Application of MoMtg1 protein and coding gene thereof in regulation and control of growth and development and pathogenicity of magnaporthe oryzae

Abstract

The invention discloses application of MoMtg1 protein and a coding gene thereof in regulation and control of growth, development and pathogenicity of magnaporthe oryzae. The deletion of the MoMTG1 gene can lead to slow growth of magnaporthe oryzae, reduction of sporulation quantity, conidium malformation and great reduction of pathogenicity to susceptible rice. The micromolecular compound mebendazole obtained through virtual screening by taking the MoMTG1 coding protein as a target has a good prevention and treatment effect on rice blast. Therefore, the MoMTG1 coding protein can be used as a medicament target, and has a good application prospect in research and development of novel targeted bactericides and prevention and control of diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial genetic engineering and plant protection, and specifically relates to the application of MoMtg1 protein and its encoding gene in regulating the growth and development and pathogenicity of rice blast fungus. Background Art

[0002] Rice blast fungus Magnaporthe oryzae ) is a devastating fungal disease in rice production, causing a 10-30% reduction in global rice production each year (Yan X., & Talbot NJ. Investigating the cellbiology of plant infection by the rice blast fungus Magnaporthe oryzae .Current Opinion in Microbiology, 2016, 34: 147–153; Martin-Urdiroz M., Oses-Ruiz M., Lauren SR., et al. Investigating the biology of plant infection by the rice blast fungus Magnaporthe oryzae . Fungal Genetics and Biology, 2016,90: 61–68). In nature, the infection of rice by rice blast fungus mainly starts with the contact of conidia with the surface of rice (Wilson RA., Talbot NJ. Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nature Review Microbiology, 2009, 7: 185–195). Under high humidity conditions, conidia germinate to form germ tubes, recognize host surface signals and form attachment cells at their tips, and then differentiate into infection spikes to penetrate host epidermal cells (Hamer JE., Howard RJ., Chumley FG., et al. A mechanism for surface attachment in spores of a plant pathogenic fungus. Science, 1998, 239: 288–90). Subsequently, primary infection hyphae continue to expand in host cells, and after 4-5 days, typical spindle-shaped lesions are formed on the surface of rice leaves. Under suitable conditions, a large number of aerial hyphae and conidiophores grow on the lesions, releasing a large number of conidia to carry out the next round of infection cycle (Dean RA., Talbot NJ.,Ebbole DJ., et al. The genome sequence of the rice blast fungus Magnaporthe grisea . Nature, 2005, 434: 980–986).

[0003] At present, the main means of production to prevent and control related diseases is to plant disease-resistant varieties and apply chemical pesticides. However, the pathogenic types of pathogens in the field are complex and the population mutates quickly, which often leads to the loss of disease resistance in disease-resistant varieties. At the same time, the irrational application of chemical agents will cause environmental pollution and hidden dangers such as drug resistance of pathogens. Therefore, it is urgent to develop new targeted pesticides that are highly efficient, low-toxic, highly specific, and not prone to drug resistance. At present, the number of molecular targets of pathogens that can be used as new targeted pesticides is very limited, which is also the bottleneck and challenge faced by the field of targeted pesticide creation in my country. Therefore, revealing the development and pathogenic mechanism of pathogens at the molecular level, exploring key pathogenic proteins, and combining structural biology, pesticide science and other disciplines to screen and design small molecule compounds is expected to provide important targets and potential lead compounds for the development of new targeted pesticides, breaking through the bottleneck of new targeted pesticide creation in my country.

[0004] In the present invention, we identified a hypothetical protein encoding gene MoMTG1 from rice blast fungus. The deletion of this gene leads to slow growth and development of rice blast fungus and significantly reduced pathogenicity to rice. At the same time, this gene has no homologous genes in humans and animals and plants, suggesting that it can be developed as a drug target. A small molecule compound based on the structural screening of the MoMTG1 encoding protein has a significant inhibitory effect on the occurrence of rice blast. Therefore, the MoMtg1 protein has the potential to be used as a target for the development of new targeted fungicides and is applied to the control of plant pathogenic fungal diseases. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides the use of MoMtg1 protein and its encoding gene in regulating the growth, development and pathogenicity of rice blast fungus.

