A drug target protein sec24-2 and its use in agriculture

Abstract

The application provides a kind of drug target protein Sec24-2 and its use in agriculture, belong to the technical field of agricultural biological medicine.The application finds that the 313-351th and 648-730th amino acids of rice blast fungus Sec24-2 protein are two important functional domains, and the active pocket formed by folding can be used as a receptor region to design targeted drugs.The 240th, 241th, 243th, 257th, 358th, 364th, 560th and 754th amino acids of rice blast fungus MoSec24-2 protein have a key role in vesicle depolymerization, effector protein secretion and pathogenicity, and fungicides designed based on drug sensitivity sites can prevent and control plant fungal diseases.The application provides a pathogenic fungus Sec24-2 protein mutant, which can be used to develop targeted selective drugs or prepare screening drug materials based on its functional sites, and has a safe, efficient and broad-spectrum application prospect.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biopharmaceutical technology, specifically relating to a drug target protein Sec24-2 and its agricultural applications. Background Technology

[0002] Currently, the fungicide targets available for the creation of green pesticides are mainly concentrated on a few targets such as respiratory chain complexes II and III, and key enzymes in appressorium melanin synthesis. This results in a single site of action, severe homogenization of pesticide structures, and even minor mutations in the target protein genes can lead to loss of pesticide activity, posing a significant risk of resistance. Furthermore, there is a lack of crystal structure data for pathogen target proteins, and molecular design often relies on the crystal structures of model organisms, which cannot accurately reflect the interaction between chemical molecules and targets, leading to large errors and low efficiency in compound screening.

[0003] Therefore, by clarifying the pathogenic mechanisms of food crop pathogens, exploring potential pesticide targets, constructing reliable three-dimensional target structures, and then designing highly selective new molecules, it is hoped that the original development of novel green pesticides can be achieved.

[0004] Currently, there are no reports on bactericides that target vesicle component proteins. Therefore, it is crucial to study vesicle transport inhibitors as bactericides with novel mechanisms of action. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a COPII vesicle component protein Sec24-2 protein, which can serve as a molecular target for drug development and provide a foundation for the development of drugs for fungal diseases.

[0006] This invention first provides the application of an agent that inhibits or weakens the function or gene expression of the Sec24-2 protein in plants in the prevention and control of the spread of plant fungal diseases, wherein the plant fungal diseases are plant diseases caused by Magnaphalthe, Fusarium, Colletotrichum, Ustilaginoidea, Sclerotinia, Aspergillus, Gaeumannomyces, Verticillium, Botrytis, and Neurospora.

[0007] Preferably, the plant pathogenic fungus is rice blast fungus, Fusarium fungus, or anthracnose fungus.

[0008] More specifically, the amino acid sequence of the Sec24-2 protein is shown in SEQ ID NO: 1-3, and preferably the nucleotide sequence of the gene is shown in SEQ ID NO: 4-6.

[0009] Preferably, the plant is wheat, rice, or soybean.

[0010] More specifically, the plant diseases caused by *Bacillus oryzae* are rice blast, the plant diseases caused by *Fusarium* are wheat scab and / or rice bakanae disease, and the plant diseases caused by *Anthracnose* are soybean anthracnose and / or grape anthracnose.

[0011] In a specific embodiment, the reagent is a small molecule compound, protein inhibitor, natural product, such as siRNA, antibody, or a reagent that can block or weaken the function of the Sec24-2 protein or gene transcription, or achieve the purpose using gene editing.

[0012] This invention also provides a mutant of the drug target Sec24-2 protein, which is based on the amino acid sequence shown in SEQ ID NO: 2, by performing domain deletion mutations or point mutations to reduce or inactivate its activity; preferably, the deletion of the domain segment is: the gene or protein sequence of the functional domain formed by amino acids 313-351 and 648-730 is completely or partially deleted; the point mutation is a mutation at one or more sites among K560, K754, F240, E241, H243, Q257, Q358 and R364, more specifically, a combination point mutation of K560R, K754R, F240+E241+H243+Q257 or a combination point mutation of Q358+R364.

[0013] Furthermore, this invention provides the application of the drug target Sec24-2 protein in the design or screening of specific agents for the prevention and control of plant fungal pathogens, wherein the plant fungal pathogens are rice blast fungus, Fusarium wilt fungus, and anthracnose fungus; preferably, the design or screening is performed targeting the key sites K560, K754, F240, E241, H243, Q257, Q358, and R364 of the drug target Sec24-2 protein.

[0014] More specifically, the tertiary structures of Sec24-2 proteins from *Bacillus oryzae*, *Fusarium oxysporum*, and *Agrobacterium anthracnose* were predicted using AlphaFold2, and candidate small molecule compounds were screened using AutoDockvina software. The PDB files of the predicted Sec24-2 tertiary structures and the MOL2 files of the small molecule compounds were submitted to AutoDockvina software. Using the software's virtual docking function, compounds with binding free energies ranging from -18.0 kcal / mol to -8.0 kcal / mol and a mean RMSD of [value missing] were selected. The small molecule compound is used as a candidate plant fungal pathogen-specific agent; optionally, it also includes further verification of the inhibitory effect of the candidate plant fungal pathogen-specific agent on the plant fungal pathogen to finally determine whether it is a plant fungal pathogen-specific agent.

