An anti-fungal peptide targeting mps1 protein and its application in preventing and treating rice blast

Abstract

This invention discloses an antifungal peptide targeting the Mps1 protein and its application in the control of rice blast disease, relating to the field of plant disease control technology. Ten antifungal peptides targeting the Mps1 protein of rice blast fungus were obtained de novo using BindCraft, with amino acid sequences shown in SEQ ID No. 1~10. Among them, XH-4 and XH-8 exhibit the best affinity and outstanding antifungal activity. Experiments show that the antifungal peptide can significantly inhibit rice blast fungus infection at concentrations of 50~200 μM, and significantly alleviate rice blast symptoms at a concentration of 200 μM, without affecting the formation of appressoriums. It blocks the host penetration process only by competitively binding to the Mps1-Mkk2 interaction interface. At a high concentration of 400 μM, it has no significant side effects on rice seed germination and seedling growth, demonstrating good safety. It is highly targeted, environmentally friendly, and easy to apply, and can be used to prepare a green pesticide for the control of rice blast disease.

Description

Technical Field

[0001] This invention relates to the field of plant disease control technology, specifically to an antifungal peptide targeting the Mps1 protein and its application in the control of rice blast. Background Technology

[0002] Plant pathogenic fungi and bacteria continue to cause significant losses in rice yield and quality globally. Among them, rice blast, caused by Magnaporthe oryzae, is one of the most serious and widespread fungal diseases affecting rice and is considered a major biological threat to safe rice production. Therefore, conducting molecular-level precision intervention research focusing on key pathogenic links of rice blast is of significant scientific importance and practical value for achieving green control of rice diseases.

[0003] The infection process of rice blast fungus is highly ordered, and its infection cycle provides important clues for identifying key pathogenic factors and potential control targets. During infection, rice blast fungus conidia germinate to form germ tubes and differentiate into specialized infection structures—appendage cells. Appendage cells generate high turgor pressure by accumulating osmotic regulators such as glycerol, driving infection pins to penetrate the host epidermis and enter the host cells. Successful penetration of the host is the decisive step for rice blast fungus to complete infection, and the molecular elements that play a key role in this stage are ideal targets for precise intervention.

[0004] Protein kinases play a central regulatory role in signal transduction in eukaryotes. The highly conserved MAPK cascade pathway in filamentous fungi, composed of the MAPKKK, MAPKK, and MAPK sequences, participates in regulating cell wall integrity, osmotic response, and pathogenicity-related development. In *Bacillus oryzae*, three major MAPK cascade pathways have been identified, corresponding to MAPKs Mps1, Osm1, and Pmk1. Mps1 plays a crucial role in appressorium penetration of the host epidermis but does not affect appressorium formation; knockout of Mps1 leads to complete loss of pathogenicity in *Bacillus oryzae*, thus Mps1 is considered a highly promising molecular target for the control of rice blast.

[0005] Research on small-molecule inhibitors targeting Mps1 has made some progress, such as A378-0, PLX-4720, TAK-733, and natural small-molecule melatonin derivatives. These compounds can target Mps1 and reduce the virulence of rice blast fungus. However, existing inhibitors mainly focus on ATP-binding pockets or relatively conserved regulatory regions, and still have limitations in terms of target selectivity, resistance risk, and ability to intervene at protein-protein interaction interfaces. Therefore, developing Mps1 regulatory strategies with complementary mechanisms of action and novel intervention modes remains an urgent scientific problem to be solved.

[0006] In recent years, small peptides, as emerging bio-based plant protection factors, have gradually attracted attention due to their advantages such as strong targeting, environmental friendliness, and high structural designability. Antifungal peptides (AFPs) have been shown to inhibit the growth and infection processes of plant pathogenic fungi through multiple mechanisms. For example, the small peptide SNP-D4 targets calmodulin of *Bacillus oryzae* to inhibit spore germination, NoPv1 inhibits cellulase activity of *Botrytis cinerea*, and the phytodefensin MtDef4 shows significant antifungal activity against *Botrytis cinerea*. These studies indicate that small peptide molecules have good potential in the control of plant fungal diseases.

