the numerical ranges below do not only have strict meanings but also include substantially equivalent ranges, for example, the amount of a protein (for example, the number or concentration) or a range thereof is measured.

[0074] In addition, in this specification, both bacterial body and cell refer to one individual of cyanobacteria.

[0075] (Implementation Method)

[0076] In this specification, the homology of the base sequence and the amino acid sequence is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by performing a pairwise analysis using the BLAST algorithm that can be used on the website of NCBI (National Center for Biotechnology Information) (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information about cyanobacterial genes and proteins encoded by the genes is disclosed in, for example, the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequence of the target protein and the base sequence of the genes encoding these proteins can be obtained from these databases.

[0077] [1. Plant disease resistance inducers]

[0078] First, the plant disease resistance inducer involved in this embodiment is described. The plant disease resistance inducer refers to a drug used to induce plant resistance to diseases and prevent plant diseases. Since the plant disease resistance inducer is used for the purpose of preventing diseases, it can also be applied before the period when the disease occurs. As the method of using the plant disease resistance inducer, it can be any of spreading, powdering, dipping, powder coating, coating, fumigation, fumigation, and infusion. As a specific use method, a method of spreading or coating the agent on the plant, a method of dipping the seeds of the plant in a liquid containing the agent, a method of spreading the agent in a farmland where the disease is occurring or may occur, or a method of mixing the agent with the soil, etc. can be listed. In addition, the amount of the plant disease resistance inducer used can be appropriately set according to the type of the target plant, the growth and reproduction stage of the target plant, the nature of the soil, the type of dosage form, the application method, the application period, etc. The plants that become the application objects of the plant disease resistance inducer can list all cultivated plants, which can be monocotyledons or dicotyledons. Examples include, but are not limited to, cruciferous plants such as cabbage, gramineous plants such as rice, corn, barley and wheat, solanaceous plants such as tomato, eggplant, potato and tobacco, cucurbitaceous plants such as cucumber, melon and pumpkin, legumes such as soybean, pea, green bean, alfalfa, and peanut, rosaceous plants such as strawberry, apple and pear, moraceae plants such as mulberry, malvaceae plants such as cotton, umbelliferae plants such as carrot, parsley and celery, Asteraceae plants such as burdock and lettuce, and grapes.

[0079] Plant disease resistance inducing agent comprises the secretion of cyanobacteria.This cyanobacteria is for example, the function of the protein (hereinafter, also referred to as in conjunction with associated protein) that participates in the combination of outer membrane and cell wall in cyanobacteria (hereinafter, also referred to as parental cyanobacteria) is suppressed or lost.In addition, about cyanobacteria (that is, parental cyanobacteria) and transformation cyanobacteria narrates later.

[0080] Plant disease resistance inducers include secretions secreted by the above-mentioned cyanobacteria and involved in the induction of plant disease resistance. The so-called induction of plant disease resistance includes not only direct induction of plant disease resistance, but also indirect (in other words, auxiliary) induction of plant disease resistance. Thus, plant disease resistance inducers can effectively induce plant resistance to diseases, and thus by applying plant disease resistance inducers to plants, the yield and quality of plants can be improved.

[0081] The secretion includes proteins and metabolites produced in the body of cyanobacteria (hereinafter, also referred to as in vivo produced substances). The in vivo produced substances include substances that induce plant resistance to diseases (so-called plant disease resistance inducing substances).

[0082] Intracellular substances produced in the bacterium are, for example, organic substances degrading enzymes such as peptidases, nucleases, or phosphatases, DNA metabolism-related substances such as adenosine or guanosine, intracellular molecules such as p-aminobenzoic acid or spermidine that participate in nucleic acid (e.g., DNA or RNA) synthesis promotion, ketone bodies such as 3-hydroxybutyric acid, or organic acids such as gluconic acid. The secretion of cyanobacteria can be a mixture of these intracellular substances produced in the bacterium.

[0083] [2. Method for producing a plant disease resistance inducer]

[0084] Next, the method for producing the plant disease resistance inducer according to the present embodiment is described with reference to Figure 1 While explaining. Figure 1 This is a flow chart showing an example of a method for producing a plant disease resistance inducer according to the present embodiment.

[0085] The manufacture method of the plant disease resistance inducing agent that the present embodiment is related to comprises the step (step S01) of preparing cyanobacteria and the step (step S02) of making this cyanobacteria secrete the secretion that participates in the disease resistance induction of plant.As mentioned above, cyanobacteria is for example, the function of the protein that participates in the combination of outer membrane and cell wall in cyanobacteria (so-called parent cyanobacteria) is suppressed or lost.Therefore, in step S01, can prepare to transform cyanobacteria.In this case, in step S02, make this transformation cyanobacteria secrete the secretion that participates in the disease resistance induction of plant.As mentioned above, this secretion comprises the above-mentioned protein and metabolite (that is, substance produced in the thalline) produced in the thalline of cyanobacteria.Comprise the substance (that is, plant disease resistance inducing substance) that participates in the disease resistance induction of plant in these thalline substances.

[0086] Hereinafter, each step will be described in more detail.

[0087] In step S01, prepare above-mentioned cyanobacteria.So-called preparation of cyanobacteria, refer to the state that the state of cyanobacteria is adjusted to the state that cyanobacteria can secrete secretions, for example, can recover thalline by the freeze-dried body of cyanobacteria or glycerine stock solution, also can reclaim the cyanobacteria that has secreted plant disease resistance material in step S02.In addition, cyanobacteria is under the situation of transformation cyanobacteria, so-called preparation of cyanobacteria, for example, can be that parental cyanobacteria is carried out genetic modification and make transformation cyanobacteria, can also be that recover thalline by the freeze-dried body of transformation cyanobacteria or glycerine stock solution, also can be that reclaim the transformation cyanobacteria that has secreted plant disease resistance inductive substance in step S02.

[0088] In step S02, make cyanobacteria secrete the secretion that participates in the disease resistance induction of plant.Cyanobacteria in the present embodiment is owing to be the transformation cyanobacteria that the function of the protein that participates in the combination of outer membrane and cell wall in cyanobacteria (that is, parental cyanobacteria) is suppressed or loses, thereby the protein and metabolites that produce in thalline are easily secreted outside the outer membrane (that is, outside thalline).These thalline bodies produce in material and also comprise the material that participates in the disease resistance induction of plant.Therefore, in step S02, by transforming cyanobacteria and cultivating under the condition of regulation, thereby participate in the disease resistance induction of plant and produce in thalline material and be secreted outside thalline.

[0089] The cultivation of cyanobacteria can generally be implemented based on the liquid culture or its transformation method using BG-11 to cultivate (with reference to Table 2). Therefore, the cultivation of transformed cyanobacteria can also be implemented in the same manner. In addition, as the incubation time of the cyanobacteria for the manufacture of plant disease resistance inducers, as long as the time carried out in the mode of accumulating in high concentrations of protein and metabolites under the condition of sufficient proliferation of thalline, for example, can be 1 to 3 days, can also be 4 to 7 days. In addition, the cultural method can be, for example, aeration stirring culture or shaking culture.

[0090] By cultivating under the above-mentioned conditions, the blue bacteria are transformed to produce protein and metabolites (that is, substances produced in the body) in the thalline, and the substances produced in the thalline are secreted into the culture fluid. The substances produced in the thalline include substances produced in the body that participate in the disease resistance induction of plants (that is, plant disease resistance inducing substances). In the case of the substances produced in the body that are secreted into the culture fluid, by filtering the culture fluid or centrifugation, etc., solid components such as cells (that is, thalline) can be removed from the culture fluid, and the culture supernatant is recovered. According to the manufacture method of the plant disease resistance inducing agent involved in the present embodiment, because the secretion that includes substances produced in the body that participate in the disease resistance induction of plants (that is, plant disease resistance inducing substances) is secreted into the cell of the blue bacteria, it is not necessary to break cells in order to reclaim the plant disease resistance inducing substances. Therefore, the remaining transformed blue bacteria after the recovery of the plant disease resistance inducing substances can be reused, and the manufacture of the plant disease resistance inducing agent is carried out.

[0091] In addition, the recovery method of the plant disease resistance inducing substance secreted into the culture solution is not limited to the above-mentioned example, can cultivate and transform cyanobacteria, reclaim the plant disease resistance inducing substance in the culture solution.For example, by using the permeation membrane that makes protein permeate, thereby can reclaim the plant disease resistance inducing substance that has permeated the permeation membrane.Like this, owing to can cultivate and transform cyanobacteria and reclaim the plant disease resistance inducing substance in the culture solution, thereby the processing of removing the thalline of transformation cyanobacteria from the culture solution becomes unnecessary.Therefore, can more simply and efficiently manufacture plant disease resistance inducing agent.

[0092] In addition, by being unnecessary by the recovery process of the thalline of culture fluid and the fragmentation process of the thalline, thereby can reduce the damage and the stress that transformation cyanobacteria is subjected to.Therefore, the secretion productivity of the plant disease resistance inducing substance of transformation cyanobacteria becomes difficult to reduce, can use transformation cyanobacteria for a longer time.

