Polypeptide, plant immune resistance inducer and application of plant immune resistance inducer

Abstract

The invention provides a polypeptide, a plant immune resistance inducer and application of the plant immune resistance inducer. The polypeptide has 10 or 11 amino acid residues; the first amino acid of the polypeptide is serine, and the ninth amino acid and the tenth amino acid of the polypeptide are cysteine; if the polypeptide has 11 amino acid residues, the 11th amino acid of the polypeptide is asparagine. The method can solve the problem of lack of small peptides secreted by gramineous plants in the prior art, and is suitable for the field of plant immune resistance inducers.

Description

Technical Field

[0001] The present invention relates to the field of plant immune inducers, and in particular to a polypeptide, a plant immune inducer and applications thereof. Background Art

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

[0003] Plant elicitors, including compounds and biological agents, are becoming a key strategy for modern agriculture to address disease challenges by activating the plant immune system to enhance its disease resistance. With global climate change, agricultural intensification, and the increasing complexity of diseases, the use of plant elicitors is becoming increasingly important. Among them, plant-secreted peptides, as a class of endogenous signaling molecules, play a crucial role in regulating plant stress responses and growth and development. Their high efficiency, broad spectrum, and environmental friendliness make them a preferred option for modern disease management.

[0004] Specifically, small peptides have the following advantages: First, high efficiency - they can activate plant immunity at extremely low concentrations and enhance the defense capabilities of crops against multiple diseases; second, broad spectrum - compared with relying on a single disease-resistant gene, small peptides can deal with multiple diseases at the same time, simplifying the prevention process; third, environmental protection - as naturally occurring molecules in plants, small peptides are easily degraded, safe and harmless to the environment and humans, and are consistent with the goals of green agriculture and sustainable agriculture. In view of these advantages, small peptides can be coordinated with disease-resistant breeding, biological control and chemical control to form a comprehensive disease management program, enhancing disease prevention effects while reducing dependence on a single method.

[0005] Research on small peptides such as systemin, Pep1, ZIP1, PIP1, PSK, and SCOOP12 demonstrates that endogenous plant peptides are highly effective in inducing disease resistance. However, to date, there have been no reports of the application of secreted peptides from the grass family for plant disease control. Gramineae encompasses most major food crops, and improving disease control technologies for these plants is crucial for ensuring food security and sustainable agricultural development. Summary of the Invention

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

[0007] In order to achieve the above-mentioned object, according to the first aspect of the present invention, a polypeptide is provided, which 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.

[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 one of SEQ ID NOs: 15-31.

[0010] Furthermore, the polypeptide may contain chemical modifications.

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

[0012] In order to achieve the above-mentioned purpose, according to the second aspect of the present invention, a plant immune inducer is provided, which comprises a polypeptide; 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; 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 one of SEQ ID NOs: 15-31.

[0015] Furthermore, the polypeptide may contain chemical modifications.

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

[0017] To achieve the above object, according to the third aspect of the present invention, a method for improving plant disease resistance is provided, which comprises applying the above polypeptide or the above plant immunity inducer to target plants; the target plants include grass plants.

[0018] Furthermore, the application method includes one or more of spraying, smearing, soaking, leaf cutting or injection.

[0019] Furthermore, the grass plant includes one or more of wheat, rice, corn, millet, barley or sorghum.

[0020] In order to achieve the above objectives, according to the fourth aspect of the present invention, there is provided a use of the above polypeptide, or any one of the above plant immunity inducers, in improving plant immunity and / or disease resistance.

[0021] In order to achieve the above object, according to the fifth aspect of the present invention, there is provided a use of the above polypeptide in the preparation of an agent for improving plant immunity and / or disease resistance.

[0022] Applying the technical solution of the present invention, a polypeptide is provided in the present application, which has 10 or 11 amino acid residues, the amino acid at position 1 is serine, and the amino acids at positions 9 and 10 are both cysteine. When the polypeptide has 11 amino acid residues, the amino acid at position 11 is asparagine. The polypeptide in the present application has immune activity, breaking through the problem of lack of secreted small peptides in Gramineae plants, and has broad-spectrum disease resistance. When used as a plant inducer, it has high thermal stability and is not easy to degrade. It can be applied to Gramineae plants, significantly improving the practicality and durability of plant immune inducers, and opening up a new path for the green prevention and control of agricultural diseases. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 1 The results of pDCCs expression induced by immune elicitors and pathogens and the alignment of pDCCs protein sequences in Example 1 of the present application are shown; wherein, Figure 1 A in the middle is a heat map of the induced expression of pDCCs genes after wheat was treated with the immune elicitors flg22 and chitin and the pathogens F. graminearum, X. translucens, and Z. tritici. Figure 1 Figure B shows the amino acid sequence alignment results of 13 secreted small peptide precursor proteins pDCCs from wheat.