[0007] Artificial intelligence (AI) technology has made groundbreaking progress in the field of protein structure prediction and design. Deep learning models, such as AlphaFold2, RoseTTAFold, and RFdiffusion, have significantly improved the accuracy of protein structure modeling and binding interface design. Structure-guided generative design platforms, such as BindCraft, have been successfully used to design high-affinity protein binders from scratch, showing high success rates in various systems. However, current research mainly focuses on small proteins with stable folded backbones. Whether these methods can be extended to short peptide systems with highly flexible structures and used to obtain small peptide binders with well-defined functions still lacks experimental validation.

[0008] In summary, existing Mps1 inhibitors suffer from limitations such as target selectivity, resistance risk, and insufficient ability to intervene in protein-protein interaction interfaces. While small peptides show potential in the control of plant fungal diseases, the discovery and optimization of lead peptides are constrained by screening efficiency and rational design capabilities. Therefore, there is an urgent need to propose an antifungal peptide targeting the Mps1 protein and its application in the control of rice blast, combining artificial intelligence technology to achieve efficient and precise small peptide binding agent design, thereby addressing the aforementioned problems of existing technologies. Summary of the Invention

[0009] The purpose of this invention is to provide an antifungal peptide targeting the Mps1 protein and its application in the prevention and control of rice blast, so as to solve the problems of low efficiency and insufficient targeting of existing antifungal peptide design and screening, which makes it difficult to achieve efficient and precise control of rice blast.

[0010] To achieve the above objectives, the present invention provides the following technical solution: an antifungal peptide targeting the Mps1 protein, wherein the amino acid sequence of the antifungal peptide is shown in any one of SEQ ID No. 1 to SEQ ID No. 10:

[0011] XH-1(SEQ ID No.1): SDWWDMLRRWDEMEREIWA;

[0012] XH-2(SEQ ID No.2): KDIWDILEEMEKENRAL;

[0013] XH-3(SEQ ID No.3): GVWDELERMEEENRL;

[0014] XH-4(SEQ ID No.4): DPHDKDMEDMKNFEKKLAE;

[0015] XH-5(SEQ ID No.5): DPHDKDMKDMENFEKKLAE;

[0016] XH-6(SEQ ID No.6): MSEFEKYLMGLNEMEEEEQLK;

[0017] XH-7(SEQ ID No.7): MDIWDILEEMEKENKAL;

[0018] XH-8(SEQ ID No.8): MDEYEREDEALREFLGE;

[0019] XH-9(SEQ ID No.9): DWFKYLEEMNKMEKEDWEM;

[0020] XH-10(SEQ ID No.10): DWFDYLEEMNKMEEEDWAM.

[0021] Further, the antifungal peptide is XH-4, with the amino acid sequence shown in SEQ ID No. 4; or XH-8, with the amino acid sequence shown in SEQ ID No. 8.

[0022] The present invention also discloses the application of an antifungal peptide targeting the Mps1 protein in the preparation of a drug for preventing and controlling rice blast fungus.

[0023] Furthermore, the application is the use of antifungal peptides in the preparation of drugs that inhibit rice blast fungus infection of the host.

[0024] Furthermore, the application is the use of antifungal peptides in the preparation of drugs that inhibit the penetration of rice blast fungus appressoriums into the host epidermis.

[0025] Furthermore, the concentration of the antifungal peptide used in the application is 50–200 μM.

[0026] The present invention also discloses a drug for controlling rice blast fungus, wherein the active ingredient comprises the aforementioned antifungal peptide.

[0027] Furthermore, the concentration of the antifungal peptide in the drug is 50–200 μM.

[0028] Furthermore, the drug also contains agriculturally acceptable excipients, specifically surfactants or stabilizers.

