Polypeptides, plant immune inducers, and methods to improve plant disease resistance

Abstract

This invention provides a polypeptide, a plant immune inducer, and a method for improving plant disease resistance. The polypeptide has proline at position 1 and cysteine ​​at positions 13 and 25; the polypeptide has 27 amino acid residues. This invention addresses the problems of scarcity, narrow disease resistance spectrum, and poor stability of existing peptide inducers, and is applicable to the field of plant immune inducers.

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 a method for improving plant disease resistance. Background Technology

[0002] Grasses are not only a major source of carbohydrates in the human diet, but also provide essential dietary fiber, vitamins, and minerals. Wheat, rice, and maize, the three main grasses, support the food needs of a large portion of the world's population and have a profound impact on food security and economic stability. Given their widespread cultivation and significant economic value, the healthy growth and disease management of grasses are crucial for ensuring global food supply and promoting sustainable agricultural development. Therefore, exploring effective control strategies for grass diseases has become a key research topic in agricultural science.

[0003] Wheat, as a widely cultivated and important food crop among the grasses, makes a significant contribution to the supply of protein and energy in the human diet. Despite its irreplaceable economic and nutritional value, wheat often faces challenges from diseases during cultivation, especially bacterial leaf streak, Fusarium head blight, and stem rot. The frequent occurrence of these diseases not only directly weakens the growth vigor of wheat and reduces crop yield and quality, but also poses a threat to global food security.

[0004] The Food and Agriculture Organization of the United Nations (FAO) points out that pathogens cause enormous agricultural losses every year, highlighting the urgency of disease control. While traditional chemical pesticides can effectively suppress diseases in the short term, long-term reliance on them brings significant negative effects: pathogens gradually develop resistance, weakening the long-term effectiveness of pesticides; pesticide residues can not only pollute the soil and introduce heavy metals, but also disrupt the balance of ecosystems, leading to a decline in biodiversity; more seriously, residual chemicals pose a potential risk to human health.

[0005] Given the limitations of chemical pesticides, exploring environmentally friendly disease control strategies is particularly urgent. Plant immune inducers, which can stimulate plants' natural defense mechanisms, are considered a kind of "plant vaccine" and have the potential to be transformed into a new generation of green pesticides to combat crop diseases. Research in this field has identified various types of immune inducers, including oligosaccharides, proteins, small molecule compounds, and peptides. However, each type of inducer has its inherent limitations: oligosaccharides, such as chitosan oligosaccharides, have high activation concentrations and short-lived effects; proteins, such as elicitor proteins, face complex production processes and poor environmental stability; small molecule compounds, such as purine bases, have narrow disease resistance spectra and may induce abnormal plant metabolism. In contrast, peptide inducers, with their smaller molecular weight and strong activity (even at 10...), offer advantages. -9Its advantages (it remains effective even at M concentrations) and ease of industrial production stand out.

[0006] However, the development of peptide immune inducers still requires breakthroughs. Currently, the types of bioactive peptides are relatively limited, with only a few, such as AtPep1, SlPep, and CEP14, mentioned in relevant patents. Furthermore, peptide inducers often target specific receptors; for example, the specificity of the PEPR2 receptor limits their breadth of disease resistance. Finally, peptide compounds are susceptible to degradation in outdoor environments, affecting their stability and durability in practical field applications. Summary of the Invention

[0007] The main objective of this invention is to provide a polypeptide, a plant immune inducer, and a method for improving plant disease resistance, in order to solve the problems of scarcity, narrow disease resistance spectrum, and poor stability of peptide inducers in the prior art.

[0008] To achieve the above objective, according to a first aspect of the present invention, a polypeptide is provided, wherein the amino acid at position 1 is proline, and the amino acids at positions 13 and 25 are both cysteine; the polypeptide has 27 amino acid residues.

[0009] Furthermore, the polypeptide has the amino acid sequence shown in SEQ ID NO: 1.

[0010] Furthermore, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 20-24.

[0011] 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 amino acid at position 1 of the polypeptide is proline, and the amino acids at positions 13 and 25 are both cysteine; the polypeptide has 27 amino acid residues.

[0012] Furthermore, the polypeptide has the amino acid sequence shown in SEQ ID NO: 1.

[0013] Furthermore, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 20-24.

[0014] 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.

[0015] Furthermore, the application methods include one or more of the following: spraying, smearing, soaking, leaf cutting, or injection.

[0016] Furthermore, grasses include one or more of wheat, rice, maize, switchgrass, or millet.

[0017] To achieve the above objectives, according to a fourth aspect of the present invention, the application of the above-mentioned polypeptide or any of the above-mentioned plant immune inducers in improving plant immunity and disease resistance is provided.

[0018] 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.

[0019] Applying the technical solution of this invention, this application provides a polypeptide with proline at position 1 and cysteine ​​at positions 13 and 25, having a length of 27 amino acid residues. Proteins with this sequence characteristic possess immunogenicity, enriching the types of immunogenic peptides and overcoming the problem of narrow disease resistance caused by excessively strong receptor specificity. Moreover, it has broad spectrum; when used as a plant resistant inducer, it has high thermal stability and is not easily degraded, making it applicable to a variety of 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

[0020] 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:

[0021] Figure 1 The heatmap shows the expression levels of the PAMPs and 18 TaDEP peptide-encoding genes under pathogen treatment in Example 1 of this application.

[0022] Figure 2 This paper presents a schematic diagram of the evolutionary analysis of TapreDEPs from Embodiment 1 of this application and a sequence characteristic diagram of their conserved domains related to immune activity; wherein, Figure 2 A in the diagram is a schematic diagram of the evolutionary analysis of the 18 TapreDEPs mentioned above. Figure 2 The diagram in B is a sequence analysis diagram of the conserved segments of 18 TapreDEPs.

[0023] Figure 3 The diagram shows the results of the predictive analysis of the secondary structure of the TapreDEP2 precursor protein in Example 1 of this application specification.

[0024] Figure 4 The diagram shows the MAPK activation capability analysis results of TaDEPs in Embodiment 2 of this application specification.

[0025] Figure 5 This document shows the qRT-PCR results of Example 2 of this application demonstrating that TaDEPs secreted peptides significantly induced the expression of immune-related genes; wherein, Figure 5 Figure A shows the qRT-PCR results of TaWRKY23 expression induced by TaDEPs secreted peptides. Figure 5 Figure B shows the qRT-PCR results of TaWRKY28 expression induced by TaDEPs secreted peptides. Figure 5 The middle image (C) shows the qRT-PCR results of TaWRKY53 expression induced by TaDEPs secreted peptides. Figure 5 The middle image (D) shows the qRT-PCR results of TaPUB23 expression induced by TaDEPs secreted peptides. Figure 5 Figure E shows the qRT-PCR results of TaPR1 expression induced by TaDEPs secreted peptides. Figure 5 The middle F figure shows the qRT-PCR results of TaPR5 expression induced by TaDEPs secreted peptides.

