Vaccine-like plant immunity inducer

Abstract

To provide a novel compound suitable as a vaccine-like plant immune inducer, a vaccine-like plant immune inducer comprising the compound as an active ingredient, and a plant disease control method using the compound.SOLUTION: A compound comprises a cyclic peptide comprising an amino acid sequence represented by the formula (1). In the amino acid sequence, an α-amino group at the amino terminus and a carboxyl group at the carboxyl terminus are joined by a peptide bond. Ser-Gly-Pro-Xaa1-Xaa2-Xaa3-Gln (1) (where, Xaa1 is Ser or Thr, Xaa2 is Arg, Thr, Trp, Cys or Asn, Xaa3 is Gln, Ser, Trp, Asn, Ile, Asp or Leu).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a vaccine-like plant immunity inducer. [Background technology]

[0002] Although plants lack a defensive response to pathogen invasion, similar to animal innate immunity (acquired immunity), they do induce a defense response called plant immunity, which is similar to animal innate immunity. When a pathogen invades, plants detect it at the molecular level and combat the disease through defense responses such as hypersensitive reactions, production of reactive oxygen species, production of antibacterial substances, hardening of cell walls, and construction of a defense barrier against the invasion. Chemicals that induce plant disease resistance are called plant disease resistance inducers. Compared to chemicals that act directly on pathogens, such as fungicides, they have advantages such as a broad target disease spectrum, a lower risk of drug-resistant bacteria emerging, and a lower environmental impact. Known plant disease resistance inducers include probenazole (trade name: Oryzemate®), acibenzolar-S-methyl (trade name: Bayonet®), tiadinil (trade name: V-get®), isotianil (trade name: Stout®), and diclobentiazox (trade name: Boon®). These agents remain in the plant body and activate the plant's defense responses.

[0003] The development of plant disease resistance inducers has long been carried out using complex systems that involve actual plants. However, in recent years, it has been reported that a test substance and a pathogenic bacterium were added to cultured plant cells, and hypersensitive cell death, a disease resistance response in plant cells, was used as an indicator to screen for substances that enhance hypersensitive cell death as plant disease resistance inducers (Patent Document 1 and Non-Patent Document 1). Another study reported that a test substance and an elicitor were added to cultured plant cells in which the jasmonic acid-dependent and salicylic acid-dependent defense pathways of the plant defense system can function independently, and the reactive oxygen species level in the plant cells was used as an indicator to screen for substances that increase the reactive oxygen species level as plant defense activators (Patent Document 2). In these methods, an external stimulus was applied in the presence of the test substance, and substances that directly activate plant resistance were selected.

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

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

[0006] [Non-Patent Document 1] Yoshiteru Noutoshi et al. The Plant Cell, 24(9): 3795-3804 (2012) Summary of the Invention [Problem to be solved by the invention]

[0007] To ensure a stable food supply and reduce environmental impact, it is desirable to develop new pesticides with different mechanisms of action that utilize the defense responses that plants naturally possess. Unlike pesticides that directly activate plant disease resistance, pesticides that strongly induce defense responses in plants when the plant is exposed to a stimulus such as pathogen invasion after a period of time has passed since application of the pesticide, i.e., vaccine-like plant immunity inducers, would be useful as a new disease control method. Thus, the present invention provides a novel compound useful as a vaccine-like plant immunity inducer, a vaccine-like plant immunity inducer containing the compound as an active ingredient, and a method for controlling plant diseases using the compound. [Means for solving the problem]

[0008] The inventors have discovered that the vaccine-like plant immunity-inducing activity 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 an incompatible pathogenic bacterium, and using hypersensitive cell death, one of the plant disease resistance responses, as an indicator.They have also discovered a cyclic peptide with vaccine-like plant immunity-inducing activity.