[0029] The present invention also discloses a method for preventing and controlling rice blast disease, which involves spraying the aforementioned drug for controlling rice blast pathogens onto the leaves of rice plants.

[0030] Compared with existing technologies, the antifungal peptide targeting the Mps1 protein and its application in the prevention and control of rice blast provided by this invention have the following beneficial effects:

[0031] (1) This invention achieves de novo design of antifungal peptides targeting Mps1 protein through BindCraft. All 10 candidate antifungal peptides can specifically bind to Mps1, with a design success rate of 100%, which significantly improves the efficiency of rational design of antifungal peptides and solves the technical defects of traditional small peptide screening cycle and poor targeting.

[0032] (2) The antifungal peptides XH-4 and XH-8 provided by the present invention can significantly inhibit the infection of rice by rice blast fungus in the concentration range of 50 to 200 μM. At a concentration of 200 μM, they can significantly reduce the symptoms of rice blast disease. The antifungal effect is stable and the control efficiency is high.

[0033] (3) The antifungal peptide provided by the present invention does not affect the formation of rice blast fungus appressorium, but specifically blocks the host penetration process mediated by Mps1. It has a precise action site and strong target selectivity, which can reduce the risk of drug resistance and make up for the problem that existing Mps1 inhibitors have a single action interface and are prone to resistance.

[0034] (4) The antifungal peptides XH-4 and XH-8 provided by this invention have no obvious side effects on rice seed germination and seedling growth at a high concentration of 400 μM. They are safe and non-toxic to rice, have good environmental compatibility, and meet the requirements for the development of green pesticides.

[0035] (5) The antifungal peptide provided by the present invention is a short synthetic peptide that is easy to prepare and flexible in application. It can be used to control rice blast by foliar spraying, providing a new lead molecule for green, efficient and precise control of rice blast, and has important application value for the creation of new biopesticides. Attached Figure Description

[0036] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0037] Figure 1 A design flowchart of the antifungal peptide provided in an embodiment of the present invention;

[0038] Figure 2 This is a diagram illustrating the ranking and complex prediction model of antifungal peptides designed based on the Mps1 protein, provided in an embodiment of the present invention.

[0039] Figure 3 The graph shows the purification of Mps1 protein and the in vitro affinity detection of antifungal peptides with Mps1 protein provided in the embodiments of the present invention.

[0040] Figure 4 This is a graph showing the in vitro interaction and binding curves between the antifungal peptides XH-4 and XH-8 and the Mps1 protein provided in this embodiment of the invention.

[0041] Figure 5 The figure shows the results of the antifungal effect test of the antifungal peptide provided in this embodiment of the invention on detached rice leaves.

[0042] Figure 6 The figure shows the results of spray antibacterial effect determination of antifungal peptides XH-4 and XH-8 on live rice provided in the embodiments of the present invention.

[0043] Figure 7 The figure shows the effect of antifungal peptides XH-4 and XH-8 on the formation of appressorium of rice blast fungus provided in the embodiments of the present invention.

[0044] Figure 8 This is a structural prediction and related structural superposition comparison diagram of the Mps1-antifungal peptide complex provided in the embodiments of the present invention;

[0045] Figure 9 The safety evaluation diagram of antifungal peptides XH-4 and XH-8 provided in the embodiments of the present invention on rice is shown. Detailed Implementation

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0047] The rice blast fungus used in this invention is the wild-type strain Guy11, purchased from the American Technology and Culture Center (ATCC®201236™). The culture medium used for the strain is complete medium (CM), with the following formula: glucose 10 g, peptone 140 2 g, casein amino acids 1 g, yeast extract 1 g, NaNO3 6 g, KH2PO4 1.52 g, KCl 0.52 g, MgSO4·7H2O 0.52 g, biotin 0.1 mg, vitamin B 0.1 mg, thiamine 0.1 mg, riboflavin 0.1 mg, niacin 0.1 mg, para-aminobenzoic acid 0.1 mg, Na2MoO4·5H2O 1.5 mg, CuSO4·5H2O 1.6 mg, CoCl2·6H2O 1.7 mg, MnCl2·4H2O 5 mg, FeSO4·7H2O 5 mg, H3BO3 11 mg, ZnSO4·7H2O 22 mg, Na4EDTA·2H2O 50 mg, agar powder 15g; adjust pH to 6.5 with NaOH, add deionized water to a final volume of 1 L, autoclave at 121℃ for 30 min, and set aside.