[0026] Figure 6 The diagram shows the results of interspecies evolutionary analysis of TapreDEPs and the conservation analysis of the immune activity of TaDEP2 in Example 2 of this application; wherein, Figure 6 Figure A in the diagram represents the results of interspecific evolutionary analysis of TapreDEPs. Figure 6 Figure B shows the results of the analysis of the conservation of immune activity.

[0027] Figure 7 The diagram shows the effect of TaDEP2 key site mutations on immune activity in Example 2 of this application.

[0028] Figure 8 The diagram shows the results of the in vitro thermal stability test of the immunologically active small peptide TaDEP2 in Example 2 of this application specification.

[0029] Figure 9 The figure shown is a graph illustrating the results of resistance detection analysis of wheat leaves treated with TaDEPs in Example 3 of this application against Xtu Kn5, the pathogen of wheat bacterial leaf streak; wherein, Figure 9 Image A shows the water-soaked phenotype of diseased wheat leaves. Figure 9 Figure B is a statistical chart of pathogen biomass.

[0030] Figure 10 The figure shown is a graph illustrating the results of resistance detection and analysis of wheat leaves treated with TaDEP2 to the pathogen FgPH-1 of wheat scab, as described in Example 3 of this application. Figure 10 Image A shows the morphology of wheat leaves infected by the pathogen that causes wheat scab. Figure 10 Figure B shows a statistical chart of the area of ​​diseased spots on wheat leaves.

[0031] Figure 11 The figure shown is a graph illustrating the results of resistance detection and analysis of FgPH-1, the pathogen of wheat scab, in wheat ears treated with TaDEP2 according to Example 3 of this application; wherein, Figure 11 Image A shows the morphology of a wheat ear infected by the pathogen that causes wheat scab. Figure 11 Figure B shows a statistical chart of the severity of disease in wheat ears.

[0032] Figure 12 The figure shown is a graph illustrating the results of the TaDEP2 treatment of wheat stem rot pathogen Fpg resistance detection in Example 3 of this application; wherein, Figure 12 Image A shows the morphology of wheat coleoptiles infected by the pathogen causing wheat stem rot. Figure 12 Figure B shows a statistical chart of the length of lesions on wheat coleoptiles. Detailed Implementation

[0033] 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.

[0034] Terminology Explanation:

[0035] Bacterial wheat stripe: A bacterial disease of wheat caused by Xanthomonas translucenspv. undulosa (Xtu) (also known as: Bacterial wheat stripe bacterium).

[0036] Fusarium head blight of wheat: It is one of the most common fungal diseases in wheat production and planting. This disease is caused by infection by a variety of Fusarium fungi, among which Fusarium graminearum (Fg) is the main pathogen.

[0037] Wheat stem base rot: This is a disease that occurs in wheat and is caused by fungi such as Fusarium pseudograminearum (Fpg). It mainly infects the base of wheat stems and can cause damage throughout the entire growth period of wheat.

[0038] 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.

[0039] As mentioned in the background section, existing peptide inducers are scarce, have a narrow spectrum of disease resistance, and are unstable. Based on this, the inventors in this application have discovered a new immune polypeptide that can be applied to the development of peptide inducers for plants, and thus proposed a series of protection schemes in this application.

[0040] In a first typical embodiment of this application, a polypeptide is provided, wherein the amino acid at position 1 is proline (P), and the amino acids at positions 13 and 25 are both cysteine ​​(C); the polypeptide has 27 amino acid residues.

[0041] The sequence of the above polypeptide is: PXXXXXXXXXXXCXXXXXXXXXXXCXX.

[0042] Here, "X" represents any naturally occurring amino acid or non-natural amino acid. That is, these positions containing "X" can be occupied by any amino acid, but this does not affect 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), 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. The amino acid represented by "X" in the amino acid sequence of SEQ ID NO: 1 of this application is the same as described herein.

[0043] Plant secretory peptides are a class of small polypeptide molecules (typically 2-100 amino acids) synthesized by plant cells and released extracellularly via the secretory pathway. They play important signal transduction and regulatory roles in plant growth, development, stress resistance, and immune responses. These secretory peptides are usually 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.

[0044] The mechanism of action of plant-secreted small peptides mainly relies on their interaction with receptor kinases, including signal recognition, signal transduction, and the generation of physiological effects. Specifically: signal recognition refers to the binding of secreted small peptides to receptor kinases on the cell membrane, such as the binding of PSK to PSKR; signal transduction refers to the transmission of signals through downstream signaling pathways (such as the MAPK cascade) after the receptor kinase is activated, regulating gene expression and cellular responses; the resulting physiological effects ultimately manifest as promoting growth, enhancing stress resistance, or activating immune responses.

[0045] This application identifies a polypeptide (i.e., the aforementioned plant-secreting peptide) with a sequence length of 27 amino acid residues. The first amino acid is proline (P), and the 13th and 25th amino acids are cysteine ​​(C). This application analyzes the polypeptide TaDEP2 containing the above sequence characteristics and performs site mutation analysis on two conserved cysteine ​​(C) residues within its conserved active region. The results indicate that the two discontinuous conserved cysteine ​​(C) residues "CXC" are crucial for maintaining the immune activity of TaDEP2, revealing the structural-functional basis of this type of polypeptide and laying a theoretical foundation for subsequent research and applications of peptide immune activity.

[0046] This application successfully discovered an immune inducer based on a peptide with the above-mentioned structural characteristics. This not only fills the gap in the field of plant immune inducer peptides due to the lack of molecular diversity, but also solves the problem of narrow disease resistance spectrum caused by receptor specificity. At the same time, it improves the stability of the peptide in complex field environments, providing an innovative, green, and efficient solution for crop disease control. It has broad application prospects and significant economic benefits, and is expected to promote agriculture towards a healthier and more environmentally friendly direction.

[0047] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0048] SEQ ID NO: 1: PXGXXGAGAXNGCTNNPXHPXGGKCXG.

[0049] Furthermore, the aforementioned polypeptide also includes a polypeptide having the amino acid sequence shown in SEQ ID NO: 1. Specifically, the amino acid at position 1, position 13, and position 25 of the amino acid sequence in SEQ ID NO: 1 is identical to the amino acid sequence shown in the aforementioned plant secretory peptide. In addition, the conserved amino acids at other sites in SEQ ID NO: 1 are: glycine (G) at positions 3 and 6, alanine (A) at position 7, glycine (G) at position 8, alanine (A) at position 9, asparagine (N) at position 11, glycine (G) at position 12, threonine (T) at position 14, asparagine (N) at positions 15 and 16, proline (P) at position 17, histidine (H) at position 19, proline (P) at position 20, glycine (G) at positions 22 and 23, lysine (K) at position 24, and glycine (G) at position 27.

