Polypeptide, plant immune resistance inducer and method for improving disease resistance of plants

Abstract

The invention provides a polypeptide, a plant immune resistance inducer and a method for improving the disease resistance of plants. The first amino acid of the polypeptide is proline, and the 13th amino acid and the 25th amino acid of the polypeptide are cysteine; the polypeptide has 27 amino acid residues. The plant immune resistance inducer can solve the problems of scarce types, narrow disease-resistant spectrum and poor stability of peptide resistance inducers 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 a method for improving plant disease resistance. Background Art

[0002] Grass crops are not only a major source of carbohydrates in the human diet, but also provide essential dietary fiber, vitamins, and minerals. Three grass crops—wheat, rice, and corn—support the food needs of a large portion of the world's population, profoundly impacting food security and economic stability. Given their widespread cultivation areas and significant economic value, the healthy growth and disease management of grass crops are crucial for ensuring global food supply and promoting sustainable agricultural development. Therefore, exploring effective prevention and control strategies for grass plant diseases has become a key topic in agricultural research.

[0003] Wheat, a widely cultivated and important cereal crop in the grass family, contributes significantly to the protein and energy supply of the human diet. Despite its irreplaceable economic and nutritional value, wheat often faces disease challenges during cultivation, particularly bacterial leaf streak, fusarium head blight, and stem rot. The frequent occurrence of these diseases not only directly weakens wheat growth, 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) notes that pathogens cause enormous agricultural losses each year, highlighting the urgency of disease prevention and control. While traditional chemical pesticides can effectively suppress diseases in the short term, long-term reliance on them carries significant negative consequences: The accumulation of resistance in pathogens weakens the long-term effectiveness of pesticides; pesticide residues can contaminate soil, introduce heavy metals, disrupt ecosystem balance, and lead to a decline in biodiversity; and, more seriously, residual chemicals pose a potential risk to human health.

[0005] In view of the limitations of chemical pesticides, it is particularly urgent to explore environmentally friendly disease control strategies. Plant immune inducers, because they can stimulate the natural defense mechanism of plants, are regarded as a kind of "plant vaccine" and have the potential to be transformed into a new generation of green pesticides for use against crop diseases. Research in this field has identified many 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 a high activation concentration and a short duration of effectiveness; proteins such as elicitor proteins face complex production processes and poor environmental stability; small molecule compounds such as purine bases have a narrow disease resistance spectrum and may induce abnormal plant metabolism. Relatively speaking, peptide inducers have a small molecular weight and strong activity (even at 10 -9M concentration) and its ease of industrial production stand out.

[0006] However, the development of peptide immune inducers still requires breakthroughs. Currently, the types of active peptides are relatively limited, with only a few mentioned in relevant patents, such as AtPep1, SlPep, and CEP14. Furthermore, peptide inducers often target specific receptors, such as the PEPR2 receptor, whose specificity limits the breadth of their disease resistance spectrum. 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 purpose of the present invention is to provide a polypeptide, a plant immune inducer and a method for improving plant disease resistance, so as 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 object, according to the 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 an amino acid sequence as shown in SEQ ID NO: 1.

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

[0011] In order to achieve the above object, according to the second aspect of the present invention, a plant immune inducer is provided, which comprises 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 an amino acid sequence as shown in SEQ ID NO: 1.

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

[0014] 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.

[0015] Furthermore, the application method includes one or more of spraying, smearing, soaking, leaf cutting or injection.

[0016] Furthermore, the grass plant includes one or more of wheat, rice, corn, switchgrass or millet.

[0017] In order to achieve the above object, 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 disease resistance.

[0018] 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.

[0019] By applying the technical solution of the present invention, the present application provides a polypeptide with a length of 27 amino acid residues, in which the amino acid at the 1st position is proline, the amino acids at the 13th and 25th positions are both cysteine, and the polypeptide has such sequence characteristics. The protein of the polypeptide has immune activity, enriches the types of immune active peptides, overcomes the problem of narrow disease resistance caused by excessive specificity of the target receptor; and has broad spectrum. When used as a plant inducer, it has high thermal stability, is not easy to degrade, and can be applied to a variety of plants, which significantly improves the practicality and durability of plant immune inducers, and opens up a new path for the green prevention and control of agricultural diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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:

[0021] Figure 1 The calorimetric expression of 18 TaDEPs small peptide encoding genes under the treatment of PAMPs and pathogens in Example 1 of the present application is shown.