[0015] The above can also be considered a method for designing or screening specific agents for controlling plant fungal pathogens. The steps include: predicting the tertiary structures of Sec24-2 proteins from *Bacillus oryzae*, *Fusarium oxysporum*, and *Amanita phalloides* using AlphaFold2; screening candidate small molecule compounds using AutoDockvina software; submitting the predicted Sec24-2 tertiary structure PDB file and the small molecule compound MOL2 file to AutoDockvina software; and using the software's virtual docking function to select compounds with binding free energies ranging from -18.0 kcal / mol to -8.0 kcal / mol and a mean RMSD of [value missing]. Small molecule compounds are candidate specific agents for plant fungal pathogens.

[0016] This invention also provides the application of the drug target Sec24-2 protein in reducing the pathogenicity of plant fungal pathogens; the plant fungal pathogens are *Strombus oryzae*, *Fusarium*, and *Anthracnose*. Preferably, this is achieved by mutations at key sites K560, K754, F240, E241, H243, Q257, Q358, and R364 of the drug target Sec24-2 protein in the plant fungal pathogens. More specifically, the mutations occur at one or more sites among K560, K754, F240, E241, H243, Q257, Q358, and R364, and more specifically, at the K560R, K754R, F240+E241+H243+Q257 combination point mutations or the Q358+R364 combination point mutations. Specifically, the reduction of plant fungal pathogen pathogenicity includes inhibiting the growth and development of plant pathogenic fungi and reducing toxin production.

[0017] The above can also be considered a method for reducing the pathogenicity of plant fungal pathogens; the plant fungal pathogens are rice blast fungus, Fusarium wilt fungus and anthracnose fungus, which includes the steps of mutating the key sites K560, K754, F240, E241, H243, Q257, Q358 and R364 of the drug target Sec24-2 protein in the plant fungal pathogen, and the steps of obtaining the mutated plant fungal pathogen mutant.

[0018] This invention discovers that amino acids 313-351 and 648-730 of the *MoSec24-2* protein, the blast fungus, are two important functional domains. The active pockets formed by their folding can be used as receptor regions for targeted drug design. Amino acids 240, 241, 243, 257, 358, 364, 560, and 754 of the *MoSec24-2* protein play crucial roles in vesicle disaggregation, effector protein secretion, and pathogenicity. As fungicide sites designed for drug sensitivity testing, these sites can control plant fungal diseases. This invention provides mutants of the pathogenic fungus *Sec24-2* protein, which can be used to develop targeted selective drugs or prepare screening drug materials based on their functional sites, showing promising applications that are safe, efficient, and broad-spectrum. Attached Figure Description

[0019] Figure 1 These are the Western blot results of ubiquitination detection of MoSec24-2 rice blast fungus and its ubiquitination site point mutant proteins. The upper part of the image shows the ubiquitination detection (antibody: Anti-Ub), and the lower part shows the total protein detection (antibody: Anti-GFP).

[0020] Figure 2 This is the result of pathogenicity analysis of the MoSEC24-2 gene knockout and ubiquitination site point mutation of rice blast fungus. A shows the leaf disease 7 days after inoculation with rice blast fungus; B shows the statistical results of disease lesion area (DLA) on the leaves; C shows the statistical results of relative fungal growth in the diseased leaves.

[0021] Figure 3 This is the result of pathogenicity analysis of the MoSEC24-2 gene knockout and its interaction site point mutation mutants of rice blast fungus. A shows the leaf disease 7 days after inoculation with rice blast fungus; B shows the statistical results of disease lesion area (DLA); C shows the statistical results of relative fungal growth in diseased leaves.

[0022] Figure 4 This is the result of an analysis of the ability of the rice blast fungus MoSEC24-2 gene knockout and ubiquitination site point mutants to infect rice leaf sheaths. A shows the grading criteria for different types of infecting hyphae; B shows the statistical results of classification based on different types of infecting hyphae.

[0023] Figure 5 This is an analysis of the ability of rice blast fungus MoSEC24-2 gene knockout and ubiquitination site point mutation mutants to inhibit reactive oxygen species (ROS) in the host. A shows images of rice ROS accumulation caused by infection with different mutants and wild-type strains observed under a microscope; B shows the statistical results of rice cells with ROS accumulation.

[0024] Figure 6 These are the results of pathogenicity analysis of the CgSEC24-2 gene knockout mutant of anthracnose fungus. A shows images of leaf disease 7 days after inoculation with anthracnose fungus; B shows the statistical results of leaf lesion diameter.

[0025] Figure 7 These are the pathogenicity analysis results of the Fusarium head blight FgSEC24-2 gene knockout mutant. A shows images of wheat disease 14 days after inoculation with Fusarium head blight; B shows the statistical results of the number of diseased ears.

[0026] Figure 8 These are the results of MST analysis of the binding affinity of anthrax pathogen CgSec24-2 and Fusarium head blight pathogen FgSec24-2 proteins and their mutant proteins to compound 1438. Note: The smaller the Kd value, the stronger the binding affinity.

[0027] Figure 9 These are the results of the analysis on the inhibition of rice blast by compound 1438. A shows images of leaf disease after 24 hours of treatment with different concentrations of compound 1438, followed by 7 days of inoculation with rice blast fungus; B shows the statistical results of disease lesion area (DLA) on leaves; C shows the statistical results of relative fungal growth in diseased leaves.

[0028] Figure 10 These are the results of the analysis on the inhibition of anthracnose and Fusarium head blight by compound 1438. A and C show the leaf disease incidence images 7-14 days after inoculation with anthracnose and Fusarium head blight 7 days after treatment with different concentrations of compound 1438 for 24 hours; B shows the statistical results of the leaf disease area 7 days after inoculation with anthracnose; B shows the statistical results of the number of diseased ears 14 days after inoculation with Fusarium head blight.