[0050] Preferably, the meaning of "X" at the amino acid positions 2, 4, 5, 10, 18, 21, and 26 of SEQ ID NO: 1 is the same as described above; more preferably, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid at "X" is: the amino acid at position 2 includes, but is not limited to, glycine (G) or arginine (R); the amino acid at position 4 includes, but is not limited to, glycine (G) or serine (S); the amino acid at position 5 includes, but is not limited to, glycine (G), proline (P), or threonine (T); the amino acid at position 10 includes, but is not limited to, proline (P) or serine (S); the amino acid at position 18 includes, but is not limited to, asparagine (N) or lysine (K); the amino acid at position 21 includes, but is not limited to, alanine (A) or proline (P); and the amino acid at position 26 includes, but is not limited to, histidine (H) or asparagine (N).

[0051] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 20-24.

[0052] SEQ ID NO: 20 (TaDEP1): PGGSPGAGAPNGCTNNPKHPAGGKCHG.

[0053] SEQ ID NO: 21 (TaDEP2): PRGGGSAGAPNGCTNNPKHPPGGKCHG.

[0054] SEQ ID NO: 22 (TaDEP3): PGGSPGAGASNGCTNNPNHPPGGKCNG.

[0055] SEQ ID NO: 23 (TaDEP4): PGGSPGAGAPNGCTNNPKHPPGGKCHG.

[0056] SEQ ID NO: 24 (TaDEP5): PGGSTGAGAPNGCTNNPKHPPGGKCHG.

[0057] The aforementioned polypeptides specifically include polypeptides having amino acid sequences as shown in any of SEQ ID NOs: 20-24. These polypeptides can induce the expression of wheat TaWRKY23, TaWRKY28, TaWRKY53, TabUB23, TaPR1, and TaPR5 genes, all of which have been shown to play important positive regulatory roles in the early or late stages of the wheat immune response. The increased expression levels of these polypeptides on these positively regulatory genes of wheat immunity indicate that they also play a positive role in the wheat immune response.

[0058] This application employs a reverse genetics strategy to screen and identify immunologically active TapreDEPs from a wheat endogenous secretory peptide library. First, by analyzing dynamic transcriptome data of wheat leaves treated with pathogen-associated model molecules (PAMPs), we screened for genes encoding secretory peptides significantly induced by PAMPs. Subsequently, based on the conservation of amino acid sequences and functional prediction, we synthesized TaDEPs containing immunologically active regions to verify their immune-activating capabilities. Taking TaDEP2 as an example, its precursor protein, TapreDEP2 (the full-length amino acid sequence of which is shown in SEQ ID NO: 19), contains a typical secretory peptide structure, consisting of an N-terminal signal peptide, a variable region, and a C-terminal conserved region. In the endoplasmic reticulum-Golgi apparatus secretion pathway, after protease treatment, the anterior signal peptide and variable region are cleaved, releasing the mature and active TaDEP2 peptide (amino acid sequence shown in SEQ ID NO: 22), which can be effectively transported to the exoplasm to exert its immunoregulatory effect.

[0059] SEQ ID NO: 19: TraesCS4A02G024100 (TapreDEP2):

[0060] MARAVVMAVLLMQCCNVVLAARLLEGDGGWLRGGVGAAGALIMQVLPRGGGSAGAPNGCTNNPKHPPGGKCHG.

[0061] In a preferred embodiment, the above-mentioned polypeptide is heated at 10~100℃ (including but not limited to 10℃, 11℃, 12℃, 13℃, 14℃, 15℃, 16℃, 17℃, 18℃, 19℃, 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, 36℃, 37℃, 38℃, 39℃, 40℃, 41℃, 42℃, 43℃, 44℃, 45℃, 46℃, 47℃, 48℃, 49℃, 50℃, 51℃, 52℃, 5...). It can maintain its activity even under treatment at 3℃, 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℃, 88℃, 89℃, 90℃, 91℃, 92℃, 93℃, 94℃, 95℃, 96℃, 97℃, 98℃, 99℃ or 100℃.

[0062] This application also conducted thermal stability experiments on the above-mentioned peptides. The immune activity of the above-mentioned peptides remained stable under the above-mentioned temperature range, which provides a basis for their application in complex and variable field environments and improves their stability and biological activity in practical applications.

[0063] Furthermore, this application, through the detection of MAPK phosphorylation levels, found that TaDEP1-TaDEP5 could significantly induce an immune response in wheat, with TaDEP2 exhibiting particularly high immune activation efficiency. In addition, real-time quantitative PCR results showed that TaDEP1 and TaDEP2 could effectively promote changes in the expression levels of immune-related genes (TaWRKY23, TaWRKY28, TaWRKY53, TabUB23, TaPR1, and TaPR5), demonstrating that the aforementioned peptides can induce an immune response in wheat, strengthen the wheat's immune response mechanism, and thus enhance the crop's resistance to diseases.

[0064] The discovery of these peptides in this application not only expands the molecular diversity of plant immune-active peptides, but also enables broad-spectrum control of various crop diseases through the specific structure of their conserved C-terminal regions. Furthermore, these peptides exhibit good thermal stability and environmental adaptability in field applications, making them suitable and durable as spray-applied immune inducers in practical agricultural production.

[0065] In a second typical embodiment of this application, a plant immune inducer is provided, which comprises a polypeptide; the amino acid at position 1 of the polypeptide is proline (P), and the amino acids at positions 13 and 25 are both cysteine ​​(C); the polypeptide has 27 amino acid residues.

[0066] Plant immune inducers are substances that can induce immune defense responses 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.

[0067] The plant immune inducer prepared using the above-mentioned polypeptides has been demonstrated in field trials of this application to effectively enhance the resistance of wheat to bacterial leaf streak, fungal Fusarium head blight, and stem rot, thereby reducing the use of chemical pesticides, mitigating environmental pollution, and ensuring food security and quality.

[0068] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0069] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any of SEQ ID NOs: 20-24.

[0070] In a preferred embodiment, the working concentration of the plant immune inducer is 1-5 μM, including but not limited to 1, 2, 3, 4 or 5 μM.

[0071] 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%.

[0072] 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.

[0073] 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.

[0074] It should be noted that this enhancement of plant disease resistance is a broad-spectrum disease resistance. It improves plant immunity by activating the response of the plant's MAPK signaling pathway and inducing changes in the expression levels of genes in the immune signaling pathway, thereby enhancing the plant's resistance to a variety of diseases, including but not limited to resistance to bacterial and fungal diseases.

