Evaluation or selection method for vaccine-like plant immunizers

Abstract

To provide a method for evaluating or selecting a vaccine-like plant immunity inducer.SOLUTION: Provided is a method for evaluating or selecting a vaccine-like plant immunity inducer, comprising: contacting a plant cultured cell with a test substance; subculturing the plant cultured cell for a certain period of time; contacting a pathogen with the plant cultured cell after culturing; and measuring hypersensitive cell death in the plant cultured cell after pathogen contact.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for evaluating or selecting a vaccine-like plant immunity inducer.

Background Art

[0002] Although plants do not have a defense response such as acquired immunity based on the antigen-antibody reaction of animals against the invasion of pathogens, they induce a defense response called plant immunity similar to the innate immunity of animals. When a pathogen invades, a plant senses it at the molecular level and combats the disease by defense responses such as hypersensitive response, production of reactive oxygen species, production of antibacterial substances, hardening of the cell wall, construction of an invasion defense wall, etc. Agents that induce such resistance of plants to diseases are called plant disease resistance inducers, and compared with agents that directly act on pathogens such as fungicides, they have advantages such as a wide range of target diseases, a low risk of emergence of drug-resistant bacteria, and a low environmental load. Known plant disease resistance inducers include probenazole (trade name: Orizemate (registered trademark)), acibenzolar-S-methyl (trade name: Bion), thiazinyl (trade name: Buiget (registered trademark)), isothianyl (trade name: Stout (registered trademark)), diclobentiazox (trade name: Boone (registered trademark)), etc. These agents remain in the plant body and activate the plant's defense response.

[0003] The development of plant disease resistance inducers has long been carried out using cumbersome systems involving actual plants. However, in recent years, it has been reported that by adding a test substance and pathogenic bacteria to cultured plant cells and using hypersensitive cell death, one of the disease resistance responses in plant cells, as an indicator, substances that enhance hypersensitive cell death have been screened as plant disease resistance inducers (Patent Document 1 and Non-Patent Document 1). Furthermore, it has been reported that by adding a test substance and an elicitor to cultured plant cells in which the jasmonic acid-dependent defense pathway and the salicylic acid-dependent defense pathway of the plant defense system can act independently, substances that increase the reactive oxygen species level in plant cells have been screened as plant defense activators, using the reactive oxygen species level in plant cells as an indicator (Patent Document 2). In these methods, external stimuli are applied in the presence of the test substance, and substances that directly activate plant disease resistance have been selected.

[0004] On the other hand, in plants, it is known that defensive responses to external stimuli are memorized (priming), allowing them to respond more strongly to subsequent stimuli. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] Japanese Patent Publication No. 2018-76275 [Patent Document 2] International Public Gazette No. 2009 / 119915 [Non-patent literature]

[0006] [Non-Patent Document 1] Yoshiteru Noutoshi et al. The Plant Cell, 24(9): 3795-3804 (2012) [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] To ensure a stable food supply and reduce environmental impact, there is a need to create new pesticides with different mechanisms of action that utilize the plant's inherent defense responses. Unlike agents that directly activate plant disease resistance, a vaccine-like plant immunity inducer—that is, a agent that strongly induces a defense response in a plant when it is stimulated by pathogen invasion or other factors after a certain period following application—would be useful as a new disease control method. Therefore, the present invention provides a method for evaluating or selecting vaccine-like plant immunizers. [Means for solving the problem]

[0008] The inventors have found that the vaccine-like plant immunity-inducing effect of a test substance can be evaluated by subculturing plant cells treated with the test substance for a certain period of time, then treating them with incompatible pathogenic bacteria, and using hypersensitive cell death, one of the plant's disease resistance responses, as an indicator.

[0009] In other words, the present invention relates to the following 1). 1) A step of bringing the test substance into contact with plant culture cells, A step of subculturing the plant culture cells for a certain period of time. A step of contacting plant culture cells after subculturing with a pathogen, and A process for measuring hypersensitive cell death in plant culture cells after contact with a pathogen. A method for evaluating or selecting vaccine-like plant immunotherapeutic agents, including those mentioned above. [Effects of the Invention]

[0010] According to the present invention, the vaccine-like plant immunity-inducing effect of a substance can be easily evaluated, and it becomes possible to select an excellent vaccine-like plant immunity-inducing agent. [Brief explanation of the drawing]

[0011] [Figure 1] A figure showing the immunostimulatory effect of cyclic peptides on cultured Arabidopsis thaliana cells. [Modes for carrying out the invention]

[0012] All patent, non-patent, and other publications cited herein are incorporated herein by reference in their entirety.