[0029] Figure 11 These are the results of evolutionary analysis and protein sequence similarity (identity) of the Sec24-2 protein sequence in different fungi. Detailed Implementation

[0030] The present invention will be further described below through specific embodiments in order to better understand the present invention, but this does not constitute a limitation on the present invention.

[0031] Example 1: Knockout of the SEC24-2 gene

[0032] 1. Construction of the SEC24-2 gene knockout vector

[0033] To investigate the function of the SEC24-2 gene in *MoSEC24-2*, *F. cyathea*, and *C. anthracnose*, predicted nucleic acid sequences encoding accession numbers MGG_06569, MU270241.1, and FGRAMPH1_01T20499 were obtained from the NCBI database https: / / www.ncbi.nlm.nih.gov (as shown in SEQ ID Nos: 4 to 6, and the encoded amino acid sequences as shown in SEQ ID Nos: 1-3, respectively).

[0034] Using the wild-type strain Guy11 of rice blast fungus as a template, approximately 1 kb upstream and approximately 1 kb downstream flanking sequences of the MoSEC24-2 gene were amplified using primers: MoSEC24-2-p1-F(5'-CCGCTCGAGGTCTAAGTAGCCATAGTC-3') / MoSEC24-2-p2-R(5'-CCGATATCGGAGCTCACTGCATCTGC-3'); MoSEC24-2-p3-F(5'-CGGGATCCCGGTCAGGGATGCAGTGTCACC-3') / MoSEC24-2-p4-R(5'-GGACTAGTCCTGGCGTCATCAGAGCAACT-3'). The MoSEC24-2-p2 / MoSEC24-2-p3 primers contain EcoR V restriction sites. Two flanking sequences were ligated using MoSEC24-2-p1-F / MoSEC24-2-p4-R, and the cloned 2kb fragment was purified and constructed into the pMD19-T vector. The approximately 1.4kb hygromycin HPH gene was amplified using FL1111 (5'-GGAGGTCAACACATCAATG-3') / FL1112 (5'-CTCTATTCCTTTGCCCTCG-3'), and the fragment was inserted into the pMD-MoSEC24-2 plasmid using EcoR V. The 3.4kb fragment was amplified using the MoSEC24-2-p1-F / MoSEC24-2-p4-R compound for protoplast transformation.

[0035] Using Fusarium strain PH-1 as a template, approximately 1 kb of upstream flanking sequence and approximately 1 kb of downstream flanking sequence of the FgSEC24-2 gene were amplified using primers FgSEC24-2-p1-F(5'-CATCACTCATGTTGATCAG-3') / FgSEC24-2-p2-R(5'-GGCGGTGTCGAGGTTCGGGA-3') and FgSEC24-2-p3-F(5'-GAACAGGTGGTGACTT-3') / FgSEC24-2-p4-R(5'-TAGTGGGTGTTGTCAAGC-3'). The FgSEC24-2-p2 / FgSEC24-2-p3 primers contain EcoR V restriction sites. Two flanking sequences were ligated using FgSEC24-2-p1-F / FgSEC24-2-p4-R, and the cloned 2kb fragment was purified and constructed into the pMD19-T vector. The approximately 1.4kb hygromycin HPH gene was amplified using FL1111 (5'-GGAGGTCAACACATCAATG-3') / FL1112 (5'-CTCTATTCCTTTGCCCTCG-3'), and the fragment was inserted into the pMD-MoSEC24-2 plasmid using EcoR V. A 3.4kb fragment was amplified using FgSEC24-2-p1-F / FgSEC24-2-p4-R for protoplast transformation.

[0036] Using anthrax strain SMCG1#C as a template, approximately 1 kb of upstream flanking sequence and approximately 1 kb of downstream flanking sequence of the CgSEC24-2 gene were amplified using primers CgSEC24-2-p1-F(5'-TAGTATCTTGAAGC-3') / CgSEC24-2-p2-R(5'-GGTTGCAGGAGGTGTGCG-3') and CgSEC24-2-p3-F(5'-GAAAAAGGTTACTAAAGTG-3') / CgSEC24-2-p4-R(5'-CCATGTCAATGTCTCGTTC-3'). The CgSEC24-2-p2 / CgSEC24-2-p3 primers contain EcoR V restriction sites. Two flanking sequences were ligated using CgSEC24-2-p1-F / CgSEC24-2-p4-R, and the cloned 2kb fragment was purified and constructed into the pMD19-T vector. The approximately 1.4kb hygromycin HPH gene was amplified using FL1111 (5'-GGAGGTCAACACATCAATG-3') / FL1112 (5'-CTCTATTCCTTTGCCCTCG-3'), and the fragment was inserted into the pMD-MoSEC24-2 plasmid using EcoR V. A 3.4kb fragment was amplified using FgSEC24-2-p1-F / FgSEC24-2-p4-R for protoplast transformation.

[0037] The approximately 1kb upstream flanking sequence of the rice blast fungus MoSEC24-2 mentioned above is shown in SEQ ID NO: 7. The approximately 1kb downstream flanking sequence of the rice blast fungus MoSEC24-2 gene mentioned above is shown in SEQ ID NO: 8. The approximately 1kb upstream flanking sequence of the Fusarium graminearum FgSEC24-2 mentioned above is shown in SEQ ID NO: 9. The approximately 1kb downstream flanking sequence of the Fusarium graminearum FgSEC24-2 gene mentioned above is shown in SEQ ID NO: 10. The approximately 1kb upstream flanking sequence of the anthracnose fungus CgSEC24-2 mentioned above is shown in SEQ ID NO: 11. The approximately 1kb downstream flanking sequence of the anthracnose fungus CgSEC24-2 gene mentioned above is shown in SEQ ID NO: 12.