[0025] Figure 2 The vector structure diagram of the transformation vector pLGY-OE3-pDCC1 in Example 1 of the present application is shown.

[0026] Figure 3 The following figure shows the identification of mature DCC1 peptide and alignment of DCC homologous peptide sequences in Gramineae plants in Example 1 of the present application; wherein, Figure 3 Figure A in the middle is the mass spectrometry analysis result of the interstitial fluid extract from the leaves of the pDCC1 highly expressed transgenic plants. Figure 3 Middle B is the sequence alignment result of mature small peptide at the C-terminus of DCC1 homologous proteins in Poaceae plants. Figure 3 Figure C in the middle is a statistical comparison of different amino acid sites of the C-terminal mature peptides of Poaceae plants.

[0027] Figure 4 The DCC1 core sequence and key amino acid activity identification results of Example 2 of the present application are shown; wherein, Figure 4 A in the middle is a schematic diagram of small peptide sequences of different lengths. Figure 4 Middle B is the result of MAPK phosphorylation detection after wheat was treated with small peptides of different lengths. Figure 4 Middle C shows the results of MAPK phosphorylation detection after wheat was treated with different peptides after the cysteine ​​on the DCC peptide was mutated to serine.

[0028] Figure 5 The figure shows the results of MAPK phosphorylation activation by different DCC1 concentrations in Example 2 of the present application.

[0029] Figure 6 The figure shows the thermal stability test results of DCC1 in Example 2 of the present application.

[0030] Figure 7 The figure shows the detection result of improving DCC1 activity by selenocysteine ​​modification in Example 2 of the present application.

[0031] Figure 8 The figure shows the results of the detection of DCC1-like peptide immunoreactivity in gramineous crops in Example 2 of the present application specification; wherein, Figure 8 Middle A is the MAPK phosphorylation detection result of wheat treated with DCC1, DCC2, DCC5 and DCC6. Figure 8 Middle B is a schematic diagram of the MAPK phosphorylation detection results of rice treated with OsDCC1, OsDCC2, OsDCC3 and OsDCC4. Figure 8 Middle C is a schematic diagram of the MAPK phosphorylation detection results of DCC1 in wheat, rice, corn, Arabidopsis and tobacco. Figure 8D in the middle is a schematic diagram of the MAPK phosphorylation detection results in rice, wheat, corn, Arabidopsis and tobacco after OsDCC1 treatment. Figure 8 Middle E is a schematic diagram of the MAPK phosphorylation detection results of maize, rice, wheat, Arabidopsis and tobacco treated with ZmDCC1.

[0032] Figure 9 The results of the test of DCC1 and DCC2 enhancing wheat resistance to bacterial leaf streak disease in Example 3 of the present application are shown; wherein, Figure 9 A in the middle is the diseased morphology of wheat leaves. Figure 9 Middle B is a statistical chart of the number of colonies on wheat leaves.

[0033] Figure 10 The figure shows the test results of OsDCC4 and DCC1 enhancing the resistance of rice to bacterial leaf streak in Example 3 of the present application; wherein, Figure 10 A in the middle is the diseased morphology of rice leaves. Figure 10 Middle B is a statistical chart of leaf spot length.

[0034] Figure 11 The figure shows the test results of OsDCC4 enhancing rice resistance to bacterial blight in Example 3 of the present application specification; wherein, Figure 11 A in the middle is the diseased morphology of rice leaves. Figure 11 Middle B is a statistical chart of leaf spot length.

[0035] Figure 12 The results of the test of DCC1 enhancing wheat fusarium head blight resistance in Example 3 of the present application are shown, wherein: Figure 12 A in the middle is a morphological diagram of symptoms of wheat head fusarium wilt. Figure 12 Middle B is the statistical chart of the ergot disease index. DETAILED DESCRIPTION

[0036] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0037] Explanation of terms:

[0038] Plant immune inducers are substances that can induce immune defense responses in plants against pests and diseases. Even at low concentrations, they are recognized by plants as signaling substances, inducing and enhancing their own immune responses, thereby improving their resistance to diseases and delaying or mitigating the onset and progression of diseases. 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 metabolism. Types include proteins, oligosaccharides, lipids, small molecule metabolites, and microbial agents.