[0048] The antifungal peptides XH-1 to XH-10 were all artificially synthesized by Genscript Biotech Inc. The preparation method of the antifungal peptide stock solution is as follows: the synthesized antifungal peptide powder is dissolved in DMSO to prepare a stock solution with a final concentration of 50 mM. It is stored at -20℃ in the dark. When using, it is diluted with the appropriate buffer or aqueous solution to the required working concentration.

[0049] Example 1:

[0050] Please see Figure 1 and Figure 2 An antifungal peptide targeting the Mps1 protein, the amino acid sequence of which is shown in any one of SEQ ID No. 1 to SEQ ID No. 10:

[0051] XH-1(SEQ ID No.1): SDWWDMLRRWDEMEREIWA;

[0052] XH-2(SEQ ID No.2): KDIWDILEEMEKENRAL;

[0053] XH-3(SEQ ID No.3): GVWDELERMEEENRL;

[0054] XH-4(SEQ ID No.4): DPHDKDMEDMKNFEKKLAE;

[0055] XH-5(SEQ ID No.5): DPHDKDMKDMENFEKKLAE;

[0056] XH-6(SEQ ID No.6): MSEFEKYLMGLNEMEEEEQLK;

[0057] XH-7(SEQ ID No.7): MDIWDILEEMEKENKAL;

[0058] XH-8(SEQ ID No.8): MDEYEREDEALREFLGE;

[0059] XH-9(SEQ ID No.9): DWFKYLEEMNKMEKEDWEM;

[0060] XH-10(SEQ ID No.10): DWFDYLEEMNKMEEEDWAM.

[0061] The specific implementation method is as follows: de novo design of antifungal peptides targeting the Mps1 protein based on the artificial intelligence tool BindCraft, including the following steps:

[0062] (1) Preparation of target protein structure:

[0063] The three-dimensional structural data of the Mps1 protein in rice blast fungus, which has been structurally resolved, were obtained and used as a target template for targeted peptide design. The data were then input into the locally deployed BindCraft design platform.

[0064] (2) Small peptide generation and parameter settings:

[0065] Initiate the BindCraft workflow, set the peptide length to 10-20 amino acids, and optimize the peptide sequence and binding conformation through a three-step process: backpropagation, de novo generation, and iterative optimization. The peptide design workflow is as follows: Figure 1 As shown in the figure. After multiple iterations of design and calculation, a total of 50 candidate antifungal peptide sequences were generated.

[0066] (3) Antifungal peptide scoring and screening:

[0067] Using BindCraft's officially recommended ipTM and pLDDT as the core scoring indicators, 50 candidate antifungal peptides were comprehensively ranked, and the top 10 antifungal peptides were selected as lead peptides. Figure 2 As shown in Figure A, they are named XH-1, XH-2, XH-3, XH-4, XH-5, XH-6, XH-7, XH-8, XH-9, and XH-10 in sequence. (The rest of the text appears to be a continuation of the previous sentence and can be left as is.) Figure 2The prediction model of the Mps1-small peptide complex of B shows that the 10 high-scoring antifungal peptides generated are all located in close spatial positions, suggesting that the 10 seed peptides have similar mechanisms of action.

[0068] (4) Synthesis of antifungal peptides:

[0069] The above 10 antifungal peptide sequences were chemically synthesized by GenScript. After synthesis, the purity was verified by mass spectrometry and HPLC, and the results met the requirements for subsequent in vitro experiments.