[0038] 2. Obtaining the SEC24-2 gene knockout mutant

[0039] 1) Preparation of culture medium

[0040] CM culture medium preparation method: Measure 50 ml of 20× nitrate (120 g sodium nitrate, 10.4 g potassium chloride, 10.4 g magnesium sulfate heptahydrate, 30.4 g potassium dihydrogen phosphate, dissolved in distilled water to 1 L), and 50 ml of 1000× trace elements (2.2 g zinc sulfate heptahydrate, 1.1 g boric acid, 0.5 g manganese chloride tetrahydrate, 0.5 g ferric sulfate heptahydrate, 0.17 g cobalt chloride hexahydrate, 0.16 g copper sulfate pentahydrate, 0.15 g sodium manganate dihydrate, 5 g... 1 ml of tetrasodium EDTA (dissolved in distilled water to 100 ml), 1 ml of vitamin solution (0.01 g biotin, 0.01 g vitamin B6, 0.01 g vitamin B1, 0.01 g riboflavin, 0.01 g para-aminobenzoic acid, 0.01 g niacin, dissolved in distilled water to 100 ml), 10 g glucose, 2 g peptone, 1 g yeast extract, 1 g casein amino acids, 15 g agar powder, add distilled water to a final volume of 1 L, dispense into Erlenmeyer flasks, sterilize at 121 °C for 20 minutes, and cool before use.

[0041] Sporulation medium preparation method: Prepare with corn flour and rice straw. Weigh 100g of rice straw, add 1L of water and boil for 30 minutes. Then add 40g of corn flour and 15g of agar powder and boil for 20 minutes. Finally, make up to 1L with distilled water, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and cool before use.

[0042] PDA medium: Boil 200g of potatoes, filter to remove residue, add 20g of glucose, 15g of agar, and pure water to a final volume of 1L, then autoclave.

[0043] YEPD medium: 3g yeast extract, 10g peptone, 20g glucose, diluted with pure water to 1L, and sterilized by high temperature and high pressure.

[0044] Preparation method of 1×STC: Weigh 20% sucrose by mass and volume, weigh 50mM Tris·Cl pH8.0, 50mM calcium chloride, add distilled water to a final volume of 1L, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and cool before use.

[0045] Preparation method of TB3 culture medium: Weigh 3g yeast extract, 3g Casamino Acids and 20% sucrose, add distilled water to a final volume of 1L, dispense into Erlenmeyer flasks, sterilize at 121℃ for 20 minutes, and cool before use.

[0046] Preparation method of PTC: Weigh 60% PEG4000 and dissolve it in 1×STC, filter it with a bacterial filter and set aside.

[0047] Preparation of enzyme solution: Dissolve the enzyme (lysing enzymes from Trichoderma harzianum, sigma) in 0.7M NaCl solution to a concentration of 7.5-10 mg / ml. Filter to sterilize and prepare immediately before use.

[0048] 2) Preparation of protoplasts

[0049] a. Activating the strain: Inoculate mycelial blocks onto CM or PDA plates and grow for 3-4 days, with colonies approximately 3 cm in diameter. The strain should not be too old.

[0050] b. Cut off colonies with a diameter of about 3cm, chop them as finely as possible, and place them in about 100ml of CM or YPD liquid culture medium (containing 50μg / ml Amp). Incubate at 28℃ and 150rpm for 2 days (36-48h).

[0051] c. Collect mycelia using 1-2 layers of Miracloth filtration, rinse twice with sterile water, and gently blot dry with absorbent paper.

[0052] d. Place the mycelia in a 50ml centrifuge tube containing 10-20ml of enzyme solution (containing 50μg / ml Amp) for enzymatic digestion. Lay the centrifuge tube flat and incubate at 30℃ and 60rpm for 1.5-2 hours. During the digestion process, aspirate the bacterial solution for microscopic examination to check the release of protoplasts.

[0053] e. The following steps are performed at 4°C. The 0.7M NaCl solution and 1×STC solution are pre-cooled at 4°C.

[0054] f. Take out the enzyme hydrolysate, add a small amount of 0.7M NaCl solution, shake gently, pour onto three layers of sterile lens paper for filtration, rinse gently 1-2 times with 0.7M NaCl solution to remove residue, and collect the filtrate in a 50ml centrifuge tube.

[0055] g. Centrifuge at 3000 rpm and 4°C for 10 min.

[0056] h. Carefully discard the supernatant, add 10-20ml of 1×STC to suspend, gently blow with a pipette tip that has been cut off, and then centrifuge at 3000rpm and 4℃ for 10min.

[0057] i. Repeat step 8 twice.

[0058] j. Add an appropriate amount (approximately 300 μl) of 1×STC for suspension, count the protoplasts, and bring the final concentration to 10. 8 Quantity / ml, aliquoted into 150μl / tube. Invert immediately or store at -70℃ (generally not stored; if storage is required, add 7% DMSO).

[0059] 3) Protoplast transformation

[0060] a. Add 2 μg DNA (5-10 μl) to 150 μl of protoplasts, mix gently, and let stand at room temperature for 25 min. The DNA concentration needs to be high enough and the DNA must be clean.

[0061] b. Add 1 ml of PTC in 2-3 portions, mix gently, and let stand at room temperature for 25 minutes. Do not let stand for too long, as PTC is toxic to protoplasts. After adding the PTC, thaw the TB3 or YEPD solid medium.

[0062] c. Add the protoplasts to approximately 10 ml of melted TB3 or YEPD solid medium (containing 50 μg / ml Amp) cooled to 45-50°C, mix gently, and pour into a petri dish. Incubate at 28°C in the dark for 24 hours. Optional: Alternatively, add the protoplasts to 5-10 ml of TB3 or YEPD (containing 50 μg / ml Amp) liquid and incubate overnight at 28°C with gentle shaking.

