Polypeptides, plant immunological inducers and uses thereof

Abstract

The application provides a polypeptide, a plant immune elicitor and application thereof. The polypeptide has 10 or 11 amino acid residues; the amino acid at the first position of the polypeptide is serine, and the amino acids at the 9th and 10th positions are both cysteine; if the polypeptide has 11 amino acid residues, the amino acid at the 11th position of the polypeptide is asparagine. The polypeptide can solve the problem that there is no small peptide secreted by gramineae plants in the prior art, and is suitable for the field of plant immune elicitors.

Description

Technical Field

[0001] This invention relates to the field of plant immune inducers, and more specifically, to a polypeptide, a plant immune inducer, and its applications. Background Technology

[0002] In agricultural production, crop diseases pose a significant threat to global food security and agricultural sustainability, causing economic losses of hundreds of billions of dollars annually. Traditionally, chemical pesticides have been widely used to control diseases, but their frequent use not only pollutes the environment and damages ecosystems but also impacts human health through the food chain. While crop disease-resistant breeding is an effective strategy, it is limited by long cycles, high costs, and limited disease-resistant gene resources. Furthermore, the rapid evolution of pathogens reduces the durability of disease-resistant genes.

[0003] Plant inducers, including compounds and biological agents, enhance disease resistance by activating the plant's immune system, and are gradually becoming a key strategy for modern agriculture to address disease challenges. With global climate change, agricultural intensification, and the increasing complexity of diseases, the application of plant inducers is particularly important. Among them, plant-secreted small peptides, as a class of endogenous signaling molecules, play an important regulatory role in plant stress responses and growth and development, exhibiting high efficiency, broad spectrum, and environmental friendliness, thus becoming one of the preferred solutions for modern disease management.

[0004] Specifically, small peptides offer the following advantages: First, high efficiency—they can activate plant immunity at extremely low concentrations, enhancing crops' defense against multiple diseases; second, broad-spectrum activity—compared to relying on a single disease-resistant gene, small peptides can simultaneously address multiple diseases, simplifying the control process; third, environmental friendliness—as molecules naturally occurring in plants, small peptides are easily degraded, safe and harmless to the environment and humans, aligning with the goals of green and sustainable agriculture. Given these advantages, small peptides can be synergistically combined with disease-resistant breeding, biological control, and chemical control to form comprehensive disease management programs, enhancing disease prevention effectiveness while reducing reliance on single methods.

[0005] Research on small peptides such as systemic peptides, Pep1, ZIP1, PIP1, PSK, and SCOOP12 has shown that endogenous small peptides in plants have a significant effect on inducing disease resistance. However, to date, there are no reports on the application of secreted small peptides derived from the Poaceae family in plant disease control. The Poaceae family covers most major food crops, and the improvement of their disease control technologies is crucial for ensuring food security and sustainable agricultural development. Summary of the Invention

[0006] The main objective of this invention is to provide a polypeptide, a plant immune inducer, and its application, in order to solve the problem of the lack of small peptides secreted by grass plants in the prior art.

[0007] To achieve the above objective, according to a first aspect of the present invention, a polypeptide is provided having 10 or 11 amino acid residues; the amino acid at position 1 of the polypeptide is serine, and the amino acids at positions 9 and 10 are both cysteine; if the polypeptide has 11 amino acid residues, the amino acid at position 11 of the polypeptide is asparagine.

[0008] Furthermore, the second amino acid of the polypeptide is glycine, arginine, valine, or asparagine; the third amino acid is serine, glycine, arginine, or proline; the fourth amino acid is serine or asparagine; the fifth amino acid is proline, glycine, serine, threonine, or arginine; the sixth amino acid is proline, arginine, glycine, aspartic acid, serine, or histidine; the seventh amino acid is threonine, serine, or glycine; and the eighth amino acid is histidine, threonine, or serine.

[0009] Furthermore, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 15-31.

[0010] Furthermore, peptides also contain chemical modifications.

[0011] Furthermore, the chemical modification includes replacing all -SH residues on cysteine ​​residues in the polypeptide with -SeH residues.

[0012] To achieve the above objective, according to a second aspect of the present invention, a plant immune inducer is provided, the plant immune inducer comprising a polypeptide; the polypeptide having 10 or 11 amino acid residues; the amino acid at position 1 of the polypeptide being serine, and the amino acids at positions 9 and 10 being cysteine; when the polypeptide has 11 amino acid residues, the amino acid at position 11 of the polypeptide is asparagine.

[0013] Furthermore, the second amino acid of the polypeptide is glycine, arginine, valine, or asparagine; the third amino acid is serine, glycine, arginine, or proline; the fourth amino acid is serine or asparagine; the fifth amino acid is proline, glycine, serine, threonine, or arginine; the sixth amino acid is proline, arginine, glycine, aspartic acid, serine, or histidine; the seventh amino acid is threonine, serine, or glycine; and the eighth amino acid is histidine, threonine, or serine.

[0014] Furthermore, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 15-31.

[0015] Furthermore, peptides also contain chemical modifications.

[0016] Furthermore, the chemical modification includes replacing all -SH residues on cysteine ​​residues in the polypeptide with -SeH residues.

[0017] To achieve the above objectives, according to a third aspect of the present invention, a method for improving plant disease resistance is provided, the method comprising applying the above-mentioned polypeptide or the above-mentioned plant immune inducer to a target plant; the target plant includes grasses.

[0018] Furthermore, the application methods include one or more of the following: spraying, smearing, soaking, leaf cutting, or injection.

[0019] Furthermore, grasses include one or more of wheat, rice, corn, millet, barley, or sorghum.

[0020] To achieve the above objectives, according to a fourth aspect of the present invention, the use of the above-mentioned polypeptide or any of the above-mentioned plant immune inducers in enhancing plant immunity and / or disease resistance is provided.

[0021] To achieve the above objectives, according to a fifth aspect of the present invention, the use of the above-mentioned polypeptide in the preparation of a reagent for improving plant immunity and / or disease resistance is provided.

[0022] Applying the technical solution of this invention, this application provides a polypeptide having 10 or 11 amino acid residues, with the first amino acid being serine, and the 9th and 10th amino acids being cysteine. When the polypeptide has 11 amino acid residues, the 11th amino acid is asparagine. The polypeptide in this application has immunomodulatory activity, overcoming the problem of the lack of secreted small peptides in gramineous plants. Moreover, it has broad-spectrum disease resistance and, when used as a plant resistant inducer, exhibits high thermal stability and is not easily degraded, making it suitable for use in gramineous plants. This significantly improves the practicality and longevity of plant immune resistant inducers, opening up new avenues for green control of agricultural diseases. Attached Figure Description

[0023] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0024] Figure 1 This document shows the expression results of pDCCs induced by immune elicitors and pathogens in Example 1 of this application, as well as a pDCCs protein sequence alignment diagram; wherein, Figure 1 A represents wheat immune elicitors flg22 and chitin, as well as pathogen F. . graminearum , X. translucens and Z. tritici Heatmap of pDCCs gene-induced expression after treatment. Figure 1Figure B shows the amino acid sequence alignment results of 13 wheat secretory peptide precursor proteins pDCCs.