[0039] Plant secretory peptides are a class of small polypeptides (usually composed of 2-100 amino acids) synthesized by plant cells and released into the extracellular space through the secretory pathway. They play an important role in signal transduction and regulation in plant growth and development, stress resistance, and immune response. These secretory peptides are usually encoded by plant genes. They are synthesized into peptide chains according to specific gene sequence information through the action of organelles such as ribosomes in plant cells. They then undergo a series of post-translational modifications, such as glycosylation and phosphorylation, to form biologically active secretory peptides. These secretory peptides can be stored in vesicles within plant cells. When exposed to external stimuli (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 peptides mainly depends on their interaction with receptor kinases, namely signal recognition (secreted peptides bind 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 signal pathways (such as MAPK cascade reactions) to regulate gene expression and cell responses) and produce 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, particularly playing a key role in responses to environmental stimuli and immune responses. Activation of the MAPK pathway is often accompanied by phosphorylation.

[0042] As mentioned in the background art, the existing technology lacks secretory peptides of the Poaceae family. Therefore, in this application, the inventors attempted to explore a new immune polypeptide that can be used in the development of peptide inducers for Poaceae plants, and thus proposed a series of protection schemes of this application.

[0043] In a first typical embodiment of the present application, a polypeptide is provided, which has 10 or 11 amino acid residues, the amino acid at position 1 is serine, and the amino acids at positions 9 and 10 are both cysteine; if the polypeptide has 11 amino acids, the amino acid at position 11 of the polypeptide is asparagine.

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

[0045] SEQ ID NO: 1: SXXXXXXXXCCN.

[0046] The above "X" represents any naturally occurring amino acid or non-natural amino acid, that is, the positions containing "X" can be occupied by any amino acid without affecting the overall function or characteristics 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), 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 with 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] Faced with the current lack of effective immune elicitors for grass crops, this application aims to fill this technological gap. Through an innovative reverse genetics screening strategy, a novel immune-activating peptide (the aforementioned peptide)—DCC1 (DUAL CYSTEINES IN C-TERMINUS)—was successfully identified from endogenous secretory peptides in wheat, as well as its homologous peptides in grasses. These peptides share three unique conserved amino acid features: a serine (S) at the beginning of the sequence and two cysteines (C) at the end, located at positions 1, 9, and 10, respectively. This structural feature is shared by members of the DCC family and confers stability and high efficiency in immune activation.

[0048] DCC1 and its analogous peptides demonstrate cross-species immune activation capabilities, inducing immune responses in gramineous crops such as wheat, rice, and corn. This demonstrates the versatility of the DCC1 small peptide structure and lays the foundation for its broad-spectrum disease control in gramineous crops. By revealing the immunological activity of the DCC1 small peptide and its analogs, this paper provides a safe and environmentally friendly disease control strategy for gramineous crops. Through multiple technical optimizations, it can achieve breakthroughs in the molecular diversity, broad-spectrum disease resistance, and field applicability of plant immune elicitors, providing strong biotechnology 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 above-mentioned conserved sites at positions 1, 9, and 10, the amino acids at other positions of the polypeptide of the present application are also conserved to a certain extent: for example, the amino acid at position 2 is glycine (most of which is glycine, according to the 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 amino acid at position 3 is mainly serine or glycine, with a few being arginine or proline; the amino acid at position 4 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, according to the 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.

[0051] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any one 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 uses a reverse genetics strategy to analyze transcriptome changes in wheat after exposure to the immune elicitors flg22 and chitin, as well as pathogens such as Fusarium graminearum, Xanthomonas translucens, and Zymoseptoria tritici. By comparing and analyzing the expression data of genes encoding precursor proteins of secreted small peptides (pDCCs) in response to these responses, candidate genes that are highly expressed after immune stimulation and pathogen infection were precisely identified.

[0070] The applicant then further employed mass spectrometry to successfully identify the key sequence of the mature DCC1 peptide in the interstitial fluid of wheat, providing a foundation for subsequent molecular design and functional validation. During this process, the applicant noted the importance of two conserved cysteines at the C-terminus of the peptide, which not only ensure its structural stability but also are key to its activity. The applicant validated the minimal active fragment of DCC1 through MAPK phosphorylation analysis.

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

[0072] In a preferred embodiment, the above polypeptide further contains chemical modifications.

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

[0074] The present application also discovered that chemical modification of such small peptides can further optimize their activity and application efficiency. In a specific embodiment of the present application, the chemical modification technology of selenocysteine ​​was adopted. By replacing the sulfur in the cysteine ​​thiol group in the small peptide with selenium, the activity of the immunologically active small peptide was successfully improved. The seleno-modified small peptide (Se-DCC1) can trigger an immune response equivalent to that of unmodified DCC1 at a lower concentration. The characteristics of this type of polypeptide 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 means in agricultural disease prevention and control. Those skilled in the art can flexibly select the type of chemical modification of the above-mentioned polypeptide according to actual needs to improve the activity and immune ability of the polypeptide.