[0063] d. Pour another 10 ml of TB3 or YEPD solid medium (containing 50 μg / ml Amp) containing 300 μg / ml Hygromycin B / Bleomycin into the petri dish.

[0064] e. Culture in the dark at 28℃ for 7-10 days. Generally, the success of the transformation can be seen after 5 days.

[0065] f. When the diameter of the above-mentioned transformant colonies is about 5 mm, pick a small piece of mycelium from the colony and transfer it to CM solid medium containing 150 μg / ml Hygromycin B / Bleomycin for screening transformants.

[0066] g. Further verification of transformants was performed using PCR and Southern blotting. The internal probe was amplified using primers SEC24-2-p5-F / SEC24-2-p6-R, and the HPH probe was amplified using FL1111 / FL1112.

[0067] h. Analyze the phenotype of the mutant.

[0068] Example 2: Construction of complementary vectors

[0069] The fragment containing its own promoter (approximately 1.5 kb upstream) was amplified using primers MoSEC24-2-p7-F(5'-GGCATTGTAAGCCGTGATG-3') / MoSEC24-2-p2-R, FgSEC24-2-p7-F(5'-TGTCGCTGTAGAGAGCAGAGATG-3') / FgSEC24-2-p2-R, and CgSEC24-2-p7-F(5'-CTGTGGCCCGCATTATAGGCAG-3') / CgSEC24-2-p2-R. This fragment was then inserted into the pYF11 vector (bleomycin resistant) via yeast transformation to form the pYF11-MoERS1 vector. The vector was then introduced into the ΔMosec24-2, ΔFgsec24-2, and ΔCgsec24-2 mutants via protoplast transformation to obtain complementary strains of ΔMosec24-2 / MoSEC24-2, ΔFgsec24-2 / FgSEC24-2, and ΔCgsec24-2 / CgSEC24-2. Transformants were first screened based on phenotype, then verified by PCR, and finally, the complementation was confirmed by pathogenicity, growth, and other phenotypic assays.

[0070] At the same time, use the primer MoSEC24-2-p8-F(5'-ACTCACTATAGGGCGAATTGGGTACTCAAATTGGTTGTTGAGCGGAGGCGAGAACG-3') / MoSEC24-2-p9-R(5'-CAATCT CGCCACCAGGGTGGCGGCCATCATCCCTCATGAAG-3'), FgSEC24-2-p10-F(5'-CTTCATGAGGGATGATGGCCGCCACCCTGGTGGCGAGATTG-3') / FgSEC 24-2-p11-R(5'-GCGGGCGAGGATGGAATAAACC-3'), CgSEC24-2-p12-F(5'-GGTTTATTCCATCCTCGCCCGCGAAGCTGCAACAAAGCTTG-3') / CgSEC24-2-p13-R(5'-CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCCAGTACCCAG The gene was used to amplify the functional domain formed by amino acids 313-351 and 648-730, as well as point mutations at the K560R and K754R ubiquitination sites, and point mutations in the F240+E241+H243+Q257 or Q358+R364 combination. These fragments were then inserted into the pYF11 vector (bleomycin resistant) via yeast transformation to form the pYF11-MoSEC24-2 vector. This vector was then introduced into the ΔMosec24-2 mutant via protoplast transformation to obtain strains with complementary deletions and point mutations in different functional domains. Transformants were first screened based on phenotype, then verified by PCR, and finally, the complementation was verified based on pathogenicity, growth, and other phenotypic characteristics.

[0071] Example 3: In vivo ubiquitination of the MoSec24-2 protein of rice blast fungus

[0072] 1. Protein Extraction: Prepare protein lysis buffer. For each sample, add 1 ml of lysis buffer + 10 μl of cocktail (1%) + 10 μl of PMSF (1 mM) to a 2.0 ml EP tube and place on ice. Multiple samples can be mixed together before aliquoting. Collect hyphae, rinse twice with sterile water, press dry with absorbent paper, grind into powder with liquid nitrogen, add an appropriate amount of powder to the pre-chilled protein lysis buffer, vortex to mix, and place on ice for 30 min, vortexing every 10 min (the sample will turn slightly red). Centrifuge at maximum speed of 4 degrees Celsius for 10 min, and aspirate 500 μl of supernatant to a new 1.5 ml EP tube. Centrifuge at maximum speed of 4 degrees Celsius for 10 min, and aspirate 200 μl of supernatant to a new 1.5 ml EP tube. Prepare dilution buffer (can be done during centrifugation in steps 3-4). Each sample requires 800 μl of dilution buffer = diluent + 1% cocktail + 1 mM. For PMSF, multiple samples can be mixed together before aliquoting; dilute 200ul of protein supernatant to 1ml (200ul + 800ul) with dilution buffer and place on ice, then aspirate one portion as a total protein control (the purpose of dilution is to reduce the concentration of detergent NP-40 to below 0.2%).

[0073] 2. Bead Incubation: Place 25 μL of beads into a new 1.5 ml EP tube and wash three times with 500 μL of WB washing buffer, 2500 g for 2 min each time. Discard the supernatant. For the last wash, use a yellow pipette tip to aspirate any remaining liquid, being careful not to aspirate the beads. Mix the protein from step 6 with the washed beads and place in a 4°C refrigerator with rotation (2 h to overnight). Remove the sample and incubate at 4°C for 2500 g for 2 min. Aspirate a portion of the supernatant as a supernatant protein control (i.e., protein that has not bound to the beads). Discard the remaining supernatant. Wash the beads: Add 500 μL of WB washing buffer and incubate at 4°C for 2500 g for 2 min. Repeat the washing process 6 to 8 times, gently tapping the tube each time to mix the beads. For the final wash, use a yellow pipette tip to aspirate the remaining liquid. Transfer the beads to a new 1.5 ml EP tube using a cut pipette tip.