[0025] Figure 2 A schematic diagram of the vector structure of the conversion vector pLGY-OE3-pDCC1 in Embodiment 1 of this application is shown.

[0026] Figure 3 The diagram shows the identification results of mature DCC1 small peptides from Example 1 of this application and the sequence alignment results of DCC homologous small peptides in gramineous plants; wherein, Figure 3 Figure A shows the mass spectrometry analysis results of interstitial fluid extract from leaves of pDCC1-overexpressing transgenic plants. Figure 3 Figure B shows the sequence alignment results of the mature small peptide at the C-terminus of the DCC1 homologous protein from grasses (Poaceae). Figure 3 The figure shows the comparative statistics of different amino acid sites of the C-terminal mature small peptides of grass plants.

[0027] Figure 4 The diagram shows the DCC1 core sequence and key amino acid activity identification results of Example 2 of this application; wherein, Figure 4 In diagram A, we see a schematic of small peptide sequences of different lengths. Figure 4 Figure B shows the results of MAPK phosphorylation detection in wheat treated with small peptides of different lengths. Figure 4 The figure in C represents the results of MAPK phosphorylation detection in wheat after different small peptides were treated with cysteine ​​to serine after the mutation of cysteine ​​on the DCC small peptide.

[0028] Figure 5 The diagram shows the results of MAPK phosphorylation activation at different DCC1 concentrations in Example 2 of this application.

[0029] Figure 6 The diagram shows the thermal stability test results of DCC1 in Embodiment 2 of this application.

[0030] Figure 7 The graph shows the detection results of the selenocysteine ​​modification improving DCC1 activity in Example 2 of this application specification.

[0031] Figure 8 The diagram shows the results of immunoassay of DCC1-like peptides in gramineous crops according to Example 2 of this application; wherein, Figure 8 Figure A shows the results of MAPK phosphorylation detection in wheat treated with DCC1, DCC2, DCC5, and DCC6. Figure 8 Figure B is a schematic diagram showing the MAPK phosphorylation detection results of rice treated with OsDCC1, OsDCC2, OsDCC3, and OsDCC4. Figure 8The diagram in C shows the results of MAPK phosphorylation detection in wheat, rice, maize, Arabidopsis thaliana, and tobacco treated with DCC1. Figure 8 The diagram in Figure D shows the results of MAPK phosphorylation detection in rice, wheat, maize, Arabidopsis thaliana, and tobacco treated with OsDCC1. Figure 8 E is a schematic diagram showing the MAPK phosphorylation detection results of ZmDCC1 treatment in maize, rice, wheat, Arabidopsis thaliana, and tobacco.

[0032] Figure 9 The diagram shows the test results of DCC1 and DCC2 enhancing wheat resistance to bacterial leaf streak in Example 3 of this application; wherein, Figure 9 Image A shows the morphological characteristics of diseased wheat leaves. Figure 9 Figure B shows the bacterial colony count on wheat leaves.

[0033] Figure 10 The diagram shows the test results of OsDCC4 and DCC1 enhancing rice resistance to bacterial leaf streak in Example 3 of this application; wherein, Figure 10 Image A shows the morphological characteristics of diseased rice leaves. Figure 10 Figure B shows a statistical chart of leaf lesion length.

[0034] Figure 11 The diagram shows the test results of OsDCC4-enhanced rice resistance to bacterial blight in Example 3 of this application; wherein, Figure 11 Image A shows the morphological characteristics of diseased rice leaves. Figure 11 Figure B shows a statistical chart of leaf lesion length.

[0035] Figure 12 The diagram shows the test results of DCC1-enhanced wheat scab resistance in Example 3 of this application, wherein... Figure 12 Image A shows the morphological symptoms of wheat scab on the ear. Figure 12 Figure B shows the statistical chart of the incidence index of Fusarium head blight. Detailed Implementation

[0036] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0037] Terminology Explanation:

[0038] Plant immune inducers are substances that can induce an immune defense response in plants against pests and diseases. Even at low concentrations, they can be recognized by plants as signaling molecules, inducing and enhancing the plant's own immune response, thereby improving the plant's resistance to diseases and delaying or mitigating their occurrence and development. Their mechanisms of action can be categorized as: activating defense gene expression, regulating hormone balance, inducing the synthesis of disease-resistant proteins, and improving secondary metabolic processes. They include: proteins, oligosaccharides, lipids, small molecule metabolites, and microbial agents.

[0039] Plant secretory peptides are a class of small polypeptide molecules (usually composed of 2-100 amino acids) synthesized by plant cells and released into the extracellular space through the secretory pathway. They play important signal transduction and regulatory roles in plant growth and development, stress resistance, and immune responses. These secretory peptides are typically encoded and synthesized by plant genes. Within plant cells, they are synthesized into peptide chains according to specific gene sequence information through the action of organelles such as ribosomes. Then, after a series of post-translational modifications, such as glycosylation and phosphorylation, biologically active secretory peptides are formed. These secretory peptides can be stored in vesicles within plant cells. When stimulated by external factors (such as pathogen infection or environmental stress signals), the vesicles fuse with the cell membrane, releasing the secretory peptides into the extracellular space.

[0040] The mechanism of action of plant secreted small peptides mainly depends on their interaction with receptor kinases, namely signal recognition (the secreted small peptide binds to receptor kinases on the cell membrane, such as the binding of PSK to PSKR), signal transduction (after the receptor kinase is activated, it transmits signals through downstream signaling pathways (such as the MAPK cascade reaction) to regulate gene expression and cellular responses) and produces physiological effects (ultimately manifested as promoting growth, enhancing stress resistance or activating immune responses).

[0041] MAPK (Mitogen-Activated Protein Kinase): This signaling pathway regulates multiple physiological and biochemical processes in plants, playing a crucial role, particularly in responses to environmental stimuli and immune responses. Activation of the MAPK pathway is usually accompanied by its phosphorylation.

[0042] As mentioned in the background section, the prior art lacks secretory peptides of grasses. Therefore, in this application, the inventors attempted to discover a new immune polypeptide that can be applied to the development of peptide inducers for grasses, and thus proposed a series of protection schemes in this application.

[0043] In a first typical embodiment of this application, a polypeptide is provided having 10 or 11 amino acid residues, wherein the first amino acid is serine, and the 9th and 10th amino acids are both cysteine; if the polypeptide has 11 amino acids, the 11th amino acid of the polypeptide is asparagine.

[0044] The polypeptide has a sequence such as SXXXXXXXCC (with 10 amino acid residues) or an amino acid sequence as shown in SEQ ID NO: 1 (with 11 amino acid residues).

[0045] SEQ ID NO: 1: SXXXXXXXXCCN.

[0046] The "X" above represents any naturally occurring or non-natural amino acid. That is, any amino acid can occupy the "X" position without affecting the overall function or properties of the sequence. "X" includes, but is not limited to, alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), and histidine (His; H). Isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), or valine (Val; V), or modified, substituted or substituted A, N, D, R, C, E, Q, G, H, I, L, K, M, F, P, S, T, W, Y, or V.