[0009] That is, the present invention relates to the following 1) to 3). 1) A compound which is a cyclic peptide consisting of an amino acid sequence represented by the following formula (1), in which the α-amino group at the amino terminal of the amino acid sequence and the carboxyl group at the carboxyl terminal of the amino acid sequence are linked by a peptide bond: Ser-Gly-Pro-Xaa1-Xaa2-Xaa3-Gln (1) (In formula (1), Xaa1 represents Ser or Thr, Xaa2 represents Arg, Thr, Trp, Cys or Asn, and Xaa3 represents Gln, Ser, Trp, Asn, Ile, Asp or Leu.) 2) A vaccine-like plant immunity inducer containing at least one compound according to 1) as an active ingredient. 3) A method for controlling plant diseases, which comprises applying the vaccine-like plant immunity inducer according to 2) to a plant or a field. [Effects of the Invention]

[0010] The present invention provides a vaccine-like plant immunity inducer that can induce a strong defense response in a plant when the plant is exposed to a stimulus such as pathogen invasion after a period of time has passed since application. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows the immunostimulatory effect of cyclic peptides on cultured Arabidopsis cells. DETAILED DESCRIPTION OF THE INVENTION

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

[0013] The compound of the present invention is a cyclic peptide consisting of an amino acid sequence represented by the following formula (1), in which the α-amino group at the amino terminal of the amino acid sequence and the carboxyl group at the carboxyl terminal are linked by a peptide bond. Ser-Gly-Pro-Xaa1-Xaa2-Xaa3-Gln (1) (In formula (1), Xaa1 represents Ser or Thr, Xaa2 represents Arg, Thr, Trp, Cys or Asn, and Xaa3 represents Gln, Ser, Trp, Asn, Ile, Asp or Leu.) Preferably, the compound of the present invention is a cyclic peptide consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 9 below, in which the α-amino group at the amino terminal and the carboxyl group at the carboxyl terminal of the amino acid sequence are linked by a peptide bond. Ser-Gly-Pro-Ser-Arg-Gln-Gln (SEQ ID NO: 1) Ser-Gly-Pro-Ser-Arg-Ser-Gln (SEQ ID NO: 2) Ser-Gly-Pro-Ser-Thr-Trp-Gln (SEQ ID NO: 3) Ser-Gly-Pro-Ser-Arg-Asn-Gln (SEQ ID NO: 4) Ser-Gly-Pro-Ser-Trp-Ile-Gln (SEQ ID NO: 5) Ser-Gly-Pro-Ser-Arg-Asp-Gln (SEQ ID NO: 6) Ser-Gly-Pro-Ser-Arg-Leu-Gln (SEQ ID NO: 7) Ser-Gly-Pro-Thr-Cys-Gln-Gln (SEQ ID NO: 8) Ser-Gly-Pro-Ser-Asn-Asn-Gln (SEQ ID NO: 9) Such cyclic peptides can all be synthesized by known chemical synthesis methods.

[0014] As shown in the Examples below, when plant culture cells treated with the compounds of the present invention were subcultured and cultured for 6 days, and then inoculated with incompatible plant pathogenic bacteria, hypersensitive cell death of the plant cells was enhanced compared to untreated plants. On the other hand, when plant culture cells treated with the compounds of the present invention 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 untreated plants. Conversely, a substance that enhanced hypersensitive cell death of plant cells using the latter method compared to untreated plants did not enhance hypersensitive cell death of plant cells using the former method compared to untreated plants. Furthermore, as shown in the Examples below, in plant culture cells treated with the compounds of the present invention, the expression of genes involved in pathogen response was significantly enhanced 1 hour after treatment, but the enhanced expression was transient, revealing that the compounds of the present invention do not directly activate defense responses over a long period of time. Furthermore, when plant cultured cells treated with the compounds of the present invention were subcultured and cultured for six days, and then inoculated with plant pathogenic bacteria, significant changes in the expression of genes involved in defense responses against infection, such as wound response-related genes and pathogen response-related genes, were observed one hour after inoculation. The plant cultured cells on the sixth day of subculture were progeny of the initial cultured cells stimulated by the compounds of the present invention, which had undergone multiple cell divisions, and these progeny cells exhibited rapid response to pathogens. These results demonstrate that, unlike the plant disease resistance inducers described in Patent Document 1 and Non-Patent Document 1, the compounds of the present invention are able to strongly induce the plant's inherent defense responses when invaded by pathogens after a period of time following compound treatment. This effect is thought to be mediated by the plant's immune memory mechanism.