[0075] In a preferred embodiment, the polypeptide is heated at 10-100°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 ℃, 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 polypeptide can fully activate the immune pathway at a concentration of 10 nM.

[0077] The DCC1 and similar peptides described in this application maintain thermal stability under extreme temperature conditions and maintain their immune-activating activity within the aforementioned temperature range. This property enhances the applicability and stability of these small peptides in variable agricultural environments. Furthermore, the authors discovered that even at an extremely low concentration of 10 nM, the DCC1 peptide can fully activate plant immune signaling pathways and induce the activation of defense mechanisms. This discovery demonstrates the high efficiency of these small peptides, reduces application costs, simplifies agricultural operations, and facilitates large-scale application.

[0078] In a second typical embodiment of the present 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 one of SEQ ID NOs: 15-31.

[0081] In a preferred embodiment, the above polypeptide further contains chemical modifications.

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

[0083] This application uses ultrapure water as a solvent to prepare a working solution of a protein pure compound of the aforementioned polypeptide (DCC) to prepare a plant immune inducer with the immune function of the polypeptide. This plant immune inducer can induce a MAPK signaling pathway response, activate the immune activity of grass plants, and enhance 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 comprises a surfactant; preferably, the surfactant comprises one or more of Silwet L-77, Tween 20, Tween 80, Sodium Lauryl Sulfate (SLS), Kinetic or Breaker735; 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 immunity inducer of the present application also includes the above-mentioned surfactant, and surfactant is a substance that can significantly reduce the surface tension of a liquid. Adding surfactant to the plant immunity inducer of the present application can improve the adhesion ability of the medicament, so that the plant immunity inducer can be attached to the leaves containing wax, thereby improving the utilization rate of the immune inducer of the present application, and also can promote the immune inducer of the present application to better penetrate into the inside of plant organs and tissues, thereby more effectively activating the immune system of the plant. Preferably, the present application controls the protein concentration and surfactant within the above-mentioned range, can make the two act synergistically, so that the above-mentioned plant immunity inducer can better play a role, improve the immune disease resistance of the target plant. Those skilled in the art can flexibly select surfactant and its concentration according to actual conditions.

[0087] In a third typical embodiment of the present 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 comprises a grass plant.

[0088] When the plant immune inducer of the present application is applied to the target plant through an exogenous source, the immune disease resistance of the target plant can be significantly improved. The present application found that when the above-mentioned plant immune inducer is applied to the organ tissue of the target plant, it can activate the MAPK signaling pathway response of the target plant, further improve the immune ability of the plant, and can effectively prevent and control bacterial diseases and fungal diseases, and promote the development of crop disease prevention and control. The above-mentioned application method can be flexibly selected by those skilled in the art for any conventional exogenous application method to treat the organs of the target plant according to actual conditions, and the effect of the present application can be achieved.

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

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

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

[0092] It should be noted that this improvement in plant disease resistance is broad-spectrum, enhancing plant immunity by activating the MAPK signaling pathway, thereby improving resistance to a variety of diseases, including but not limited to resistance to bacterial and fungal diseases. Preferably, the bacterial diseases 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 include but are not limited to wheat head blight.

[0093] In this application, when the above-mentioned DCC and similar peptides are used to prepare plant immune inducers, the prepared plant immune inducers have broad spectrum and compatibility. The immunologically active DCC can significantly induce immune responses in grass crops, and has potential application value in preventing grass crop diseases. When the above-mentioned plant immune inducers are sprayed exogenously on grass crops, the disease resistance of grass crops is enhanced, which can prevent the occurrence of grass crop diseases in agricultural production and protect grass crops from pesticide contamination.

[0094] In a fifth typical embodiment of the present application, there is provided a use of the above polypeptide in the preparation of an agent for improving plant immunity and / or disease resistance.