[0047] In response to the current lack of effective inducers of resistance in gramineous crops, this application aims to fill this technological gap. Through an innovative reverse genetics screening strategy, a novel immune-activating peptide (i.e., the aforementioned polypeptide) – DCC1 (DUAL CYSTEINES IN C-TERMINUS) – and its homologous peptides in gramineous plants were successfully identified from endogenously secreted small peptides in wheat. These small peptides possess three unique conserved amino acid features: a serine (S) at the sequence initiation and two cysteine ​​residues (C) at the end, located at positions 1, 9, and 10, respectively. This structural feature is shared by members of the DCC family and exhibits stable and efficient immune-activating function.

[0048] DCC1 and its analogues exhibit cross-species immune activation capabilities, inducing immune responses in gramineous crops such as wheat, rice, and maize. This demonstrates the versatility of the DCC1 small peptide structure and lays the foundation for its broad-spectrum disease control in gramineous crops. This invention, by revealing the immunomodulatory activity of DCC1 small peptides and their analogues, provides a safe and green disease control strategy for gramineous crops. Through various technological optimizations, breakthroughs can be achieved in the molecular diversity, broad-spectrum disease resistance, and field applicability of plant immune inducers, providing strong biotechnological support for the sustainable development of modern agriculture.

[0049] In a preferred embodiment, the second amino acid of the polypeptide is glycine, arginine, valine, or asparagine; the third amino acid is serine, glycine, arginine, or proline; the fourth amino acid is serine or asparagine; the fifth amino acid is proline, glycine, serine, threonine, or arginine; the sixth amino acid is proline, arginine, glycine, aspartic acid, serine, or histidine; the seventh amino acid is threonine, serine, or glycine; and the eighth amino acid is histidine, threonine, or serine.

[0050] In addition to the conserved sites at positions 1, 9, and 10 mentioned above, the amino acids at other positions of the polypeptide in this application also exhibit a certain degree of conservation: for example, the second amino acid is glycine (most commonly glycine; based on statistics of the amino acid types at this site in DCC1 homologous peptides from wheat, barley, rice, millet, corn, and sorghum, glycine accounts for 86.36%), arginine, valine, or asparagine; the third amino acid is mainly serine or glycine, with a few being arginine or proline; the fourth amino acid is serine or asparagine. (Polar and uncharged); the 5th amino acid is mainly proline or glycine, with a few being serine, threonine, or arginine; the 6th amino acid is mainly proline or arginine, with a few being glycine, aspartic acid, serine, or histidine; the 7th amino acid is threonine, serine, or glycine; the 8th amino acid is histidine (mostly histidine, based on the amino acid type of DCC1 homologous peptides at this site in wheat, barley, rice, millet, corn, and sorghum, histidine accounts for 86.36%), threonine, or serine.

[0051] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 15-31.

[0052] SEQ ID NO: 15: SGSSPPTHCC.

[0053] SEQ ID NO: 16: SGSSGRTHCC.

[0054] SEQ ID NO: 17: SRSSTRTHCC.

[0055] SEQ ID NO: 18: SGGSGGTHCC.

[0056] SEQ ID NO: 19:SVSSPPTHCC.

[0057] SEQ ID NO: 20: SGSSRDTHCC.

[0058] SEQ ID NO: 21: SNGSGGGHCC.

[0059] SEQ ID NO: 22: SGGGNGSGTCC.

[0060] SEQ ID NO: 23: SGGGNRGGTCC.

[0061] SEQ ID NO: 24:SGGNSPTHCC.

[0062] SEQ ID NO: 25: SGRNPRTHCC.

[0063] SEQ ID NO: 26: SGSNPPSHCCN.

[0064] SEQ ID NO: 27: SGPSPPSSCCN.

[0065] SEQ ID NO: 28:SGSNPRTHCC.

[0066] SEQ ID NO: 29: SSGSNGSSHCC.

[0067] SEQ ID NO: 30: SGSNGSSHCCN.

[0068] SEQ ID NO: 31: SGSNSHTHCCN.

[0069] This application utilizes a reverse genetics strategy to analyze wheat's response to immune elicitors flg22, chitin, and pathogens such as... Fusarium graminearum , Xanthomonas translucens and Zymoseptoria tritici Transcriptome changes after attack. By comparing and analyzing the expression data of secretory small peptide precursor protein encoding genes (pDCCs) under these responses, candidate genes highly expressed after immune stimulation and pathogen infection were precisely located.

[0070] Subsequently, this application further employed mass spectrometry to successfully identify the key sequence of the mature DCC1 small peptide in the intercellular fluid of wheat, providing a foundation for subsequent molecular design and functional verification. During this process, the applicant recognized the importance of the two conserved cysteine ​​residues at the C-terminus of the small peptide, which not only ensure the stability of the peptide structure but are also crucial for its activity. This application verified the minimum effective active fragment of DCC1 through MAPK phosphorylation analysis.

[0071] This application also discovered several polypeptides highly homologous to DCC1, such as OsDCCs in rice, ZmDCCs in maize, HvDCCs in barley, SiDCCs in millet, and SbDCCs in sorghum. The discovery of these similar peptides has greatly expanded the molecular diversity of the plant immune-activating peptide family, opened up new possibilities for integrated crop disease control strategies, and in particular enhanced biological control methods against bacterial diseases, meeting the green and efficient needs of sustainable agricultural development.

[0072] In a preferred embodiment, the above-mentioned polypeptide also contains chemical modifications.

[0073] In a preferred embodiment, the chemical modification includes replacing -SH on cysteine ​​residues in the polypeptide with -SeH.

[0074] This application also discovered that chemical modification of these small peptides can further optimize their activity and application efficiency. In a specific embodiment of this application, a selenocysteine ​​chemical modification technique was employed. By replacing the sulfur in the sulfhydryl group of cysteine ​​in the small peptide with selenium, the activity of the immunomodulatory small peptide was successfully improved. The selenomodified small peptide (Se-DCC1) can trigger an immune response equivalent to that of unmodified DCC1 at a lower concentration. The characteristics of these peptides can further reduce the dosage requirements for applying small peptides, thereby reducing application costs, promoting the development of green agriculture, and responding to the urgent need for efficient, safe, and environmentally friendly methods in agricultural disease control. Those skilled in the art can flexibly select the type of chemical modification for the above-mentioned peptides according to actual needs to improve their activity and immunomodulatory capacity.

[0075] In a preferred embodiment, the above-mentioned polypeptide is subjected to temperatures ranging from 10 to 100°C (including but not limited to 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 32°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C). It can maintain its activity even under treatment at 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, 60℃, 61℃, 62℃, 63℃, 64℃, 65℃, 66℃, 67℃, 68℃, 69℃, 70℃, 71℃, 72℃, 73℃, 74℃, 75℃, 76℃, 77℃, 78℃, 79℃, 80℃, 81℃, 82℃, 83℃, 84℃, 85℃, 86℃, 87℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃.