[0093] As described above, by using the modified cyanobacteria in this embodiment, a plant disease resistance inducer can be obtained simply and efficiently.

[0094] Hereinafter, cyanobacteria (so-called parent cyanobacteria) and modified cyanobacteria will be described. Hereinafter, parent cyanobacteria will be referred to as cyanobacteria, and cyanobacteria in the present embodiment will be referred to as modified cyanobacteria.

[0095] [3. Cyanobacteria]

[0096] Cyanobacteria, also known as blue algae or blue bacteria, are a group of prokaryotes that capture light energy with chlorophyll and perform photosynthesis while electrolyzing water to produce oxygen using the energy obtained. Cyanobacteria are rich in diversity. For example, regarding cell shape, there are single-celled species such as Synechocystis sp. PCC 6803 and multi-celled filamentous species such as Anabaena sp. PCC 7120. Regarding the growth and reproduction environment, there are also thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcuse longatus, and freshwater species such as Synechocystis. In addition, there are many species such as Microcystis aeruginosa that have air sacs and produce toxins, and species such as Gloeobacterviolaceus that do not have thylakoids but have proteins called phycobilisomes as light-harvesting antennas in the plasma membrane that have unique characteristics.

[0097] Figure 2 is a schematic diagram showing the cell surface layers of cyanobacteria. Figure 2As shown, the cell surface layer of cyanobacteria is composed of a protoplasmic membrane (also referred to as an inner membrane 1), a peptidoglycan 2, and a lipid membrane 5 forming the outermost layer of the cell in order from the inside. Peptidoglycan 2 is covalently bonded with a sugar chain 3 composed of glucosamine and mannosamine, etc., and pyruvic acid is bound to these covalently bonded sugar chains 3 (non-patent literature 2: Jurgens and Weckesser, 1986, J. Bacteriol., 168: 568-573). In this specification, the cell wall 4 is referred to as the cell wall 4, including the peptidoglycan 2 and the covalently bonded sugar chain 3. In addition, the gap between the protoplasmic membrane (i.e., the inner membrane 1) and the outer membrane 5 is called the periplasm, and there are various enzymes involved in the decomposition of proteins or the formation of a three-dimensional structure, the decomposition of lipids or nucleic acids, or the uptake of nutrients outside the cell.

[0098] SLH domain-retaining outer membrane proteins (e.g., Slr1841 in the figure) are composed of a C-terminal region integrated into the lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 protruding from the lipid membrane, and are widely distributed in bacteria belonging to the Negativicutes class (Firmicutes), which is a group of cyanobacteria and Gram-negative bacteria (Non-patent document 3: Kojima et al., 2016, Biosci. Biotech. Biochem., 10: 1954-1959). The region integrated into the lipid membrane (i.e., outer membrane 5) forms a channel for the outer membrane to allow hydrophilic substances to pass through, while the SLH domain 7 has the function of binding to the cell wall 4 (Non-patent document 4: Kowata et al., 2017, J. Bacteriol., 199: e00371-17). In order for the SLH domain 7 to bind to the cell wall 4, the covalently bonded sugar chain 3 in the peptidoglycan 2 needs to be modified with pyruvic acid (Non-patent document 5: Kojima et al., 2016, J. Biol. Chem., 291: 20198-20209). Examples of genes encoding SLH domain-retaining outer membrane proteins 6 include slr1841 or slr1908 retained by Synechocystis sp. PCC 6803, or oprB retained by Anabaena sp. 90.

[0099] An enzyme that catalyzes the pyruvate modification reaction of the covalently bonded sugar chain 3 in the peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modification enzyme 9) was identified in Bacillus anthracis, a Gram-positive bacterium, and was named CsaB (Non-patent document 6: Mesnage et al., 2000, EMBO J., 19: 4473-4484). Among cyanobacteria whose genome base sequences have been published, many species retain genes encoding homologous proteins with an amino acid sequence identity of 30% or more to CsaB. Examples include slr0688 retained by Synechocystis sp. PCC 6803 and syn7502_03092 retained by Synechococcus sp. 7502.

[0100] In cyanobacteria, CO2 fixed by photosynthesis is converted into various amino acids and precursors of intracellular molecules through multi-stage enzyme reactions. Using these as raw materials, proteins and metabolites are synthesized in the cytoplasm of cyanobacteria. Some of these proteins and metabolites function in the cytoplasm, while others are transported from the cytoplasm to the periplasm and function in the periplasm. However, cases of active secretion of proteins and metabolites outside the cell have not been reported in cyanobacteria so far.

[0101] Since cyanobacteria have high photosynthesis ability, they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, in cyanobacteria, the outer membrane 5 only has very little Figure 2 For example, in Synechocystis sp. PCC 6803, the organic matter channel protein 8 that permeates organic matter is only about 4% of the total protein in the outer membrane 5. On the other hand, in order to efficiently take in inorganic ions required for growth and reproduction into the cell, cyanobacteria have a large number of organic matter channel proteins such as Slr1270 in the outer membrane 5. Figure 2 Ion channel proteins that only permeate inorganic ions, such as the SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) of Synechocystis sp. PCC 6803, for example, account for about 80% of the total proteins in the outer membrane 5.

[0102] As described above, in cyanobacteria, since there are very few channels in the outer membrane 5 through which organic substances such as proteins can pass, it is generally considered difficult to actively secrete proteins and metabolites produced in the bacteria to the outside of the bacteria.

[0103] [4. Transformation of cyanobacteria]

[0104] Next, regarding the transformation of cyanobacteria, Figure 2 While explaining.

[0105] The cyanobacteria in the present embodiment is a modified cyanobacteria in which the function of the protein (so-called binding-related protein) participating in the combination of the outer membrane 5 and the cell wall 4 in the cyanobacteria (so-called parent cyanobacteria) is suppressed or lost. More specifically, for example, the total amount of the protein (i.e., binding-related protein) participating in the combination of the outer membrane 5 and the cell wall 4 in the modified cyanobacteria is suppressed to more than 30% and less than 70% of the total amount of the protein in the parent strain (i.e., the parent cyanobacteria). For example, the so-called "the total amount of binding-related protein is suppressed to 30% of the total amount of the protein in the parent strain" refers to the state in which 70% of the total amount of the protein in the parent strain is lost and 30% remains. Thus, in the modified cyanobacteria, the combination (e.g., binding amount and binding force) of the outer membrane 5 and the cell wall 4 is partially reduced, so that the outer membrane 5 becomes easy to partially break away from the cell wall 4. Therefore, in the modified cyanobacteria, the secretion productivity of the substances produced in the thalli such as proteins and metabolites produced in the thalli to the thalli outside the thalli is improved. As mentioned above, comprise the induction of the disease resistance of participating in plant in the thalline material and produce material in the thalline (that is, plant disease resistance inducing substance).Therefore, in the transformation cyanobacteria, the secretion productivity of the plant disease resistance inducing substance produced in the thalline to the plant disease resistance inducing substance outside the thalline is also improved.In addition, owing to not needing to break the thalline and reclaim the plant disease resistance inducing substance, thus even after reclaiming the plant disease resistance inducing substance, also can reuse the transformation cyanobacteria.In addition, in this manual, transforming cyanobacteria to make protein and metabolite in the thalline is called generation, and the secretion of the protein and metabolite produced to the thalline is called secretion production outside the thalline.

[0106] The protein involved in the binding of the outer membrane 5 to the cell wall 4 can be, for example, at least one of the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9. In the present embodiment, in the modified cyanobacteria, for example, the function of at least one of the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 is inhibited or lost. For example, in the modified cyanobacteria, the function of at least one of the SLH domain-maintained outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 can be inhibited or lost, and (ii) the expression of the SLH domain-maintained outer membrane protein 6 bound to the cell wall 4 and the expression of the enzyme (i.e., the cell wall-pyruvate modification enzyme 9) that catalyzes the pyruvate modification reaction of the bound sugar chain on the surface of the cell wall 4 can be inhibited. As a result, the binding (i.e., the binding amount and binding force) of the SLH domain 7 of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 and the covalently bonded sugar chain 3 on the surface of the cell wall 4 is reduced. Therefore, the outer membrane 5 becomes easy to detach from the cell wall 4 in these weakly bound parts. The outer membrane 5 partially detaches from the cell wall 4, so that the substances produced in the cells of the modified cyanobacteria, especially the proteins and metabolites in the periplasm, become easy to leak out of the cells (outside the outer membrane 5). Thus, the secretion productivity of the plant disease resistance inducing substances produced in the bacteria to the outside of the bacteria by the modified cyanobacteria is improved.

[0107] The following describes in more detail a cyanobacterium modified such that the outer membrane 5 is partially separated from the cell wall 4 by suppressing the function of at least one of the binding-related proteins of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9.

