JP7849686B2 - Evaluation or selection method for vaccine-like plant immunizers - Google Patents

Description

This invention relates to a method for evaluating or selecting vaccine-like plant immunizers.

While plants do not possess a defensive response to pathogen invasion like the acquired immunity based on antigen-antibody reactions in animals, they induce a defensive response called plant immunity, which is similar to the innate immunity of animals. When a pathogen invades, plants sense it at the molecular level and counteract the disease through defensive responses such as hypersensitivity reactions, production of reactive oxygen species, production of antimicrobial substances, hardening of the cell wall, and construction of an invasion defense wall. Drugs that induce such resistance to plant diseases are called plant disease resistance inducers, and compared to drugs that act directly on pathogens, such as fungicides, they have advantages such as a broader range of target diseases, a lower risk of drug-resistant strains emerging, and lower environmental impact. Known plant disease resistance inducers include probenazole (trade name: Oryzemate®), acibenzolar-S-methyl (trade name: Bion), thiadinil (trade name: V-Get®), isothianil (trade name: Stout®), and diclobentiazox (trade name: Boon®). These chemicals remain in the plant and activate its defense response.

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 and salicylic acid-dependent defense pathways of the plant defense system can act independently, substances that increase reactive oxygen species levels in plant cells have been screened as plant defense activators (Patent Document 2). These methods involve applying external stimuli in the presence of the test substance, and substances that directly activate plant disease resistance have been selected.

On the other hand, in plants, it is known that defensive responses to external stimuli are memorized (priming), allowing for a stronger response to subsequent stimuli.

Japanese Patent Publication No. 2018-76275 International Public Gazette No. 2009/119915

Yoshiteru Noutoshi et al. The Plant Cell, 24(9): 3795-3804 (2012)

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.

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

In other words, the present invention relates to 1) below.
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 step of measuring hypersensitive cell death in plant culture cells after contact with the pathogen.
A method for evaluating or selecting vaccine-like plant immunotherapeutic agents, including those mentioned above.

According to the present invention, the vaccine-like plant immunity-inducing effect of a substance can be easily evaluated, making it possible to select a superior vaccine-like plant immunity-inducing agent.

A figure showing the immunostimulatory effect of cyclic peptides on cultured Arabidopsis thaliana cells.

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

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 hypersensitive reactions, production of reactive oxygen species, production of antimicrobial substances, hardening of cell walls, and construction of invasion defense walls, with hypersensitive reactions being preferred and hypersensitive cell death more preferred. The aforementioned period is not limited, but is preferably from one day after application, more preferably from two days after application, and preferably within six months, more preferably within one month, and even more preferably within one week. Furthermore, the period after application is preferably from 1 day to 6 months, more preferably from 2 days to 6 months, even more preferably from 2 days to 1 month, and even more preferably from 2 days to 1 week. Therefore, in this invention, the vaccine-like plant immunity inducer can contribute to inducing a defense response against pathogen invasion in plants, 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 even more preferably from 2 days to 1 week, making it useful for controlling plant diseases. While the agent may transiently activate the plant's defense response immediately after application, this activation usually subsides within a few hours to less than a day.

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 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 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 specified substance were subcultured for six 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 one hour after inoculation. The plant culture cells on day six of subculture were descendant cells that had undergone multiple cell divisions from the initial culture cells stimulated by the specified substance, and these descendant cells exhibited a rapid response to the pathogen. These results demonstrate that, unlike the plant disease resistance inducers described in Patent Document 1 and Non-Patent Document 1, the specified substance can strongly induce the plant's inherent defense response when pathogens invade after a period of time has elapsed since treatment with the compound. This effect is thought to be mediated through the plant's immune memory mechanism. Therefore, the specified substance can serve as a vaccine-like plant immunity inducer.

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.
In other words, in the method for evaluating or selecting vaccine-like plant immune inducers of the present invention (hereinafter also simply referred to as the method of the present invention), the vaccine-like plant immune inducer effect of the test substance is evaluated using hypersensitive cell death in plant culture cells that have been exposed to a pathogen after being exposed to the test substance and subjected to subculturing for a certain period of time as an indicator. Furthermore, based on this evaluation, a substance having a vaccine-like plant immune inducer effect is selected as a vaccine-like plant immune inducer.

More specifically, the method of the present invention includes the steps of contacting plant culture cells with a test substance, subculturing the plant culture cells for a certain period of time, contacting the subculturized plant culture cells with a pathogen, and measuring hypersensitive cell death in the plant culture cells after pathogen contact.