[0074] 3. Elution: Add 50 μL of 0.2 M glycine (pH 2.5), gently tap for 30 seconds, incubate at 2500 g for 2 minutes at 4 degrees Celsius, then aspirate 45 μL of supernatant into a new tube and add 5 μL of 1 M Tris base (pH 10.4).

[0075] 4. Ubiquitination detection: The proteins eluted in step 3 above were subjected to SDS-PAGE gel electrophoresis. After electrophoresis, the proteins on the gel block were transferred to a PVDF membrane, blocked with skim milk powder for 2 hours, and then washed with 1xTBST 3-5 times. Anti-Ub primary antibody (catalog number: AUB01, brand: Cytoskeleton) was prepared in 20 ml of 3% skim milk powder buffer, incubated for 1-2 hours, and then washed with 1xTBST 3-5 times. Finally, the results were observed by staining with ECL luminescence reagent (P0018AS, Beyotime).

[0076] Reagent preparation:

[0077] Lysis buffer (CoIP): 10mM Tris / Cl pH 7.5; 150mM NaCl; 0.5mM EDTA; 0.5% NP-40

[0078] Wash buffer:10mM Tris / Cl pH 7.5; 150mM NaCl; 0.5mM EDTA

[0079] Elution buffer:200mM glycine pH 2.5

[0080] For neutralization:1M Tris base(pH 10.4)

[0081] Experimental results showed that MoSec24-2 underwent ubiquitination in vivo, and this ubiquitination was dependent on the UBA domain of the blast fungus depolymerization factor MoSwa2. Mutations at the K560 and K754 ubiquitination sites (ΔMosec24-2 / MoSEC24-2) further mutated this process. 2R The ubiquitination of MoSec24-2 disappears. Figure 1 This result indicates that K560 and K754 are two important active sites of MoSec24-2, which are crucial for its normal biochemical function and for fungicide screening.

[0082] Example 4: Pathogenicity determination of MoSEC24-2 gene deletion and point mutation mutants of rice blast fungus

[0083] 1. The method of spraying rice leaves with rice blast fungus spore liquid is as follows:

[0084] 1) First, induce the production of conidia by the rice blast fungus Guy11. Inoculate the mycelial blocks of rice blast fungus Guy11 on CM medium onto SDC medium and incubate in the dark at 28°C for 4 days. Then, scrape off the surface mycelium and induce conidia under black light for 3 days to obtain conidia. For the preparation method of the conidial medium, please refer to Example 1.2.

[0085] 2) Rice seedlings cultured in the greenhouse for 14 days were used for spray inoculation experiments. 4 ml of conidia were collected from the SDC plates, at a concentration of 5 × 10⁻⁶. 4 The solution, containing 0.2% (w / v) gelatin per ml, was sprayed onto rice leaves and cultured in the dark with humidity for 24 hours, followed by alternating light and dark culture for 5-7 days.

[0086] 3) Calculate the affected area and the number of lesions.

[0087] 4) Each treatment is repeated three times.

[0088] 2. The method of inoculating rice seedlings with rice blast fungus spore liquid is as follows:

[0089] 1) First, induce the rice blast fungus to produce conidia. For specific methods, see Implementation Case 3.1.

[0090] 2) Rice seedlings cultured in a greenhouse for 20 days were used for inoculation experiments. Conidia were collected from the SDC plates at a concentration of 1×10⁻⁶. 5 Inject the cells / ml into the stems of rice seedlings, culture in the dark with moisture for 24 hours, and then culture in alternating light and dark conditions for 5-7 days.

[0091] 3) Calculate the affected area and the number of lesions.

[0092] 4) Each treatment is repeated three times.

[0093] The results of rice spray inoculation experiments showed that the ΔMosec24-2 mutant, the functional domain formed by amino acids 313-351, the functional domain formed by amino acids 648-730, and the point mutations at the K560R and K754R ubiquitination sites (ΔMosec24-2 / MoSEC24-2) were effective. 2R ), F240+E241+H243+Q257 combinatorial point mutation (ΔMosec24-2 / MoSEC24-2) L4 ) or Q358+R364(ΔMosec24-2 / MoSEC24-2) L12 The mutant with combined point mutations showed a significant reduction in lesions compared to the wild-type Guy11 and complementary strains. Figure 2 China A and Figure 3(A); and, the mutant showed significantly lower disease lesion area (DLA) than the wild-type Guy11 and its complementary strain (A). Figure 2 China B and Figure 3 (B); Simultaneously, the relative fungal growth of pathogens in the leaves was also statistically analyzed, revealing that the mutant strain was significantly lower than the wild-type Guy11 and its complementary strain. Figure 2 C and Figure 3 (C). This result indicates that MoSec24-2 regulates the pathogenicity of rice blast fungus, and that K560, K754, F240, E241, H243, Q257, Q358, and R364 are important active sites of MoSec24-2, which are crucial for its normal biochemical function and fungicide screening.