[0075] In a preferred embodiment, the application method includes, but is not limited to, one or more of the following: spraying, smearing, soaking, leaf cutting, or injection.

[0076] 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 in the target plants, induce changes in the expression levels of genes in the immune signaling pathway, further enhance the plant's immunity, and effectively control bacterial and fungal diseases, 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 the actual situation, and all can achieve the effects of this application.

[0077] In a preferred embodiment, the grasses include one or more of wheat, rice, maize, switchgrass, or millet.

[0078] 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.

[0079] It should be noted that this enhancement of plant disease resistance is a broad-spectrum resistance, achieved by activating the MAPK signaling pathway and inducing changes in gene expression levels along immune signaling pathways, thereby improving plant immunity and enhancing resistance to various diseases, including but not limited to resistance to bacterial and fungal diseases. Preferably, the bacterial diseases mentioned above include, but are not limited to, wheat bacterial leaf streak; preferably, the fungal diseases mentioned above include, but are not limited to, wheat scab or wheat stem rot.

[0080] In this application, the aforementioned polypeptides and similar peptides are used to prepare plant immune inducers. The resulting plant immune inducers possess broad-spectrum and compatibility, and these immunologically active TaDEPs can significantly induce immune responses in gramineous crops, demonstrating potential application value in the prevention of gramineous crop diseases. When these plant immune inducers are exogenously sprayed onto gramineous crops, they can enhance the disease resistance of gramineous crops, prevent the occurrence of crop diseases in agricultural production, and simultaneously protect crops from pesticide contamination.

[0081] 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.

[0082] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0083] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0084] Example 1: Screening and Identification of TaDEP2 Immunologically Active Small Peptides

[0085] 1. Transcriptome Data Acquisition and Analysis

[0086] RNA-seq datasets of genes encoding potential immunoreactive peptides (TaDEPs) in wheat leaves treated with PAMPs were obtained from the wheat genomics database (WheatOmic, http: / / wheatomics.sdau.edu.cn / ).

[0087] Homologous gene BLAST search analysis identified 18 members of this family (TapreDEPs) in the wheat genome. Based on transcriptome data published in the wheat genomics database, the expression patterns of these 18 TapreDEPs were analyzed using bioinformatics methods. The expression heatmaps of the peptide-encoding genes of the 18 TaDEPs under AMP and pathogen treatments are shown below. Figure 1 As shown in the figure. The results indicate that most of them were significantly induced by PAMPs, Xanthomonas translucens (Xt), and Fusarium graminearum (Fg), suggesting their important role in wheat immune regulation.

[0088] 2. Screening and identification of TaDEPs immunomodulatory peptides

[0089] 2.1 Screening and prediction of TaDEPs immunomodulatory peptides

[0090] Evolutionary and amino acid sequence conservation analyses of 18 TapreDEPs (SEQ ID NO: 2-19) were performed using bioinformatics techniques. The analyses revealed highly conserved regions, including two conserved but discontinuous cysteine ​​residues (C). This site is predicted to be essential for the immune activity of the conserved regions. The evolutionary analysis of TapreDEPs, encoding wheat immune-active secretory peptides, is related to their immune activity. The sequence characteristics of the conserved domains are shown in the figure below. Figure 2 As shown, where, Figure 2 A is a schematic diagram of the evolutionary analysis of the 18 TapreDEPs mentioned above, where "Bootstrap percentage (BP)" means the percentage of the bootstrap value. Figure 2 Figure B is a schematic diagram of the sequence analysis of the conserved regions of the 18 TapreDEPs mentioned above. In this diagram, “Signal peptide” means signal peptide, “Variable region” means variable region, and “Conserved region” means conserved region.

[0091] The underlined regions in the following amino acid sequences represent the conserved C-terminal regions.

[0092] SEQ ID NO: 2: TraesCS4D02G277600:

[0093] MTRAVVVAVLLVQCCNVVLAARLLEGDRGWLHGGVGAAGALIMQVLMARTVVVALLLMQFCDVVLAARLLEGDGAWLQGGVGAAGALIMHILPGGS PGAGAPNGCTNNPKHPAGGKCHG GR.

[0094] SEQ ID NO:3:TraesCS4A02G025000:

[0095] MARAVVVAVLLVQCCNVVLAARLLEGDRGWLHGGVGTAGALIMQVL PGGSPGAGTPNGCTNNPNHPPG GPCNG GR。

[0096] SEQ ID NO:4:TraesCS4D02G277700:

[0097] MKISMGSSATMARAVLIAAVLLVQCCNVVVAARLLEGDRGWLHGGVGAAGALIMQVL PGGSPGAGTPN GCTNNPNHPPGGPCNG GR。

[0098] SEQ ID NO:5:TraesCS4A02G024400:

[0099] MARAVVVALLLVQCCNVVVAARLLEGDHGWLHGGVGPAGALIMQVL PGGSPGAGTPNGCTNNPNHPPG GPCNG GR。

[0100] SEQ ID NO:6:TraesCS4B02G279100:

[0101] MARAVVIAAVLLVQCCNVVLAARLLEGDRGWLHGGVGAAGALIMQVL PGGSPGAGTPNGCTNNPNHPP GGACNG GR。

[0102] SEQ ID NO:7:TraesCS4B02G462100LC:

[0103] MVKAVVVAVLLVQCCNVVLAARLLEGDGGWLHGGAAGALIMQIL AGHSPGAGTPNPCTHDPINNNPPG A GFRESMEPYW。

[0104] SEQ ID NO:8:TraesCS4A02G019700LC:

[0105] MGCFSTACFSVVVVFFDQSSILFFLLPLQARHSSIQPRISNSCSTVRTVRVCSETESSTRDTYMATMVRAVVLAVLLVQCCNVVLAARLLEGDGGWLHGDVGAAGALIMQTL AGHSPGAGAPNPCTHDPVNNNPPGA RYSVTNGARDVKEMTLNDRQMLAYRSATNGPEQRAQSGSKVLGMPWKSFCTG。

[0106] SEQ ID NO:9:TraesCS4B02G279500:

[0107] MVRAVVVAVLLVQCCSVVLAARLLEGDGGWLHGGVGAAGALIMQIL PSGSPGVGTPNPYTHDPINNNP PGQ 。

[0108] SEQ ID NO:10:TraesCS4B02G279300:

[0109] MARAVVVAVLLVQCCNVALAARLLEGDGGWLHGDIGTAGALIMQVL PGGSPGAGASNGCTNNPNHPPG GKCNG 。

[0110] SEQ ID NO:11:TraesCS4A02G024200(TapreDEP3):