[0013] In the present invention, "vaccine-like plant immunity inducer" refers to an agent that, when a plant is subjected to a stimulus such as pathogen invasion after a predetermined period following application of the agent, induces a stronger defense response in the plant than in a control plant (e.g., a plant without application). Here, stimuli such as pathogen invasion include external stimuli such as contact with pathogens, pathogen invasion, pathogen infection, and contact with foreign substances. The defense response is not particularly limited as long as it is a response induced by external stimuli such as contact with pathogens, pathogen invasion, pathogen infection, and contact with foreign substances, but examples include hypersensitivity reactions, production of reactive oxygen species, production of antimicrobial substances, hardening of cell walls, and construction of invasion defense walls, with hypersensitivity reactions being preferred and hypersensitivity cell death more preferred. The aforementioned period is not limited, but is preferably from 1 day after application, more preferably from 2 days after application, and preferably within 6 months, more preferably within 1 month, and even more preferably within 1 week after application. Furthermore, the period is preferably from 1 day after application to within 6 months, more preferably from 2 days after application to within 6 months, even more preferably from 2 days after application to within 1 month, and even more preferably from 2 days after application to within 1 week. Therefore, in the present invention, the vaccine-like plant immunity inducer can contribute to inducing a defense response to pathogen invasion in plants, etc., preferably from 1 day after application to within 6 months, more preferably from 2 days after application to within 6 months, even more preferably from 2 days after application to within 1 month, and even more preferably from 2 days after application to within 1 week, and is useful for controlling plant diseases. It should be noted that the agent may transiently activate the plant's defense response immediately after application, but this activation usually subsides within a few hours to less than 1 day.

[0014] As shown in the examples below, when plant culture cells treated with a specific substance were subcultured for 6 days and then inoculated with incompatible plant pathogenic bacteria, hypersensitive cell death of the plant cells was enhanced compared to the untreated case. On the other hand, when plant culture cells treated with the specific substance were inoculated with plant pathogenic bacteria 1 hour after treatment (corresponding to the methods described in Patent Document 1 and Non-Patent Document 1), hypersensitive cell death of the plant cells was not enhanced compared to the untreated case. Conversely, the substance that enhanced hypersensitive cell death of plant cells compared to the untreated case using the latter method did not enhance hypersensitive cell death of plant cells compared to the untreated case using the former method. Furthermore, as shown in the examples below, although the expression of gene groups involved in the pathogen response significantly improved 1 hour after treatment in plant culture cells treated with the specific substance, the improvement in expression was transient, and it became clear that the compounds of the present invention do not directly activate the defense response over a long period of time. Furthermore, when plant culture cells treated with the specific substance were subcultured for 6 days and then inoculated with plant pathogenic bacteria, the expression of genes involved in infection defense responses, such as injury response-related genes and pathogen response-related genes, significantly changed 1 hour after inoculation. The plant culture cells on the 6th day of subculture were progeny cells that had undergone multiple cell divisions from the initial culture cells stimulated by the specific substance, and these progeny cells exhibited a rapid response to the pathogen. These results indicate that, unlike the plant disease resistance inducers described in Patent Document 1 and Non-Patent Document 1, the specific substance can strongly induce the plant's inherent defense response when pathogens invade after a period of time has passed since treatment with the compound. This effect is thought to be mediated through the plant's immunological memory mechanism. Therefore, the specific substance can serve as a vaccine-like plant immunity inducer.

[0015] Based on these findings, it can be said that vaccine-like plant immunoinducing agents can be evaluated or selected using hypersensitive cell death, which is one of the plant's disease resistance responses, as an indicator. That is, in the method for evaluating or selecting a vaccine-like plant immune inducer of the present invention (hereinafter, also simply referred to as the method of the present invention), a test substance is brought into contact with plant cultured cells, subcultured for a certain period, and then the hypersensitive cell death in the plant cultured cells contacted with the pathogen is used as an index to evaluate the vaccine-like plant immune inducing action of the test substance. Further, based on the evaluation, a substance having a vaccine-like plant immune inducing action is selected as a vaccine-like plant immune inducer.