[0094] Example 5: Determination of the infection and mycelial expansion ability of MoSEC24-2 gene deletion mutant

[0095] The experimental method for rice blast fungus infection is detailed in Example 3. To explain the above-mentioned ΔMosec24-2 mutant, and the K560R and K754R ubiquitination site point mutations (ΔMosec24-2 / MoSEC24-2), further details are provided. 2R The decrease in pathogenicity is due to a reduction in the number of typical lesions and their inability to spread normally. Infection experiments were conducted in rice leaf sheaths. After 24 and 48 hours of infection, 100 appressorium infection sites were observed and graded (Grade I: no infection; Grade II: formation of primary infection hyphae; Grade III: formation of secondary infection hyphae, but without spreading to adjacent cells; Grade IV: infection hyphae spread to adjacent cells). Figure 4 (A). In the wild-type neutral and complementary strains, 80% of infection sites formed grade III and IV infectious hyphae, while in the mutant, less than 30% of infection sites formed grade III and IV infectious hyphae. Furthermore, the mutant hyphae failed to spread to neighboring cells within 48 hours of infection, whereas over 80% of the wild-type hyphae spread to neighboring cells. Figure 4 (B) The above results indicate that K560 and K754 are important active sites of MoSec24-2 and are essential for the growth of infecting hyphae, while the normal expansion of infecting hyphae is key to the formation of typical lesions of rice blast fungus.

[0096] Example 6: The MoSEC24-2 gene deletion mutant cannot inhibit the accumulation of reactive oxygen species in the host.

[0097] DAB (3,3′-diamino-benzidine) staining method: For the specific method of infecting rice leaf sheaths with rice blast fungus, please refer to Example 4. Rice leaf sheaths infected for 24 hours were stained with 1 mg / ml DAB (pH 3.5) for 8 hours in the dark at room temperature, and then destained with ethanol / acetic acid (94:4) for 1 hour. The inner epidermis of the leaf sheath was then torn off with tweezers for microscopic observation.

[0098] DAB staining of infected rice cells with reactive oxygen species (ROS) revealed that no ROS were produced in the leaf sheaths of wild-type strain Guy11, while a large amount of ROS accumulated in the leaf sheath cells of rice infected with the ΔMosec24-2 mutant. Figure 5 (A); Statistical analysis of 100 infection sites revealed that less than 15% of the leaf sheaths in the wild type produced reactive oxygen species (ROS), while more than 50% of the leaf sheath cells in the mutant produced ROS. Figure 5 (B). The results showed that MoSec24-2 could inhibit the production of reactive oxygen species in the host and promote the spread of infectious hyphae.

[0099] Example 7: Pathogenicity determination of Fusarium head blight FgSEC24-2 gene deletion and point mutation mutants

[0100] For details on the preparation of the sporulation medium, please refer to Example 1.2. For details on the method for inducing sporulation, please refer to 4.1, except that the SDC medium is replaced with PDA medium. In a spray chamber, wheat (cultivar Zhengmai 0943) at the flowering stage was injected with a suspension of wild-type strain PH-1 and ΔFgsec24-2 mutant spores (1×10⁻⁶). 5 The plants were inoculated with 20 seedlings per mL. The inoculated test plants were cultured in a greenhouse at 25°C and 90% relative humidity. The number of diseased spikelets per plant was counted 7-14 days after inoculation.

[0101] Experimental results showed that the number of spikelets infected with the ΔFgsec24-2 mutant after inoculation of wheat ears was significantly reduced compared to the wild type, and the lesions could not expand normally. Figure 6 This result indicates that the FgSEC24-2 gene regulates the pathogenicity of Fusarium.

[0102] Example 8: Pathogenicity determination of anthrax bacteria CgSEC24-2 gene deletion and point mutation mutants

[0103] The preparation method for the sporulation medium is detailed in Example 1.2. The method for inducing sporulation is detailed in 3.1, except that the SDC medium is replaced with PDA medium. In a spray chamber, soybean seedlings at the 3-leaf stage (cultivar Hefeng 47) were injected with a suspension of wild-type strain SMCG1#C and ΔCgsec24-2 mutant spores (1×10⁻⁶). 5The plants were inoculated with 20 seedlings per mL. The inoculated test plants were cultured in a greenhouse at 25°C and 90% relative humidity. The diseased area of ​​each plant type was counted 5-7 days after inoculation.

[0104] Experimental results showed that the ΔCgsec24-2 mutant, when inoculated onto soybean leaves, significantly reduced the diseased area compared to the wild type, and the lesions did not expand normally. Figure 7 (A); Statistical analysis revealed that the average number of diseased ears in the mutant strain was 2, while the average number in the wild-type and complementary strains was 11. Figure 7 (B) This result indicates that the CgSEC24-2 gene regulates the pathogenicity of Fusarium.

[0105] Example 9: Designing and screening fungicides using rice blast fungus, Fusarium wilt fungus, and anthracnose fungus Sec24-2 as targets.