[0015] Therefore, the compounds of the present invention can be used as vaccine-like plant immunity inducers and can be used for inducing plant immunity, specifically for inducing a defense response, or for controlling plant diseases. The compounds of the present invention may be used alone or in combination of two or more.

[0016] In the present invention, the term "vaccine-like plant immunity inducer" refers to an agent that, when applied to a plant and exposed to a stimulus such as pathogen invasion a predetermined period of time, induces a stronger defense response in the plant than in a control (e.g., untreated) plant. Here, "stimuli such as pathogen invasion" include external stimuli such as contact with a pathogen, pathogen invasion, pathogen infection, and contact with a foreign substance. The defense response is not particularly limited as long as it is induced by an external stimulus such as contact with a pathogen, pathogen invasion, pathogen infection, or contact with a foreign substance. Examples of the defense response include hypersensitive responses, production of reactive oxygen species, production of antibacterial substances, hardening of cell walls, and construction of a defense wall against invasion. Hypersensitive responses are preferred, and hypersensitive cell death is more preferred. The aforementioned period is not limited, but is typically at least one day, more preferably at least two days, after application, and is preferably within six months, more preferably within one month, and even more preferably within one week after application. Furthermore, the activation period 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 after application. Thus, in the present invention, the vaccine-like plant immunity inducer can contribute to the induction of a defense response against pathogen invasion into a plant, 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 after application, and is useful for plant disease control. Furthermore, the agent may transiently activate the plant's defense response immediately after application, but the activation usually subsides within a few hours to less than a day.

[0017] The vaccine-like plant immunity inducer of the present invention may be one in which the compound of the present invention is used as is, or may be used as a composition containing the compound as an active ingredient (e.g., pesticide, plant supplement, fertilizer, cultivation substrate, etc.). The composition may be in the form of a liquid or gel composition, or may be in a solid state (block, granular, powder, etc.). In the case of a liquid composition, it may be a concentrated type that can be used as is or after dilution. In the case of a solid composition, it can be used by dissolving it in water.

[0018] The composition may contain optional components in addition to the compound of the present invention. Such components include solvents (e.g., water, buffer solution, culture medium, isopropyl alcohol, xylene, cyclohexane, methylnaphthalene, etc.), carriers (diatomaceous earth, vermiculite, perlite, peat moss, activated carbon, humus, talc, zeolite, clay, carbon black, pulp, straw, soybean meal, bentonite, kaolin, montmorillonite, alumina, calcium carbonate, hydrated lime, silica sand, ammonium sulfate, urea, etc.), surfactants, spreading agents, pH adjusters, preservatives, antioxidants, thickeners, plant essential nutrients, amino acids, nucleic acids, sugars, etc. The composition can be prepared by appropriately combining the compound of the present invention with these components according to a standard method.

[0019] The content of the compound of the present invention in the composition can be appropriately set to suit the application amount. In the case of a solid composition, the content is typically 0.01 wt% or more, more preferably 0.1 wt% or more, and preferably 70 wt% or less, more preferably 50 wt% or less, based on the total weight of the composition. It is also preferably 0.01 to 70 wt%, more preferably 0.1 to 70 wt%, even more preferably 0.1 to 50 wt%. In the case of a liquid or gel composition, the content is typically 1 wt% or more, more preferably 5 wt% or more, and preferably 90 wt% or less, more preferably 80 wt% or less, based on the total weight of the composition. It is also preferably 1 to 90 wt%, more preferably 5 to 90 wt%, even more preferably 5 to 80 wt%.