[0076] In a preferred embodiment, the above-mentioned polypeptide can fully activate the immune pathway at a concentration of 10 nM.

[0077] The DCC1 peptides and similar peptides described in this application maintain thermal stability under extreme temperature conditions and retain their immune-activating activity within the aforementioned temperature range. This characteristic enhances the applicability and stability of these small peptides in variable agricultural environments. Furthermore, this application found that only an extremely low concentration of 10 nM is required for the aforementioned DCC1 small peptides to fully activate plant immune signaling pathways and induce the activation of defense mechanisms. This discovery demonstrates the high efficiency of these small peptides, which also implies reduced application costs, simplifies agricultural operations, and makes them easier to promote and apply on a large scale.

[0078] In a second typical embodiment of this application, a plant immune inducer is provided, wherein the polypeptide has 10 or 11 amino acid residues; the amino acid at position 1 of the polypeptide is serine, and the amino acids at positions 9 and 10 are both cysteine; if the polypeptide has 11 amino acid residues, the amino acid at position 11 of the polypeptide is asparagine.

[0079] In a preferred embodiment, the second amino acid of the polypeptide is glycine, arginine, valine, or asparagine; the third amino acid is serine, glycine, arginine, or proline; the fourth amino acid is serine or asparagine; the fifth amino acid is proline, glycine, serine, threonine, or arginine; the sixth amino acid is proline, arginine, glycine, aspartic acid, serine, or histidine; the seventh amino acid is threonine, serine, or glycine; and the eighth amino acid is histidine, threonine, or serine.

[0080] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 15-31.

[0081] In a preferred embodiment, the above-mentioned polypeptide also contains chemical modifications.

[0082] In a preferred embodiment, the chemical modification includes replacing all -SH residues on cysteine ​​residues in the polypeptide with -SeH residues.

[0083] This application describes the preparation of a working solution using ultrapure water as a solvent from the protein-purified complex of the aforementioned polypeptide (DCC), which can yield a plant immune inducer possessing the immune function of the polypeptide. This plant immune inducer can induce a response in the MAPK signaling pathway, thereby inducing and activating the immune activity of gramineous plants and enhancing cellular immune function.

[0084] In a preferred embodiment, the working concentration of the plant immune inducer is 1~10 μM, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 μM.

[0085] In a preferred embodiment, the plant immune inducer further includes a surfactant; preferably, the surfactant includes one or more of Silwet L-77, Tween 20, Tween 80, Sodium Lauryl Sulfate (SLS), Kinetic, or Breaker 735; preferably, the volume content of the surfactant is 0.015~0.025%, including but not limited to 0.015%, 0.020%, or 0.025%.

[0086] The plant immune inducer of this application also includes the aforementioned surfactant, which is a substance capable of significantly reducing the surface tension of a liquid. Adding a surfactant to the plant immune inducer of this application improves the adhesion of the agent, allowing it to adhere to waxy leaves, thereby increasing the utilization rate of the immune inducer. It also promotes better penetration of the immune inducer into the interior of plant organs and tissues, thus more effectively activating the plant's immune system. Preferably, by controlling the protein concentration and surfactant within the aforementioned ranges, the two work synergistically, enabling the plant immune inducer to function better and enhance the target plant's immune resistance. Those skilled in the art can flexibly select the surfactant and its concentration according to the actual situation.

[0087] In a third typical embodiment of this application, a method for improving plant disease resistance is provided, the method comprising applying the above-mentioned polypeptide or the above-mentioned plant immune inducer to a target plant; the target plant includes grasses.

[0088] When the plant immune inducer of this application is applied exogenously to target plants, it can significantly improve the target plants' immune resistance to diseases. This application finds that when the aforementioned plant immune inducer is applied to the organs and tissues of target plants, it can activate the MAPK signaling pathway response of the target plants, further enhancing plant immunity and effectively controlling bacterial and fungal diseases, thus promoting the development of crop disease control. Those skilled in the art can flexibly choose any conventional exogenous application method to treat the organs of the target plants according to actual conditions, and all methods can achieve the effects of this application.

[0089] It should be noted that this enhancement of plant disease resistance is a broad-spectrum disease resistance, which improves plant immunity by activating the MAPK signaling pathway, thereby enhancing resistance to a variety of diseases, including but not limited to resistance to bacterial and fungal diseases.

[0090] In a preferred embodiment, the application method includes one or more of the following: spraying, smearing, soaking, leaf cutting, or injection. In a preferred embodiment, the grass family includes one or more of wheat, rice, corn, millet, barley, or sorghum.

[0091] In a fourth typical embodiment of this application, the application of the above-mentioned polypeptide or the above-mentioned plant immune inducer is provided in improving plant immunity and disease resistance.

[0092] It should be noted that this enhancement of plant disease resistance is a broad-spectrum resistance, achieved by activating the MAPK signaling pathway to improve plant immunity and thus enhance resistance to a variety of diseases, including but not limited to resistance to bacterial and fungal diseases. Preferably, the bacterial diseases mentioned above include, but are not limited to, one or more of wheat bacterial leaf streak, rice bacterial leaf streak, or rice bacterial blight; preferably, the fungal diseases mentioned above include, but are not limited to, wheat scab.

[0093] In this application, the aforementioned DCC and similar peptides are used to prepare plant immune inducers. The resulting plant immune inducers possess broad-spectrum and compatibility, and the immunomodulatory DCC can significantly induce immune responses in gramineous crops, demonstrating potential application value in the prevention of gramineous crop diseases. Exogenous spraying of the aforementioned plant immune inducers on gramineous crops enhances their disease resistance, prevents the occurrence of gramineous crop diseases in agricultural production, and simultaneously protects gramineous crops from pesticide contamination.

[0094] In a fifth typical embodiment of this application, the above-mentioned polypeptide is provided for use in the preparation of a reagent for improving plant immunity and / or disease resistance.

[0095] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0096] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0097] Example 1: Screening and Identification of Immune-Stimulating Peptide DCC

[0098] 1. Transcriptome Data Analysis of Wheat under Biological Stress

[0099] 1.1 Acquisition and analysis of transcriptome data of wheat after biotic stress

[0100] Transcriptome data of wheat under biological stress were obtained from the wheat genome database (WheatOmics 1.0, http: / / 202.194.139.32 / ). The results of pDCCs expression induced by immune elicitors and pathogens, and pDCCs protein sequence alignment are as follows: Figure 1 As shown, Figure 1 The red marker in the middle (A) indicates an upregulated gene. Bioinformatics analysis revealed that a class of genes encoding secretory small peptide precursor proteins were affected by pathogenic immune elicitors flg22 and chitin, as well as pathogens. F. graminearum , X. translucen s and Z. tritici Induced upregulation of expression (e.g.) Figure 1 (See the heatmap of A). In wheat, this type of secretory small peptide precursor protein comprises 13 members. The amino acid sequences of these 13 members are compared (SEQ ID NOs: 2~14 sequence comparison diagram is shown in the figure). Figure 1 As shown in Figure B, where the boxed portion represents the conserved region of these sequences), we found that its C-terminus contains two conserved cysteine ​​residues. Based on this characteristic, we named this type of protein pDCCs (DUALCYSTEINES IN C-TERMINUS precursors, DCC precursor proteins, where A, B, and D represent wheat subgenomes; pDCC1A, B, and D represent the precursor proteins or gene sequences of DCC1 in wheat subgenomes A, B, and D, respectively).