[0108] The type of cyanobacteria (i.e., parent cyanobacteria) that becomes the parent microorganism of the modified cyanobacteria in the present embodiment, before at least one of the expression of SLH domain-retaining outer membrane protein 6 and the expression of cell wall-pyruvate modifying enzyme 9 is inhibited or lost, is not particularly limited, and can be all types of cyanobacteria. For example, the parent cyanobacteria can be Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, wherein it can be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0109] The amino acid sequences of the SLH domain-retaining outer membrane protein 6 and the enzyme that catalyzes the cell wall-pyruvate modification reaction (i.e., cell wall-pyruvate modification enzyme 9) in these parental cyanobacteria, the base sequences of the genes encoding these binding-related proteins, and the locations of the genes on the chromosomal DNA or plasmid can be confirmed in the above-mentioned NCBI database and Cyanobase.

[0110] In addition, the SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 whose functions are inhibited or lost in the modified cyanobacteria involved in this embodiment can be substances of any parent cyanobacteria as long as they are retained by the parent cyanobacteria, and are not restricted by the location of the genes encoding them (for example, on chromosomal DNA or on a plasmid).

[0111] For example, when the parent cyanobacteria is Synechocystis, the SLH domain-maintaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042, etc., when the parent cyanobacteria is Synechococcus, it may be NIES970_09470, etc., when the parent cyanobacteria is Anabaena, it may be Anacy_5815 or Anacy_3458, etc., when the parent cyanobacteria is Microcystis, it may be A0A0F6U6F8_MICAE, etc., when the parent cyanobacteria is Cyanothece, It can be A0A3B8XX12_9CYAN etc., when the parent cyanobacteria is Leptolyngbya, it can be A0A1Q8ZE23_9CYAN etc., when the parent cyanobacteria is Calothrix, A0A1Z4R6U0_9CYAN can be mentioned, when the parent cyanobacteria is Nostoc, it can be A0A1C0VG86_9NOSO etc., when the parent cyanobacteria is Crocosphaera, it can be B1WRN6_CROS5 etc., when the parent cyanobacteria is Pleurocapsa, it can be K9TAE4_9CYAN etc.

[0112] More specifically, the SLH domain-retaining outer membrane protein 6 may be, for example, Slr1841 (sequence number 1) of Synechocystis sp. PCC6803, NIES970_09470 (sequence number 2) of Synechococcus sp. NIES-970, or Anacy_3458 (sequence number 3) of Anabaena cylindrica PCC 7122. In addition, it may be a protein having an amino acid sequence that is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

[0113] Thus, in the modified cyanobacteria, for example, (i) the function of any SLH domain-maintained outer membrane protein 6 shown in the above sequence numbers 1 to 3 or a protein having an amino acid sequence of 50% or more identical to any of the above SLH domain-maintained outer membrane proteins 6 can be inhibited or lost, and (ii) the expression of any SLH domain-maintained outer membrane protein 6 shown in the above sequence numbers 1 to 3 or a protein having an amino acid sequence of 50% or more identical to any of the above SLH domain-maintained outer membrane proteins 6 can be inhibited. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-maintained outer membrane protein 6 is inhibited or lost, or (ii) the expression level of the SLH domain-maintained outer membrane protein 6 in the outer membrane 5 or a protein having the same function as the SLH domain-maintained outer membrane protein 6 is reduced. As a result, in the modified cyanobacteria, the amount and strength of the binding domain (e.g., SLH domain 7) of the outer membrane 5 for binding to the cell wall 4 are reduced, and thus the outer membrane 5 becomes easy to partially detach from the cell wall 4. Thus, in the modified cyanobacteria, substances produced in the bacteria become easy to leak out of the bacteria, and thus plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria.

[0114] Generally, if the amino acid sequence of a protein is more than 30% identical, the homology of the three-dimensional structure of the protein is high, so it can be said that the possibility of having the same function as the protein is high. Therefore, as the SLH domain-retaining outer membrane protein 6 whose function is inhibited or lost, it can be, for example, a protein or polypeptide composed of an amino acid sequence having an identity of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further more preferably more than 80%, and preferably more than 90% with any of the amino acid sequences of the SLH domain-retaining outer membrane protein 6 shown in the above-mentioned sequence numbers 1 to 3, and having the function of binding to the covalently bonded sugar chain 3 of the cell wall 4.

[0115] In addition, for example, cell wall-pyruvate modification enzyme 9 can be Slr0688 when the parent cyanobacterium is Synechocystis, can be Syn7502_03092 or Synpcc7942_1529 when the parent cyanobacterium is Synechococcus, can be ANA_C20348 or Anacy_1623 when the parent cyanobacterium is Anabaena, can be CsaB (NCBI access ID: TRU80220) when the parent cyanobacterium is Microcystis, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, can be CsaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Anabaena. When the parent cyanobacterium is Spirulina, it can be CsaB (NCBI access ID: WP_026079530.1) and the like; when the parent cyanobacterium is Calothrix, it can be CsaB (NCBI access ID: WP_096658142.1) and the like; when the parent cyanobacterium is Nostoc, it can be CsaB (NCBI access ID: WP_099068528.1) and the like; when the parent cyanobacterium is Crocosphaera, it can be CsaB (NCBI access ID: WP_012361697.1) and the like; when the parent cyanobacterium is Pleurocapsa, it can be CsaB (NCBI access ID: WP_036798735) and the like.

[0116] More specifically, the cell wall-pyruvate modification enzyme 9 may be, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. PCC 7942, or Anacy_1623 (SEQ ID NO: 6) of Anabaena cylindrica PCC 7122. In addition, it may be a protein having an amino acid sequence 50% or more identical to that of these cell wall-pyruvate modification enzymes 9.

[0117] Thus, in the modified cyanobacteria, for example, (i) the function of any of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having 50% or more of the same amino acid sequence as any of the cell wall-pyruvate modification enzymes 9 can be inhibited or lost, and (ii) the expression of any of the cell wall-pyruvate modification enzymes 9 shown in the above sequence numbers 4 to 6 or a protein having 50% or more of the same amino acid sequence as any of the cell wall-pyruvate modification enzymes 9 can be inhibited. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modification enzyme 9 or a protein having the same function as the enzyme is inhibited or lost, or (ii) the expression level of the cell wall-pyruvate modification enzyme 9 or a protein having the same function as the enzyme is reduced. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvate, and thus the binding amount and binding force of the sugar chain 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 are reduced. Therefore, in the modified cyanobacteria involved in the present embodiment, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less likely to be modified by pyruvic acid, so the binding force between the cell wall 4 and the outer membrane 5 is weak, and the outer membrane 5 becomes easy to partially detach from the cell wall 4. As a result, in the modified cyanobacteria, substances produced in the bacteria become easy to leak out of the bacteria, and thus the plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria.

[0118] In addition, as described above, if the amino acid sequence of a protein is 30% or more identical, it can be said that there is a high possibility that it has the same function as the protein. Therefore, the cell wall-pyruvate modification enzyme 9 whose function is inhibited or lost can be, for example, a protein or polypeptide composed of an amino acid sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further more preferably 80% or more, and further preferably 90% or more to any of the amino acid sequences of the cell wall-pyruvate modification enzyme 9 shown in the above sequence numbers 4 to 6, and having a function of catalyzing a reaction in which the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 is modified with pyruvic acid.

[0119] In addition, in the present specification, the inhibition or loss of the function of the SLH domain-retaining outer membrane protein 6 means the inhibition or loss of the ability of the protein to bind to the cell wall 4, the inhibition or loss of the transport of the protein to the outer membrane 5, or the inhibition or loss of the ability of the protein to be integrated into the outer membrane 5 and perform its function.

[0120] Inhibiting or losing the function of the cell wall-pyruvate modification enzyme 9 means inhibiting or losing the function of the protein to modify the covalently bonded sugar chain 3 of the cell wall 4 with pyruvate.

[0121] The means for inhibiting or losing the functions of these proteins are not particularly limited as long as they are means commonly used in inhibiting or losing the functions of proteins. The means may be, for example, deletion or inactivation of the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibition of transcription of these genes, inhibition of translation of the transcription products of these genes, or administration of inhibitors that specifically inhibit these proteins.

[0122] In the present embodiment, gene deletion or inactivation of the protein that expresses the combination of outer membrane 5 and cell wall 4 is participated in transformation in cyanobacteria.Thus, transformation cyanobacteria is suppressed because the expression of the protein that participates in the combination of cell wall 4 and outer membrane 5, or because the function of this protein is suppressed or lost, thereby the combination (that is, binding capacity and bonding force) of cell wall 4 and outer membrane 5 is partially reduced.As a result, in transformation cyanobacteria, because outer membrane 5 becomes easy to partly break away from cell wall 4, thereby transformation cyanobacteria produces in thalline protein and metabolite etc. and produces material in thalline and becomes easy to leak outside outer membrane 5, i.e. outside thalline.Therefore, transformation cyanobacteria will secrete the plant disease resistance inducing substance produced in thalline to the secretion productivity of the plant disease resistance inducing substance outside thalline to improve.Thus, the extraction process of producing material in thalline such as broken thalline becomes unnecessary, thereby the physiological activity reduction and yield reduction of producing material in thalline become difficult to occur. Therefore, the physiological activity reduction and yield reduction of the plant disease resistance inducing substance produced in the bacterium are also less likely to occur, thereby being able to manufacture a plant disease resistance inducing agent with improved plant disease resistance inducing effect. In addition, since the extraction process of the above-mentioned substance produced in the bacterium becomes unnecessary, even after the substance is recovered, the modified cyanobacteria can be reused to produce the plant disease resistance inducing substance.