[0020] The vaccine-like plant immunity inducer of the present invention is preferably applied before the onset of disease. The method for supplying the vaccine-like plant immunity inducer of the present invention is not particularly limited, as long as it is applied so that the immunity induction effect can be exerted in the plant when necessary. That is, there are no particular limitations on the manner in which the vaccine-like plant immunity inducer of the present invention is contacted with or delivered to the plant or the soil in the plant's rhizosphere. Such manners may include spraying, dusting, dipping, dressing, painting, fumigation, smoking, irrigation, etc. Specific embodiments include a method of spraying or applying an agent to a plant, a method of dipping plant seeds in a liquid containing an agent, a method of applying or smearing an agent on seeds, a method of spraying an agent in a field, a method of mixing an agent with soil, a method of mixing an agent with fertilizer, etc.

[0021] The application amount of the vaccine-like plant immunity inducer of the present invention may vary depending on the application method, application time, plant species, cultivation density, growth stage, formulation of the agent, etc. For example, the amount of the compound of the present invention to be used is 10,000 m 2 The concentration of the compound of the present invention is usually preferably 1 g or more, more preferably 5 g or more, and preferably 5,000 g or less, more preferably 1,000 g or less, per unit weight. It is also preferably 1 to 5,000 g, more preferably 5 to 5,000 g, and even more preferably 5 to 1,000 g. When used as a liquid composition, the concentration of the compound of the present invention is usually preferably 0.01 ppm or more, more preferably 0.1 ppm or more, and 10,000 ppm or less, more preferably 3,000 ppm or less. It is also preferably 0.01 to 10,000 ppm, more preferably 0.1 to 10,000 ppm, and even more preferably 0.1 to 3,000 ppm. The vaccine-like plant immunity inducer of the present invention may be applied in an amount within the above range at once, or may be applied in divided doses several times.

[0022] The target plant for the vaccine-like plant immunity inducer of the present invention is not particularly limited, and may be either a monocotyledonous or dicotyledonous plant as long as it is a cultivated plant. Examples include 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, kidney bean, alfalfa, peanut, etc.), Brassicaceae family (radish, Chinese cabbage, cabbage, 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.), and Vitaceae family (grape, etc.).

[0023] The vaccine-like plant immunity inducer of the present invention is useful for controlling plant diseases because it can induce a strong defense response in a plant when the plant is exposed to a stimulus such as invasion by a pathogen after a period of time has elapsed since application of the agent. Plant diseases can be caused by filamentous fungi, bacteria, viruses, etc. Specifically, for example, rice blast fungus (Magnaporthe oryzae), rice seedling blight fungus (Burkholderia plantarii), rice leaf blight fungus (Cochliobolus miyabeanus), rice sheath blight fungus (Rhizoctonia solani), rice bacterial leaf blight fungus (Xanthomonas oryzae), potato powdery scab fungus (Spongospora subterranea), potato late blight fungus (Phytophthora infestans), potato black blight fungus (Rhizoctonia solani), potato scab fungus (Streptomyces scabies), barley powdery mildew fungus (Eryshiphe graminis f. sp. hordei), wheat head blight fungus (Gibberella zeae), wheat snow blight fungus (Sclerotinia borealis), wheat leaf rust (Puccinia recondita), wheat powdery mildew (Erysiphe graminis), wheat root rot (Rhizoctonia solani), soybean downy mildew (Peronospora manshurica), soybean purple spot (Cercospora kikuchii), pea ascochyta (Mycosphaerella pinodes), corn smut (Ustilago maydis), sweet potato fusarium wilt (Fusarium oxysporum f. sp. batatas), melon fusarium wilt (Fusarium oxysporum f. sp. melonis), lettuce root rot (Fusarium oxysporum f. sp. lactucae), tomato wilt (Fusarium oxysporum f. sp.lycopersici, Verticillium dahliae, Colletotrichum phomoides, Pseudomonas syringae pv. tomato, Fusarium oxysporum f. sp. spinaciae, Plasmodiophora brassicae, Pythium debaryanum, Botrytis cinerea, and the like. In a preferred embodiment, the vaccine-like plant immunity inducer of the present invention is used to control diseases caused by filamentous fungi or bacteria, i.e., pathogenic fungi. In a more preferred embodiment, the vaccine-like plant immunity inducer of the present invention is used to control diseases caused by pathogenic bacteria.