[0016] More specifically, the method of the present invention includes a step of bringing a test substance into contact with plant cultured cells, a step of subculturing the plant cultured cells for a certain period, a step of bringing a pathogen into contact with the plant cultured cells after subculture, and a step of measuring the hypersensitive cell death in the plant cultured cells after contact with the pathogen.

[0017] The plant cultured cells used in the method of the present invention may be cultured cells of any plant. For example, cruciferous plants (Arabidopsis thaliana, cabbage, rapeseed, etc.), gramineous plants (rice, corn, barley, wheat, etc.), solanaceous plants (tomato, eggplant, potato, tobacco, etc.), cucurbitaceous plants (cucumber, melon, pumpkin, etc.), leguminous plants (soybean, pea, kidney bean, alfalfa, peanut, etc.), cruciferous plants (radish, Chinese cabbage, cabbage, Arabidopsis thaliana, etc.), rosaceous plants (strawberry, apple, pear, etc.), moraceae plants (mulberry, etc.), malvaceae plants (cotton, etc.), umbelliferous plants (carrot, parsley, celery, etc.), asteraceae plants (burdock, sunflower, chrysanthemum, lettuce, etc.), vitaceae plants (grape, etc.), etc. Among these, from the viewpoints of easy availability and easy handling, the cultured cells of Arabidopsis thaliana are preferable, and the Arabidopsis thaliana MM2d cultured cells are more preferable.

[0018] The test substance used in the method of the present invention is not particularly limited as long as it is a substance desired to be used as a vaccine-like plant immunity inducer. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, etc., and may be a compound, a composition or a mixture. The form of the test substance is not particularly limited and may be any form such as solid, semi-solid, gel, liquid, gas, etc. As the test substance, a cyclic peptide is preferable, and a cyclic peptide consisting of an amino acid sequence containing 7 amino acid residues, in which the α-amino group at the amino terminus and the carboxyl group at the carboxyl terminus of the amino acid sequence are linked by a peptide bond, is more preferable.

[0019] In the method of the present invention, the test substance is brought into contact with plant cultured cells. The method of bringing the test substance into contact with plant cultured cells is not particularly limited, and examples thereof include culturing plant cultured cells in a medium containing the test substance; adding the test substance to a medium containing plant cultured cells; directly adding the test substance to plant cultured cells by coating, spraying, scattering, dropping, etc. The concentration and the amount of contact of the test substance may be appropriately set based on the form, chemical properties, cytotoxicity, etc. of the test substance. For example, a predetermined amount of the test substance diluted to an appropriate concentration may be brought into contact with plant cultured cells under the conditions of 15 to 35°C, preferably for 1 to 24 hours, more preferably for 6 to 24 hours, and even more preferably for 12 to 24 hours.

[0020] Subsequently, the plant cultured cells that have been brought into contact with the test substance in the above procedure are subcultured for a certain period. The plant cultured cells may be subcultured at least once after being brought into contact with the test substance for the above-mentioned time, and may be subcultured a plurality of times according to the growth status of the plant cultured cells. Here, the certain period is usually preferably 1 day or more, more preferably 2 days or more, even more preferably 5 days or more, and preferably 2 weeks or less, more preferably 10 days or less, even more preferably 1 week or less. Also, it is preferably 1 day to 2 weeks, more preferably 2 to 10 days, even more preferably 2 days to 1 week, and still more preferably 5 days to 1 week.

[0021] The method for culturing plant cells should follow general plant cell culture methods, but it is generally preferable to carry out the culture under aerobic conditions such as shaking culture in liquid culture or aerated stirring culture. Commercially available media such as LS medium, MS medium, Gamborg B5 medium, White medium, Chu(N6) medium, DKW medium, Hoagland medium, McCown medium, and SH medium can be used for culturing plant cells. It is preferable to add plant hormones commonly used for culturing plant cells to the media. Examples of plant hormones that can be used include auxins such as 2,4-D, NAA, IAA, and IBA; cytokinins such as kinetin, BA, and 2iP; and gibberellins such as gibberellin A3 (GA3). Other culture conditions for plant cells should follow the normal conditions for culturing plant cells. For example, plant cells can be cultured using the media at a temperature of preferably 15-35°C, more preferably 20-30°C, for the time described above.