[0006] The technical solution adopted by the present invention to solve the technical problem is:

[0007] In a first aspect, the present invention first provides a use of the MoMtg1 protein of rice blast fungus.

[0008] In a specific embodiment, the present invention provides a use of MoMtg1 protein in any one of the following (A1)-(A10):

[0009] (A1) Application in the control of rice blast;

[0010] (A2) Use in the preparation of products for preventing and controlling rice blast;

[0011] (A3) Application in screening fungicides against rice blast;

[0012] (A4) Application in the preparation of products for screening rice blast fungicides;

[0013] (A5) Application in breeding rice blast fungus with reduced pathogenicity;

[0014] (A6) Use of the method in the preparation of a product for cultivating rice blast fungus with reduced pathogenicity;

[0015] (A7) Application in the cultivation of rice blast fungus with reduced conidia yield;

[0016] (A8) Use of the method in preparing a product for cultivating rice blast fungus with reduced conidia yield;

[0017] (A9) Application in cultivating rice blast fungus with reduced growth rate;

[0018] (A10) Use in the preparation of a product for cultivating rice blast fungus with a reduced growth rate;

[0019] The MoMtg1 protein is any of the following proteins:

[0020] (D1) a protein having an amino acid sequence as shown in SEQ ID No. 1;

[0021] (D2) a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 1;

[0022] (D3) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 1;

[0023] (D4) A protein having 75% or more homology with the amino acid sequence shown in SEQ ID No. 1 and having the same function.

[0024] In a specific embodiment, in (D3), the substitution and / or deletion and / or addition of one or several amino acid residues is the substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0025] In a specific embodiment, in (D3), the protein can be artificially synthesized, or its encoding gene can be synthesized first and then expressed biologically.

[0026] In a specific embodiment, in (D3), the coding gene of the protein can be obtained by deleting one or several codons of amino acid residues in the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutation of one or several base pairs, and / or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0027] In a specific embodiment, "homology" includes an amino acid sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more homology with the amino acid sequence shown in SEQ ID No. 1 of the present invention.

[0028] In a second aspect, the present invention also provides a biomaterial related to the aforementioned protein for use in any one of the following (A1)-(A10):

[0029] (A1) Application in the control of rice blast;

[0030] (A2) Use in the preparation of products for preventing and controlling rice blast;

[0031] (A3) Application in screening fungicides against rice blast;

[0032] (A4) Application in the preparation of products for screening rice blast fungicides;

[0033] (A5) Application in breeding rice blast fungus with reduced pathogenicity;

[0034] (A6) Use of the method in the preparation of a product for cultivating rice blast fungus with reduced pathogenicity;

[0035] (A7) Application in the cultivation of rice blast fungus with reduced conidia yield;

[0036] (A8) Use of the method in preparing a product for cultivating rice blast fungus with reduced conidia yield;

[0037] (A9) Application in cultivating rice blast fungus with reduced growth rate;

[0038] (A10) Use in preparing a product for cultivating rice blast fungus with a reduced growth rate.

[0039] In a specific embodiment, the biological material is any one of the following:

[0040] (B1) a nucleic acid molecule encoding the protein described above;

[0041] (B2) an expression cassette containing the nucleic acid molecule described in (B1);

[0042] (B3) a recombinant vector containing the nucleic acid molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2);

[0043] (B4) a recombinant microorganism containing the nucleic acid molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B2);

[0044] (B5) a transgenic plant cell line containing the nucleic acid molecule described in (B1), or a transgenic plant cell line containing the expression cassette described in (B2);

[0045] (B6) transgenic plant tissue containing the nucleic acid molecule described in (B1), or transgenic plant tissue containing the expression cassette described in (B2);

[0046] (B7) a transgenic plant organ containing the nucleic acid molecule described in (B1), or a transgenic plant organ containing the expression cassette described in (B2);

[0047] (C1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein mentioned above;

[0048] (C2) A gene encoding the nucleic acid molecule expressed in (C1).