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

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

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

[0098] 1. Analysis of wheat transcriptome data under biotic stress

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

[0100] The transcriptome data of wheat after biological stress were obtained from the wheat genome database (WheatOmics 1.0, http: / / 202.194.139.32 / ). The expression of pDCCs induced by immune elicitors and pathogens and the alignment results of pDCCs protein sequences were shown in Figure 2. Figure 1 As shown, Figure 1 The red markers in A indicate upregulated genes. Bioinformatics analysis revealed that a class of secretory peptide precursor protein encoding genes were upregulated by pathogenic immune elicitors flg22 and chitin, as well as pathogens F. graminearum, X. translucens, and Z. tritici (e.g. Figure 1 In wheat, this type of secretory peptide precursor protein includes 13 members. The amino acid sequences of these 13 members were aligned (SEQ ID NOs: 2-14 sequence alignment diagram shown in Figure 1). Figure 1 The C-terminus of the two proteins was found to contain two conserved cysteines. Based on this feature, we named this type of protein pDCCs (DUALCYSTEINES IN C-TERMINUS precursors, DCC precursor proteins, A, B, D represent wheat subgenomes, respectively; pDCC1A, B, D represent the precursor proteins or gene sequences of DCC1 in wheat A, B, D subgenomes, respectively).

[0101] 1.2 Identification of the size of the active DCC peptide fragment

[0102] Small peptide precursor proteins in plants are expressed and translated into amino acids within the cell, secreted outside the cell under the guidance of the N-terminal signal peptide, and then processed by proteases in the extracellular matrix into active small peptides. To identify the fragment size of the active small peptide DCC1 of 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 (the schematic diagram of the pLGY-OE3-pDCC1 vector is shown in the figure). Figure 2 As shown, Figure 2The vector was then transformed into Agrobacterium competent cells EHA105, and positive Agrobacterium clones were selected to infect the wheat variety Fielder to obtain pDCC1 overexpressing transgenic plants. The results of the identification of mature DCC1 small peptides and the sequence alignment of DCC homologous small peptides in Gramineae plants in this embodiment are shown in Figure 2. Figure 3 shown.

[0103] The interstitial fluid of the leaves of the 2-week-old pDCC1 high-expressing transgenic plants was extracted, and the 0.3-5 kDa small peptides in the interstitial fluid were subsequently analyzed by mass spectrometry. A small peptide of 10 amino acids was identified (the results are shown in Figure 3 (shown in A), located at the C-terminal position 54-63 of pDCC1 ( Figure 1 The amino acid sequence of the conserved region of the pDCC protein sequence of B is SGSSPPTHCC (SEQ ID NO: 15), indicating that this small peptide is likely to be the active mature small peptide DCC1.

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

[0105] Furthermore, the C-terminal mature peptide sequence (the 10 amino acid sequence at the C-terminus) was aligned, and the alignment statistics are shown in the figure below. Figure 3As shown in Figure 3, we found that the first serine (S) at the C-terminus and the 9th and 10th cysteine ​​(C) residues are highly conserved across all grasses. The amino acids at other positions are also somewhat conserved. For example: the second amino acid is glycine (most of them are glycine, according to the 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, and a few are arginine or proline; the fourth amino acid is serine or asparagine (polar and uncharged); the fifth amino acid is mainly proline or glycine, and a few are serine, threonine or arginine; the sixth amino acid is mainly proline or arginine, and a few are glycine, aspartic acid, serine or histidine; the seventh amino acid is threonine, serine or glycine; the eighth amino acid is histidine (most of them are histidine, according to the 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 homologous proteins in C4 plants (C4 plants refer to plants that use the C4 photosynthesis pathway, including but not limited to millet, maize, and sorghum) have an additional asparagine N at the C-terminus. These results indicate that the C-terminal peptide of the pDCC1 protein is highly conserved among grasses, especially the serine and cysteine ​​residues at key sites, which may be related to the activity of the peptide.

[0107] The amino acid sequence of the pDCC1 precursor protein is shown in SEQ ID NOs: 2-14. The first underlined portion of the following sequence represents the signal peptide sequence, and the second underlined portion represents the mature peptide sequence. The nucleotide sequence encoding the pDCC1 precursor protein 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 Analysis of the immunological activity 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 immunoreactive peptide fragments, DCC1 peptides of varying lengths were prepared based on the amino acid sequence of the pDCC1 precursor protein. These peptides also included peptides with key amino acid mutations and DCC homologous peptides from rice and maize. The specific peptides, along with their sequences, lengths, and sequence information, are shown in Table 1. Underlined fragments in Table 1 indicate amino acid mutation sites. Purity was >95% (synthesized and prepared by Shanghai Qiangyao Biotechnology Co., Ltd.).

[0125] Table 1

[0126]

[0127] Preparation of small peptide mother solution and working solution: Take the DCC1 small peptide protein pure complex and use ultrapure water as solvent to prepare a mother solution with a concentration of 1 mM, and 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 the DCC1 peptide that activates plant immune responses

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

[0130] The MAPK phosphorylation detection steps are as follows: take one-week-old wheat variety Fielder leaves, cut them into 1 cm leaf 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 them at room temperature overnight.