[0022] Next, the plant culture cells that have been subcultured using the procedure described above are brought into contact with pathogens. Examples of pathogens include filamentous fungi, bacteria, and viruses that cause plant diseases. Specifically, for example, rice blast fungus (Magnaporthe oryzae), rice seedling blight bacterial disease fungus (Burkholderia plantarii), rice leaf spot disease fungus (Cochliobolus miyabeanus), rice sheath blight fungus (Rhizoctonia solani), rice bacterial leaf spot fungus (Xanthomonas oryzae), potato powdery scab fungus (Spongospora subterranea), potato late blight fungus (Phytophthora infestans), potato black scurf fungus (Rhizoctonia solani), potato scab fungus (Streptomyces scabies), barley powdery mildew fungus (Eryshiphe graminis f. sp. hordei), wheat red mold fungus (Gibberella zeae), wheat snow mold large nodule sclerotinia fungus (Sclerotinia) (borealis), wheat rust (Puccinia recondita), wheat powdery mildew (Erysiphe graminis), wheat root rot (Rhizoctonia solani), soybean downy mildew (Peronospora manshurica), soybean purple spot (Cercospora kikuchii), pea brown spot (Mycosphaerella pinodes), corn smut (Ustilago maydis), sweet potato wilt (Fusarium oxysporum f. sp. batatas), melon wilt (Fusarium oxysporum f. sp. melonis), lettuce root rot (Fusarium oxysporum f. sp. lactucae), tomato wilt (Fusarium oxysporum f. sp. (lycopersici), tomato verticillium wilt (Verticillium dahliae), tomato anthracnose (Colletotrichum phomoides), tomato bacterial leaf spot (Pseudomonas syringae pv.)Examples of pathogens include, but are not limited to, tomato, spinach wilt (Fusarium oxysporum f. sp. spinaciae), cruciferous root-knot (Plasmodiophora brassicae), cucumber seedling damping-off (Pythium debaryanum), and strawberry gray mold (Botrytis cinerea). In one preferred embodiment, a filamentous fungus or bacteria, i.e., a pathogenic bacterium, is used as the pathogen. In a more preferred embodiment, bacteria are used as the pathogen. In an even more preferred embodiment, tomato bacterial leaf spot disease is used as the pathogen.

[0023] There are no particular limitations on the method for bringing plant culture cells into contact with pathogens, but examples include culturing plant culture cells in a medium containing pathogens; adding pathogens to a medium containing plant culture cells; and directly applying pathogens to plant culture cells by coating, spraying, scattering, dropping, etc. The amount of pathogen contact can be appropriately determined based on the form and properties of the pathogen. For example, a predetermined amount of pathogen is usually brought into contact with plant culture cells under conditions of 15 to 35°C, preferably for 6 to 24 hours, more preferably for 12 to 24 hours.

[0024] Next, hypersensitive cell death is measured in plant culture cells that have been exposed to the pathogen using the procedure described above. Here, hypersensitive cell death is one of the plant's disease resistance responses, and refers to programmed cell death that plants induce to contain pathogens. Hypersensitive cell death can be measured as a level of hypersensitive cell death, using the amount or percentage of dead cells as an indicator, according to methods known in the field. For example, hypersensitive cell death can be measured by dead cell staining using Evans blue, trypan blue, propidium iodide (PI), etc. In a preferred embodiment, hypersensitive cell death is measured by staining dead cells with Evans blue. Specifically, for example, plant culture cells that have been in contact with a pathogen are stained with an aqueous Evans blue solution to a final concentration of 1% by weight, and then washed. Next, the dye is eluted from the cells with 50% by volume methanol and 1% by weight SDS, the supernatant is collected, and the amount of cells that have undergone cell death can be measured by measuring the absorbance at 595 nm using a plate reader or the like. Furthermore, since Evans blue staining cannot determine whether cell death is programmed cell death or not, it may produce a false positive result if the test substance itself is toxic to plant cultured cells. However, such false positives can be ruled out by preparing plant cultured cells under the same conditions as described above, except that they are not exposed to pathogens, and measuring cell death in those plant cultured cells.