[0049] In a specific embodiment, the nucleic acid molecule (B1) is a gene as shown in 1) or 2) or 3) as follows: 1) the nucleotide sequence is the gene shown in SEQ ID No: 2; 2) a gene that has 75% or more identity with the nucleotide sequence defined in 1) and encodes the MoMtg1 protein; 3) a gene that hybridizes with the nucleotide sequence defined in 1) or 2) under stringent conditions and encodes the MoMtg1 protein.

[0050] The term "identity" as used herein refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in SEQ ID No. 1 of the present invention. Identity can be evaluated by the naked eye or computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences. The above-mentioned 75% or more identity can be 80%, 85%, 90% or more identity.

[0051] The loss of the MoMTG1 gene will lead to slower vegetative growth, reduced asexual reproduction ability, and abnormal conidia morphology of the rice blast fungus. Further studies have found that the pathogenicity of the ∆Momtg1 mutant is significantly reduced, the number of lesions caused by it on rice leaves is significantly reduced, and the lesions cannot expand normally. These results indicate that the MoMTG1 gene is a gene that plays an important role in the growth, development and pathogenicity of the rice blast fungus.

[0052] The present invention found that the MoMTG1 gene is highly conserved only in filamentous ascomycetes and does not exist in humans, animals and plants, indicating that MoMtg1 is also conservative in function and is a conservative pathogenic factor.

[0053] In specific embodiments, the vector may be a plasmid, a cosmid, a phage or a viral vector.

[0054] In a specific embodiment, the microorganism may be a fungus, a bacterium, such as rice blast fungus.

[0055] In a specific embodiment, the screening of rice blast fungicides is to use the aforementioned MoMtg1 protein as a drug target to screen rice blast fungicides.

[0056] In a specific embodiment, the fungicide is mebendazole.

[0057] The present invention found that the fungicide small molecule compound mebendazole obtained by virtual screening based on the predicted structure of MoMtg1 protein specifically binds to MoMtg1 and can significantly inhibit the infection of rice blast fungus to rice. This shows that MoMtg1 protein has the potential to be developed as a new fungicide target and can be used for the screening and design of targeted fungicides. At the same time, mebendazole may have good prospects for application in the prevention and control of rice blast.

[0058] In a third aspect, the present invention provides a method for cultivating transgenic rice blast fungi with reduced pathogenicity and / or reduced conidia yield and / or reduced growth rate, the method comprising the step of obtaining transgenic rice blast fungi by reducing the expression level and / or activity of the MoMtg1 protein mentioned above, wherein the reduction of the expression level and / or activity of the MoMtg1 protein mentioned above is achieved by knocking out, inhibiting or silencing the gene encoding the MoMtg1 protein in the recipient rice blast fungi.

[0059] In a specific embodiment, the gene encoding the MoMtg1 protein described above in the recipient rice blast fungus is knocked out by homologous recombination.

[0060] In a more specific embodiment, the method of knocking out the gene encoding the MoMtg1 protein in the recipient rice blast fungus by homologous recombination is to introduce the homologous recombination fragment used for homologous recombination into the protoplasts of the recipient rice blast fungus.

[0061] In a specific embodiment, the homologous recombination fragment also falls within the protection scope of the present invention.

[0062] In a fourth aspect, the present invention also protects the use of mebendazole in any of the following (E1)-(E2):

[0063] (E1) Application in controlling rice blast;

[0064] (E2) Use in the preparation of products for preventing and controlling rice blast.