[0024] In relation to the above-described embodiment, the present invention further discloses the following aspects. <1> A compound which is a cyclic peptide consisting of an amino acid sequence represented by the following formula (1), in which the α-amino group at the amino terminal of the amino acid sequence and the carboxyl group at the carboxyl terminal of the amino acid sequence are linked by a peptide bond. Ser-Gly-Pro-Xaa1-Xaa2-Xaa3-Gln (1) (In formula (1), Xaa1 represents Ser or Thr, Xaa2 represents Arg, Thr, Trp, Cys or Asn, and Xaa3 represents Gln, Ser, Trp, Asn, Ile, Asp or Leu.) <2> A cyclic peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 9, in which the α-amino group at the amino terminal and the carboxyl group at the carboxyl terminal of the amino acid sequence are linked by a peptide bond. <1> The compound described. <3> <1> or <2> A vaccine-like plant immunity inducer comprising at least one of the compounds described above as an active ingredient. <4> contributes to the induction of a defense response in plants when invaded by a pathogen, preferably at least one day after application, more preferably at least two days after application, and preferably within six months, more preferably within one month, even more preferably within one week after application, and preferably at least one day after application but within six months, more preferably at least two days after application but within six months, even more preferably at least two days after application but within one month, even more preferably at least two days after application but within one week, <3> A vaccine-like plant immunity inducer as described above. <5> a solid composition, wherein the content of the compound is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 70% by weight or less, more preferably 50% by weight or less, preferably 0.01 to 70% by weight, more preferably 0.1 to 70% by weight, and even more preferably 0.1 to 50% by weight, based on the total weight of the composition; <3> or <4> A vaccine-like plant immunity inducer as described above. <6> A liquid or gel composition, in which the content of the compound is preferably 1% by weight or more, more preferably 5% by weight or more, and preferably 90% by weight or less, more preferably 80% by weight or less, preferably 1 to 90% by weight, more preferably 5 to 90% by weight, and even more preferably 5 to 80% by weight, based on the total weight of the composition. <3> or <4> A vaccine-like plant immunity inducer as described above.

[0025] <7> <3> ~ <6> A method for controlling plant diseases, which comprises applying the vaccine-like plant immunity inducer according to any one of the above to a plant or a field. <8> <3> ~ <6> A method for inducing immunity in plants, comprising applying the vaccine-like plant immunity inducer according to any one of the above to a plant or a field. <9> The composition controls plant diseases preferably at least one day after application, more preferably at least two days after application, and preferably within six months, more preferably within one month, and even more preferably within one week after application. The composition also controls plant diseases preferably at least one day after application but within six months, more preferably at least two days after application but within six months, more preferably at least two days after application but within one month, and even more preferably at least two days after application but within one week. <7> The method described. <10> The composition contributes to the induction of a defense response in a plant against pathogen invasion, preferably at least one day after application, more preferably at least two days after application, and preferably within six months, more preferably within one month, and even more preferably within one week after application, and preferably at least one day after application but within six months, more preferably at least two days after application but within six months, even more preferably at least two days after application but within one month, and even more preferably at least two days after application but within one week, <8> The method described. <11> To the plant body <3> ~ <6> The vaccine-like plant immunity inducer according to any one of the above is sprayed or applied. <3> ~ <6> immersing plant seeds in a solution containing the vaccine-like plant immunity inducer according to any one of the preceding claims; <3> ~ <6> a field to which the vaccine-like plant immunity inducer according to any one of the preceding claims is applied or smeared; <3> ~ <6> a vaccine-like plant immunity inducer according to any one of the preceding claims, <3> ~ <6> or adding to a fertilizer <3> ~ <6> and mixing the vaccine-like plant immunity inducer according to any one of the above. <7> ~ <10> The method according to any one of the preceding claims. <12> The amount of the compound used is 10,000 m 2 per serving, it is preferably 1 g or more, more preferably 5 g or more, and preferably 5,000 g or less, more preferably 1,000 g or less, preferably 1 to 5,000 g, more preferably 5 to 5,000 g, and even more preferably 5 to 1,000 g. <7> ~ <11> The method according to any one of the preceding claims. <13> The amount of the compound used is, in terms of concentration, preferably 0.01 ppm or more, more preferably 0.1 ppm or more, and 10,000 ppm or less, more preferably 3,000 ppm or less, preferably 0.01 to 10,000 ppm, more preferably 0.1 to 10,000 ppm, and even more preferably 0.1 to 3,000 ppm. <7> ~ <11> The method according to any one of the preceding claims.