[0022] Figure 2 The following is a schematic diagram of the evolutionary analysis of TapreDEPs in Example 1 of the present application and a sequence characteristic result diagram of the conserved domains related to immune activity; wherein, Figure 2 Figure A is a schematic diagram of the evolutionary analysis of the 18 TapreDEPs mentioned above. Figure 2 Figure B is a schematic diagram of the sequence analysis of the conserved segments of 18 TapreDEPs.

[0023] Figure 3 The figure shows the result of the prediction analysis of the secondary structure of the TapreDEP2 precursor protein in Example 1 of the present application.

[0024] Figure 4 The figure shows the results of the MAPK activation ability analysis of TaDEPs in Example 2 of the present application.

[0025] Figure 5 The qRT-PCR results of Example 2 of the present application specification showing that the TaDEPs secreted peptide significantly induced the expression of immune-related genes; wherein, Figure 5 A in the middle is the qRT-PCR result of TaDEPs secreted peptide inducing TaWRKY23 expression. Figure 5 Middle B is the qRT-PCR result of TaDEPs secreted peptide inducing TaWRKY28 expression. Figure 5 Middle C is the qRT-PCR result of TaDEPs secreted peptide inducing TaWRKY53 expression. Figure 5 Figure D shows the qRT-PCR results of TaDEPs secreted peptides inducing TaPUB23 expression. Figure 5 Figure E in the middle is the qRT-PCR result of TaDEPs secreted peptide inducing TaPR1 expression. Figure 5 Figure F shows the qRT-PCR results of TaDEPs secreted peptides inducing TaPR5 expression.

[0026] Figure 6 The results of the interspecies evolution analysis of TapreDEPs and the conservation analysis of the immune activity of TaDEP2 in Example 2 of the present application are shown; wherein, Figure 6 Figure A in the middle is the result of interspecies evolutionary analysis of TapreDEPs. Figure 6 Middle B is the result of immunoreactivity conservation analysis.

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

[0028] Figure 8 The figure shows the results of in vitro thermal stability analysis of the immunologically active small peptide TaDEP2 in Example 2 of the present application.

[0029] Figure 9 The figure shows the results of the resistance analysis of wheat leaves treated with TaDEPs in Example 3 of the present application to the pathogen Xtu Kn5 of wheat bacterial leaf streak; wherein, Figure 9 A in the middle is the water-soaked phenotype of diseased wheat leaves. Figure 9 Middle B is the statistical chart of pathogen biomass.

[0030] Figure 10 The figure shows the results of the resistance analysis of wheat leaves treated with TaDEP2 in Example 3 of the present application to the pathogen FgPH-1 of wheat fusarium scaber; wherein, Figure 10 A in the middle is a morphological picture of wheat leaves infected by wheat fusarium scaber pathogen. Figure 10 Middle B is a statistical chart of the area of ​​wheat leaf spots.

[0031] Figure 11 The figure shows the results of the resistance analysis of wheat head blight pathogen FgPH-1 in wheat ears treated with TaDEP2 in Example 3 of the present application specification; wherein, Figure 11 A in the middle is a morphological picture of wheat ears infected by wheat fusarium scaber pathogen. Figure 11 Middle B is a statistical chart of the severity of disease in wheat ears.

[0032] Figure 12 The figure shows the results of the analysis of the resistance of wheat stem rot pathogen Fpg to wheat stems treated with TaDEP2 in Example 3 of the present application; wherein, Figure 12 A in the middle is a morphological picture of wheat coleoptiles infected by wheat stem rot pathogen. Figure 12 Middle B is a statistical chart of the lesion length of wheat coleoptile. DETAILED DESCRIPTION

[0033] 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.

[0034] Explanation of terms:

[0035] Wheat bacterial leaf streak: a bacterial disease of wheat caused by Xanthomonas translucenspv. undulosa (Xtu) (also known as wheat bacterial leaf streak).

[0036] Wheat fusarium wilt: It is one of the most common fungal diseases in wheat production and cultivation. The disease is caused by infection by various Fusarium species, among which Fusarium graminearum (Fg) is the main pathogen.

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

[0038] 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.

[0039] As mentioned in the background art, the peptide elicitors in the prior art are scarce, have a narrow disease resistance spectrum and poor stability. Based on this, in this application, the inventors have discovered a new immune polypeptide that can be used in the development of plant peptide elicitors, and thus proposed a series of protection schemes of this application.

[0040] In a first typical embodiment of the present application, a polypeptide is provided, wherein 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.

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

[0042] Wherein, "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 without affecting the overall function or properties of the sequence. "X" includes, but is not limited to, alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine ​​(Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), 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 the present application is the same as described herein.