[0123] The gene expressing the protein involved in the combination of the outer membrane 5 and the cell wall 4 can be, for example, at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. In the modified cyanobacteria, at least one of the genes encoding the SLH domain-maintaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 is deleted or inactivated. Therefore, in the modified cyanobacteria, for example, the expression of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 (i) is inhibited, or the function of at least one of the SLH domain-maintaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 (ii) is inhibited or lost. Therefore, the SLH domain 7 of the SLH domain-maintaining outer membrane protein 6 in the outer membrane 5 and the covalently bonded sugar chain 3 on the surface of the cell wall 4 (i.e., the binding amount and binding force) are reduced. Thus, in the part where the outer membrane 5 is weakly bound to the cell wall 4, the outer membrane 5 becomes easy to detach from the cell wall 4. As a result, the outer membrane 5 of the modified cyanobacteria is reduced by the combination of the outer membrane 5 and the cell wall 4, so that the outer membrane 5 becomes easy to partially break away from the cell wall 4, and thus the protein and metabolites produced in the bacterium become easy to leak out of the bacterium. Thus, the plant disease resistance inducing substance produced in the bacterium of the modified cyanobacterium also becomes easy to leak out of the bacterium.

[0124] In the present embodiment, in order to inhibit or lose the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modification enzyme 9 in cyanobacteria, for example, the transcription of at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modification enzyme 9 can be inhibited.

[0125] For example, the gene encoding the SLH domain-maintaining outer membrane protein 6 can be slr1841, slr1908, or slr0042 when the parent cyanobacterium is Synechocystis, can be nies970_09470 when the parent cyanobacterium is Anabaena, can be anacy_5815 or anacy_3458 when the parent cyanobacterium is Microcystis, can be A0A0F6U6F8_MICAE when the parent cyanobacterium is Cyanothece, can be For example, A0A3B8XX12_9CYAN, A0A1Q8ZE23_9CYAN, A0A1Z4R6U0_9CYAN, A0A1C0VG86_9NOSO, B1WRN6_CROS5, and K9TAE4_9CYAN when the parent cyanobacteria is Crocosphaera, and Pleurocapsa, etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0126] More specifically, the gene encoding SLH domain-retaining outer membrane protein 6 can be slr1841 (sequence number 7) of Synechocystis sp. PCC 6803, nies970_09470 (sequence number 8) of Synechococcus sp. NIES-970, anacy_3458 (sequence number 9) of Anabaena cylindrica PCC 7122, or a gene whose amino acid sequence is more than 50% identical to these genes.

[0127] Thus, in the modified cyanobacteria, any gene encoding the SLH domain-maintaining outer membrane protein 6 shown in the above sequence numbers 7 to 9 or a gene having a base sequence more than 50% identical to any of the above genes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any of the above proteins is inhibited, or (ii) the function of any of the above SLH domain-maintaining outer membrane proteins 6 or a protein having the same function as any of the above proteins is inhibited or lost. As a result, in the modified cyanobacteria, the binding amount and binding force of the binding domain (e.g., SLH domain 7) used for the outer membrane 5 to bind to the cell wall 4 are reduced, so that the outer membrane 5 becomes easy to partially detach from the cell wall 4. As a result, the proteins and metabolites produced in the bacteria become easy to leak out of the bacteria, and thus the plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria.

[0128] As described above, if the amino acid sequence of a protein is more than 30% identical, it can be said that there is a high possibility that it has the same function as the protein. Therefore, if the base sequence of a gene encoding a protein is more than 30% identical, it can be considered that there is a high possibility that a protein with the same function as the protein is expressed. Therefore, as a gene encoding an SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost, it can be, for example, a base sequence having an identity of more than 40%, preferably more than 50%, more preferably more than 60%, further preferably more than 70%, further more preferably more than 80%, and preferably more than 90% with any of the base sequences of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in the above-mentioned sequence numbers 7 to 9, and a gene encoding a protein or polypeptide having a function of binding to a covalently bonded sugar chain 3 of a cell wall 4.

[0129] In addition, for example, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 when the parent cyanobacterium is Synechocystis, syn7502_03092 or synpcc7942_1529 when the parent cyanobacterium is Synechococcus, ana_C20348 or anacy_1623 when the parent cyanobacterium is Anabaena, csaB (NCBI access ID: TRU80220) when the parent cyanobacterium is Microcystis, csaB (NCBI access ID: WP_107667006.1) when the parent cyanobacterium is Cyanothece, and csaC (NCBI access ID: WP_107667006.2) when the parent cyanobacterium is Cyanothece. When the cyanobacteria is Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc., when the parent cyanobacteria is Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., when the parent cyanobacteria is Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc., when the parent cyanobacteria is Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., when the parent cyanobacteria is Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735), etc. The base sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0130] More specifically, the gene encoding cell wall-pyruvate modification enzyme 9 may be slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or anacy_1623 (SEQ ID NO: 12) of Anabaena cylindrica PCC 7122. In addition, the gene may be one having a base sequence 50% or more identical to these genes.

[0131] Thus, in the modified cyanobacteria, any gene encoding the cell wall-pyruvate modification enzyme 9 shown in the above sequence numbers 10 to 12 or a gene with a base sequence more than 50% identical to that of the gene encoding any of the above enzymes is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modification enzymes 9 or a protein having the same function as any of the above enzymes is inhibited, or (ii) the function of any of the above cell wall-pyruvate modification enzymes 9 or a protein having the same function as any of the above enzymes is inhibited or lost. Thus, the covalently bonded sugar chain 3 on the surface of the cell wall 4 becomes less susceptible to modification by pyruvic acid, thereby reducing the binding amount and binding force of the sugar chain 3 of the cell wall 4 and the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. Therefore, in the modified cyanobacteria involved in the present embodiment, the amount of sugar chain 3 of the cell wall 4 used for binding to the outer membrane 5 modified by pyruvic acid is reduced, so the binding force between the cell wall 4 and the outer membrane 5 is weak, and the outer membrane 5 becomes easy to partially detach from the cell wall 4. As a result, proteins and metabolites produced in the bacteria become easy to leak out of the bacteria, and thus plant disease resistance inducing substances produced in the bacteria also become easy to leak out of the bacteria.

[0132] As described above, if the base sequence of the gene encoding the protein is 30% or more identical, it can be considered that there is a high possibility that a protein having the same function as the protein is expressed. Therefore, as a gene encoding a cell wall-pyruvate modification enzyme 9 whose function is suppressed or lost, for example, it can be composed of a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, further preferably 70% or more, further more preferably 80% or more, and preferably 90% or more with any of the base sequences of the genes encoding the cell wall-pyruvate modification enzyme 9 shown in the above-mentioned sequence numbers 10 to 12, and encoding a protein or polypeptide having a function of catalyzing a reaction in which the covalently bonded sugar chain 3 of the peptidoglycan 2 of the cell wall 4 is modified with pyruvic acid.

[0133] [5. Method for producing modified cyanobacteria]

[0134] Next, the method for producing the modified cyanobacteria in the present embodiment will be described. The method for producing the modified cyanobacteria includes the step of inhibiting or losing the function of a protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacteria.

[0135] In the present embodiment, the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 .

[0136] In addition, the means for inhibiting or losing the function of a protein is not particularly limited and may be, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering inhibitors that specifically inhibit these proteins.

[0137] The means for deleting or inactivating the above-mentioned gene can be, for example, the introduction of mutations into one or more bases in the base sequence of the gene, the replacement of the base sequence with other base sequences or the insertion of other base sequences, or the deletion of part or all of the base sequence of the gene.

[0138] The means for inhibiting the transcription of the above-mentioned gene can be, for example, introduction of mutations into the promoter region of the gene, inactivation of the promoter caused by substitution to other base sequences or insertion of other base sequences, or CRISPR interference method (non-patent document 7: Yao et al., ACS Synth. Biol., 2016, 5: 207-212), etc. The specific method of the above-mentioned introduction of mutations or substitution or insertion of base sequences can be, for example, ultraviolet irradiation, site-directed mutagenesis, or recombination method, etc.

[0139] In addition, means for inhibiting the translation of the transcription products of the above genes include, for example, RNA (ribonucleic acid) interference method and the like.