[0070] Example 2:

[0071] Please see Figure 3 and Figure 4 Prokaryotic expression, purification, and in vitro affinity (SPR) assay of Mps1 protein with antifungal peptides:

[0072] (1) Construction of Mps1-6×His fusion protein:

[0073] A prokaryotic expression vector was constructed, the Mps1 gene was fused with a 6×His tag, and transformed into an Escherichia coli expression strain. The heterologous expression of the Mps1-6×His fusion protein was achieved by IPTG induction.

[0074] (2) Protein purification:

[0075] The expression product was initially purified by nickel column affinity chromatography, and the eluent was collected. Further purification was then performed by size exclusion chromatography to obtain high-purity, homogeneous Mps1-6×His fusion protein for subsequent SPR assays. 1 μg of protein was subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The results are as follows: Figure 3 As shown in Figure A, where M represents the Marker; the results show a single specific band at the 48 kDa position, and the protein purity meets the requirements for SPR detection.

[0076] (3) Surface plasmon resonance (SPR) affinity determination:

[0077] Purified Mps1-6×His protein was used as a ligand and immobilized on the surface of an SPR sensor chip. Antifungal peptides XH-1 to XH-10 were diluted to specific concentrations and flowed through the chip surface as analytical streams. Binding and dissociation signals were monitored in real time, and the equilibrium dissociation constant K was calculated using a fitting method. D value.

[0078] The results are as follows Figure 3 As shown in B, all 10 antifungal peptides could specifically bind to the Mps1 protein in vitro, achieving a design success rate of 10 / 10 (100%); among them, the two with the highest affinity were:

[0079] XH-8: KD =1.17 μM;

[0080] XH-4: K D =1.19 μM.

[0081] Figure 4 A, Figure 4 B represents the binding / dissociation kinetics curve and affinity fitting curve of the antifungal peptide XH-4, respectively. Figure 4 C Figure 4 D represents the binding / dissociation kinetics curve and affinity fitting curve of the antifungal peptide XH-8, respectively; Figure 4 Analysis of the in vitro interaction curves of the antifungal peptides XH-4 and XH-8 with the target protein Mps1 revealed that the binding and dissociation modes of XH-4 and XH-8 with Mps1 are very similar, both exhibiting a rapid binding and rapid dissociation mechanism. This result is consistent with... Figure 2 The prediction of high spatial overlap between the antifungal peptide B and Mps1. However, XH-1 exhibits non-specific binding, K... D The value is an estimate, approximately 42.0 μM.

[0082] Example 3:

[0083] Please see Figure 5 Determination of the antifungal activity of antifungal peptides on isolated rice leaves:

[0084] To determine the antibacterial activity of 10 designed lead peptides against rice blast fungus, this embodiment uses the rice spore hanging drop method to determine pathogenicity and screen for the lead peptide with the best antibacterial effect.

[0085] (1) Preparation of spore suspension:

[0086] The rice blast fungus strain Guy11 was inoculated onto CM medium and cultured until a large number of conidia were produced. The conidia were then rinsed with sterile water, filtered through multiple layers of gauze to remove mycelia, and the conidia concentration was adjusted to 5 × 10⁻⁶. 4 cfu / mL, for later use.

[0087] (2) Inoculation and treatment of detached leaves:

[0088] Select healthy, uniformly growing rice leaves, disinfect them, and place them in a moist culture dish. Use 4‰ DMSO as a blank solvent control. Dilute the antifungal peptides XH-1 to XH-10 to a working concentration of 200 μM, mix them with an equal amount of spore suspension, and inoculate them onto the surface of the rice leaves using the hanging drop method.