[0025] By comparing the measured level of hypersensitive cell death with that of a control, the effect of the test substance on hypersensitive cell death induced in response to pathogens can be investigated. More specifically, it is examined whether the level of hypersensitive cell death in the group exposed to the test substance is higher than that of the control. For example, if the level of hypersensitive cell death in the group exposed to the test substance is statistically significantly higher than that of the control, it can be concluded that the hypersensitive cell death was enhanced by the test substance. Alternatively, if the level of hypersensitive cell death in the group exposed to the test substance is preferably 105% or higher, more preferably 110% or higher, and even more preferably 115% or higher than that of the control, it can be concluded that the hypersensitive cell death was enhanced by the test substance.

[0026] As controls used to compare the levels of hypersensitive cell death described above, plant culture cells cultured under the same conditions as described above, except for the contact conditions with the test substance, can be used. For example, plant culture cells that have not been in contact with the test substance (e.g., plant culture cells that have been subcultured for a certain period without contact with the test substance and then contacted with a pathogen after subculture, plant culture cells that have been in contact with a control substance such as the solvent of the test substance, subcultured for a certain period, and then contacted with a pathogen after subculture, etc.), and plant culture cells that have been in contact with a low concentration of the test substance, subcultured for a certain period, and then contacted with a pathogen after subculture, etc. However, the types of controls are not limited to these, as long as the effects of the test substance can be compared. In a preferred embodiment, plant culture cells cultured under the same conditions as described above are used as a control for comparing the levels of hypersensitive cell death, except that the solvent of the test substance is used instead of the test substance.

[0027] In the method of the present invention, the level of hypersensitive cell death measured as described above is used as an indicator to evaluate the vaccine-like plant immunity-inducing effect of the test substance. For example, if the level of hypersensitive cell death in the group exposed to the test substance is higher than in the control group, it can be concluded that the test substance strongly induced a defensive response in the plant cells when they were stimulated after a period of time following contact with the test substance. Therefore, the test substance can be selected as a vaccine-like plant immunizer.

[0028] If necessary, the test substances selected in the above procedure may be further evaluated. For example, as in the method described in Patent Document 1 and Non-Patent Document 1, the test substance and pathogen are brought into contact with plant cultured cells, and the disease resistance-inducing effect of the test substance is evaluated using hypersensitive cell death as an indicator. Test substances that do not enhance hypersensitive cell death in this evaluation, i.e., do not show a disease resistance-inducing effect, can be evaluated as more preferable as vaccine-like plant immunoinducing agents.

[0029] The substance selected by the method of the present invention can be used as an active ingredient in a vaccine-like plant immune inducer for inducing plant immunity, specifically for inducing a protective response, or for controlling plant diseases.