[0140] By using any of the above means, the function of the protein involved in the binding of the outer membrane 5 and the cell wall 4 in the cyanobacteria can be inhibited or lost, thereby producing a modified cyanobacterium.

[0141] Thus, in the transformation cyanobacteria manufactured by above-mentioned manufacture method, the combination (that is, binding capacity and bonding force) of cell wall 4 and adventitia 5 partially reduces, thereby adventitia 5 becomes easy to partially break away from cell wall 4.As a result, in transformation cyanobacteria, the protein produced in thalline and metabolites etc. thalline produce material and become easy to leak outside adventitia 5 (that is, outside thalline), thereby participate in the material (that is, plant disease resistance inducing substance) of plant disease resistance induction and also become easy to leak outside thalline.Therefore, according to the manufacture method of transformation cyanobacteria in the present embodiment, the transformation cyanobacteria that the secretion productivity of plant disease resistance inducing substance has improved can be provided.

[0142] In addition, in the transformation cyanobacteria manufactured by the manufacture method among the present embodiment, the plant disease resistance inducing substance that produces in the thalline leaks out into outside the thalline, thereby does not need to break the thalline in order to reclaim this material.For example, as long as the transformation cyanobacteria is cultivated under suitable condition, then reclaim the plant disease resistance inducing substance that is secreted into the nutrient solution, thereby also can cultivate the transformation cyanobacteria while reclaiming the plant disease resistance inducing substance in the nutrient solution.Therefore, if use the transformation cyanobacteria that obtains by this manufacture method, then can implement the production of efficient microbiological plant disease resistance inducing substance.Therefore, according to the manufacture method of the transformation cyanobacteria among the present embodiment, even can provide the transformation cyanobacteria that the utilization efficiency that can be reused is high after reclaiming the plant disease resistance inducing substance.

[0143] [6. Methods for inducing plant disease resistance]

[0144] The plant disease resistance induction method involved in this embodiment uses the above-mentioned plant disease resistance inducer. As described above, the plant disease resistance inducer involved in this embodiment is a plant disease resistance inducer with improved plant disease resistance induction effect, so by using the above-mentioned plant disease resistance inducer, plant disease resistance can be effectively induced.

[0145] The above-mentioned plant disease resistance inducer can certainly be used directly, or it can be concentrated or diluted and used. When the plant disease resistance inducer is applied to plants, the concentration and application method of the plant disease resistance inducer can be appropriately determined according to the type of plant, the nature of the soil, and the purpose, etc. The plant disease resistance inducer, for example, can be the culture solution of the transformed cyanobacteria itself, or it can be a solution in which the thalline of the transformed cyanobacteria is removed from the culture solution, or it can be an extract obtained by extracting the culture solution through membrane technology, etc. In addition, the dosage form of the plant disease resistance inducer can be liquid, or it can be a powder obtained by drying the liquid plant disease resistance inducer through technologies such as spray drying. In addition, the application method of the plant disease resistance inducer to plants can be, for example, spraying, watering, or mixing of plants or soil, etc. More specifically, for example, a few milliliters can be added to the root of the plant body once a week for each plant body, or it can be spread on the leaves.

[0146] Example

[0147] Hereinafter, the modified cyanobacteria, the method for producing the modified cyanobacteria, the plant disease resistance inducer, the method for inducing plant disease resistance, and the method for producing the plant disease resistance inducer of the present disclosure will be specifically described by way of examples, but the present disclosure is not limited to the following examples and is not subject to any limitation of the following examples.

[0148] In the following examples, as a method for partially separating the outer membrane of cyanobacteria from the cell wall, the expression of the slr1841 gene encoding the SLH domain-maintaining outer membrane protein (Example 1) and the expression of the slr0688 gene encoding the cell wall-pyruvate modifying enzyme (Example 2) were inhibited, and two kinds of modified cyanobacteria were produced. Then, the secretion productivity of the protein of these modified cyanobacteria was measured and the secreted in vivo production substances (here, proteins and intracellular metabolites) were identified. In addition, the cyanobacterial species used in this example is Synechocystis sp.PCC 6803 (hereinafter, only referred to as "cyanobacteria").

[0149] (Example 1)

[0150] In Example 1, a modified cyanobacterium in which the expression of the slr1841 gene encoding the SLH domain-retaining outer membrane protein was suppressed was produced.

[0151] (1) Construction of a cyanobacterial strain in which the expression of the slr1841 gene is suppressed

[0152] As a gene expression inhibition method, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method is used. In this method, the expression of slr1841 gene can be inhibited by introducing the gene encoding dCas9 protein (hereinafter referred to as dCas9 gene) and slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria.

[0153] The construction of gene expression inhibition using this method is as follows.

[0154] First, a complex is formed between a Cas9 protein lacking nuclease activity (dCas9) and an sgRNA (slr1841_sgRNA) that complementarily binds to the base sequence of the slr1841 gene.

[0155] Next, the complex recognizes the slr1841 gene on the chromosomal DNA of the cyanobacteria and specifically binds to the slr1841 gene. The binding forms a steric hindrance, thereby inhibiting the transcription of the slr1841 gene. As a result, the expression of the slr1841 gene of the cyanobacteria is inhibited.

[0156] Hereinafter, the method of introducing the above-mentioned two genes into the chromosomal DNA of cyanobacteria will be specifically described.

[0157] (1-1) Introduction of dCas9 gene

[0158] Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (reference to non-patent literature 7) as a template, the primers psbA1-Fw (sequence number 13) and psbA1-Rv (sequence number 14) described in Table 1 were used to amplify the dCas9 gene and the operating element gene for controlling the expression of the dCas9 gene, as well as the spectinomycin resistance marker gene that serves as a mark for gene introduction by the PCR (Polymerase Chain Reaction) method. In addition, in the LY07 strain, the above-mentioned three genes are inserted into the psbA1 gene on the chromosomal DNA in a connected state, so that they can be amplified as one DNA fragment by the PCR method. Here, the resulting DNA fragment is described as a "psbA1::dCas9 box". Using the In-Fusion PCR cloning method (registered trademark), the psbA1::dCas9 box is inserted into the pUC19 plasmid to obtain the pUC19-dCas9 plasmid.

[0159]

[0160] The obtained pUC19-dCas9 plasmid 1 μg was mixed with cyanobacterial culture fluid (cell concentration OD730 = about 0.5), and the pUC19-dCas9 plasmid was introduced into the cells of cyanobacteria by natural transformation. The transformed cells were grown and propagated on BG-11 agar culture containing 20 μg / mL of spectinomycin for screening. In the screened cells, homologous recombination occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and the psbA1 downstream fragment region on the pUC19-dCas9 plasmid. Thus, a Synechocystis dCas9 strain with a dCas9 box inserted in the psbA1 gene region was obtained. In addition, the composition of the BG-11 culture used is shown in Table 2.

[0161] Table 2

[0162] Element Content (mg / L) EDTA-Na 1 Ammonium Ferric Citrate 6 <![CDATA[NaNO3]]> 1500 <![CDATA[MgSO4]]> 75 <![CDATA[K2HPO4]]> 39 <![CDATA[CaCl2]]> 28.6 <![CDATA[H3BO4]]> 2.8 <![CDATA[MnCl2]]> 1.8 <![CDATA[ZnSO4]]> 0.2 <![CDATA[CuSO4]]> 0.08 <![CDATA[Na2MoO4]]> 0.02 <![CDATA[Co(NO3)2]]> 0.005 TES-KOH (pH 7.5) 4580

[0163] (1-2) Introduction of slr1841_sgRNA gene

[0164] In the CRISPR interference method, a sequence of about 20 bases complementary to the target sequence is introduced into a region called protospacer on the sgRNA gene, so that the sgRNA specifically binds to the target gene. The protospacer sequence used in this example is shown in Table 3.

[0165] Table 3

[0166] Protospacer target genes Base sequence Serial Number slr1841 5′-ACAGCAGGGCTCACTAACA-3′ 21 slr0688 5′-TGCAGCAAACAGACTAAAA-3′ 22

[0167] In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are inserted into the slr2030-slr2031 gene on the chromosomal DNA in a connected form. Therefore, by adding a protospacer sequence (sequence number 21) complementary to the slr1841 gene (sequence number 7) to the primers used when amplifying the sgRNA gene by PCR, it is possible to easily obtain an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841.

[0168] First, using the chromosomal DNA of the LY07 strain as a template, the two DNA fragments were amplified by PCR using the set of primers slr2030-Fw (sequence number 15) and sgRNA_slr1841-Rv (sequence number 16) and the set of sgRNA_slr1841-Fw (sequence number 17) and slr2031-Rv (sequence number 18) recorded in Table 1.

[0169] Next, the mixed solution of the above DNA fragments was used as a template and the primers slr2030-Fw (SEQ ID NO. 15) and slr2031-Rv (SEQ ID NO. 18) described in Table 1 were used for amplification by PCR, thereby obtaining a DNA fragment (slr2030-2031::slr1841_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were sequentially connected. The slr2030-2031::slr1841_sgRNA was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-slr1841_sgRNA plasmid.