[0043] Plant secretory peptides are a class of small polypeptides (typically 2-100 amino acids) synthesized by plant cells and released into the extracellular space through the secretory pathway. They play important roles in signal transduction and regulation in plant growth and development, stress resistance, and immune responses. These secretory peptides are typically 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.

[0044] The mechanism of action of plant-secreted peptides primarily relies on their interaction with receptor kinases, encompassing signal recognition, signal transduction, and the production of physiological effects. Specifically, signal recognition refers to the binding of secreted peptides to receptor kinases on the cell membrane, such as the binding of PSK to PSKR. Signal transduction involves the activation of receptor kinases, which transmit signals through downstream signaling pathways (such as the MAPK cascade) to regulate gene expression and cellular responses. The resulting physiological effects ultimately manifest as growth promotion, enhanced stress resistance, or immune response activation.

[0045] This application discovered a polypeptide (i.e., the aforementioned plant-secreted small peptide) with a sequence length of 27 amino acid residues, with the first amino acid being proline (P) and the 13th and 25th amino acids being cysteine ​​(C). This application analyzed the peptide TaDEP2 containing the aforementioned sequence features and performed site mutation analysis on two conserved cysteines (C) within its conserved active region. The results showed that the two discontinuous conserved cysteines (C) in its conserved "CXC" region are important for maintaining TaDEP2's immune activity, revealing the structural and functional basis of this type of peptide and laying a theoretical foundation for the subsequent research and application of peptide immune activity.

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

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

[0048] SEQ ID NO: 1: PXGXXGAGAXNGCTNNPXHPXGGKCXG.

[0049] Furthermore, the polypeptide also includes a polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. In the amino acid sequence of SEQ ID NO: 1, the amino acid at position 1, the amino acid at position 13, and the amino acid at position 25 are consistent with the amino acid sequence of the plant secretory peptide. In addition, the conserved amino acids at other positions 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 meanings of "X" at amino acids at positions 2, 4, 5, 10, 18, 21 and 26 of SEQ ID NO: 1 are the same as those described above; more preferably, in the amino acid sequence shown in SEQ ID NO: 1, the amino acid at position "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 one 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 an amino acid sequence as set forth in any one of SEQ ID NOs: 20-24. These polypeptides are capable of inducing the expression of wheat genes TaWRKY23, TaWRKY28, TaWRKY53, TaPUB23, TaPR1, and TaPR5, all of which have been shown to play important positive regulatory roles in the early or late stages of wheat immune responses. The increased expression levels of these wheat positive immune regulatory genes indicated that these polypeptides also play a positive role in wheat immune responses.

[0058] This application employed a reverse genetics strategy to screen and identify immunologically active TapreDEPs from a library of endogenous secretory peptides from wheat. First, by analyzing dynamic transcriptome data from wheat leaves treated with pathogen-associated pattern molecules (PAMPs), we identified genes encoding secreted small peptides that were significantly induced by PAMPs. Subsequently, based on amino acid sequence conservation and functional predictions, TaDEPs containing immunologically active regions were synthesized to validate their immune-activating abilities. For example, the precursor protein, TapreDEP2 (the full-length amino acid sequence of the precursor protein is shown in SEQ ID NO:19), possesses 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 secretory pathway, protease treatment removes the front signal peptide and variable region, releasing the mature, active TaDEP2 small peptide (amino acid sequence shown in SEQ ID NO:22), which is effectively transported to the exoplasm to exert its immune-regulating effects.

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

[0060] MARAVVMAVLLMQCCNVVLAARLLEGDGGWLRGGVGAAGALIMQVLPRGGGSAGAPNGCTNNPKHPPGGKCHG.

[0061] 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, 34°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°C, ℃, 3°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C, 70°C, 71°C, 72°C, 73°C, 74°C, 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, 81°C, 82°C, 83°C, 84°C, 85°C, 86°C, 87°C, 88°C, 89°C, 90°C, 91°C, 92°C, 93°C, 94°C, 95°C, 96°C, 97°C, 98°C, 99°C or 100°C).

[0062] This application also conducted a thermal stability experiment on the above-mentioned polypeptide. The immune activity of the above-mentioned polypeptide can remain stable when treated in the above-mentioned temperature range, which provides a basis for its application in complex and changeable field environments and improves its stability and biological activity in practical applications.