[0101] 1.2 Identification of the size of DCC small peptide active fragments

[0102] In plants, small peptide precursor proteins are expressed and translated into amino acids within the cell, then secreted extracellularly under the guidance of an N-terminal signal peptide. They are then processed by proteases in the intercellular matrix into active small peptides. To identify the fragment size of the active small peptide DCC1 from wheat pDCC1, we constructed the pDCC1 gene coding sequence (SEQ ID NO: 39) into the overexpression vector pLGY-OE3 containing the ubi promoter to obtain the transformation vector pLGY-OE3-pDCC1 (see schematic diagram of the pLGY-OE3-pDCC1 vector). Figure 2 As shown, Figure 2In this study, "repeat" refers to a repetitive sequence, "operator" refers to an operating element, "promotor" refers to a promoter, and "binding site" refers to a binding site. This vector was then transformed into Agrobacterium competent cells EHA105. Positive Agrobacterium clones were selected and used to infect the wheat variety Fielder to obtain pDCC1 overexpressing transgenic plants. The identification of mature DCC1 small peptides and the sequence alignment results of DCC homologous small peptides in gramineous plants in this study are as follows: Figure 3 As shown.

[0103] Leaves from 2-week-old pDCC1-overexpressing transgenic plants were collected, and interstitial fluid was extracted. Mass spectrometry analysis was then performed on small peptides of 0.3-5 kDa in the interstitial fluid, identifying small peptides of 10 amino acids (results are shown below). Figure 3 As shown in Figure A), it is located at bit 54-63 of the C terminal of pDCC1 ( Figure 1 The conserved region of the pDCC protein sequence in B is SGSSPPTHCC (SEQ ID NO: 15). This indicates that this small peptide is likely the active mature peptide DCC1.

[0104] BLAST alignment analysis of the pDCC1 protein sequence using the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) revealed that pDCC1 homologs are found only in grasses (Poaceae), suggesting that this protein may have unique biological functions within this group. The amino acid sequences of mature peptides of DCC1 homologs in grasses (wheat, barley, rice, millet, maize, and sorghum) are shown in SEQ ID NOs: 15-31 above. Specifically, the amino acid sequences of HvDCC1 in barley are identical to those of DCC1 / 3 / 4 in wheat; the amino acid sequence of SbDCC1 in sorghum is identical to that of ZmDCC2 in maize; and the amino acid sequence of SbDCC2 is identical to that of SiDCC2 in millet. A schematic diagram illustrating the alignment of C-terminal mature peptides of DCC1 homologs in grasses is shown below. Figure 3 As shown in B.

[0105] Furthermore, the C-terminal mature peptide sequence (the 10 amino acid sequences at the C-terminus) was compared, and the comparison statistics are shown in the diagram below. Figure 3As shown in Figure C, we found that the serine (S) at position 1, and the cysteine ​​(C) at positions 9 and 10 at the C-terminus are highly conserved in all grasses. Meanwhile, amino acids at other positions also exhibit some degree of conservation. For example: the second amino acid is glycine (most commonly glycine; based on statistics of the amino acid types of DCC1 homologous peptides at this site in wheat, barley, rice, millet, corn, and sorghum, glycine accounts for 86.36%), arginine, valine, or asparagine; the third amino acid is mainly serine or glycine, with a few being arginine or proline; the fourth amino acid is serine or asparagine (polar and uncharged); the fifth amino acid is mainly proline or glycine, with a few being serine, threonine, or arginine; the sixth amino acid is mainly proline or arginine, with a few being glycine, aspartic acid, serine, or histidine; the seventh amino acid is threonine, serine, or glycine; and the eighth amino acid is histidine (most commonly histidine; based on statistics of the amino acid types of DCC1 homologous peptides at this site in wheat, barley, rice, millet, corn, and sorghum, histidine accounts for 86.36%), threonine, or serine.

[0106] Notably, DCC homologs in C4 plants (C4 plants refer to plants that employ the C4 photosynthetic pathway, including but not limited to millet, maize, and sorghum) exhibit an additional asparagine N at the C-terminus. These results suggest that the C-terminal peptide of the pDCC1 protein is highly conserved in gramineous plants, particularly the serine and cysteine ​​residues at key sites, which may be related to peptide activity.

[0107] The amino acid sequence of the pDCC1 precursor protein is shown in SEQ ID NO: 2-14. In the following sequences, the first underlined portion represents the signal peptide sequence, and the second underlined portion represents the mature small peptide sequence. The nucleotide sequence encoding the precursor protein pDCC1 is shown in SEQ ID NO: 39.

[0108] pDCC1-A (SEQ ID NO: 2): MAATAAALMRMVLLVVLLVQMLSVMAVSA RTLKGDAWLTDGIGMVMEMFGDLK SGSSPPTHCC .

[0109] pDCC1-B (SEQ ID NO: 3): MAATATTLMRMVLLVVLLVQMLNVMTVSA RTLKGDAWLKDGIGMVMEMLGDLK SGSSPPTHCC .

[0110] pDCC1-D (SEQ ID NO: 4): MAATATTLMRMVLLVVLLVQMLNVMAVSA RTLKGGAWLKDGIGMVMEMLGDLK SGSSPPTHCC .