[0170] The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain by the same method as described above (1-1), and the transformed cells were screened on BG-11 agar culture containing 30 μg / mL kanamycin. Thus, a transformant Synechocystis dCas9 slr1841_sgRNA strain (hereinafter also referred to as slr1841 suppression strain) in which slr2030-slr2031 gene on chromosomal DNA was inserted with slr1841_sgRNA was obtained.

[0171] (1-3) Suppression of slr1841 gene

[0172] The promoter sequences of the dCas9 gene and the slr1841_sgRNA gene were designed so that expression was induced in the presence of anhydrotetracycline (aTc). In this example, the expression of the slr1841 gene was inhibited by adding aTc to a final concentration of 1 μg / mL during culture.

[0173] (Example 2)

[0174] In Example 2, the following steps were used to obtain a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modification enzyme was suppressed.

[0175] (2) Construction of a cyanobacterial strain in which the expression of the slr0688 gene was suppressed

[0176] By the same steps as above (1-2), the sgRNA gene containing the protospacer sequence (sequence number 22) complementary to the slr0688 gene (sequence number 4) was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9slr0688_sgRNA strain. In addition, using the set of primers slr2030-Fw (sequence number 15) and sgRNA_slr0688-Rv (sequence number 19) and the set of sgRNA_slr0688-Fw (sequence number 20) and slr2031-Rv (sequence number 18) described in Table 1, and using the In-Fusion PCR cloning method (registered trademark), (i) slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were connected in sequence. The DNA fragment (slr2030-2031::slr0688_sgRNA) was inserted into the pUC19 plasmid to obtain the pUC19-slr0688_sgRNA plasmid. Except for this, the same conditions as those in the above (1-2) were used.

[0177] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in the above (1-3).

[0178] (Comparative Example 1)

[0179] In Comparative Example 1, the Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

[0180] Next, observation of the cell surface state and protein secretion productivity test were performed on the strains obtained in Example 1, Example 2 and Comparative Example 1. The details are described below.

[0181] (3) Observation of the cell surface status of the strain

[0182] Ultrathin sections were prepared for the modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (i.e., slr1841 inhibition strain), the modified cyanobacterium Synechocystis dCas9slr0688_sgRNA strain obtained in Example 2 (hereinafter also referred to as slr0688 inhibition strain), and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter also referred to as control strain), and the state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0183] (3-1) Cultivation of strains

[0184] The slr1841 inhibitor strain of Example 1 was inoculated into a BG-11 culture medium containing 1 μg / mL aTc at an initial bacterial cell concentration of OD730 = 0.05 and then cultured under a light intensity of 100 μmol / m 2 The culture was shaken at 30°C for 5 days at 40 °C. The slr0688-inhibited strain of Example 2 and the control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

[0185] (3-2) Preparation of Ultrathin Sections of Strain

[0186] The culture solution obtained in (3-1) above was centrifuged at 2,500g for 10 minutes at room temperature to recover the cells of the slr1841 inhibitor strain of Example 1. Next, the cells were quickly frozen with liquid propane at -175°C and then fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, the dehydrated cells were soaked in propylene oxide, and then sank in a resin (Quetol-651) solution. The resin was then allowed to stand at 60°C for 48 hours to solidify, thereby embedding the cells with the resin. The cells in the resin were thinly sliced ​​into 70nm thickness using an ultracut to prepare ultrathin sections. The ultrathin sections were stained with 2% uranyl acetate and 1% lead citrate solution to prepare a transmission electron microscope sample of the slr1841 inhibitor strain of Example 1. In addition, the same operation was performed on the slr0688-suppressed strain of Example 2 and the control strain of Comparative Example 1, and samples for transmission electron microscopy were prepared.

[0187] (3-3) Observation using an electron microscope

[0188] The ultrathin sections obtained in (3-2) were observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in Figure 3 to Figure 8 .

[0189] First, the slr1841-suppressed strain of Example 1 will be described. Figure 3 This is a TEM (Transmission Electron Microscope) image of the slr1841-suppressed strain of Example 1. Figure 4 yes Figure 3 A magnified image of the dashed area A. Figure 4 (a) is Figure 3 The enlarged TEM image of the dashed area A is shown in Figure 1. Figure 4 (b) depicts Figure 4 (a) is an enlarged TEM image of the sample.

[0190] like Figure 3 As shown, in the slr1841-suppressed strain of Example 1, the outer membrane was partially peeled off from the cell wall (ie, the outer membrane was partially peeled off), and the outer membrane was partially bent.

[0191] In order to confirm the state of the cell surface in more detail, the dotted area A was magnified and observed. Figure 4 (a) and Figure 4 As shown in (b), it is possible to confirm that the outer membrane is partially peeled off (the dot-dash areas a1 and a2 in the figure). In addition, a greatly bent portion of the outer membrane can be confirmed next to the dot-dash area a1. It is considered that this portion is a portion where the outer membrane is weakly bonded to the cell wall, and the culture fluid penetrates from the outer membrane into the periplasm, so the outer membrane expands outward and bends.

[0192] Next, the slr0688-suppressed strain of Example 2 will be described. Figure 5 This is a TEM image of the slr0688 inhibitor strain of Example 2. Figure 6 yes Figure 5 A magnified image of the dashed area B. Figure 6 (a) is Figure 5 The enlarged TEM image of the dashed area B is shown in Figure 1. Figure 6 (b) depicts Figure 6 (a) is an enlarged TEM image of the sample.

[0193] like Figure 5 As shown, in the slr0688-inhibited strain of Example 2, the outer membrane was partially peeled off from the cell wall, and the outer membrane was partially bent. In addition, in the slr0688-inhibited strain, it was confirmed that the outer membrane was partially separated from the cell wall.

[0194] In order to confirm the state of the cell surface in more detail, the dotted area B was magnified and observed. Figure 6 (a) and Figure 6 As shown in (b), it is possible to confirm a portion where the outer membrane is greatly bent (the dotted line region b1 in the figure) and a portion where the outer membrane is partially peeled off (the dotted line regions b2 and b3 in the figure). In addition, it is possible to confirm a portion where the outer membrane is detached from the cell wall near each of the dotted line regions b1, b2 and b3.

[0195] Next, the control strain of Comparative Example 1 will be described. Figure 7 This is a TEM image of the control strain of Comparative Example 1. Figure 8 yes Figure 7 A magnified image of the dashed area C. Figure 8 (a) is Figure 7 The enlarged TEM image of the dashed area C, Figure 8 (b) depicts Figure 8 (a) is an enlarged TEM image of the sample.

[0196] like Figure 7 As shown, the cell surface of the control strain of Comparative Example 1 was flat, and the inner membrane, cell wall, outer membrane, and S layer were stacked in order. That is, in the control strain, the outer membrane was not observed to be separated from the cell wall, the outer membrane was peeled off (i.e., peeled off) from the cell wall, and the outer membrane was not bent as in Examples 1 and 2.

[0197] (4) Protein secretion productivity test

[0198] The slr1841 inhibitor strain of Example 1, the slr0688 inhibitor strain of Example 2, and the control strain of Comparative Example 1 were cultured separately, and the amount of protein secreted outside the cell (hereinafter, also referred to as the amount of secreted protein) was measured. The secretory productivity of the protein of each of the above-mentioned strains was evaluated by the amount of protein in the culture medium. In addition, the so-called secretory productivity of protein refers to the ability to produce protein by secreting the protein produced in the cell outside the cell. The specific method is described below.

[0199] (4-1) Cultivation of strains

[0200] The slr1841-suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. The culture was performed independently three times. In addition, the strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as the strain of Example 1.

[0201] (4-2) Quantification of proteins secreted outside cells

[0202] The culture solution obtained in (4-1) above was centrifuged at 2,500g for 10 minutes at room temperature to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22μm to completely remove the cells of the slr1841 inhibitor strain of Example 1. The amount of total protein contained in the filtered culture supernatant was quantified by the BCA (Bicinchoninic Acid) method. This series of operations was performed on three culture solutions obtained by independent culture, and the average value and standard deviation of the amount of protein secreted outside the cells of the slr1841 inhibitor strain of Example 1 were calculated. In addition, for the strains of Example 2 and Comparative Example 1, the protein in three culture solutions was quantified under the same conditions, and the average value and standard deviation of the amount of protein in the three culture solutions were calculated.

[0203] The results are shown in Fig. 9 . Fig. 9 It is a bar graph showing the amount of protein in the culture supernatant of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1 (n=3, error bar=SD).

[0204] like Fig. 9 As shown, the amount of protein secreted into the culture supernatant (mg / L) of both the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2 was increased by about 25 times compared with the control strain of Comparative Example 1.