[0111] MARAVVVAVLLVQFCNVVIAARLLEGDGGWLHGDIGAAGALIMQVL PGGSPGAGASNGCTNNPNHPPG GKCNG 。

[0112] SEQ ID NO:12:TraesCS4D02G277900:

[0113] MARAVVLAVLLMQCCNVVLAARLLEGDGGWLHGDIGVAGALIMQVL PGGSPGAGASNGCTNNPNHPPG GKCNG 。

[0114] SEQ ID NO:13:TraesCS4B02G279200(TapreDEP5):

[0115] MARVVVVALLLMQFCNVVMAARLLEGDGAWLQGGVGAAGALIMQIL PGGSTGAGAPNGCTNNPKHPPG GKCHG 。

[0116] SEQ ID NO:14:TraesCS4A02G024300(TapreDEP1):

[0117] MARTVVVALLLMQFCDVVLAARLLEGDGAWLQGGVGAAGALIMHIL PGGSPGAGAPNGCTNNPKHPAG GKCHG 。

[0118] SEQ ID NO:15:TraesCS4D02G277800(TapreDEP4):

[0119] MARAVVVALLLMQFCDVVLAARLLEGDGGWLHLHGGVGAAGALILQIL PGGSPGAGAPNGCTNNPKHP PGGKCHG 。

[0120] SEQ ID NO:16:TraesCS4B02G279400:

[0121] MARAVMMTVLLMQCCNVVLAARLLEGDGGWLHGGVGAAGALIMQVL PRGGGSAGAPNGCTNNPKHPPG GKCHG 。

[0122] SEQ ID NO:17:TraesCS4D02G278000:

[0123] MASAERITRAALLVTAAVLMQCCNAALAARLLEGDGGWLRR GAGSLIMEVLPRCRQLVLERSQTPP VVGELPRAASNLAVADMLV。

[0124] SEQ ID NO:18:TraesCS4B02G279600:

[0125] MASTARITRAALLVAAVALMQCCNAVLAARLLEGDGVDVWLRQGAGSLIMQAL PRGGSPPGAGNSCWN DPKHPPSSGSWL ARRIAPPDVDKLV。

[0126] SEQ ID NO:19:TraesCS4A02G024100(TapreDEP2):

[0127] MARAVVMAVLLMQCCNVVLAARLLEGDGGWLRGGVGAAGALIMQVL PRGGGSAGAPNGCTNNPKHPPG GKCHG 。

[0128] 2.2 Secondary structure prediction analysis of TapreDEP2 precursor protein

[0129] The secondary structure of the TapreDEP2 precursor protein was predicted and analyzed using AlphaFold 2 software. The results are as follows: Figure 3 As shown. The results indicate that the conserved C-terminal region of TapreDEP2 (i.e., TaDEP2) forms a ring structure through two conserved cysteine ​​residues (C), further suggesting the importance of these two conserved cysteine ​​residues (C) for the function of TaDEP2.

[0130] 2.3 Screening and synthesis of TaDEPs with immune-activating activity

[0131] Based on expression pattern analysis and conserved segment sequence analysis, five members (TapreDEP1-5) were selected from 18 TapreDEPs that were significantly induced by PAMPs and pathogens and had relatively high induction levels for verification of the immunomodulatory activity of the proteins encoded by this family of genes. The conserved regions exerting immunomodulatory activity were predicted and named TaDEP1-TaDEP5, respectively. These five small peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., and their amino acid sequences are SEQ ID NOs: 20-24.

[0132] Example 2: Analysis of the immunomodulatory activity and evolutionary conservation of TaDEPs small peptides

[0133] 1. Validation of the immunomodulatory activity of TaDEPs

[0134] (1) Analysis of phosphorylation level of MAPK

[0135] The MAPK (Mitogen-Activated Protein Kinase) cascade signaling pathway is a highly conserved signaling pathway in eukaryotes. It plays a crucial role in plant growth, development, and stress responses, particularly in plant immune responses, typically regulating immunity through phosphorylation. Therefore, the phosphorylation level of MAPK can serve as one indicator for assessing the activation status of plant cellular immunity.

[0136] Experimental design: TaDEP1-5 and the immune-activating peptide positive control flg22 were both synthesized and prepared by Shanghai Qiangyao Biotechnology Co., Ltd., and were respectively prepared into working solutions with a concentration of 1 μM using ddH2O as the solvent. Sterile water treatment served as a blank control.

[0137] flg22 (Flagellin 22) is a highly conserved 22-amino acid peptide at the N-terminus of bacterial flagellin that can activate the immune response in plants as a pathogen-associated molecular pattern (PAMP).

[0138] The flg22 sequence is: QRLSTGSRINSAKDDAAGLQIA (SEQ ID NO: 39).

[0139] Wheat leaves (Fielder) aged 7-10 days were cut into 1 cm radius segments and placed in cell culture dishes containing 1 mL ddH2O. After adding 0.02% Triton-X100, the cells were subjected to vacuum permeation treatment. The leaves were then washed twice with ddH2O, and another 1 mL ddH2O was added to the cell culture dishes. The cells were incubated overnight at room temperature. The leaf segments were treated with TaDEP1-5 peptides using working solution, with sterile water treatment as a blank control and flg22 treatment as a positive control. Samples taken 15 min after treatment were rapidly frozen in liquid nitrogen for later use. Total protein was extracted from the samples to detect the activation level of MAPK by the aforementioned peptides.

[0140] The steps for extracting total protein are as follows: Place the dried leaf segments into a 2 mL centrifuge tube, freeze in liquid nitrogen, homogenize, add 100 μL of plant protein extraction solution (25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10 mM NaF, 1 mM PMSF, 0.1% SDS, 1% Triton X-100, 0.1% SDS, 5 mM DTT), vortex to mix, and incubate on ice for 10 min to help plant cells lyse fully; centrifuge at 12000 rpm at 4℃ for 5-10 min. Take 80 μL of supernatant into a 1.5 mL centrifuge tube, add 20 μL of 5× SDS loading buffer, mix thoroughly, and boil in a 95℃ metal bath for 5 min. Add the denatured protein to the wells of a 10% SDS-PAGE protein gel, and perform electrophoresis at a constant voltage of 90 V for about 20 min until the sample is completely located at the boundary between the stacking gel and the separating gel. Continue electrophoresis at a constant voltage of 140 V for about 50 min (until the protein pre-stained markers are completely separated), and then stop electrophoresis. Perform membrane transfer. After transfer, block the PVDF membrane in 5% BSA at room temperature for 1 h, and then incubate it overnight at 4℃ with anti-pERK1 / 2 antibody. The next day, wash three times with 1×TBST at room temperature for 10 min each time, incubate with rabbit secondary antibody at room temperature for 1 h, and then wash three times with 1×TBST again. Subsequently, PVDF membranes were developed using ECL chemiluminescent substrates and photographed using a Tanon 5200 series fully automated chemiluminescence imaging system to evaluate the immune-inducing activity of TaDEPs.