[0026] <14> For producing a vaccine-like plant immunosuppressant, <1> or <2> Use of the compounds described. <15> The vaccine-like plant immunosuppressant contributes to the induction of a defense response in a plant when invaded by a pathogen, preferably at least one day after application, more preferably at least two days after application, and preferably within six months, more preferably at least one month, and even more preferably at least one week after application, and preferably at least one day after application but within six months, more preferably at least two days after application but within six months, even more preferably at least two days after application but within one month, and even more preferably at least two days after application but within one week. <14> Use as described. <16> the vaccine-like plant immunosuppressant is a solid composition, and the content of the compound is preferably 0.01% by weight or more, more preferably 0.1% by weight or more, and preferably 70% by weight or less, more preferably 50% by weight or less, preferably 0.01 to 70% by weight, more preferably 0.1 to 70% by weight, and even more preferably 0.1 to 50% by weight, based on the total weight of the composition; <14> or <15> Use as described. <17> The vaccine-like plant immunosuppressant is a liquid or gel composition, and the content of the compound is preferably 1% by weight or more, more preferably 5% by weight or more, and preferably 90% by weight or less, more preferably 80% by weight or less, preferably 1 to 90% by weight, more preferably 5 to 90% by weight, and even more preferably 5 to 80% by weight, based on the total weight of the composition. <14> or <15> Use as described. [Example]

[0027] The present invention will be described in more detail below based on examples, but the present invention is not limited to these examples.

[0028] Example 1 Immunostimulatory effect of cyclic peptides on plant cells The immunostimulatory effects of cyclic peptides on plant cells were evaluated using two different assay methods: conventional and passage systems, as shown below. (1) Conventional system Five-day-old Arabidopsis thaliana MM2d cultured cells were dispensed into 96-well plates in 60 μL aliquots and treated for 1 hour with 100 μM cyclic peptide (DMSO) or DMSO and 100 μM sodium salicylate (positive control). Then, 40 μL of Pst DC3000 (Pseudomonas syringae pv. tomato DC3000 avrRpm1), an incompatible plant pathogen, was added and inoculated. After 20 hours, dead cells were stained with Evans blue (Noutoshi & Shirasu, Methods in Molecular Biology, 1795:39-47 (2018)). Specifically, Evans blue solution was added to the pathogen-treated cell solution to a final concentration of 1% by weight and allowed to stand for 1 hour with occasional stirring. The Evans blue solution was then removed and the cells were washed three times with 250 μL of water. 200 μL of elution solution (50% by volume methanol, 1% by weight SDS) heated to 55°C was added, and the amount of cells that had undergone cell death was assessed by measuring the absorbance (595 nm) of the extracted Evans blue dye diluted 4-fold with the elution solution. (2) Subculture As with the conventional system, the cyclic peptide was added and left overnight, after which the entire cell suspension was subcultured in 1.2 mL of MM2d subculture medium in a 24-well plate. After 6 days of shaking culture, 60 μL of each culture was transferred to a 96-well plate and inoculated with 40 μL of Pst DC3000 solution. After 20 hours, dead cells were stained with Evans blue.

[0029] Cell death is a defense response induced by plants in response to the recognition of incompatible pathogenic bacteria. Here, we quantitatively measured cell death and calculated the cell death enhancement rate (%) to evaluate the plant immune response. The cell death enhancement rate (%) was calculated as the ratio of the Evans blue staining value for each treatment, with the Evans blue staining value for DMSO-treated (no peptide treatment) cells set at 100%. Each treatment was performed in triplicate, and the average was calculated.