[0030] With regard to the embodiments described above, the present invention further discloses the following embodiments. <1> The process of bringing the test substance into contact with plant culture cells, A step of subculturing the plant culture cells for a certain period of time. A step of contacting plant culture cells after subculturing with a pathogen, and A process for measuring hypersensitive cell death in plant culture cells after contact with a pathogen. Methods for evaluating or selecting vaccine-like plant immunotherapeutic agents, including <2> The process further includes comparing the level of hypersensitive cell death with a control. <1> Method of description. <3> The process further includes selecting the test substance as a vaccine-like plant immunoinducer if the level of hypersensitive cell death is higher compared to the control. <2> Method of description. <4> The aforementioned period is preferably 1 day or more, more preferably 2 days or more, even more preferably 5 days or more, and preferably 2 weeks or less, more preferably 10 days or less, even more preferably 1 week or less, and also preferably 1 day to 2 weeks, more preferably 2 to 10 days, even more preferably 2 days to 1 week, and even more preferably 5 days to 1 week. <1> ~ <3> The method described in any one of the items. <5> The test substance is preferably a cyclic peptide, more preferably a cyclic peptide consisting of an amino acid sequence containing 7 amino acid residues, wherein the α-amino group at the amino terminus and the carboxyl group at the carboxyl terminus of the amino acid sequence are linked by a peptide bond. <1> ~ <4> The method described in any one of the items. <6> The plant culture cells are preferably Arabidopsis thaliana culture cells, and more preferably Arabidopsis thaliana MM2d culture cells. <1> ~ <5> The method described in any one of the items. <7> The plant culture cells are brought into contact with the test substance for preferably 1 to 24 hours, more preferably 6 to 24 hours, and even more preferably 12 to 24 hours. <1> ~ <6> The method described in any one of the items. <8> The pathogen is preferably brought into contact with the subcultured plant cells for 6 to 24 hours, more preferably for 12 to 24 hours. <1> ~ <7> The method described in any one of the items. <9> The pathogen is preferably a pathogenic fungus, more preferably a bacterium, and even more preferably Pseudomonas syringae pv. tomato. <1> ~ <8> The method described in any one of the items. <10> The aforementioned hypersensitive cell death is measured by Evans blue staining. <1> ~ <9> The method described in any one of the items. <11> The aforementioned vaccine-like plant immunity inducer contributes to inducing a plant's defense response to pathogen invasion preferably 1 day after application, more preferably 2 days after application, and preferably within 6 months after application, more preferably within 1 month, even more preferably within 1 week, and also preferably from 1 day to 6 months after application, more preferably from 2 days to 6 months, even more preferably from 2 days to 1 month, and most preferably from 2 days to 1 week. <1> ~ <10> The method described in any one of the items. [Examples]

[0031] The present invention will be described in more detail below based on examples, but the present invention is not limited thereto.

[0032] Example 1: Immunostimulatory effect of cyclic peptide on plant cells The immunostimulatory effects of cyclic peptides on plant cells were evaluated using two different assay methods, one conventional and one passaged, as described below. (1) Conventional system Arabidopsis thaliana MM2d cultured cells, 5 days after subculturing, were dispensed in 60 μL portions into 96-well plates. These were then treated for 1 hour with either 100 μM cyclic peptide (dissolved in DMSO) or DMSO and 100 μM sodium salicylate as a positive control. Subsequently, 40 μL of Pst DC3000 (Pseudomonas syringae pv. tomato DC3000 avrRpm1) solution was added as an incompatible plant pathogenic bacterium, and dead cell staining with Evans blue was performed after 20 hours (Noutoshi & Shirasu, Methods in Molecular Biology, 1795:39-47 (2018)). Specifically, Evans blue aqueous solution was added to the pathogenic bacteria-treated cell solution to a final concentration of 1% by weight, and the solution was allowed to stand for 1 hour with occasional stirring. Afterward, the Evans blue aqueous solution was removed, and the cells were washed three times with 250 μL of water. The amount of cells that underwent cell death was evaluated by adding 200 μL of eluate (50% methanol by volume, 1% SDS by weight) warmed to 55°C, and measuring the absorbance (595 nm) of the extracted Evans blue dye diluted fourfold with the eluate. (2) Succession As with the conventional system, cyclic peptides were added and allowed to stand overnight. The entire cell solution was then subcultured into a 24-well plate containing 1.2 mL of MM2d subculture medium. After 6 days of shaking culture, 60 μL of each culture medium was transferred to a 96-well plate, inoculated with 40 μL of Pst DC3000 solution, and dead cell staining with Evans blue was performed after 20 hours.

[0033] Cell death is one of the defense responses that plants induce themselves when they recognize incompatible pathogenic bacteria. Here, we evaluated the immune response of plants by quantitatively measuring cell death and calculating the cell death enhancement rate (%). The cell death enhancement rate (%) was calculated as the ratio of the Evans blue staining values ​​of cells treated with DMSO (without peptide treatment) to 100%. Each treatment was performed with n=4, and the average value was calculated.

[0034] From the evaluation of 320 candidate cyclic peptides, nine positive peptides that reacted only in the passaged system were obtained. Additionally, five peptides that reacted only in the conventional system were also obtained. These selected peptides were re-evaluated in both the conventional and passaged systems (Tables 1 and 2, and Figure 1). The nine positive peptides found in the passaged system did not show immunoactivating effects in the conventional system, but were confirmed to show immunoactivating effects only in the passaged system. These results demonstrate the successful acquisition of nine cyclic peptides with long-term immunostimulatory effects.