[0141] The analysis results of TaDEPs' MAPK activation capability are as follows: Figure 4As shown, wheat TaDEP1-5 peptides significantly activated the phosphorylation of MAPK in leaves, and the activation intensity was roughly the same as flg22, indicating that TaDEPs can significantly activate the wheat immune response.

[0142] (2) Detection of expression levels of immune-related genes

[0143] Many genes play important roles in the regulation of plant immune responses, such as TaWRKY23, TaWRKY28, TaWRKY53, TabUB23, TaPR1, and TaPR5. To further examine the relationship between TaDEPs and wheat immune responses, we tested the expression levels of these immune-related genes.

[0144] Based on the aforementioned research results, TaDEP1-5 showed roughly the same activation level of MAPK in wheat leaves, with TaDEP1 and TaDEP2 exhibiting the lowest and highest MAPK activation abilities, respectively. Therefore, TaDEP1 and TaDEP2 were selected as representatives of the TaDEPs family of small peptides to verify their immunomodulatory functions.

[0145] The leaf fragments treated with TaDEP1 and TaDEP2 were used to extract total RNA from the treated samples using the Trizol method, and reverse transcription was performed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novizan, Nanjing, China) according to the manufacturer's instructions.

[0146] Real-time quantitative PCR (qRT-PCR) was performed using a QuantStudio™ 3 real-time quantitative PCR system. Each reaction volume (10 µL) contained 5 μL of 2× Q3 SYBR qPCR Master Mix (Universal), 0.2 μL of primer F (10 μM), 0.2 μL of primer R (10 μM), 2 μL of template DNA / cDNA, and ddH2O added to 10 μL. PCR conditions were as follows: reaction at 95°C for 30 seconds, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C per cycle. TaActin was used as an internal control, and qRT-PCR analysis was performed using three biological replicates. Using qRT-PCR and 2... -∆∆Ct Methods were used to analyze the expression data of relevant genes.

[0147] The reaction primers are as follows:

[0148] TaWRKY23:

[0149] F: GAGCGTAGACGTCAGCACCA (SEQ ID NO: 25);

[0150] R: CACGGATGCTAATGGCCACC (SEQ ID NO: 26).

[0151] TaWRKY28:

[0152] F: CGAAAGTACGGCCAGAAGGT (SEQ ID NO: 27);

[0153] R: TCTTCTTCACCTGGCACGAC (SEQ ID NO: 28).

[0154] TaWRKY53:

[0155] F:CCCTTCAGGACCAACACCAA (SEQ ID NO: 29);

[0156] R: CCAAGAACCACGAGACACCA (SEQ ID NO: 30).

[0157] TaPUB23:

[0158] F: CGTTCATCAGAATGCTCAGCTG (SEQ ID NO: 31);

[0159] R: TTCTCTTTGTAGGCACGAACCA (SEQ ID NO: 32).

[0160] TaPR1:

[0161] F: CAGAACTCGCCTCAGGACTAC (SEQ ID NO: 33);

[0162] R: CGTAGTTGTAGTCCTTCTTCTC (SEQ ID NO: 34).

[0163] TaPR5:

[0164] F: CATCAAGAACAACTGCGGCTC (SEQ ID NO: 35);

[0165] R: CGTAGAAGTCCTGGGTGCTG (SEQ ID NO: 36).

[0166] TaDEPs secreted peptides significantly induce the expression of immune-related genes. qRT-PCR data are shown below. Figure 5 As shown, Figure 5The vertical axis “Relative expression” in the figure refers to the relative expression level. The results show that both TaDEP1 and TaDEP2 can significantly induce the expression of immune-related genes, that is, they can significantly activate the immune response in wheat leaves. At the same time, the data also show that TaDEP2 has a relatively stronger immune activation ability in wheat.

[0167] In summary, TaDEP2 and other members of its family have strong immune-activating activities, not only significantly inducing the activation of MAPK in wheat leaves, but also significantly inducing the expression of immune-related genes.

[0168] 2. Evolutionary analysis of TapreDEPs and verification of the immune activity of TaDEP2 in different species

[0169] Subsequently, we searched for homologous proteins of TapreDEPs in gramineous crops (rice, switchgrass, millet, and maize) and conducted evolutionary analysis. The results of interspecies evolutionary analysis of TapreDEPs and conservation analysis of the immunomodulatory activity of TaDPE2 are as follows: Figure 6 As shown. The evolutionary analysis results are as follows. Figure 6 As shown in Figure A, preDEPs exhibit high evolutionary affinity in wheat and several other common gramineous crops. Figure 6 In Chinese A, "Specie" means species, "Oryza sativa" means rice, "Panicum virgatun" means willow sorghum, "Setaria italica" means millet, "Sorghum bictor" means sorghum, "Triticum aestivum" means wheat, and "Zea mays" means corn.

[0170] Based on the aforementioned research results, TaDEP2 possesses relatively high immune-activating activity. Therefore, we focused our investigation on TaDEP2 and further validated its broad-spectrum immune activity in various wheat varieties, other gramineous crops (rice and maize), and Arabidopsis thaliana. MAPK activity was detected in leaves using the aforementioned methods. A schematic diagram of the MAPK activity detection results is shown below. Figure 6As shown in Figure B, "Wheat" refers to wheat, with the subsection representing wheat varieties; "Arabidopsis" refers to Arabidopsis, with the subsection representing Arabidopsis varieties; "Maize" refers to maize, with the subsection representing maize varieties; and "Rice" refers to rice, with the subsection representing maize varieties. TaDEP2 treatment significantly induced MAPK activation in the leaves of multiple wheat lines, as well as rice and maize, but did not induce MAPK activation in the leaves of the dicotyledonous plant Arabidopsis. Combined with... Figure 6 The phylogenetic tree analysis shown in A demonstrates that TaDEP2 possesses broad-spectrum immune-inducing properties in gramineous crops.

[0171] Two conserved cysteine ​​residues (C) on TaDEP2 were mutated, and the resulting peptide was synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. The amino acid sequence characteristics of the mutated TaDEP2 are as follows:

[0172] TaDEP2 CC / SS :PRGGGSAGAPNGSTNNPKHPPGGKSHG (SEQ ID NO: 37), that is, the cysteine ​​(C) at positions 13 and 25 are both mutated to serine (S).

[0173] TaDEP2 C25S :PRGGGSAGAPNGCTNNPKHPPGGKSHG (SEQ ID NO: 38), that is, the cysteine ​​(C) at position 25 is mutated to serine (S).