[0111] pDCC2-A (SEQ ID NO:5): MAATVNMVKAVLLLLLVIQISSVLA AAARPFVGDDGRWLENGIGMVTQMLGGVKQ SGSSGRTHCC 。

[0112] pDCC2-B(SEQ ID NO:6): MAATANMAKAVLLLLPVIQISSVLA AAARPFVGDDGQWLQNGIGMVTQMLGGVKQ SGSSGRTHCC 。

[0113] pDCC2-D (SEQ ID NO:7): MAATANMAKVVLLLLLVIQISSVLA AAARPFVGDDGQWLQNGIGMVTQMLGGVKQ SGSSGRTHCC 。

[0114] pDCC3-A (SEQ ID NO:8): MAALMRMVLLVVFLVHMFSVVAPVSA RTLKGDASWLRDGIGMVAEMLRDLK SGSSPPTHCC 。

[0115] pDCC3-B(SEQ ID NO:9): MAALMKTLLLVVFLVHMFNVIAPVSA RALKGDASWLKDGIGMVVEMLGDLK SGSSPPTHCC 。

[0116] pDCC3-D (SEQ ID NO:10): MAALIRMVLLVVFLVYMFSVMAPASA RTLKGDASWLSDGIGMVVEMLGDLK SGSSPPTHCC 。

[0117] pDCC4-A (SEQ ID NO:11): MTTARIVEVMLLLMFLTLIFSVHLASA ARLLEGWREGGIGTVTRMLGGIKQ SGSSPPTHCC 。

[0118] pDCC5-B(SEQ ID NO:12): MAAAAAKVVVLLLLVMQILSIIVG AARPLEGDHGWTGNGIETVTEMLSAAK SRSSTRTHCC 。

[0119] pDCC5-D (SEQ ID NO:13): MAAAAAKVVVLLLLVIHVLGVVVG AARPLEGDHGWTGNGIEMVTQMLSAAK SRSSTRTHCC 。

[0120] pDCC6-A (SEQ ID NO:14): MAKQALLAVILVHICGVMA AASRTLRGDDWLEDSVQTVVMQIFGGSK SGGSGGTHCC 。

[0121] pDCC1 CDS (SEQ ID NO: 39): ATGGCGGCGACCGCGGCGGCGTTGATGAGGATGGTGCTGCTGGTGGTGCTCTTGGTGCAGATGCTCAGCGTCATGGCCGTCTCGGCGAGGACGTTGAAGGGGGACGCCTGGCTCACGGACGGCATCGGGATGGTGATGGAGATGTTCGGCGACCTGAAATCAGGGTCCAGCCCTCCCACGCACTGCTGCTAA.

[0122] Example 2: Immunomodulatory activity analysis of the immune-stimulating peptide DCC and similar peptides

[0123] 1. DCC peptide synthesis and working solution preparation

[0124] To further verify the size of immunologically active small peptide fragments, small peptides of different lengths were prepared based on the amino acid sequence of the pDCC1 precursor protein, including small peptides with key amino acid mutations and small peptides homologous to DCC from rice and maize. Specific small peptides, their sequences, lengths, and sequence details are shown in Table 1. The underlined portions in Table 1 indicate amino acid mutation sites, and the purity is >95% (synthesized and prepared by Shanghai Qiangyao Biotechnology Co., Ltd.).

[0125] Table 1

[0126]

[0127] Preparation of small peptide stock solution and working solution: Take DCC1 small peptide protein pure compound and use ultrapure water as solvent to prepare a stock solution with a concentration of 1 mM. Dilute it to a working solution of appropriate concentration before use for experimental treatment.

[0128] 2. Identification of the core fragment and key amino acids of DCC1 small peptide activating plant immune response

[0129] MAPK (Mitogen-Activated Protein Kinase) phosphorylation plays a central role in plant immunity, regulating plant defense responses through cascade signal transduction. Detecting MAPK phosphorylation levels in plants can determine whether an immune response has been activated.

[0130] The steps for detecting MAPK phosphorylation are as follows: Take leaves of 1-week-old wheat variety Fielder, cut them into 1 cm segments, place them in ddH2O containing 0.2% Tween-20, vacuum for 5 minutes until the leaves are completely soaked, then wash them twice with ddH2O and incubate overnight at room temperature.

[0131] Wheat leaf segments were treated with ddH2O (blank control group) and small peptides for 15 minutes respectively. The leaf segments were then placed in 2 mL centrifuge tubes containing steel balls, frozen in liquid nitrogen, and then ground into powder using a grinder.

[0132] Add 200 μL of protein extraction buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 25% glycerol, 1 mM NaF, 1 mM Na3VO4, 2 mM DTT, 1× protease inhibitor (Roche, catalog number: 05056489001)), vortex thoroughly, incubate on ice for 10 minutes, and centrifuge at 12000 rpm at 4°C for 10 minutes. Take 40 μL of the supernatant, add 10 μL of 5× SDS-PAGE loading buffer, mix well, and heat at 95°C for 5 minutes.

[0133] Load 10 μL of protein sample onto 10% SDS-PAGE for protein electrophoresis.

[0134] After electrophoresis, the target protein on the SDS-PAGE was transferred to a PVDF membrane. The PVDF membrane was blocked with 5% BSA (prepared with 1×TBST buffer) at room temperature for 1 hour. Then, it was incubated overnight at 4°C with anti-pERK1 / 2 antibody (1:2000). The next day, it was washed three times with 1×TBST at room temperature for 5 minutes each time. It was then incubated with anti-Rabbit secondary antibody (1:10000) at room temperature for 1 hour. After washing three times with 1×TBST, MAPK phosphorylation was detected by imaging.

[0135] DCC1 core sequence (such as) Figure 4 As shown in Figure A) and the results of the identification of key amino acid activities are as follows: Figure 4 As shown.

[0136] Based on the pDCC1 protein sequence, small peptides of different lengths were synthesized, with sequences as follows: Figure 4 As shown in Figure A, wheat leaves were treated with 10 nM and 100 nM small peptides, respectively. The results showed that the small peptides with a length of 10 amino acids in the conserved region had the strongest immunogenicity. Figure 4 As shown in Figure B), both excessively long and excessively short peptide fragments exhibit reduced activity, consistent with the mature small peptide fragments in the interstitial fluid identified by previous mass spectrometry.

[0137] Sequence alignment of DCC small peptides revealed that the C-terminal cysteine ​​residue is highly conserved. Figure 3 (Results from B) After mutating cysteine ​​(C) to serine (S), MAPK phosphorylation could not be activated (results as shown in Figure B). Figure 4 As shown in Figure C), it lacks immunogenicity; therefore, the two cysteine ​​residues at the C-terminus are crucial for the immunogenicity of the DCC peptide.

[0138] 3. The minimum concentration of DCC to activate immunity

[0139] Treatment of wheat leaves with different concentrations of DCC1 peptide revealed that even as low as 10 nM significantly activated MAPK phosphorylation, and different DCC1 concentrations activated immune responses such as... Figure 5 As shown.

[0140] 4. Thermal stability analysis of DCC1

[0141] Thermal stability testing: The DCC1 small peptide stock solution was treated at 22℃ and 50℃ for 6 hours, and at 95℃ for 1 hour, respectively. It was then used to treat wheat leaves for 15 minutes to detect MAPK phosphorylation activation. The results of the DCC1 thermal stability testing are shown in the diagram below. Figure 6 As shown in the figure. The results showed that the DCC1 peptide still had the activity to activate MAPK phosphorylation after treatment at 50℃ for 6 hours and at 95℃ for 1 hour, indicating that the DCC1 peptide can maintain stable immune-inducing activity under high temperature conditions.

[0142] 5. Chemical modification enhances the activity of DCC small peptides.

[0143] Se-DCC1 was synthesized by replacing the sulfur (S) in two cysteine ​​residues in the DCC1 peptide with selenium (Se), a member of the same family. MAPK phosphorylation assays revealed that Se-DCC1 possessed stronger immunomodulatory activity; 1 nM Se-DCC1 showed comparable activity to 5 nM DCC1 (the results of selenocysteine ​​modification enhancing DCC1 activity are shown in the figure). Figure 7 (As shown). This may be because selenium has a larger atomic radius, selenocysteine ​​is more reactive under physiological conditions, and Se-DCC1 is more likely to bind to receptors than DCC1, thereby activating the immune response.

[0144] 6. Immunoassay of DCC small peptides in grasses (wheat, rice, and maize)

[0145] DCC small peptides from wheat, rice, and maize were detected by MAPK phosphorylation using the same method as described above. Arabidopsis thaliana and Nicotiana bungeana were used as negative controls.