[0063] Furthermore, the present application found that TaDEP1-TaDEP5 can significantly induce the immune response of wheat through the detection of MAPK phosphorylation levels, and TaDEP2 in particular showed extremely high immune activation efficiency. In addition, the results of real-time fluorescence quantitative PCR showed that TaDEP1 and TaDEP2 can effectively promote the changes in the expression levels of immune-related genes (TaWRKY23, TaWRKY28, TaWRKY53, TaPUB23, TaPR1 and TaPR5), proving that the above-mentioned polypeptides can induce the immune response of wheat and strengthen the immune response mechanism of wheat, thereby enhancing the crop's resistance to disease.

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

[0065] In a second typical embodiment of the present 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 elicitors are substances that can induce immune 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. They include proteins, oligosaccharides, lipids, small molecule metabolites, and microbial agents.

[0067] The plant immune inducer prepared using the above-mentioned polypeptide has been confirmed through field trials in this application to be able to effectively enhance the resistance of wheat to bacterial leaf streak disease, fungal ergot disease and stem base rot, thereby reducing the use of chemical pesticides, alleviating environmental pollution, and ensuring food safety and quality.

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

[0069] In a preferred embodiment, the polypeptide has an amino acid sequence as shown in any one 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 comprises a surfactant; preferably, the surfactant comprises 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 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.

[0073] 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 immunity inducer to a target plant; the target plant comprises a grass plant.

[0074] It should be noted that this improvement in plant disease resistance is a broad-spectrum disease resistance. By activating the response of the plant MAPK signaling pathway and inducing changes in the expression of genes in the immune signaling pathway, the plant's immune ability is improved, thereby achieving an improvement in resistance to a variety of diseases, including but not limited to the ability to resist bacterial diseases and fungal diseases.

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

[0076] 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 finds 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, induce the expression of genes on the immune signaling pathway to change, further improve the immune capacity 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.

[0077] In a preferred embodiment, the grass plant includes one or more of wheat, rice, corn, switchgrass or millet.

[0078] 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.

[0079] It should be noted that this improvement in plant disease resistance is broad-spectrum, enhancing plant immunity by activating the MAPK signaling pathway and inducing changes in the expression of genes in the immune signaling pathway, thereby improving plant 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 wheat bacterial leaf streak; preferably, the fungal diseases include but are not limited to wheat head blight or wheat stem rot.

[0080] In this application, when the above-mentioned polypeptides and similar peptides are used to prepare plant immune inducers, the prepared plant immune inducers have broad spectrum and compatibility, and the immunologically active TaDEPs can significantly induce immune responses in grass crops, showing potential application value in preventing grass crop diseases. When the above-mentioned plant immune inducers are sprayed exogenously on grass crops, they can enhance the disease resistance of grass crops, prevent the occurrence of crop diseases in agricultural production, and protect crops from pesticide contamination.

[0081] In a fifth typical embodiment of the present application, there is provided a use of the above-mentioned polypeptide in the preparation of an agent for improving plant immunity and / or disease resistance.

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

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

[0084] Example 1 Screening and identification of TaDEP2 immunoreactive peptides

[0085] 1. Transcriptome Data Acquisition and Analysis

[0086] The RNA-seq dataset of genes encoding potential immunoreactive peptides (TaDEPs) in the DEP (Delta-like phytocytokines) family of small peptides in wheat leaves treated with PAMPs was obtained from the Wheat Omic database (http: / / wheatomics.sdau.edu.cn / ), that is, the genes encoding TaDEPs induced by PAMPs were obtained.

[0087] Through BLAST search analysis of homologous genes, 18 members of this family (TapreDEPs) were obtained in the wheat genome. Based on the transcriptome data published by the wheat genomics database, the expression patterns of these 18 TapreDEPs were analyzed by bioinformatics methods. The expression calorimetry of the 18 TaDEPs small peptide encoding genes under AMPs and pathogen treatment is shown in the figure below. Figure 1 The results showed 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 immunoreactive peptides

[0089] 2.1 Screening of TaDEPs immunoactive peptides and prediction of active segments

[0090] Through bioinformatics analysis, evolutionary analysis and amino acid sequence conservation analysis of 18 TapreDEPs (SEQ ID NO: 2-19) were performed. It was found that their conserved segments were highly conserved and contained two conserved and discontinuous cysteines (C). It was predicted that the characteristics of this site were necessary for the conserved segment to exert immune activity. The evolutionary analysis of wheat immune-active small peptide secretion peptide encoding genes TapreDEPs was related to immune activity. The sequence characteristics of the conserved domain were shown in the figure. Figure 2 As shown, Figure 2 Figure 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 segments of the above 18 TapreDEPs, where "Signal peptide" means signal peptide, "Variable region" means variable region, and "Conserved region" means conserved region.