[0106] This experiment used AlphaFold2 to predict the tertiary structure of Sec24-2 proteins from *Oryza sativa*, *Fusarium*, and *Agropyron cristatum*. Figure 8 The study used AutoDockvina software to screen a library of 100,000 small molecule compounds. In this embodiment, the PDB file of the predicted Sec24-2 tertiary structure and the MOL2 file of the small molecule compound were submitted to AutoDockvina software. Utilizing the software's virtual docking function (including calculations of binding free energy and RMSD values; lower values ​​indicate a higher probability of binding), a small molecule compound 1438 (binding free energy -13.0 kcal / mol, mean RMSD value) capable of binding to and inhibiting Sec24-2 function was successfully screened. (Compound of Formula I). ​​Simultaneously, Sec24-2 was cloned from the cDNA of *Bacillus oryzae*, *Fusarium*, and *Anthracis* strains and constructed into the pET15b vector (Novagen, Madison, WI, USA). The above vector plasmid was expressed in *E. coli* BL21, and cultured in liquid LB medium (containing 100 μg / ml ampicillin) at 37°C until the OD600 reached 0.4-0.8. The culture temperature was lowered to 16°C, and 0.4 mM IPTG (isopropylβ-D-1-thiogalactopyranoside) was added to induce Sec24-2 protein expression. After 12 hours, the bacterial culture was collected by centrifugation at 5300g for 15 minutes. The precipitate was resuspended and lysed with 1 mM lysis buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl, 10 mM imidazole), followed by sonication. The supernatant was collected as the Sec24-2 protein. Protein expression was detected by Western blotting, and the binding of small molecule compound 1438 to Sec24-2 was verified by microscale thermophoresis (MST). MST results showed that the binding constant Kd of compound 1438 to FgSec24-2 was 0.17 μM, significantly lower than that of FgSec24-2. F322A / Q354A Mutant; similarly, the binding constant Kd of 1438 to CgSec24-2 was 0.43 μM, significantly lower than that of FgSec24-2. F324A / Q355A mutant ( Figure 8 The result indicates that 1438 can effectively bind to FgSec24-2 and CgSec24-2. Note: The smaller the Kd value, the stronger the binding ability.

[0107]

[0108] Example 10: Method for using compound 1438 to control or prevent plant pathogenic microbial infection

[0109] In a spray chamber, rice (susceptible variety CO39), wheat (cultivar Zhengmai 0943), and soybean (cultivar Hefeng 47) seedlings at the three-leaf stage were sprayed with a custom-synthesized compound 1438 from ChemBridgh and a control solvent (DMSO). Spores (1×10⁻⁶) were detected 24 hours after application. 5 The plants were inoculated with a suspension of (number of plants / mL). The inoculated test plants were cultured in a greenhouse at 25°C and 90% relative humidity. When an appropriate level of disease appeared on the untreated control plants (7-14 days after application), the leaf area affected was counted and the inhibition rate was calculated as follows: Inhibition rate = (affected leaf area of ​​control group - affected leaf area of ​​treated group) / affected leaf area of ​​control group.

[0110] Experimental results showed that compound 1438 had a significant preventive effect against rice blast, and the affected area of ​​rice blast decreased significantly with increasing agent concentration. Figure 9 A) Statistical data on diseased area (DLA) showed that the average diseased area without treatment (DMSO) was nearly 50%, the average diseased area treated with 100 μM DMSO was 36%, 200 μM was 30%, 500 μM was 17%, and 1000 μM was 8%. Figure 9 In the treatment of fungal infections (B), the treatment significantly reduced the affected area. Fungal biomass analysis revealed that the average biomass was 25 μM without treatment (DMSO), 18 μM after 100 μM treatment, 15 μM after 200 μM treatment, 8 μM after 500 μM treatment, and 3 μM after 1000 μM treatment. Figure 9 (B) indicates that compound 1438 significantly inhibits fungal growth in rice. 1438 also showed significant control efficacy against anthracnose. Figure 10 In the study of lesion diameter (A), statistical data showed that the average diameter of lesions without treatment (DMSO) was 2.4 cm, the average diameter of lesions treated with 200 μM DMSO was 1.6 cm, the average diameter of lesions treated with 500 μM DMSO was 1.2 cm, and the average diameter of lesions treated with 1000 μM DMSO was 0.8 cm. Figure 10 (See Figure B). It is evident that the treatment significantly inhibited the infection of anthracnose bacteria. Similarly, 1438 also demonstrated significant control over Fusarium head blight. Figure 10 C) The statistics of the number of diseased ears showed that the average number of diseased ears without treatment (DMSO) was 11, the average number of diseased ears with 100μM DMSO treatment was 7, the average number of diseased ears with 200μM treatment was 5, the average number of diseased ears with 500μM treatment was 3, and the average number of diseased ears with 1000μM treatment was 2. Figure 9 As shown in Figure B), the treatment with the agent can significantly inhibit the infection of Fusarium graminearum.

[0111] These results indicate that compound 1438 has the potential to be developed as a fungicide, and further optimization of its skeleton is expected to yield a novel fungicide with broad-spectrum fungicidal activity against different pathogenic fungi.

[0112] Notably, the amino acid sequences of the Sec24-2 protein in different fungi were obtained through NCBI blastP search. Phylogenetic analysis was performed using MEG7 software, and sequence homology analysis was conducted using BioEdit software. The results showed that the Sec24-2 protein is highly conserved across different pathogenic fungi. Figure 10This result indicates that the Sec24-2 protein may have similar functions in different fungi as in *Oryza sativa*, regulating fungal pathogenicity. Therefore, the uses described in this invention should not be limited to the pathogens provided in the above embodiments, but can be extended to related fungi listed in the phylogenetic tree, such as *Sclerotinia*, *Aspergillus*, *Gaeumannomyces*, *Verticillium*, *Botrytis*, and *Neurospora*.

[0113] Finally, it should be noted that the above examples are merely a few specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. The use of reagents for knocking out the FgSEC24-2 gene of Fusarium in the control of wheat scab caused by Fusarium graminearum, characterized in that, The nucleotide sequence of the FgSEC24-2 gene is shown in SEQ ID NO:

5.

2. The application of reagent for knocking out CgSEC24-2 gene of anthracnose fungus in preventing and treating soybean anthracnose caused by Colletotrichum gloeosporioides, characterized in that, The nucleotide sequence of the CgSEC24-2 gene is shown in SEQ ID NO:

6.

3. Use according to claim 1 or 2, characterized in that, The reagent is used to achieve the stated purpose through gene editing.

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