The plant culture cells used in the method of the present invention may be culture cells of any plant, but examples include culture cells prepared from plants belonging to the Brassicaceae family (Arabidopsis thaliana, cabbage, rapeseed, etc.), Poaceae family (rice, corn, barley, wheat, etc.), Solanaceae family (tomato, eggplant, potato, tobacco, etc.), Cucurbitaceae family (cucumber, melon, pumpkin, etc.), Fabaceae family (soybean, pea, green bean, alfalfa, peanut, etc.), Brassicaceae family (radish, Chinese cabbage, cabbage, Arabidopsis thaliana, etc.), Rosaceae family (strawberry, apple, pear, etc.), Moraceae family (mulberry, etc.), Malvaceae family (cotton, etc.), Apiaceae family (carrot, parsley, celery, etc.), Asteraceae family (burdock, sunflower, chrysanthemum, lettuce, etc.), Vitaceae family (grape, etc.). Of these, cultured cells of Arabidopsis thaliana are preferred, and Arabidopsis thaliana MM2d cultured cells are more preferred, from the viewpoint of ease of acquisition and handling.

The test substance used in the method of the present invention is not particularly limited, as long as it is a substance desired for use as a vaccine-like plant immunizer. The test substance may be a naturally occurring substance, a substance artificially synthesized by chemical or biological methods, and may be a compound, a composition, or a mixture. The form of the test substance is not particularly limited and may be solid, semi-solid, gel, liquid, gas, or any other form. Preferably, the test substance is a cyclic peptide, more preferably a cyclic peptide consisting of an amino acid sequence containing seven amino acid residues, where 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.

In the method of the present invention, a test substance is brought into contact with plant culture cells. The method for bringing the test substance into contact with plant culture cells is not particularly limited, but examples include culturing plant culture cells in a medium containing the test substance; adding the test substance to a medium containing plant culture cells; or directly adding the test substance to plant culture cells by coating, spraying, scattering, or dropping. The concentration and amount of the test substance should be appropriately determined 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 culture cells under conditions of 15 to 35°C for preferably 1 to 24 hours, more preferably 6 to 24 hours, and even more preferably 12 to 24 hours.

Next, the plant culture cells that have been in contact with the test substance using the procedure described above are subcultured for a certain period. The plant culture cells only need to be subcultured at least once after contact with the test substance for the aforementioned time, and may be subcultured multiple times depending on the growth status of the plant culture 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, and even more preferably 1 week or less. Furthermore, it is 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.

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 these media. Examples of plant hormones 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 usual conditions for culturing plant cells. For example, plant culture cells can be cultured using the culture medium at a temperature of preferably 15 to 35°C, more preferably 20 to 30°C, for the time described above.

Next, the plant cells that have been subcultured using the procedure described above are exposed to pathogens. Examples of pathogens include filamentous fungi, bacteria, and viruses that cause plant diseases. Specifically, for example, rice blast (Magnaporthe oryzae), rice seedling blight (Burkholderia plantarii), rice leaf spot (Cochliobolus miyabeanus), rice sheath blight (Rhizoctonia solani), rice bacterial leaf spot (Xanthomonas oryzae), potato powdery scab (Spongospora subterranea), potato late blight (Phytophthora infestans), potato black scurf (Rhizoctonia solani), potato scab (Streptomyces scabies), barley powdery mildew (Eryshiphe graminis f. sp. hordei), cereal red mold (Gibberella zeae), cereal snow mold (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 vine wilt (Fusarium oxysporum f. sp. batatas), Melon vine 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) Examples of pathogens include, but are not limited to, *Pseudomonas syringae pv. tomato*, *Fusarium oxysporum f. sp. spinaciae*, *Plasmodiophora brassicae*, *Pythium debaryanum*, and *Botrytis cinerea*. In one preferred embodiment, a filamentous fungus or bacteria, i.e., a pathogenic fungus, is used as the pathogen. In a more preferred embodiment, bacteria are used as the pathogen. In an even more preferred embodiment, *Pseudomonas syringae pv. tomato* is used as the pathogen.

The method for contacting plant culture cells with pathogens is not particularly limited, 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, or dropping. The amount of pathogen contact should be appropriately determined based on the form and properties of the pathogen. For example, a predetermined amount of pathogen is usually contacted with plant culture cells under conditions of 15 to 35°C for preferably 6 to 24 hours, more preferably 12 to 24 hours.

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 undergo 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.

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 test substance exposure group is higher than that of the control. For example, if the level of hypersensitive cell death in the test substance exposure group 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 test substance exposure group 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.

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.

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.

If necessary, the test substances selected in the above procedure may be further evaluated. For example, as 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., those that do not show disease resistance-inducing effect, can be evaluated as more preferable as vaccine-like plant immunotherapeutic agents.

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.