[0091] The underlined amino acid sequence below indicates the conserved C-terminal region.

[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 Prediction and analysis of the secondary structure of TapreDEP2 precursor protein

[0129] The secondary structure of TapreDEP2 precursor protein was predicted and analyzed using AlphaFold 2 software. Figure 3 The results showed that the C-terminal conserved segment of TapreDEP2 (i.e., TaDEP2) forms a ring structure through two conserved cysteines (C), further suggesting the importance of the two conserved cysteines (C) for the function of TaDEP2.

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

[0131] Combining expression pattern analysis and conserved region sequence analysis, five members (TapreDEP1-5) from the 18 TapreDEPs that were significantly induced by PAMPs and pathogens at relatively high levels were selected to validate the immunological activity of the proteins encoded by these genes. The conserved regions that exerted immunological activity were predicted and named TaDEP1-TaDEP5, respectively. These peptides were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd., with the amino acid sequences of the five peptides being SEQ ID NOs: 20-24, respectively.

[0132] Example 2 Analysis of the immunological activity and evolutionary conservation of TaDEPs peptides

[0133] 1. Validation of the Immunoreactivity of TaDEPs

[0134] (1) Analysis of MAPK phosphorylation levels

[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 responses to environmental stress. In particular, plant immunity is regulated through phosphorylation and its transmission. Therefore, MAPK phosphorylation levels can be used as an indicator of plant cellular immune activation.

[0136] Experimental Design: TaDEP1-5 and the immune-activating peptide positive control flg22 were synthesized by Shanghai Qiangyao Biotechnology Co., Ltd. and prepared in ddH2O as a solvent to a working concentration of 1 μM. Sterile water was used as a blank control.

[0137] flg22 (Flagellin 22) is a highly conserved 22-amino acid peptide at the N-terminus of bacterial flagellin, which can act as a pathogen-associated molecular pattern (PAMP) to activate plant immune responses.

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

[0139] 7-10 day-old wheat leaves (Fielder) were cut into 1 cm radius segments and placed in a cell culture dish containing 1 mL of ddH2O. After adding 0.02% Triton-X100, the leaves were vacuum infiltrated. The leaves were then rinsed twice with ddH2O and incubated overnight at room temperature in 1 mL of ddH2O. The leaf segments were then treated with TaDEP1-5 peptides using the working solution. Sterile water served as a blank control, and flg22 was treated simultaneously as a positive control. Samples were taken 15 minutes after treatment and quickly frozen in liquid nitrogen until ready for use. Total protein was extracted from the samples and used to assay the activation of MAPKs by the peptides.

[0140] The steps for extracting total protein are as follows: place the dried leaf segments in a 2 mL centrifuge tube, freeze them in liquid nitrogen, and then homogenize them. Add 100 μL of plant protein extract (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 mix, and let it stand in an ice bath for 10 minutes to help the plant cells fully lyse; centrifuge at 12000 rpm and 4°C for 5-10 minutes. Transfer 80 μL of the supernatant to a 1.5 mL centrifuge tube, add 20 μL of 5× SDS loading buffer, mix thoroughly, and boil in a 95°C metal bath for 5 min. Add the denatured protein to the wells of a 10% SDS-PAGE gel and run electrophoresis at 90 V for approximately 20 min until the sample is completely located at the boundary between the stacking and separating gels. Continue electrophoresis at 140 V for approximately 50 min (until the protein marker is completely separated). Transfer the membrane to a membrane. After transfer, block the PVDF membrane in 5% BSA for 1 h at room temperature and incubate with anti-pERK1 / 2 antibody overnight at 4°C. Wash the membrane three times with 1× TBST (10 min each time) at room temperature, incubate with rabbit secondary antibody for 1 h at room temperature, and wash again three times with 1× TBST. Subsequently, the PVDF membrane was developed using ECL chemiluminescent substrate, and images were taken using a Tanon 5200 series fully automatic chemiluminescence image analysis system to evaluate the immune-inducing activity of TaDEPs.

[0141] The results of the MAPK activation ability analysis of TaDEPs are as follows Figure 4As shown in the figure, wheat TaDEP1-5 peptides all significantly activated the phosphorylation of MAPK in leaves, and the activation intensity was roughly consistent with flg22, that is, TaDEPs can significantly activate the wheat immune response.

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

[0143] Many genes play important roles in the regulation of plant immune responses, such as TaWRKY23, TaWRKY28, TaWRKY53, TaPUB23, TaPR1, and TaPR5. To further examine the relationship between TaDEPs and wheat immune responses, we examined the expression levels of these immune-related genes.