[0065] Beneficial Effects

[0066] The MoMtg1 protein and its encoding gene provided by the present invention are used in regulating the growth and development and pathogenicity of rice blast fungus. Through knockout experiments on wild-type rice blast fungus (Magnaporthe oryzae), it was found that the loss of the MoMTG1 gene would lead to slower vegetative growth, decreased asexual reproduction ability, and abnormal conidia morphology of rice blast fungus. The pathogenicity of the ∆Momtg1 mutant was significantly reduced, the number of lesions caused by it on rice leaves was significantly reduced, and the lesions could not expand normally, indicating that MoMTG1 is not only an important regulatory gene for the pathogenicity of rice blast fungus, but also has broad application prospects in the prevention and treatment of plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 :Evolutionary tree analysis of MoMtg1 protein and its homologous proteins.

[0068] Figure 2 : Schematic diagram of the construction of knockout mutants of the MoMTG1 gene of rice blast fungus, wherein, Figure 2Figure A is a schematic diagram of the MoMTG1 knockout strategy in the rice blast fungus genome; Figure 2 Figure B shows Southern blot analysis of gene knockout mutants using MoMTG1 gene probe and HPH probe, respectively.

[0069] Figure 3 :The vegetative growth, sporulation analysis and conidia morphology observation of wild-type strain Guy11, mutant ∆Momtg1 and complemented strain MoMTG1-C, among which, Figure 3 Panel A shows the morphology of Guy11, ΔMomtg1, and MoMTG1-C after 7 days of culture on CM plates; Figure 3 Figure B shows the conidia production and conidia morphology of the corresponding strain on a glass slide after 20 hours of induction.

[0070] Figure 4 : Pathogenicity analysis of wild-type strain Guy11, mutant ∆Momtg1 and complemented strain MoMTG1-C in rice and barley.

[0071] Figure 5 :Molecular docking mode and binding verification of small molecule mebendazole and MoMtg1, among which, Figure 5 Figure A shows the molecular docking analysis of mebendazole and MoMtg1; Figure 5 Figure B shows MoMtg1 and the protein MoMtg1 with mutation in the binding site bsm The three-dimensional structure of the samples was compared and the overall structural differences were quantified by the root mean square deviation. Figure 5 Figure C shows the affinity relationship between MoMtg1 and tobendazole analyzed by microcalorimetry experiment.

[0072] Figure 6 :The effect of small molecule mebendazole on controlling rice blast. Among them, Figure 6 Figure A shows that Guy11 conidia suspension containing different concentrations of mebendazole was sprayed onto rice leaves, and photos were taken on the 6th day after inoculation, and the proportion of lesion area on the leaves was analyzed using ImageJ; Figure 6 Figure B shows that the rice leaves were inoculated with the conidia suspension of Guy11 12 hours after the aqueous solution containing different concentrations of mebendazole was sprayed on them. Photos were taken on the 6th day after inoculation, and the proportion of the lesion area on the leaves was analyzed using ImageJ.

[0073] Figure 7 :MoMtg1 protein structure predicted based on AlphaFold3. DETAILED DESCRIPTION

[0074] In order to better understand the present invention, the following examples are further described, but the present invention is not limited thereto. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents or instruments used without indicating the manufacturer are all regarded as conventional products that can be purchased on the market.

[0075] Example 1, Phylogenetic tree analysis of MoMtg1

[0076] The amino acid sequence of MoMtg1 encoded by MoMTG1 was searched by BlastP in the NCBI database, and a phylogenetic tree was constructed in MEGA-X using the neighbor-joining method for all homologous protein amino acid sequences downloaded from Ensembl Fungi.

[0077] The results showed that MoMtg1 and its homologous proteins only exist in Ascomycota, mainly distributed in Eurotiomycetes, Dothideomycetes, Leotiomycetes and Sordariomycetes where MoMtg1 is located. They are also sporadically distributed in Pezizomycetes, Orbiliomycetes and Xylonomycetes.