[0174] TaDEP2 was detected using the aforementioned method. C25S and TaDEP2 CC / SS The effects of mutant small peptides on MAPK activation levels and the effects of mutations at key TaDEP2 sites on their immune activity are shown in the following results. Figure 7 As shown. The results indicate that a single conserved cysteine ​​(C) mutation (TaDEP2) C25S The TaDEP2 mutation significantly reduced the activation level of MAPK by TaDEP2, while two conserved cysteine ​​(C) mutations (TaDEP2) significantly reduced the activation level of MAPK by TaDEP2. CC / SS This results in the near loss of TaDEP2's ability to activate MAPK. This demonstrates the necessity of the two conserved cysteine ​​residues (C) for TaDEP2's immune activity.

[0175] 4. Thermal stability analysis of TaDEP2

[0176] To further analyze the feasibility of using TaDEP2 as an immune inducer in field production, the thermal stability of the TaDEP2 immunomodulatory peptide was analyzed in detail. TaDEP2 was treated at different temperatures (25℃, 37℃, 42℃, and 50℃ for 12 h; 95℃ for 30 min). MAPK activity was then measured in leaves using the aforementioned treatment method. The results of the in vitro thermal stability analysis of the immunomodulatory peptide TaDEP2 are as follows: Figure 8 As shown, TaDEP2 maintained its biological activity well under different heat treatment conditions ranging from 25 to 50 °C, and its biological activity only slightly decreased under high-temperature treatment at 95 °C. These results indicate that TaDEP2 maintains stable immune-inducing activity under conditions ranging from room temperature to high temperatures.

[0177] Example 3: Application of TaDEP2 immunomodulatory small peptides in inducing immunity

[0178] Based on the activation of immune responses in wheat leaves by TaDEP2, this invention discloses the application potential and method of mature small peptide TaDEP2 as a plant immune inducer in the control of wheat diseases. The specific implementation plan is as follows:

[0179] 1. Preparation of TaDEP2 Inducer

[0180] Preparation method: The artificially synthesized powdered immunomodulatory peptide TaDEP2 is prepared into a 2 μM working solution using ddH2O as a solvent for injection treatment, or a 5 μM working solution (containing 0.02% Tween-20 or Silwet L-77) is prepared for spray treatment. The resulting working solution is the plant disease resistance inducer.

[0181] 2. Analysis of TaDEP2-induced wheat disease resistance

[0182] 2.1 Experiment on the control of bacterial leaf streak in wheat

[0183] Bacterial leaf streak of wheat is one of the major bacterial diseases of wheat, caused by *Xtu*, the pathogen that enters the host through natural openings or wounds. It primarily affects wheat leaves, but in severe cases can also damage leaf sheaths, stems, glumes, and grains. Using ddH2O as a blank control, the inducing agent was injected into the leaves of Fielder plants aged 7-10 days to systematically evaluate its immune activation-mediated disease resistance effect. The specific procedure is as follows:

[0184] (1) Plants in the treatment group were injected with TaDEP1-5 inducer working solution, while plants in the control group were injected with ddH2O; 24 h later, bacterial suspension was inoculated on the upper surface of the leaves.

[0185] (2) The pathogenic fungus strain Kn5 (also known as Xtu Kn5) of wheat bacterial leaf streak was cultured overnight at 28°C using NB (nutrient broth). The bacterial cells were collected and diluted with ddH2O to a concentration of 5×10⁻⁶ for Xtu Kn5. 5 CFU / mL, injected from the front of the leaf.

[0186] (3) Four days after inoculation, the severity of waterlogging on the leaves was recorded by taking photos and the biomass of pathogens in the leaves (CFU / cm²) was determined by the gradient dilution plate counting method.

[0187] The results of the Xtu Kn5 resistance assay in wheat leaves treated with TaDEPs are as follows: Figure 9 As shown, where, Figure 9 Image A shows the morphology of a wheat leaf. Figure 9 Figure B shows the pathogen biomass statistics. It is evident that TaDEPs treatment significantly inhibited the proliferation of Xtu Kn5 pathogen, with significantly lower pathogen biomass in the leaves of the treated group compared to the control group; the degree of waterlogging in the leaves was also weaker. This indicates that TaDEPs can effectively activate the immune response of wheat plants and enhance their resistance to wheat bacterial leaf streak.

[0188] 2.2 Experiment on the control of wheat scab

[0189] Fusarium head blight is one of the major fungal diseases in the wheat industry, mainly caused by Fusarium graminearum (Fg). It can occur from the seedling stage to the heading stage of wheat. The occurrence of this disease often causes symptoms such as seedling rot, stem base rot, stalk rot and ear rot, among which ear rot is the most damaging.

[0190] 2.2.1 Spraying the leaves

[0191] Using ddH2O as a blank control, the inducer was applied to the leaves of Fielder plants by spraying. The immune activation-mediated disease resistance effect was systematically evaluated. The specific operation is as follows:

[0192] (1) Cut the first true leaf, which is 3-4 weeks old, and place it in a tray lined with water-soaked filter paper. Spray with H2O or TaDEP2 working solution. After 24 h, inoculate with Fg PH-1 pathogen spore suspension.

[0193] (2) Inoculation with Fg PH-1 pathogenic spore suspension: Collect Fg PH-1 pathogenic spores and dilute to 5×10⁻⁶. 5 Spores / mL, take 10 μL and drop it onto the leaf surface (center position), then seal the tray again with plastic wrap; then incubate under normal photoperiod.

[0194] (3) Four days after inoculation, take photos to record the disease status of the leaves and count the area of ​​lesions.

[0195] The results of the TaDEP2 treatment of wheat leaves for Fg PH-1 resistance detection are as follows: Figure 10 As shown, where, Figure 10 Image A shows the morphology of a wheat leaf. Figure 10 Figure B shows the statistical graph of lesion area. It can be seen that the lesion area on the leaves treated with TaDEP2 was significantly smaller than that in the control group, indicating that TaDEPs can effectively activate the immune response of wheat plants and enhance their resistance to wheat scab.

[0196] 2.2.2 Spraying the ear of grain

[0197] Using ddH2O as a blank control, the inducer was applied to the panicle of Fielder plants by spraying. The immune activation-mediated disease resistance effect was systematically evaluated. The specific operation is as follows:

[0198] (1) Select wheat ears in the flowering stage, spray with H2O or TaDEP2 working solution, put No. 5 transparent self-sealing bag on the pretreated ears to keep them moist, and inoculate with Fg PH-1 pathogen after 24 h.

[0199] (2) Cultivation of Fusarium graminearum (Fg PH-1): Cut the culture medium that has been inoculated in advance into small square pieces of about 2 mm, transfer them to a new culture medium and let the mycelium grow on the surface for one day.