[0205] The data record is omitted, but the absorbance of the culture medium was measured (730nm) and the amount of secretory protein per 1g dry weight of the bacteria (mg protein / g cell dry weight) was calculated. The results showed that the amount of secretory protein per 1g dry weight of the bacteria (mg protein / g cell dry weight) of the slr1841 inhibition strain of Example 1 and the slr0688 inhibition strain of Example 2 was approximately 36 times higher than that of the control strain of Comparative Example 1.

[0206] In addition, if Fig. 9As shown, compared with the slr1841 inhibition strain of Example 1 in which the expression of the gene (slr1841) encoding the SLH domain-retaining outer membrane protein was suppressed, the slr0688 inhibition strain of Example 2 in which the expression of the gene (slr0688) encoding the cell wall-pyruvate modification enzyme was suppressed, secreted a larger amount of protein into the culture supernatant. This is considered to be related to the fact that the number of covalently bonded sugar chains on the cell wall surface is greater than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, it is considered that the slr0688 inhibition strain of Example 2 has a further reduced amount and strength of binding between the outer membrane and the cell wall compared to the slr1841 inhibition strain of Example 1, and thus the amount of secreted protein is increased compared to the slr1841 inhibition strain of Example 1.

[0207] Can confirm by above result, by suppressing the protein function of the combination of adventitia and cell wall, thereby the adventitia of cyanobacteria and the combination of cell wall are partially weakened, and adventitia partially breaks away from cell wall.Also can confirm that the combination by adventitia and cell wall is a little less than, thereby the protein produced in the cell of cyanobacteria becomes easy to leak into the extracellular.Therefore show, according to the transformation cyanobacteria and manufacture method thereof that the present embodiment relates to, the secretion productivity of protein significantly improves.

[0208] (5) Identification of secreted proteins

[0209] Next, the secretory proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS / MS. The method is described below.

[0210] (5-1) Sample preparation

[0211] 8 times the amount of cold acetone was added to the culture supernatant, and after standing at 20°C for 2 hours, the protein precipitate was obtained by centrifugation at 20,000g for 15 minutes. 100mM Tris pH8.5 and 0.5% sodium dodecanoate (SDoD) were added to the precipitate, and the protein was dissolved by a closed ultrasonic crusher. After adjusting the protein concentration to 1μg / mL, dithiothreitol (DTT) was added at a final concentration of 10mM and stood at 50°C for 30 minutes. Next, iodoacetamide (IAA) was added at a final concentration of 30mM and stood at room temperature (shaded) for 30 minutes. In order to stop the reaction of IAA, cysteine ​​was added at a final concentration of 60mM and stood at room temperature for 10 minutes. 400ng of trypsin was added and stood at 37°C overnight to fragment the protein peptides. After adding 5% TFA (Trifluoroacetic Acid), the supernatant was obtained by centrifugation at 15,000g for 10 minutes at room temperature. SDoD was removed by this operation. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Then, 3% acetonitrile and 0.1% formic acid were added and the sample was dissolved using a closed ultrasonic crusher. The peptide concentration was prepared to 200 ng / μL.

[0212] (5-2) LC-MS / MS analysis

[0213] The sample obtained in the above (5-1) was analyzed under the following conditions using an LC-MS / MS device (UltiMate 3000RSLCnano LCSystem).

[0214] Sample injection amount: 200ng

[0215] Column: CAPCELL CORE MP 75μm×250mm

[0216] Solvent: Solvent A is 0.1% formic acid aqueous solution, solvent B is 0.1% formic acid + 80% acetonitrile

[0217] Gradient program: 4 minutes after sample injection, 8% solvent B, 27 minutes after solvent B, 44% solvent B, 28 minutes after solvent B, 80% solvent B, 34 minutes after the end of the measurement

[0218] (5-3) Data Analysis

[0219] The obtained data were analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0220] Software: Scaffold DIA

[0221] Database: UniProtKB / Swiss Prot database (Synechocystis sp. PCC 6803)

[0222] Fragmentation: HCD

[0223] Precursor Tolerance: 8ppm

[0224] Fragment Tolerance: 10ppm

[0225] Data Acquisition Type: Overlapping DIA

[0226] Peptide Length: 8-70

[0227] Peptide Charge: 2-8

[0228] Max Missed Cleavages: 1

[0229] Fixed Modification: Carbamidomethylation

[0230] Peptide FDR: less than 1%

[0231] Table 4 shows the 30 proteins with the largest relative quantitative values ​​among the identified proteins that are expected to have significant enzyme activity.

[0232]

[0233] These 6 kinds of proteins are all contained in the culture supernatant of the slr1841 inhibitor strain of Example 1 and the slr0688 inhibitor strain of Example 2. In all these proteins, the periplasmic (referring to the gap between the outer membrane and the inner membrane) transfer signal is maintained. According to this result, it can be confirmed that in the modified strains of Example 1 and Example 2, the outer membrane is partially separated from the cell wall, so that the protein in the periplasm becomes easy to leak out of the outer membrane (that is, outside the bacterium). Therefore, the modified cyanobacteria involved in this embodiment show that the secretion productivity of the protein is greatly improved.

[0234] (6) Identification of secreted intracellular metabolites

[0235] (6-1) Sample preparation

[0236] To 80 μl of the culture supernatant of the modified cyanobacteria was added 20 μl of an aqueous solution adjusted to a concentration of 1,000 μM of the internal standard substance, and the mixture was stirred and ultrafiltrated before use for measurement.

[0237] (6-2)CE (Capillary Electrophoresis)-TOFMS (Time-Of-Flight MassSpectrometry) analysis

[0238] In this test, measurements in the positive ion mode and the negative ion mode were performed under the conditions shown below.

[0239] [Cation mode]

[0240] Device: Agilent CE-TOFMS system

[0241] Capillary:Fused silica capillary id50μm×80cm

[0242] Determination conditions:

[0243] Run buffer:Cation buffer solution(p / n:H3301-1001)

[0244] CE voltage:Positive,30kV

[0245] MS ionization:ESI positive

[0246] MS scan range: m / z 50-1,000

[0247] [Negative ion mode]

[0248] Device: Agilent CE-TOFMS system

[0249] Capillary:Fused silica capillary id50μm×80cm

[0250] Determination conditions:

[0251] Run buffer:Anion buffer solution(p / n:H3301-1001)

[0252] CE voltage:Positive,30kV

[0253] MS ionization: ESI negative

[0254] MS scan range: m / z 50-1,000

[0255] (6-3) Data processing

[0256] For the peaks detected by CE-TOFMS, the automatic integration software MasterHands (registered trademark) ver.2.17.1.11 was used to automatically detect the peaks with a signal / noise ratio of 3 or more. For the detected peaks, based on the values ​​of the mass-to-charge ratio (m / z) and the migration time inherent to each metabolite, the values ​​of all substances registered in the metabolite library of HMT (Humman Metabolom Technologis (Strain)) were compared to search for metabolites contained in the culture supernatant of the modified cyanobacteria. The allowable error for the search was + / -0.5min for the migration time and + / -10ppm for m / z. For each metabolite identified, the concentration was calculated as a single-point correction of 100μM. The main metabolites identified are shown in Table 5.

[0257] Table 5

[0258]

[0259] All of these 12 intracellular metabolites were contained in the culture supernatant of each of the slr1841-inhibited strain of Example 1 and the slr0688-inhibited strain of Example 2. Although the data are not published, these metabolites were not contained in the culture supernatant of the control strain of Comparative Example 1. Based on this result, it can be confirmed that in the modified strains of Examples 1 and 2, the intracellular metabolites become easy to leak out of the outer membrane (i.e., outside the bacteria) due to the partial detachment of the outer membrane from the cell wall.

[0260] (7) Disease resistance induction test

[0261] Then, in order to evaluate the plant disease resistance effect of the secretion of transformation cyanobacteria (here, the culture supernatant of transformation cyanobacteria), following plant cultivation test and gene expression investigation were implemented.In the gene expression investigation, by investigating the expression change of the gene of participation in disease resistance in the plant cultivated in the plant cultivation test, the effect of plant disease resistance inducing agent was evaluated.In addition, transformation cyanobacteria is the slr0688 inhibition strain of embodiment 2, and similarly to above-mentioned (4-1), the cultivation of bacterial strain is carried out independently 3 times.That is, in embodiment 3 and 4, the culture supernatant of the transformation cyanobacteria of use embodiment 2 is as plant disease resistance inducing agent.

[0262] (7-1) Plant cultivation experiment

[0263] (7-1-1) Tomato cultivation experiment

[0264] In the tomato cultivation experiment, cherry tomatoes were cultivated by the following method. First, commercially available culture soil was added to the cultivation pots (22 cm × 16 cm), and 3 tomato seeds were sown in each pot. The cultivation was carried out at an indoor temperature of 23°C and a white light source with a light beam density of 250 μmol / m 2 / s, 16 hours of light conditions and 8 hours of dark conditions. About 1 week after the start of cultivation, thinning was performed at the stage of cotyledon expansion to make the individual sizes in each flower pot uniform. In addition, 500 mL of commercially available chemical fertilizer (500 times dilution of the original solution containing 6% total nitrogen, 10% water-soluble phosphoric acid, 5% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese, and 0.005% water-soluble boron) was applied to each flower pot once every 50 days.