[0035] [Table 1]

[0036] [Table 2]

[0037] Example 2: Analysis of the effects of cyclic peptides with long-term immune priming activity on plant cells. One of the cyclic peptides obtained in Example 1 (cyclo-SGPSWIQ) was used to treat Arabidopsis thaliana cultured cells, and comprehensive gene expression changes were analyzed by RNA-seq from samples taken after 1 hour, 6 hours, or 6 days of passage. RNA was extracted, and libraries prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolab) were subjected to next-generation sequencing analysis using MiSeq (Illumina). The obtained data were mapped to Arabidopsis thaliana cDNA information using CLC genomics workbench (Philgen), and genes that showed significant (FDR < 0.1) expression changes in a two-group comparison with DMSO (peptide solvent) treatment (control group) were defined as differentially expressed genes (DEGs) and listed using the DEseq2 package of the statistical analysis software R.

[0038] DEGs (gene numbers can be found in PANTHER, see http: / / pantherdb.org / ) extracted from data 1 hour after cyclic peptide treatment were subjected to GO enrichment analysis. The results revealed a significant increase in the expression of genes involved in pathogenic response. Table 3 shows those related to pathogenic response or DNA regulation. Specifically, genes involved in phenylpropanoid synthesis, which is involved in defense responses, immune responses, and antimicrobial substance production, were detected. Genes related to DNA and histone regulation were also detected, suggesting that the long-term immune priming effect of cyclic peptides may be mediated through epigenetic regulation.

[0039] [Table 3]

[0040] Table 4 shows the DEGs extracted from data 6 hours after cyclic peptide treatment. This differs significantly from the results obtained 1 hour after the previous peptide treatment, with very few altered gene groups observed. This result indicates that while the isolated cyclic peptide activates the expression of defense-related genes immediately after administration, its effects largely subside within 6 hours of administration. In other words, the stimulation by the cyclic peptide is transient and does not directly activate the defense response over a long period.

[0041] [Table 4]

[0042] Next, cultured cells treated with cyclic peptides were passaged for 6 days, and these were used to treat the cells with Pst DC3000 strain. The changes in gene expression after 1 hour were then examined. The results are shown in Table 5. In samples derived from cells treated with cyclic peptides, infection defense response pathways, such as injury response and pathogen response-related genes, were significantly altered. This means that, even after 6 days, the response at the early stage of 1 hour after pathogen infection was significantly enhanced in cells treated with cyclic peptides compared to the control. Cells at 6 days of passage were descendants of initial cultured cells that had undergone multiple cell divisions after being stimulated by the peptide, and these cells exhibited a rapid response to pathogenic bacteria. This confirmed, through gene responses, that the cyclic peptide obtained in this study induces a long-term priming effect.

[0043] [Table 5]

[0044] In this study, we successfully obtained nine cyclic peptides that induce long-term priming activity. Furthermore, RNA-seq analysis revealed that these peptides transiently induced a pathogen response pathway after administration, and that the cultured cells that received the peptides maintained a rapid response to pathogens even after multiple cell divisions. This indicates that the peptides obtained in this study induce long-term priming. A method for selecting peptides that can induce long-term priming mechanisms with only transient stimulation is an extremely useful technique for efficiently cultivating plants.

Claims

1. The process of bringing the test substance into contact with plant culture cells, The process involves subculturing the plant culture cells for 5 days to 2 weeks. A step of contacting plant culture cells after cultivation with a pathogen, and A process for measuring hypersensitive cell death in plant culture cells after contact with a pathogen. A method for evaluating or selecting vaccine-like plant immunotherapeutic agents, including those mentioned above.

2. The method according to claim 1, further comprising the step of comparing the level of hypersensitive cell death with a control.

3. The method according to claim 2, further comprising the step of selecting the test substance as a vaccine-like plant immunoinducer when the level of hypersensitive cell death is higher compared to the control.

4. The method according to any one of claims 1 to 3, wherein the test substance is a cyclic peptide.

5. The method according to any one of claims 1 to 4, wherein the plant cultured cells are Arabidopsis thaliana cultured cells.

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