With regard to the embodiments described above, the present invention further discloses the following embodiments.
<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 step of measuring hypersensitive cell death in plant culture cells after contact with the pathogen.
A method for evaluating or selecting a vaccine-like plant immunoinducer, including <2> the method according to <1>, further comprising the step of comparing the level of hypersensitive cell death with a control.
<3> The method according to <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 <1> to <3>, wherein the certain 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.
<5> The method according to any one of <1> to <4>, wherein the test substance is preferably a cyclic peptide, more preferably a cyclic peptide consisting of an amino acid sequence containing seven 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.
<6> The method according to any one of <1> to <5>, wherein the plant culture cells are preferably Arabidopsis thaliana culture cells, and more preferably Arabidopsis thaliana MM2d culture cells.
<7> The method according to any one of <1> to <6>, wherein the plant culture cells are preferably exposed to the test substance for 1 to 24 hours, more preferably 6 to 24 hours, and even more preferably 12 to 24 hours.
<8> The method according to any one of <1> to <7>, wherein the pathogen is brought into contact with the plant culture cells after subculturing, preferably for 6 to 24 hours, more preferably for 12 to 24 hours.
<9> The method according to any one of <1> to <8>, wherein the pathogen is preferably a pathogenic fungus, more preferably a bacterium, and even more preferably Pseudomonas syringae pv. tomato.
<10> The method according to any one of <1> to <9>, wherein the hypersensitive cell death is measured by Evans blue staining.
<11> The method according to any one of <1> to <10>, wherein the vaccine-like plant immunity inducer contributes to inducing a defense response in plants when pathogens invade, preferably one day after application, more preferably two days after application, and preferably within six months after application, more preferably within one month, even more preferably within one week, and also preferably within six months from one day after application, more preferably within six months from two days after application, even more preferably within one month from two days after application, and most preferably within one week from two days after application.

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

Example 1 Immunostimulatory effect of cyclic peptides on plant cells The immunostimulatory effect of cyclic peptides on plant cells was evaluated using two different assay methods, one conventional and one passaged, as shown below.
(1) Conventional method Arabidopsis thaliana MM2d cultured cells, 5 days after subculturing, were dispensed in 60 μL portions into 96-well plates. Then, 100 μM cyclic peptide (dissolved in DMSO), or DMSO and 100 μM sodium salicylate as a positive control, were added and treated for 1 hour. Subsequently, 40 μL of Pst DC3000 (Pseudomonas syringae pv. tomato DC3000 avrRpm1) solution was added as an incompatible plant pathogenic bacterium and inoculated. 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 cell solution treated with pathogenic bacteria to a final concentration of 1% by weight, and the solution was allowed to stand for 1 hour with occasional stirring. After that, 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) warmed to 55°C, and measuring the absorbance (595 nm) of the extracted Evans blue dye diluted fourfold with the eluate.
(2) Passaging system As with the conventional system, cyclic peptides were added and allowed to stand overnight. The entire cell solution was then passed into a 24-well plate containing 1.2 mL of MM2d passaging medium. After 6 days of shaking culture, 60 μL of each culture medium was transferred to a 96-well plate, 40 μL of Pst DC3000 solution was inoculated, and dead cell staining with Evans blue was performed after 20 hours.

Cell death is one of the defense responses that plants induce themselves when they recognize incompatible pathogenic bacteria. Here, we evaluated the plant immune response 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 for each treatment, with the Evans blue staining value for DMSO-treated cells (without peptide treatment) set to 100%. Each treatment was performed with n=4, and the average value was calculated.

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 indicate that we successfully obtained nine cyclic peptides with long-term immunostimulatory effects.

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 a library prepared using NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolab) was subjected to next-generation sequencing analysis using MiSeq (Illumina). The obtained data was mapped to Arabidopsis thaliana cDNA information using CLC genomics workbench (Philgen), and genes that showed significant (FDR < 0.1) changes in expression in a two-group comparison with DMSO (peptide solvent) treatment (control group) were defined as differentially expressed genes (DEGs) and listed.

DEGs (gene numbers can be found in PANTHER, see http://pantherdb.org/) extracted from data one 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.

Table 4 shows the DEGs extracted from data collected 6 hours after cyclic peptide treatment. This differs significantly from the results obtained 1 hour after the peptide treatment, with almost no changes observed in the gene groups. This result indicates that while the isolated cyclic peptide activates the expression of defense-related genes immediately after administration, this effect largely subsides 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.

Next, cultured cells treated with cyclic peptides were passaged for 6 days, and these were used to infect cells with the Pst DC3000 strain. Changes in gene expression after 1 hour were 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.

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 pathogenic response pathway after administration, and that the cultured cells treated with the peptides maintained a rapid response to pathogens even after multiple cell divisions. Therefore, it was determined that the obtained peptides 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 efficient plant cultivation.

Claims (5)

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 cultured plant cells with a pathogen after cultivation, and a step of measuring hypersensitive cell death in cultured plant cells after contact with the pathogen.
A method for evaluating or selecting vaccine-like plant immunotherapeutic agents, including those mentioned above. The method according to claim 1, further comprising the step of comparing the level of hypersensitive cell death with a control. 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. The method according to any one of claims 1 to 3 , wherein the test substance is a cyclic peptide. The method according to any one of claims 1 to 4 , wherein the plant cultured cells are Arabidopsis thaliana cultured cells.