[0265] (Example 3)

[0266] In Example 3, the individual sizes of the flower pots were made uniform as described above, and after the cherry tomato seedlings were planted, 10 mL of the plant disease resistance inducer diluted 20 times with water was sprayed on the leaves once every two weeks per tomato plant.

[0267] (Comparative Example 2)

[0268] In Comparative Example 2, the same procedure as in Example 3 was carried out except that water was used instead of the disease resistance inducer.

[0269] (7-1-2) Spinach cultivation experiment

[0270] First, commercially available culture soil was added to a cultivation bottle (12 cm × 10 cm), and three spinach seeds were sown in each bottle. The cultivation was carried out at a room temperature of 23°C and a white light source with a light quantum density of 100 μmol / m 2 / s, 10 hours of light and 14 hours of darkness. About 1 week after the start of cultivation, the seedlings were thinned out at the stage when the cotyledons were expanded to make the individual sizes in each bottle uniform.

[0271] (Example 4)

[0272] In Example 4, the individual sizes of the plants in each bottle were made uniform as described above, and two weeks after sowing, the plant disease resistance inducer diluted 20 times with water was sprayed on the leaves to the extent that the leaves were visually wet.

[0273] (Comparative Example 3)

[0274] In Comparative Example 3, the same procedure as in Example 4 was carried out except that water was used instead of the disease resistance inducer.

[0275] (7-2) Gene expression survey

[0276] For cherry tomatoes cultivated in Example 3 and Comparative Example 2, leaves were cut 100 days after sowing, and for spinach cultivated in Example 4 and Comparative Example 3, leaves were cut 20 days after sowing, and total RNA was prepared using Nucleospin (registered trademark) RNA plant kit (manufactured by Takara Bio). The total RNA was treated with TruSeq (registered trademark) Standard mRNA LTSample Prep Kit (manufactured by Illumina) to prepare a library, and the prepared library was subjected to transcriptome sequencing using NovaSeq (registered trademark) next-generation sequencer (manufactured by Illumina). Specifically, RNA-Seq was performed on samples of the comparative example (untreated group) and the example (treated group) using a new generation sequencer, thereby detecting transcripts (genes) with different expression levels in the comparison between the untreated group and the treated group. Then, from among the detected genes, the expression levels of NPR1 (Nonexpressor of Pathogenesis-Related 1), TGA and PR-1 (Pathogenesis-Related-1), which are genes involved in plant disease resistance (hereinafter referred to as plant disease resistance-related genes) and are widely present in plants, were analyzed to investigate the expression changes of plant disease resistance-related genes.

[0277] (result)

[0278] The results of the expression level analysis of the plant disease resistance-related genes in Examples 3 and 4 are shown in Table 6. In addition, Table 7 shows the NCBI-gene IDs of the disease resistance genes corresponding to *1 to *8 in Table 6.

[0279] Table 6

[0280]

[0281] Table 7

[0282] GeneID ※1 101245709 ※2 110805644 ※3 110775599 ※4 110780711 ※5 110786139 ※6 110805644 ※7 110775599 ※8 110777098

[0283] Table 6 shows the results of the expression level analysis of Example 3, which shows the relative values ​​of the expression levels (transcription levels) of the plant disease resistance-related genes of the cherry tomatoes of Example 3 relative to the expression levels (also called transcription levels) of the plant disease resistance-related genes of the cherry tomatoes of Comparative Example 2. In addition, Table 6 shows the results of the expression level analysis of Example 4, which shows the relative values ​​of the expression levels (transcription levels) of the plant disease resistance-related genes of the spinach cultivated in Example 4 relative to the expression levels (transcription levels) of the plant disease resistance-related genes of the spinach cultivated in Comparative Example 3. As shown in Table 6, it was confirmed that the expression levels of the NPR1 gene, the TGA gene, and the PR-1 gene in the cherry tomatoes (Example 3) and spinach (Example 4) to which the plant disease resistance inducer of the present invention was applied increased by at least 2 times. Specifically, with regard to the expression level of the NPR1 gene, both the cherry tomatoes (Example 3) and the spinach (Example 4) increased by 2 times. In addition, the expression level of the TGA gene increased by nearly 15 times in cherry tomatoes (Example 3) and increased by 4 to 53 times in spinach (Example 4). In addition, the expression level of the PR-1 gene 3 increased by 2 times in cherry tomatoes (Example 3) and increased by 3 to 4 times in spinach (Example 4).

[0284] As mentioned above, it is known that NPR1 gene, TGA gene, and PR-1 gene are widely present in plants and improve disease resistance. The above results suggest that the disease resistance inducer disclosed in the present invention has a universal effect that can be applied in various plants and significantly activates disease resistance-related genes. Therefore, it can be confirmed that the plant disease resistance inducer disclosed in the present invention can effectively induce disease resistance in plants.

[0285] Industrial Availability

[0286] According to the plant disease resistance inducer and the plant disease resistance induction method disclosed in the present invention, the disease resistance of the plant can be effectively induced, thereby improving the resistance of the plant to the disease. In addition, according to the method for manufacturing the plant disease resistance inducer disclosed in the present invention, the plant disease resistance inducer containing its secretion can be manufactured by culturing cyanobacteria, thereby making it easy and efficient to manufacture the plant disease resistance inducer. Therefore, according to the present disclosure, the resistance of the plant to the disease can be improved, and thus the increase in crop yield and the improvement in quality can be expected.

[0287] Explanation of symbols

[0288] 1. Endometrium

[0289] 2 Peptidoglycan

[0290] 3 Sugar chains

[0291] 4 Cell Wall

[0292] 5 Outer membrane

[0293] 6 SLH domain-retaining outer membrane protein

[0294] 7 SLH domain

[0295] 8 Organic matter channel protein

[0296] 9 Cell wall-pyruvate modifying enzymes

Claims

1. Plant disease resistance inducer, Contains secretions of cyanobacteria.

2. The plant disease resistance inducer according to claim 1, The cyanobacteria are modified cyanobacteria in which the function of a protein involved in the combination of the outer membrane and the cell wall is inhibited or lost.

3. The plant disease resistance inducer according to claim 2, The protein involved in the binding between the outer membrane and the cell wall is at least one selected from the group consisting of an SLH (Surface Layer Homology) domain-maintaining outer membrane protein and a cell wall-pyruvate modifying enzyme.

4. The plant disease resistance inducer according to claim 3, The SLH domain-retaining outer membrane protein is Slr1841 consisting of the amino acid sequence shown in SEQ ID NO: 1, NIES970_09470 consisting of the amino acid sequence shown in SEQ ID NO: 2, Anacy_3458 consisting of the amino acid sequence shown in SEQ ID NO: 3, or A protein having an amino acid sequence that is at least 50% identical to any of the above-mentioned SLH domain-retaining outer membrane proteins.

5. The plant disease resistance inducer according to claim 3, The cell wall-pyruvate modifying enzyme is Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, Synpcc7942_1529 consisting of the amino acid sequence shown in SEQ ID NO: 5, Anacy_1623 consisting of the amino acid sequence shown in SEQ ID NO: 6, or A protein whose amino acid sequence is at least 50% identical to any of the above cell wall-pyruvate modifying enzymes.

6. The plant disease resistance inducer according to claim 1, The cyanobacteria are modified cyanobacteria in which a gene expressing a protein involved in the combination of the outer membrane and the cell wall is deleted or inactivated.

7. The plant disease resistance inducer according to claim 6, The gene expressing a protein involved in the binding of the outer membrane to the cell wall is at least one selected from a gene encoding an SLH domain-retaining outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme.

8. The plant disease resistance inducer according to claim 7, The gene encoding the SLH domain-maintaining outer membrane protein is slr1841 consisting of the base sequence shown in SEQ ID NO:7, nies970_09470 consisting of the base sequence shown in SEQ ID NO: 8, anacy_3458 consisting of the base sequence shown in SEQ ID NO: 9, or A gene whose base sequence is at least 50% identical to any of the above genes.

9. The plant disease resistance inducer according to claim 7, The gene encoding the cell wall-pyruvate modifying enzyme is slr0688 consisting of the base sequence shown in SEQ ID NO:10, synpcc7942_1529 consisting of the base sequence shown in SEQ ID NO:11, anacy_1623 consisting of the base sequence shown in SEQ ID NO: 12, or a gene having a base sequence that is 50% or more identical to any of the above genes.

10. Methods for inducing plant disease resistance, Use of plant disease resistance inducers comprising secretions of cyanobacteria.

11. A method for producing a plant disease resistance inducer. include Steps for preparing cyanobacteria, and The step of causing the cyanobacteria to secrete a secretion that participates in the induction of disease resistance in plants.

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