[0089] (3) Culture and disease statistics:

[0090] The petri dishes were incubated at 25-28℃ under high humidity and in the dark. After obvious lesions appeared on the control leaves, the diseased area in a 5 mm × 5 mm region was counted using ImageJ software to calculate the antibacterial effect. The data were expressed as the mean ± sd of n=3 independent replicates (P<0.0001; two-tailed T test). Figure 5 Results of the assay for the antifungal effect of antifungal peptides on detached rice leaves; Figure 5 A represents the pathogenicity phenotype determined by hanging drop assay of rice leaf spores; Figure 5 B represents the statistical data on the area of ​​diseased rice leaves.

[0091] The results showed that at a concentration of 200 μM, all 10 antifungal peptides significantly inhibited rice blast fungus infection, with highly significant differences compared to the DMSO control group (P < 0.0001, two-tailed t-test). Among them, XH-4 and XH-8 showed the most prominent inhibitory effects on the diseased areas of the leaves. These results further demonstrate the reliability of the antifungal peptides designed and obtained by BindCraft.

[0092] Example 4:

[0093] Please see Figure 6 Determination of the spraying control efficacy of antifungal peptides XH-4 and XH-8 on live rice:

[0094] To further determine the actual control effect of XH-4 and XH-8 on rice blast, this embodiment uses live rice spray inoculation method to carry out antifungal peptide antibacterial activity verification test.

[0095] (1) Live rice culture:

[0096] Rice seeds were selected and germinated using conventional methods. They were then sown in nutrient soil and cultured in an artificial climate chamber until they reached the 3-4 leaf stage. Rice plants with uniform growth and consistent vigor were selected for the experiment.

[0097] (2) Experimental grouping and treatment:

[0098] The experiment was set up with the following groups:

[0099] Blank control: 4‰ DMSO;

[0100] Positive control: 10 g / L Isoprothiolane (IPT);

[0101] Treatment groups: XH-4 and XH-8 were set with three concentration gradients of 50 μM, 100 μM, and 200 μM, respectively;

[0102] Apply the pesticide evenly using a foliar spray method, mixing the pesticide with a conidial suspension (5×10⁻⁶). 4 After mixing (cfu / mL), spray to complete the inoculation.

[0103] (3) Moisturizing culture and disease statistics:

[0104] After inoculation, rice was cultured in a high-humidity artificial climate chamber at 25-28℃. After the disease symptoms stabilized, the diseased area of ​​a 5 cm leaf region was counted using ImageJ software. At least 10 rice leaves were counted for each group, and statistical analysis was performed using a two-tailed T-test. Figure 6 Results of the antifungal effects of antifungal peptides XH-4 and XH-8 on rice spray. Figure 6 A represents the phenotype of rice blast disease after spraying with antifungal peptides XH-4 and XH-8 on live rice; the blank control was 4‰ DMSO, and the positive control was 10 g / L isoprothiolane (IPT). Figure 6 B represents the statistical analysis of the diseased area of ​​live rice treated with spray; the statistical area is a rectangle with a length of 5 cm, and at least 10 leaves are counted in each group (P < 0.1; P < 0.0001; two-tailed T test).

[0105] The results showed that both XH-4 and XH-8 significantly inhibited the infection of rice blast fungus in living rice leaves within the concentration range of 50–200 μM, and the control effect gradually increased with increasing concentration. At a concentration of 200 μM, they significantly inhibited the development of rice blast, with a significant difference compared with the blank control group (P < 0.1; P < 0.0001, two-tailed T test), indicating that XH-4 and XH-8 have excellent control effects on rice blast and potential for field application.

[0106] Example 5:

[0107] Please see Figure 7 Effects of antifungal peptides on appressorium formation of rice blast fungus:

[0108] Previous studies have shown that the Mps1 gene knockout mutant of rice blast fungus can form appressoria normally, but completely loses its ability to penetrate the host epidermis for infection. To clarify the key stages of XH-4 and XH-8 in inhibiting the virulence of rice blast fungus, this example conducts an appressoria induction experiment to verify the effect of antifungal peptides on appressoria formation.