[0030] From the evaluation of 320 candidate cyclic peptides, nine positive peptides were obtained that reacted only with the passaged system. Five peptides were also obtained that reacted only with the conventional system. These selected peptides were re-evaluated in the conventional system and the passaged system (Tables 1 and 2 and Figure 1). Further testing confirmed that the nine positive peptides found in the passaged system did not exhibit immune stimulating effects in the conventional system, but did exhibit immune stimulating effects only in the passaged system. From these results, we succeeded in identifying nine cyclic peptides with long-term immunostimulatory effects.

[0031] [Table 1]

[0032] [Table 2]

[0033] Example 2: Analysis of the effect of cyclic peptides with long-term immune priming activity on plant cells Arabidopsis thaliana cultured cells were treated with one of the cyclic peptides (cyclo-SGPSWIQ) obtained in Example 1, and samples taken after 1 hour, 6 hours, or 6 days of subculture were analyzed for comprehensive gene expression changes using RNA-seq. RNA was extracted, and libraries prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) were subjected to next-generation sequencing analysis using MiSeq (Illumina). The resulting data were mapped to Arabidopsis thaliana cDNA information using CLC genomics workbench (Filgen). Genes whose expression was significantly upregulated (FDR < 0.1) in a two-group comparison with DMSO (peptide solvent) treatment (control) were defined as differentially expressed genes (DEGs) and listed using the DEseq2 package in the statistical analysis software R.

[0034] DEGs (see PANTHER for gene numbers: http: / / pantherdb.org / ) extracted from the 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 pathogen responses. Among these, those related to pathogen responses or DNA regulation are shown in Table 3. Specifically, genes involved in defense responses, immune responses, and phenylpropanoid synthesis, which are involved in antibacterial substance production, were detected. Genes related to DNA and histone regulation were also detected, suggesting that the long-term immune priming effect of the cyclic peptide may be mediated by epigenetic control.

[0035] [Table 3]

[0036] Table 4 shows the DEGs extracted from the data 6 hours after cyclic peptide treatment. Significantly different from the results obtained 1 hour after peptide treatment, almost no altered gene groups were observed. These results demonstrate that the isolated cyclic peptide activates the expression of defense-related genes immediately after administration, but this effect subsides within 6 hours of administration. This indicates that the stimulation by the cyclic peptide is only transient and does not directly activate defense responses over a long period of time.

[0037] [Table 4]

[0038] Next, the cultured cells treated with the cyclic peptide were passaged for 6 days and then infected with Pst DC3000 strain. Gene expression changes were examined 1 hour later. The results are shown in Table 5. Samples derived from cells treated with the cyclic peptide showed significant changes in defense response pathways, including genes related to injury response and pathogen response. This means that even after 6 days, the response at an early stage, 1 hour after pathogen infection, was significantly enhanced in the cyclic peptide-treated cells compared to the control. The cells on passage 6 were the descendants of the peptide-stimulated primary culture cells after multiple cell divisions, and they showed rapid response to pathogenic bacteria. This confirmed that the cyclic peptide produced in this study induces a long-term priming effect, as confirmed by the gene responses.

[0039] [Table 5]

[0040] In this study, we successfully isolated nine cyclic peptides that induce long-term priming. Furthermore, RNA-seq analysis demonstrated that the peptides transiently induced a pathogen response pathway after administration, and that the cultured cells maintained a rapid response to pathogens even after multiple cell divisions, demonstrating that the peptides induce long-term priming. This ability to induce long-term priming with only a transient stimulus is extremely useful for efficient plant cultivation.

Claims

1. A compound which is a cyclic peptide consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 9, in which the α-amino group at the amino terminal and the carboxyl group at the carboxyl terminal of said amino acid sequence are linked by a peptide bond.

2. A vaccine-like plant immunity inducer for use in monocotyledonous or dicotyledonous plants, comprising at least one compound according to claim 1 as an active ingredient.

3. A method for controlling plant diseases, which comprises applying the vaccine-like plant immunity inducer according to claim 2 to monocotyledonous or dicotyledonous plants or to a field of monocotyledonous or dicotyledonous plants.

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