[0200] (3) When inoculating with Fg PH-1, select a small spikelet in the middle of the wheat ear, gently open the glumes, place the culture medium with mycelium inside the glumes, put a No. 5 transparent self-sealing bag over the inoculated spikelet to keep it moist, and remove the self-sealing bag after 48-72 h.

[0201] (4) Three weeks after inoculation, take photos to record the disease incidence in the wheat ears and calculate the severity of the disease using the following formula:

[0202] [Disease severity = number of diseased spikelets / total number of spikelets × 100%].

[0203] The results of the TaDEP2 treatment of wheat spikes for Fg PH-1 resistance detection are as follows: Figure 11 As shown, where, Figure 11 Image A shows the morphology of a wheat ear. Figure 11 Figure B shows the statistical chart of disease severity. It is evident that the TaDEP2-treated wheat ears showed significantly less disease severity and a lower proportion of diseased spikelets compared to the control group, indicating that TaDEPs can effectively activate the immune response of wheat plants and enhance the resistance of wheat ears to Fusarium head blight.

[0204] 2.3 Experiment on the control of wheat stem base rot

[0205] Wheat stem base rot is one of the major fungal diseases of wheat caused by Fusarium pseudograminearum (Fpg) and other fungi. It mainly infects the base of wheat stems and can cause damage throughout the entire growth period of wheat.

[0206] Using ddH2O as a blank control, the inducer was applied to the coleoptiles of Fielder plants by spraying, and the immune activation-mediated disease resistance effect was systematically evaluated. The specific operation is as follows:

[0207] (1) Disinfection: Soak wheat seeds in 25% 84 disinfectant solution for 10 min and rinse them with ddH2O.

[0208] (2) Germination: Soak seeds in 0.5% H2O2 at room temperature until they turn white; add a small amount of ddH2O and place at 4℃ for 1-2 days; transfer to room temperature and place until the seeds absorb the nutrients and grow roots but do not sprout (about 3 days old).

[0209] (3) Prepare petri dishes. Place 2-4 pieces of gauze in each petri dish, pour in an appropriate amount of ddH2O or 1 / 2 Hogland nutrient solution, and place a filter paper after the gauze is wetted. Place the seeds in the petri dishes in the same direction. No need for the dish lid. Place the dish in a container and cover it with plastic wrap to keep it moist. Cultivate in a light culture room until the coleoptile grows.

[0210] (4) Pretreatment: Spray H2O or TaDEP2 working solution onto the seedlings in (3) and let them stand for 2 h under moist conditions.

[0211] (5) Cut off 1-2 mm from the tip of the pretreated coleoptile in (4) to create an opening, and adjust the concentration to 1×10 6 Shake the spore solution containing 1 spore / mL well, add Tween (final concentration 0.02%), and apply 2 μL to the coleoptile wound.

[0212] (6) Add 1 / 2 Hogland nutrient solution (10 mL) to the petri dish and add 10 mL ddH2O around the petri dish to keep it moist.

[0213] (7) Cover with plastic wrap, place in a light incubation room, take photos to record and count the disease incidence after 5-7 days, and measure the length of the lesions.

[0214] The results of Fpg resistance detection analysis of wheat coleoptiles treated with TaDEP2 are as follows: Figure 12 As shown, where, Figure 12 Image A shows the morphology of a wheat coleoptile. Figure 12Figure B shows the statistical graph of wheat lesion length. It can be seen that the lesion length on the coleoptiles treated with TaDEP2 was significantly smaller than that in the control group, indicating that TaDEPs can effectively activate the immune response of wheat plants and enhance their resistance to wheat stem rot.

[0215] The above experiments demonstrate that exogenous application of TaDEP2 immune inducer can significantly enhance the broad-spectrum resistance of wheat to bacterial and fungal diseases by activating the plant's basic immune mechanism.

[0216] As can be seen from the above description, the embodiments of the present invention achieve the following technical effects: This application successfully identified multiple TaDEPs peptides, which possess immune-activating activity and enhance wheat's resistance to various diseases. Furthermore, in the above experimental verification, TaDEP2, a member of the TaDEPs family, exhibited good thermal stability, maintaining its biological activity even under high-temperature treatment conditions. This provides a guarantee for the field application of these peptides under different climatic conditions. This application also determined the core immune-active sequence of TaDEPs and analyzed mutations at key sites, demonstrating not only the correlation between their structure and function but also providing a theoretical basis for the subsequent optimization design of peptides. This application also applied the plant immune inducers prepared from TaDEPs to wheat disease control via spraying or injection. These peptides showed effectiveness in preventing wheat bacterial leaf streak, Fusarium head blight, and stem rot, indicating that the peptides of this application can play an important role in actual agricultural production as a novel, green immune inducer, promoting healthy crop growth, reducing dependence on chemical pesticides, and having a positive significance for promoting sustainable agricultural development.

[0217] 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 polypeptide, characterized in that, The amino acid at position 1 of the polypeptide is proline, and the amino acids at positions 13 and 25 are both cysteine. The polypeptide has 27 amino acid residues; The amino acid sequence of the polypeptide is the amino acid sequence shown in SEQ ID NO:

24.

2. The use of the polypeptide or amino acid sequence of claim 1, which is any one of SEQ ID NOs: 20-23, in the preparation of a reagent for improving plant disease resistance; The plant in question is wheat; The disease resistance refers to the ability to resist bacterial and fungal diseases.

3. The use of the polypeptide of claim 1, or the polypeptide with the amino acid sequence shown in any of SEQ ID NOs: 20-23, in the preparation of a reagent for enhancing plant immunity; The plant is one or more of wheat, rice, or corn.

4. A plant immune inducer, characterized in that, The plant immune inducer includes the polypeptide as described in claim 1.

5. A method for improving plant disease resistance, characterized in that, The method includes applying the polypeptide of claim 1, or the polypeptide with the amino acid sequence shown in any of SEQ ID NOs: 20-23, or the plant immune inducer of claim 4 to the target plant; The target plant is wheat.

6. The method according to claim 5, characterized in that, The application method includes one or more of the following: spraying, smearing, soaking, leaf cutting, or injection.

7. The application of the polypeptide of claim 1, or the polypeptide with the amino acid sequence shown in any of SEQ ID NOs: 20-23, or the plant immune inducer of claim 4 in improving plant disease resistance; The plant in question is wheat; The disease resistance includes the ability to resist bacterial and fungal diseases.

8. The application of the polypeptide of claim 1, or the polypeptide with the amino acid sequence shown in any of SEQ ID NOs: 20-23, or the plant immune inducer of claim 4 in improving plant immunity; The plant is one or more of wheat, rice, or corn.

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