[0131] After treating wheat leaf segments with ddH2O (blank control group) and small peptide for 15 minutes, the leaf segments were placed in a 2 mL centrifuge tube containing steel balls, frozen with 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 No. 05056489001)), vortex thoroughly, place on ice for 10 minutes, and centrifuge at 12,000 rpm at 4°C for 10 minutes. Remove 40 μL of supernatant, add 10 μL of 5× SDS-PAGE loading buffer, mix thoroughly, and heat at 95°C for 5 minutes.

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

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

[0135] DCC1 core sequence (e.g. Figure 4 The results of key amino acid activity identification are shown in Figure 2A. Figure 4 shown.

[0136] Based on the pDCC1 protein sequence, small peptides of different lengths were synthesized, with sequences such as Figure 4 As shown in A, wheat leaves were treated with 10 nM and 100 nM of small peptides, respectively. The results showed that the small peptide with a length of 10 amino acids in the conserved region had the strongest immune activity (results Figure 4 (As shown in B), the activity of peptides that are too long or too short is reduced, which is consistent with the mature small peptide fragments in the interstitial fluid identified by mass spectrometry in the early stage.

[0137] According to the sequence alignment of DCC peptides, the C-terminal cysteine ​​is highly conserved ( Figure 3 Result in B), after cysteine ​​(C) was mutated to serine (S), MAPK phosphorylation could not be activated (results as shown in Figure 4 Therefore, the two cysteines at the C-terminus are very important for the immune activity of the DCC peptide.

[0138] 3. Minimum concentration of DCC for immune activation

[0139] Different concentrations of DCC1 peptide were used to treat wheat leaves. It was found that as low as 10 nM could significantly activate MAPK phosphorylation. Different DCC1 concentrations activated immune responses such as Figure 5 shown.

[0140] 4. DCC1 Thermal Stability Analysis

[0141] Thermal stability test: DCC1 peptide stock solution was placed at 22℃ and 50℃ for 6 hours, and 95℃ for 1 hour. Then it was used to treat wheat leaves for 15 minutes to detect MAPK phosphorylation activation. The DCC1 thermal stability test results are shown in the figure below. Figure 6 The results showed that the DCC1 peptide still had the activity of activating MAPK phosphorylation after being treated at 50℃ for 6 hours and 95℃ for 1 hour, indicating that the DCC1 peptide can still maintain stable immune induction activity under high temperature conditions.

[0142] 5. Chemical modification to enhance the activity of DCC peptides

[0143] Se-DCC1 was synthesized by replacing the sulfur (S) in the two cysteines in the DCC1 peptide with selenium (Se). MAPK phosphorylation assays revealed that Se-DCC1 possessed stronger immunoreactivity, with 1 nM Se-DCC1 having comparable activity to 5 nM DCC1 (selenocysteine ​​modification enhances DCC1 activity). Figure 7 This may be because selenium has a larger atomic radius and selenocysteine ​​is more active under physiological conditions, so Se-DCC1 is more likely to bind to receptors than DCC1 and activate immune responses.

[0144] 6. Analysis of the Immunoreactivity of DCC Peptides in Gramineae (Wheat, Rice, and Corn)

[0145] DCC peptides from the grasses wheat, rice, and corn were subjected to MAPK phosphorylation detection using the same method as above, with Arabidopsis thaliana and Nicotiana benthamiana serving as negative controls.

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

[0147] There are also four homologous peptides OsDCC in rice (sequences are shown in Table 1, OsDCC1, OsDCC2, OsDCC3 and OsDCC4), which can induce MAPK phosphorylation in two rice varieties, ZH11 and Nipponbare (the MAPK phosphorylation detection results of rice treated with OsDCC1, OsDCC2, OsDCC3 and OsDCC4 are shown in Figure 1). Figure 8 (as shown in B), activating immune activity.

[0148] The homologous peptide ZmDCC1 in maize (sequence of ZmDCC1 in Table 1) can not only activate the phosphorylation of MAPK in maize itself, but also induce the phosphorylation of MAPK in rice and wheat (the results of MAPK phosphorylation detection in maize, rice, wheat, Arabidopsis and tobacco treated with ZmDCC1 are shown in Figure 2). Figure 8 Similarly, wheat DCC1 can also induce rice MAPK phosphorylation (the results of MAPK phosphorylation detection in wheat, rice, maize, Arabidopsis and tobacco treated with DCC1 are shown in Figure 8 As shown in C), rice OsDCC1 can induce MAPK phosphorylation in wheat (the results of MAPK phosphorylation detection in rice, wheat, maize, Arabidopsis and tobacco after OsDCC1 treatment are shown in Figure 8 This may be because the grass family is closely related in evolution and the receptors that recognize small peptides are conserved.