[0144] According to the above research results, TaDEP1-5 have roughly the same activation levels of MAPK in wheat leaves, among which TaDEP1 and TaDEP2 show the relatively 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 immune regulatory functions.

[0145] Total RNA was extracted from leaf segments treated with TaDEP1 and TaDEP2 using the Trizol method and reverse transcribed using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Novozymes, Nanjing, China) according to the manufacturer's instructions.

[0146] Real-time fluorescence quantitative PCR (qRT-PCR) was performed using the QuantStudio™ 3 Real-time Fluorescence Quantitative PCR System. Each reaction system (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: 95°C for 30 seconds, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C. TaActin was used as an internal control, and qRT-PCR analysis was performed with three biological replicates. -∆∆Ct The relevant gene expression data were analyzed.

[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] The qRT-PCR data of TaDEPs secreted peptides significantly induced the expression of immune-related genes. Figure 5 As shown, Figure 5The Chinese interpretation of the vertical axis "Relative expression" is relative expression level. The results show that 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 for wheat.

[0167] In summary, TaDEP2 and other members of its family have strong immune activation activity, 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 corn) and conducted evolutionary analysis. The results of interspecies evolutionary analysis of TapreDEPs and conservation analysis of the immune activity of TaDPE2 are as follows: Figure 6 The results of evolutionary analysis are shown in Figure 6 As shown in Figure A, preDEPs have a high degree of evolutionary affinity in wheat and several other common grass crops. Figure 6 In Chinese A, the Chinese meaning of “Specie” is species, the Chinese meaning of “Oryza sativa” is rice, the Chinese meaning of “Panicum virgatun” is switchgrass, the Chinese meaning of “Setaria italica” is millet, the Chinese meaning of “Sorghum bictor” is sorghum, the Chinese meaning of “Triticum aestivum” is wheat, and the Chinese meaning of “Zea mays” is corn.

[0170] Based on the above research results, TaDEP2 has relatively high immune activation activity. Therefore, we focused on TaDEP2 and further verified the broad spectrum of TaDEP2 immune activity in various wheat lines, other grass crops (rice and corn), and Arabidopsis. The leaves were treated using the above method and MAPK activity was detected. The MAPK activity detection results are shown in the figure below. Figure 6As shown in B, the Chinese meaning of "Wheat" is wheat, and the part it covers is the name of the wheat variety; the Chinese meaning of "Arabidopsis" is Arabidopsis, and the part it covers is the name of the Arabidopsis variety; the Chinese meaning of "Maize" is corn, and the part it covers is the name of the corn variety; the Chinese meaning of "Rice" is rice, and the part it covers is the name of the corn variety; TaDEP2 treatment can significantly induce the activation of MAPK in multiple wheat lines and rice and corn leaves, but cannot cause the activation of MAPK in the leaves of the dicotyledonous plant Arabidopsis. Combined Figure 6 The phylogenetic tree analysis shown in A proves that TaDEP2 has broad-spectrum immune-inducing properties in grass crops.

[0171] The two conserved cysteines (C) on TaDEP2 were mutated and the 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), i.e., the cysteine ​​(C) at position 25 is mutated to serine (S).

[0174] TaDEP2 was detected according to the aforementioned method C25S and TaDEP2 CC / SS The effects of mutant peptides on MAPK activation levels and the effects of TaDEP2 key site mutations on its immune activity are shown in the following table. Figure 7 The results showed that a single conserved cysteine ​​(C) mutation (TaDEP2 C25S ) significantly weakened the activation level of TaDEP2 on MAPK, while the mutation of two conserved cysteine ​​(C) (TaDEP2 CC / SS ) almost abolished the ability of TaDEP2 to activate MAPK, indicating that the two conserved cysteines (C) are essential for the immunoreactivity of TaDEP2.

[0175] 4. TaDEP2 Thermal Stability Analysis

[0176] To further analyze the feasibility of using TaDEP2 as an inducer in field production, the thermal stability of TaDEP2 immunoreactive peptide was analyzed in detail. TaDEP2 was treated at different temperatures (25℃, 37℃, 42℃ and 50℃ for 12 h; 95℃ for 30 min). The leaves were treated using the above method and MAPK activity was detected. The results of the in vitro thermal stability analysis of the immunoreactive peptide TaDEP2 are shown in Figure 2. Figure 8 As shown in the results, TaDEP2 can maintain its biological activity well under different heat treatment conditions of 25-50°C. The biological activity of TaDEP2 is only slightly weakened under high temperature treatment of 95°C. This result shows that TaDEP2 can maintain stable immune-inducing activity under conditions from room temperature to high temperature.