[0146] Results of immunoassay for DCC1-like peptides in gramineous crops: Figure 8 As shown. Figure 8 The Chinese definitions of "Wheat" and "Rice" are: wheat, rice, maize, ZH11 and Nipponbare (or abbreviated as "Nip") are two rice varieties, B73 is a maize inbred line, and Col-0 is the Arabidopsis Col-0 ecotype. Nb"For the native tobacco ( Nicotiana benthamiana Thirteen pDCCs (SEQ ID NOs: 2-14) in wheat can form four mature DCC peptides (sequences are shown in Table 1 for DCC1 / 3 / 4, DCC2, DCC5, and DCC6, among which the mature peptide sequences formed by the pDCC1, pDCC3, and pDDC4 precursor proteins are identical). Treatment of wheat Fielder leaves with these four synthesized peptides (100 nM) induced MAPK phosphorylation, similar to the control immunostimulant flg22 (MAPK phosphorylation detection results for wheat treated with DCC1, DCC2, DCC5, and DCC6 are shown in Table 1). Figure 8 As shown in Figure A, DCC1 can also be considered as small peptides DCC3 and DCC4 with the same amino acid sequence, which activate its immune activity.

[0147] Four homologous peptides, OsDCC (sequences shown in Table 1: OsDCC1, OsDCC2, OsDCC3, and OsDCC4), also exist in rice. These peptides induce MAPK phosphorylation in both rice varieties ZH11 and Nipponbare (see Table 1 for the MAPK phosphorylation detection results of rice treated with OsDCC1, OsDCC2, OsDCC3, and OsDCC4). Figure 8 As shown in Figure B), it activates immune activity.

[0148] The homologous peptide ZmDCC1 in maize (sequence shown in Table 1) can activate MAPK phosphorylation in maize itself, and also induce MAPK phosphorylation in rice and wheat (the results of MAPK phosphorylation detection in maize, rice, wheat, Arabidopsis thaliana, and tobacco treated with ZmDCC1 are shown in Table 1). Figure 8 (As shown in Figure E). Similarly, wheat DCC1 can also induce MAPK phosphorylation in rice (the results of MAPK phosphorylation detection in wheat, rice, maize, Arabidopsis and tobacco treated with DCC1 are shown in Figure E). Figure 8 As shown in Figure C), OsDCC1 in rice can induce MAPK phosphorylation in wheat (the results of MAPK phosphorylation detection in rice, wheat, maize, Arabidopsis and tobacco treated with OsDCC1 are shown in Figure C). Figure 8 (As shown in D), this may be because grasses are closely related in evolution and the receptors that recognize small peptides are conserved.

[0149] Example 3: Application of DCC immunomodulatory small peptides in inducing immunity

[0150] Based on the activating properties of DCC small peptides on the plant immune system, this invention discloses a method for applying mature small peptide DCC as a plant immune inducer in disease control. Specific implementation schemes are shown in Table 2.

[0151] Table 2

[0152]

[0153] (1) Efficacy test of control against wheat bacterial leaf streak

[0154] Working solution preparation: Dilute the DCC1 and DCC2 peptides with ddH2O to prepare a 2 μM working solution. Use ddH2O as a blank control.

[0155] Injection treatment: Inject the aforementioned working solution into the first cotyledon of the Fielder wheat variety, which is about 2 weeks old.

[0156] Pathogen culture and inoculation: Inoculate with wheat bacterial leaf streak strains... Xanthomonas translucens pv. Undulosa ( Xtu Kn5 (i.e., the following) Xtu Kn5) was inoculated into 2 mL of NB medium and incubated overnight at 28°C and 200 rpm. The bacterial cells were collected and dissolved in ddH2O. Xtu Kn5 was diluted to OD600 = 0.005, and then... Xtu Kn5 was injected into the first cotyledon of pretreated wheat.

[0157] Statistical analysis of control efficacy: Four days after inoculation with live pathogens, the disease phenotype of leaves was recorded (by taking photos), and the number of colonies in the leaves was determined by the serial dilution plate counting method.

[0158] The test results for DCC1 and DCC2-enhanced wheat resistance to bacterial leaf streak are as follows: Figure 9 As shown, Figure 9 Image A shows the morphological characteristics of diseased wheat leaves. Figure 9 Figure B shows the bacterial colony count on wheat leaves. Wheat leaves pretreated with small peptides DCC1 and DCC2 were then inoculated. Xtu Four days later, the disease severity of Kn5 was milder, with a highly significant difference compared to the blank control (P<0.0001), and fewer colonies were found in the leaves, indicating that DCC peptide can effectively activate the defense response of wheat leaves and can be used for the prevention of wheat bacterial leaf streak.

[0159] (2) Control efficacy experiment against rice bacterial leaf streak

[0160] Preparation of working solution and preparation of spray inducer: 5 μM OsDCC4 and 5 μM MDCCC1 were prepared using ddH2O as a diluent; ddH2O was used as a blank control. 0.02% (V / V) of Silwet L-77 (Silwet L-77 is an organosilicon-based surfactant (polyether-modified organosilicon), developed by Momentive Performance Materials, widely used in agriculture, industry, and scientific research, mainly as a wetting agent, spreading agent, and penetrant) was added to the working solution to prepare the spray inducer.

[0161] Spraying treatment: One hour before pathogen inoculation, spray the surface of the leaves of the TP309 rice variety, which is 4 weeks old, with an inducer.

[0162] Pathogen culture and live inoculation: Bacterial leaf streak strains of rice Xanthomonas oryzae pv. Oryzicola ( Xoc RS105 (i.e., the following) Xoc RS105 was grown on PSA medium (1L: 10 g tryptone, 1 g sodium glutamate, 10 g sucrose, 12 g agar) at 28°C for 2 days, then adjusted... Xoc RS105 OD600 to 0.5, inoculated using the infiltration method, select fully expanded leaves, and inject into the rice leaf from the back of the leaf using a needleless syringe.

[0163] Statistical analysis of control efficacy: Fourteen days after inoculation with live pathogens, leaf disease incidence was investigated, and lesion length was measured. Results of OsDCC4 and DCC1 enhancing rice resistance to bacterial leaf streak are as follows: Figure 10 As shown, Figure 10 Image A shows the morphological characteristics of diseased rice leaves. Figure 10 Figure B shows a statistical graph of leaf lesion length. The disease severity of rice leaves pretreated with OsDCC4 and DCC1 peptides was significantly lower than that of the blank control (P<0.0001), indicating that pretreatment with OsDCC4 and DCC1 peptides can activate the rice defense response and improve the rice's resistance to bacterial leaf streak.

[0164] (3) Experiment on the control efficacy against rice bacterial blight

[0165] Preparation of working solution and preparation of spray inducer: 0.5 μM OsDCC4 was prepared using ddH2O as a diluent; ddH2O was used as a blank control. Tween 20 was added to the working solution at a volume content of 0.02% (V / V) to prepare the spray inducer.