[0149] Example 3: Application of DCC immunoreactive peptides for immune induction

[0150] Based on the activation properties of the DCC peptide on the plant immune system, the present invention discloses a method for using the mature peptide DCC as a plant immune inducer in disease prevention. The specific implementation scheme is shown in Table 2.

[0151] Table 2

[0152]

[0153] (1) Wheat bacterial leaf streak control efficacy experiment

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

[0155] Injection treatment: The aforementioned working solution was injected into the first cotyledon of the wheat variety Fielder, which had grown for about 2 weeks.

[0156] Pathogen culture and inoculation: The wheat bacterial leaf streak strain Xanthomonas translucens pv. Undulosa (Xtu) Kn5 (hereinafter referred to as Xtu Kn5) was inoculated into 2 mL of NB medium and cultured overnight at 28°C and 200 rpm. The bacteria were collected and diluted with ddH2O to OD600 = 0.005. Xtu Kn5 was injected into the first cotyledon of the pretreated wheat.

[0157] Statistics of control efficacy: 4 days after the pathogen was inoculated, the disease phenotype of the leaves was recorded (photographed) and the number of colonies on the leaves was determined by the gradient dilution plate count method.

[0158] The results of the test on DCC1 and DCC2 enhancing wheat resistance to bacterial leaf streak are as follows Figure 9 As shown, Figure 9 A in the middle is the diseased morphology of wheat leaves. Figure 9 Figure B shows the statistical graph of wheat leaf colony counts. Wheat leaves pretreated with the peptides DCC1 and DCC2 showed milder disease severity four days after inoculation with Xtu Kn5, with a highly significant difference compared to the blank control (P < 0.0001). The number of colonies on the leaves was also lower, indicating that the DCC peptides can effectively activate wheat leaf defense responses and can be used to prevent wheat bacterial leaf streak.

[0159] (2) Rice bacterial leaf streak prevention experiment

[0160] Working solution preparation and spray inducer preparation: 5 μM OsDCC4 and 5 μM DCC1 were prepared using ddH2O as the diluent; ddH2O was used as a blank control. Silwet L-77 (a silicone-based surfactant (polyether-modified silicone) developed by Momentive Performance Materials, USA, is widely used in agriculture, industry, and scientific research as a wetting agent, spreading agent, and penetrant) was added to the working solution at a volume concentration of 0.02% (v / v) to prepare the spray inducer.

[0161] Spraying treatment: 1 hour before pathogen inoculation, the inducer was sprayed on the surface of the leaves of 4-week-old rice variety TP309.

[0162] Pathogen culture and live inoculation: Xanthomonas oryzae pv. Oryzicola (Xoc) RS105 (hereinafter referred to as Xoc RS105) caused by bacterial leaf streak of rice was grown on PSA medium (1 L: 10 g tryptone, 1 g sodium glutamate, 10 g sucrose, 12 g agar) at 28°C for 2 days. The OD600 of Xoc RS105 was then adjusted to 0.5. Inoculation was performed by the infiltration method. Fully expanded leaves were selected and injected into the rice leaves from the back using a needleless syringe.

[0163] Statistics of control efficacy: 14 days after the pathogen was inoculated, the leaf disease was investigated and the length of the lesions was measured. Figure 10 As shown, Figure 10 A in the middle is the diseased morphology of rice leaves. Figure 10 Figure B shows the statistical graph of leaf lesion length. Rice leaves pretreated with OsDCC4 and DCC1 peptides showed significantly lower disease severity than the blank control (P < 0.0001), indicating that pretreatment with OsDCC4 and DCC1 peptides can activate rice defense responses and enhance resistance to bacterial leaf streak.

[0164] (3) Rice bacterial blight prevention experiment

[0165] Preparation of working solution and spray inducer: Prepare 0.5 μM OsDCC4 using ddH2O as the diluent; use ddH2O as a blank control. Add 0.02% (v / v) Tween 20 to the working solution to prepare the spray inducer.

[0166] Spraying treatment: 2 hours before pathogen inoculation, the inducer was sprayed on the surface of the leaves of 10-week-old rice variety ZH11.

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

[0168] Statistics of prevention effect: 140 days after the pathogen was inoculated, the disease occurrence on the leaves was investigated and the length of the lesions was measured.