[0109] (1) Appressorium induction:

[0110] A suspension of conidia of the rice blast fungus Guy11 (concentration 5 × 10⁻⁶) was prepared. 4 The mixture (cfu / mL) was thoroughly mixed with 200 μM XH-4 antifungal peptide and 200 μM XH-8 antifungal peptide, respectively; 0.4‰ DMSO was used as a solvent control. The mixture was then dropped onto a hydrophobic glass slide to simulate the hydrophobic interface of rice leaves and to induce appressorium formation.

[0111] (2) Microscopic observation and data statistics:

[0112] The rice blast fungus was cultured at 25℃ in the dark for 24 h. Spore germination and appressorium formation were observed using an optical microscope. Multiple fields of view were randomly selected for observation, and the appressorium formation rate was statistically analyzed. Each group was divided into three biological replicates. Data are expressed as mean ± standard deviation and statistically analyzed using a two-tailed t-test. Figure 7 A is a microscopic observation of appressorium formation of rice blast fungus after treatment with XH-4 and XH-8 for 24 hours (scale bar: 10 μM). Figure 7 B is a graph showing the statistical results of the rice blast fungus appressorium formation rate.

[0113] The results showed that, compared with the 0.4‰ DMSO control group, there was no significant difference in the formation rate of rice blast fungus appressorium in the XH-4 treatment group and the XH-8 treatment group (ns represents no significant difference; two-tailed T test), indicating that at a concentration of 200 μM, neither XH-4 nor XH-8 affected the normal formation of rice blast fungus appressorium.

[0114] This result is consistent with the phenotype of the Mps1 gene deletion mutant, which fully confirms that XH-4 and XH-8 do not work by inhibiting appressorium formation, but by targeting the Mps1 protein and blocking the appressorium-mediated host penetration infection step, thereby inhibiting the pathogenicity of rice blast fungus.

[0115] Example 6:

[0116] Please see Figure 8 Antifungal peptide binding site prediction and mechanism of action analysis:

[0117] (1) Structural superposition comparison:

[0118] The structure of the p38α (MAPK) and MKK6 (MAPKK) complex has been reported (PDB 8A8M). Since the Mps1 protein of rice blast fungus has a high structural similarity with the p38α protein, the predicted three-dimensional structure of the Mps1-XH-8 complex was superimposed and compared with the structure of the p38α-MKK6 complex using PyMOL software, and the root mean square deviation (RMSD) was calculated. Figure 8 A is a structural superposition comparison diagram of the Mps1-XH-8 complex and the p38α-MKK6 complex (PDB 8A8M).

[0119] The results showed that the RMSD of the Mps1-XH-8 complex and the p38α-MKK6 complex was 3.052 Å. The structural alignment results showed that the binding site of the antifungal peptide XH-8 was close to the lipid binding site of p38α and spatially occupied the binding region of MKK6 and p38α, suggesting that the antifungal peptide XH-8 may affect the interaction between MAPK and MAPKK.

[0120] (2) Interaction interface prediction:

[0121] Based on the above structural alignment results, the upstream kinase corresponding to Mps1 (MAPK) in rice blast fungus was determined to be Mkk2 (MAPKK). The three-dimensional model of the Mps1-Mkk2 complex was predicted using AlphaFold3 software, and the model evaluation scores were ipTM=0.64 and pTM=0.6. The three-dimensional structure of the antifungal peptide XH-8 was in situ superimposed into the Mps1-Mkk2 complex model. Figure 8 B is a schematic diagram of the superposition of the Mps1-Mkk2 complex with XH-8 as predicted by AlphaFold3.

[0122] The results showed that XH-8 directly invaded the protein-protein interaction interface between Mkk2 and Mps1 in space, suggesting that the antifungal peptides XH-4 and XH-8 interfered with the activation of Mps1 and downstream signal transduction by competitively binding to the Mps1-Mkk2 interaction interface, thereby blocking the penetration and infection process of rice blast fungus on the host rice.