[0177] Example 3: Application of TaDEP2 immunoreactive peptide for immune induction

[0178] Based on the fact that TaDEP2 activates immune responses in wheat leaves, the present invention discloses the potential and methods of using the mature small peptide TaDEP2 as a plant immune inducer in wheat disease prevention and control. The specific implementation plan is as follows:

[0179] 1. Preparation of TaDEP2 Inducer

[0180] Preparation plan: The artificially synthesized powdered immunoactive 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) for spraying treatment. The prepared working solution is the plant disease resistance inducer.

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

[0182] 2.1 Experiment on prevention and control of wheat bacterial leaf streak

[0183] Wheat bacterial leaf streak is a major bacterial disease of wheat, caused by X. tritici (Xtu). The pathogen invades through natural orifices or wounds in the host, primarily damaging wheat leaves. In severe cases, it can also damage sheaths, stems, glumes, and grains. Using ddH2O as a blank control, the inducer was injected into the leaves of 7-10-day-old Fielder plants to systematically evaluate its immune activation-mediated disease resistance. The specific procedures are as follows:

[0184] (1) The plants in the treatment group were injected with the working solution of TaDEP1-5 inducer, and the plants in the control group were injected with ddH2O. After 24 h, the bacterial suspension was inoculated on the front of the leaves.

[0185] (2) Cultivate the pathogen Kn5 (also known as Xtu Kn5) of Pseudomonas aeruginosa strain in NB (nutrient broth) at 28°C overnight. Collect the bacteria and dilute Xtu Kn5 with ddH2O to a concentration of 5×10 5 CFU / mL, injected from the adaxial side of the leaf.

[0186] (3) Four days after inoculation, photos were taken to record the severity of leaf waterlogging, and the pathogen biomass (CFU / cm²) in the leaves was determined by the gradient dilution plate count method.

[0187] The results of Xtu Kn5 resistance analysis in wheat leaves treated with TaDEPs are shown in Figure 2. Figure 9 As shown, Figure 9 A in the middle is the wheat leaf morphology diagram, Figure 9 Figure B shows the pathogen biomass. As shown, TaDEPs treatment significantly inhibited the proliferation of the Xtu Kn5 pathogen, with the pathogen biomass in the treated leaves significantly lower than in the control. Waterlogging was also less pronounced, indicating that TaDEPs can effectively activate the immune response of wheat plants and enhance their resistance to bacterial leaf streak.

[0188] 2.2 Wheat scab prevention and control experiment

[0189] Wheat 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 ear 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 harmful.

[0190] 2.2.1 Spray blades

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

[0192] (1) Cut the first true leaf of a 3-4 week-old plant and place it on a tray covered with water-soaked filter paper. Spray it with H2O or TaDEP2 working solution. After 24 hours, inoculate it with a spore suspension of the pathogen Fg PH-1.

[0193] (2) Inoculation of Fg PH-1 pathogen spore suspension: Collect Fg PH-1 pathogen spores and dilute to 5×10 5 spores / mL, 10 μL was dropped on the leaf surface (middle position), and the tray was sealed with plastic wrap again; then cultured under normal photoperiod.

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

[0195] The results of the Fg PH-1 resistance test in wheat leaves treated with TaDEP2 are shown in Figure 2. Figure 10 As shown, Figure 10 A in the middle is a picture of wheat leaf morphology. Figure 10 Figure B shows the statistical graph of lesion area. It can be seen that the lesion area on leaves treated with TaDEP2 is 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 head blight.

[0196] 2.2.2 Spraying the ears

[0197] Using ddH2O as a blank control, the inducer was applied to the panicles of Fielder plants by spraying to systematically evaluate the disease resistance mediated by immune activation. The specific procedures were as follows:

[0198] (1) Select wheat ears at the flowering stage, spray them with H2O or TaDEP2 working solution, put a No. 5 transparent ziplock bag on the pretreated ears to moisturize them, and inoculate Fg PH-1 pathogen 24 hours later.

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

[0200] (3) When inoculating Fg PH-1, select a spikelet in the middle of the wheat ear, slightly open the husk, place the culture medium with mycelium inside the husk, and put a No. 5 transparent ziplock bag on the inoculated ear to keep it moist. Remove the ziplock bag after 48-72 hours.