[0169] The results of OsDCC4 enhancing rice resistance to bacterial blight are as follows Figure 11 As shown, Figure 11 A in the middle is a morphological diagram of diseased rice leaves, where the red arrow points to the diseased area. Figure 11 Figure B shows the statistical graph of leaf lesion length. The severity of lesion on rice leaves pretreated with OsDCC4 peptide was significantly lower than that on the blank control (P < 0.0001), indicating that OsDCC4 peptide pretreatment can activate rice defense responses and improve rice resistance to bacterial blight.

[0170] (4) Experiment on the prevention effect of wheat fusarium head blight

[0171] Preparation of working solution and spray inducer: Prepare 5 μM DCC1 using ddH2O as the diluent; use ddH2O as a blank control. Add 0.02% (v / v) Silwet L-77 to the working solution to prepare the spray inducer.

[0172] Spraying treatment: 24 hours before pathogen inoculation, spray the inducer on the ears of the wheat variety Fielder at the heading stage, and put a plastic bag on the ears to seal and keep them moist.

[0173] Pathogen culture and inoculation: Fusarium graminearum (Fg) PH-1, the pathogen of wheat fusarium head blight, was grown for 5 days on PDA medium (1 L: 200 g of peeled, boiled, filtered potato juice, 20 g of glucose, and 15 g of agar). The culture blocks containing mycelium were then cut into 2 mm cubes and transferred to fresh medium for one day. A single spikelet was selected from the center of a wheat ear. The husk of the spikelet was gently separated, and the mycelium block was placed into the husk using tweezers. The husk was sealed in a plastic bag to maintain moisture for 2 days, after which the bag was removed.

[0174] Statistics of prevention efficacy: 21 days after the pathogen was inoculated live, the 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 A in the middle is a morphological diagram of the symptoms of wheat head scab, in which the red arrow points to the susceptible area. Figure 12 Figure B shows the scab index. The DCC1 peptide pretreatment significantly reduced the index of wheat spikelets compared to the blank control (P < 0.0001), indicating that DCC1 peptide pretreatment can activate wheat defense responses and further enhance wheat resistance to scab.

[0176] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects:

[0177] The DCC1 and its similar peptides of the present application only require an active fragment of 10 or 11 amino acids, and can be produced on a large scale at low cost through solid-phase chemical synthesis or microbial expression systems (such as yeast fermentation). Compared with traditional protein-based inducers, they have obvious cost advantages, which are conducive to promoting the development of sustainable agriculture and meeting the demand for safe and green prevention and control methods in agricultural production. In addition, the immunoactive small peptides provided by the present application can be accurately applied to plants by injection or spraying, avoiding the excessive use of traditional chemical pesticides and environmental pollution. At the same time, it also overcomes the long cycle and high cost problems in disease-resistant breeding. It can accurately prevent and control bacterial and fungal diseases, improve crop quality and yield, and provide agricultural production with a safe, green, and efficient means of crop disease prevention. It is of great significance to promote the precision and sustainability of agricultural disease management.

[0178] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that 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; When the polypeptide has 11 amino acid residues, the 11th amino acid of the polypeptide is asparagine.

2. The polypeptide according to claim 1, characterized in that 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 7th amino acid is threonine, serine, or glycine; The 8th amino acid is histidine, threonine or serine.

3. The polypeptide according to claim 2, characterized in that The polypeptide has an amino acid sequence as shown in any one of SEQ ID NOs: 15 to 31.

4. The polypeptide according to claim 1, characterized in that The polypeptide further contains a chemical modification, wherein the chemical modification includes replacing all -SH on the cysteine ​​in the polypeptide with -SeH.

5. Use of the polypeptide according to any one of claims 1 to 4 in the preparation of an agent for improving plant immunity and / or disease resistance.

6. A plant immune inducer, characterized in that The plant immunity inducer includes a polypeptide; The polypeptide is the polypeptide according to any one of claims 1 to 4.

7. A method for improving plant disease resistance, characterized in that: The method comprises applying the polypeptide according to any one of claims 1 to 4 or the plant immunity elicitor according to claim 6 to a target plant; The target plants include grass plants.

8. The method according to claim 7, characterized in that The application method includes one or more of spraying, smearing, soaking, leaf cutting or injection.

9. The method according to claim 7, characterized in that The grass plant includes one or more of wheat, rice, corn, millet, barley or sorghum.

10. Use of the polypeptide according to any one of claims 1 to 4 or the plant immunity elicitor according to claim 6 in improving plant immunity and / or disease resistance.

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