[0123] Example 7:

[0124] Please see Figure 9 Safety evaluation of antifungal peptides XH-4 and XH-8 in rice:

[0125] To further clarify the biosafety of antifungal peptides XH-4 and XH-8 in rice, this embodiment conducted safety evaluation experiments from two dimensions: seed germination and plant growth.

[0126] (1) Seed germination safety test:

[0127] Rice seeds of uniform size and full size were selected and treated with 400 μM XH-4 and 400 μM XH-8 antifungal peptide solutions for 48 h, respectively. Simultaneously, 8‰ DMSO was used as a solvent control group, and 1 g / L carbendazim was used as a conventional pesticide control group. Each treatment group contained 50 rice seeds, with three biological replicates. Seeds were cultured under identical conditions, and seed germination rates were recorded. Figure 9 A represents the effect of treatment with antifungal peptides XH-4 and XH-8 on the germination rate of rice seeds.

[0128] The results showed that at a concentration of 400 μM, the germination rate of rice seeds in both the XH-4 and XH-8 treatment groups was 100%, and there was no significant difference compared with the DMSO control group and the carbendazim control group (α represents no significant difference, two-tailed T test), indicating that high concentrations of XH-4 and XH-8 had no adverse effects on rice seed germination.

[0129] (2) Plant growth safety test:

[0130] The rice seeds after the above germination treatment were sown in nutrient soil and cultured continuously for 10 days under uniform water and fertilizer management. The plant height, growth, leaf color, and whether yellowing, dwarfing, wilting and other phenotypes of pesticide damage appeared in the rice seedlings were observed and recorded. The effect of antifungal peptides on the growth of rice seedlings was comprehensively evaluated. Figure 9 B represents the phenotypic observation of rice seedlings after treatment with antifungal peptides XH-4 and XH-8 for 10 days.

[0131] The results showed that the rice seedlings treated with XH-4 and XH-8 grew normally, with no significant difference in plant height and vigor compared to the control group. No pesticide damage, yellowing, dwarfing, or growth inhibition were observed, indicating that the antifungal peptides XH-4 and XH-8 were safe for the growth of rice seedlings.

[0132] In summary, at a high concentration of 400 μM, the antifungal peptides XH-4 and XH-8 had no significant negative impact on rice seed germination and seedling growth, and were safe and non-toxic to rice, providing a good safety basis for the development of green pesticides.

[0133] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An antifungal peptide targeting the Mps1 protein, characterized in that, The antifungal peptide is XH-8, and its amino acid sequence is shown in SEQ ID No.

8.

2. The application of the antifungal peptide targeting Mps1 protein as described in claim 1 in the preparation of a drug for preventing and controlling rice blast fungus.

3. The application of the antifungal peptide targeting Mps1 protein according to claim 2 in the preparation of a drug for controlling rice blast fungus, characterized in that, The application is the use of antifungal peptides in the preparation of drugs that inhibit the infection of the host by rice blast fungus.

4. The application of the antifungal peptide targeting Mps1 protein according to claim 3 in the preparation of a drug for controlling rice blast fungus, characterized in that, The application is the use of antifungal peptides in the preparation of drugs that inhibit the penetration of rice blast fungus appressoria into the host epidermis.

5. The application of the antifungal peptide targeting Mps1 protein according to claim 2 in the preparation of a drug for controlling rice blast fungus, characterized in that, The concentration of the antifungal peptide used in this application is 50~200 μM.

6. A drug for controlling rice blast fungus, characterized in that, The active ingredient comprises the antifungal peptide as described in claim 1.

7. The drug for controlling rice blast fungus according to claim 6, characterized in that, The concentration of the antifungal peptide in the drug is 50~200 μM.

8. A drug for controlling rice blast fungus according to claim 6 or 7, characterized in that, The drug also contains agriculturally acceptable excipients.

9. A method for preventing and controlling rice blast, characterized in that, The drug for controlling rice blast fungus as described in any one of claims 6-8 is sprayed onto the leaves of rice plants.

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