[0201] (4) Three weeks after inoculation, take photos to record the disease status of wheat ears and calculate the severity of the disease. The calculation formula is as follows:

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

[0203] The results of the Fg PH-1 resistance test analysis of wheat ears treated with TaDEP2 are shown in Figure 2. Figure 11 As shown, Figure 11 A in the middle is the morphological diagram of wheat ears. Figure 11 Figure B shows the disease severity statistics. As can be seen, the severity of disease in wheat spikelets treated with TaDEP2 was significantly less severe than in the control group, and the proportion of diseased spikelets was lower, indicating that TaDEPs can effectively activate the immune response of wheat plants and enhance resistance to wheat head blight.

[0204] 2.3 Wheat stem rot prevention and control experiment

[0205] Wheat stem rot is one of the main fungal diseases of wheat caused by Fusarium pseudograminearum (Fpg) and other fungi. It mainly infects the base of the wheat stem and may cause harm 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 to systematically evaluate its immune activation-mediated disease resistance. The specific procedures are as follows:

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

[0208] (2) Germination: Soak the seeds in 0.5% H2O2 at room temperature until they turn white; add a small amount of ddH2O and place them at 4°C for 1-2 days; transfer to room temperature and place them until the seeds absorb water and take root but do not sprout (approximately 3 days old).

[0209] (3) Prepare culture dishes. Place 2-4 pieces of gauze in each dish. Pour in an appropriate amount of ddH2O or 1 / 2 Hoagland nutrient solution. Place a piece of filter paper after soaking the gauze. Place the seeds in the culture dish in the same direction without a lid. Place them in a container and cover with plastic wrap to keep them moist. Culture in a light-lit culture room until the coleoptile grows.

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

[0211] (5) Cut off 1-2 mm from the top of the coleoptile pretreated in (4) to create a wound and adjust the concentration to 1×10 6 The spore solution with spores / mL was shaken evenly, Tween (final concentration 0.02%) was added, and 2 μL was aspirated and applied to the wound of the coleoptile.

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

[0213] (7) Cover with plastic wrap and place in a light culture room for cultivation. After 5-7 days, take photos and record the incidence of the disease, and measure the length of the lesions.

[0214] The results of the Fpg resistance analysis of wheat coleoptiles treated with TaDEP2 are shown in Figure 2. Figure 12 As shown, Figure 12 A in the middle is the morphological diagram of wheat coleoptile. Figure 12Figure B shows the statistical graph of wheat lesion length. It can be seen that the lesions on the coleoptiles treated with TaDEP2 are significantly smaller than those 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 prove that exogenous application of TaDEP2 immune inducer can significantly enhance wheat's broad-spectrum resistance to bacterial and fungal diseases by activating the plant's basic immune mechanism.

[0216] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present application successfully identified multiple TaDEPs small peptides, which have immune activation activity and enhance wheat resistance to various diseases. In addition, in the above experimental verification, TaDEPs family member TaDEP2 showed good thermal stability and could still maintain its biological activity under high temperature treatment conditions, which provides a guarantee for the field application of these small peptides under different climatic conditions. The present application also determined the core immune active sequence of TaDEPs and performed mutation analysis of key sites, which not only demonstrated the relationship between its structure and function, but also provided a theoretical basis for the subsequent optimization design of peptides. The present application also applied plant immune inducers prepared by TaDEPs by spraying or injection to wheat disease prevention and control. These peptides are effective in preventing wheat bacterial leaf streak, fusarium head blight, and stem base rot. This indicates that the small peptides of the present application can be used as a new, green immune inducer to play an important role in actual agricultural production, promote the healthy growth of crops, reduce dependence on chemical pesticides, and have positive significance for promoting the sustainable development of agriculture.

[0217] 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 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.

2. The polypeptide according to claim 1, characterized in that The polypeptide has the amino acid sequence shown in SEQ ID NO:

1.

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: 20 to 24.

4. Use of the polypeptide according to any one of claims 1 to 3 in the preparation of an agent for improving plant immunity and / or disease resistance.

5. A plant immune inducer, characterized in that: The plant immunity inducer comprises the polypeptide according to any one of claims 1 to 3.

6. A method for improving plant disease resistance, characterized in that: The method comprises applying the polypeptide according to any one of claims 1 to 3 or the plant immunity inducer according to claim 5 to a target plant; The target plants include grass plants.

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

8. The method according to claim 6 or 7, characterized in that The grass plant includes one or more of wheat, rice, corn, switchgrass or millet.

9. Use of the polypeptide according to any one of claims 1 to 3 or the plant immunity inducer according to claim 5 in improving plant immunity and disease resistance.

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