[0166] Spraying treatment: Two hours before pathogen inoculation, spray the surface of the leaves of rice variety ZH11, which is 10 weeks old, with an inducer.

[0167] Pathogen culture and live inoculation: Rice bacterial blight strain Xanthomonas oryzae pv. oryzae ( Xoo PXO99A (i.e., the following) Xoo PXO99A was grown on PSA medium (1L: 10 g tryptone, 1 g sodium glutamate, 10 g sucrose, 12 g agar) at 28°C for 2 days, then adjusted... Xoo PXO99A OD600 to 0.5, inoculation was performed using the leaf-cutting method. Fully unfolded leaves were selected, and the leaf tips were cut off 1 cm from the leaf tip with scissors dipped in bacterial solution for inoculation.

[0168] Statistical analysis of control efficacy: 140 days after inoculation with live pathogens, the disease incidence on leaves was investigated and the length of lesions was measured.

[0169] The results of OsDCC4 enhancing rice resistance to bacterial blight are as follows: Figure 11 As shown, Figure 11 Image A shows the morphological characteristics of diseased rice leaves, with red arrows pointing to the infected areas. Figure 11 Figure B shows a statistical graph of leaf lesion length. The disease severity of rice leaves pretreated with OsDCC4 peptide was significantly lower than that of the blank control (P<0.0001), indicating that OsDCC4 peptide pretreatment can activate the rice defense response and improve the rice's resistance to bacterial blight.

[0170] (4) Efficacy experiment in controlling wheat scab (Fusarium head blight)

[0171] Preparation of working solution and preparation of spray inducer: 5 μM DCC1 was prepared using ddH2O as a diluent; ddH2O was used as a blank control. Silwet L-77 at a volume content of 0.02% (V / V) was added to the working solution to prepare the spray inducer.

[0172] Spraying treatment: 24 hours before inoculation with pathogens, spray the ear of the Fielder wheat variety during the heading stage with an inducer and cover the ear with a plastic bag to seal and keep it moist.

[0173] Pathogen culture and live inoculation: The pathogen of wheat scab, Fusarium graminearum, was used for inoculation. Fusarium graminearum ( FgPH-1 was grown on PDA medium (1L: 200 g peeled and cooked potato juice, filtered, 20 g glucose, 15 g agar) for 5 days. Then, the culture block with mycelium growth was cut into 2 mm cubes and transferred to a new culture medium for one day of growth. A small spikelet was selected from the middle of a wheat ear, and the glumes of the spikelet were gently separated. The mycelium block was placed into the glumes with tweezers, sealed in a plastic bag and kept moist for 2 days, and then the plastic bag was removed.

[0174] Statistical analysis of control efficacy: 21 days after inoculation with live pathogens, wheat ears were removed, the number of diseased spikelets was counted, and the disease index was calculated (calculation formula: disease index = number of diseased spikelets / total number of spikelets).

[0175] The results of DCC1 enhancing wheat resistance to Fusarium head blight are as follows: Figure 12 As shown, Figure 12 Image A shows the morphological symptoms of wheat scab on the ear, with red arrows pointing to the infected areas. Figure 12 Figure B shows the statistical chart of Fusarium head blight incidence index. The incidence index of wheat ears pretreated with DCC1 peptide was significantly lower than that of the blank control (P<0.0001), indicating that DCC1 peptide pretreatment can activate the wheat defense response and further improve wheat resistance to Fusarium head blight.

[0176] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects:

[0177] The DCC1 and its similar peptides of this application require only 10 or 11 amino acid active fragments and can be produced at low cost on a large scale through solid-phase chemical synthesis or microbial expression systems (such as yeast fermentation). Compared with traditional protein-based inducers, they have a significant cost advantage, which is conducive to promoting the development of sustainable agriculture and meeting the demand for safe and green control methods in agricultural production. Furthermore, the immunomodulatory small peptides provided in this application can be precisely applied to plants through injection or spraying, avoiding the overuse and environmental pollution of traditional chemical pesticides. They also overcome the challenges of long cycles and high costs in disease resistance breeding, enabling precise control of bacterial and fungal diseases, improving crop quality and yield, and providing a safe, green, and efficient means of crop disease prevention and control for agricultural production. This is of great significance for promoting the precision and sustainability of agricultural disease management.

[0178] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for improving plant disease resistance, characterized in that, The method includes applying a polypeptide to a target plant; The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 24; The target plant is wheat or rice; The disease resistance refers to the ability to resist fungal or bacterial diseases. The fungal disease mentioned is wheat scab; The bacterial diseases mentioned are bacterial leaf streak of wheat, bacterial leaf streak of rice, or bacterial blight of rice; When the amino acid sequence of the polypeptide is SEQ ID NO: 15, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat, the bacterial leaf streak of rice, or the scab of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 16, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 24, when applied to the target plant, it enhances the target plant's resistance to rice bacterial leaf blight or rice bacterial leaf streak.

2. The method according to claim 1, characterized in that, The polypeptide also contains chemical modifications, namely, replacing all -SH residues on the cysteine ​​residues in the polypeptide with -SeH residues.

3. The method according to claim 1, characterized in that, The application method includes one or more of the following: spraying, smearing, soaking, leaf cutting, or injection.

4. Application of peptides in the preparation of reagents for enhancing plant immunity; The plant is wheat, rice, or corn; The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO:

24.

5. Application of polypeptides in the preparation of reagents to enhance plant disease resistance; The plant in question is either wheat or rice; The disease resistance refers to the ability to resist fungal or bacterial diseases. The fungal disease mentioned is wheat scab; The bacterial diseases mentioned are bacterial leaf streak of wheat, bacterial leaf streak of rice, or bacterial blight of rice; The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 24; in, When the amino acid sequence of the polypeptide is SEQ ID NO: 15, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat, the bacterial leaf streak of rice, or the scab of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 16, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 24, when applied to the target plant, it enhances the target plant's resistance to rice bacterial leaf blight or rice bacterial leaf streak.

6. A plant immune inducer, characterized in that, The plant immune inducer includes polypeptides; The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO:

24.

7. The use of polypeptides or the plant immune inducers of claim 6 in enhancing plant immunity; The plant is wheat, rice, or corn; The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO:

24.

8. The application of polypeptides, or the plant immune inducer as described in claim 6, in improving plant disease resistance; The plant in question is either wheat or rice; The disease resistance refers to the ability to resist fungal or bacterial diseases. The amino acid sequence of the polypeptide is SEQ ID NO: 15, SEQ ID NO: 16 or SEQ ID NO: 24; The fungal disease mentioned is wheat scab; The bacterial diseases mentioned are bacterial leaf streak of wheat, bacterial leaf streak of rice, or bacterial blight of rice; in, When the amino acid sequence of the polypeptide is SEQ ID NO: 15, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat, the bacterial leaf streak of rice, or the scab of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 16, when applied to the target plant, it enhances the target plant's resistance to the bacterial leaf streak of wheat. When the amino acid sequence of the polypeptide is SEQ ID NO: 24, when applied to the target plant, it enhances the target plant's resistance to rice bacterial leaf blight or rice bacterial leaf streak.

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