[0078] Example 2: Obtaining knockout mutants and complementation strains of MoMTG1, ChMTG1 and FgMTG1

[0079] 1) Construction of knockout fragments and PEG-mediated protoplast transformation

[0080] According to the relevant information of MoMTG1 gene (MGG_03546) in FungiDB database, the sequences of about 1000 bp upstream and downstream of MoMTG1 coding region were selected, and the genomic DNA of Guy11 was used as template, and primers MoMTG1ko-LF-1 / MoMTG1ko-LF-2 and MoMTG1ko-RF-1 / MoMTG1ko-RF-2 were used to PCR amplify the upper and lower homology arm fragments. The hygromycin phosphotransferase gene (HPH) fragment with a size of 1346 bp was obtained by PCR amplification with primers HPH-1 / HPH-2 using vector pCX62 as template. Then, the MoMTG1 upper and lower homologous arm fragments and the hygromycin phosphotransferase gene (HPH) fragment obtained in the previous step were used as templates, and the HPH fragment was connected to the upper and lower homologous arms in sequence by Overlap PCR. The primers used were MoMTG1ko-LF / MoMTG1ko-RF, and finally an LF-HPH-RF fusion fragment of about 3400 bp was obtained, which can be used for gene knockout.

[0081] The conventional PCR reaction system is:

[0082]

[0083] The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 56°C for 30 s, 72°C for 1 min, for a total of 35 cycles; 72°C for 7 min, and stored at 10°C to obtain the amplified product. The primer sequences described above are shown in Table 1.

[0084] Overlap PCR reaction system is:

[0085]

[0086] The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 54°C for 30 s, 72°C for 2 min, for a total of 35 cycles; 72°C for 7 min, and 10°C storage to obtain the amplified product. The primer sequences described above are shown in Table 1.

[0087]

[0088] For the protoplast transformation method of rice blast fungus, first prepare the mycelial pellets of wild-type strain Guy11 with 75 mL liquid CM medium, collect by filtration and press dry with absorbent paper, transfer to 0.7 M sodium chloride solution dissolved with 0.2 g chitinase, and treat at 30°C, 70 rpm for 2 h. The enzymatic hydrolyzate is filtered with a single layer of Miracloth (EMDMillipore Corp., 475855-1R) and collected by centrifugation at 3300 rpm to obtain Guy11 protoplasts. Resuspend the protoplasts with STC buffer and dilute them to an appropriate concentration. Observe and confirm the state and concentration of the protoplasts under a 20x microscope, and dispense into sterile 10 mL centrifuge tubes, 150 μl per tube. Then add no less than 4 μg of the knockout fragment or no less than 2 μg of the plasmid to each tube, mix gently and let stand at room temperature for 25-30 min. Add 1 mL of PTC dropwise to each tube and mix thoroughly immediately. Let stand at room temperature for no more than 25 min, then add 6 mL of liquid TB3 medium and expand at 30°C, 70 rpm for 1-2 h. After expansion, mix the protoplasts with 40 mL of solid TB3 medium containing the corresponding antibiotics and pour them into plates. If it is a knockout, add hygromycin B with a final concentration of 150 μg / mL. After it solidifies, add 50 mL of solid TB3 medium containing double the concentration of the corresponding antibiotics. After the plate cools and solidifies, invert and culture it in a 28°C incubator for at least 4 days. After a single colony grows on the surface of the plate, pick a few mycelial blocks and transfer them to a CM plate with the same resistance for further verification.

[0089] 2) Southern blot analysis of knockout mutants

[0090] The genomic DNA of wild-type Guy11 and candidate knockout mutants was extracted, and primers were designed and amplified according to the sequence information. The hygromycin probe Probe2 with a size of 1346 bp was amplified with primers HPH-1 / HPH-2, and the gene probe Probe1 with a size of 400 bp was amplified with primers MoMTG1ko-F / MoMTG1ko-R. The PCR reaction conditions were: 95℃ for 5 min; 95℃ for 30 s, 56℃ for 30 s, 68℃ for 30 s, for a total of 35 cycles; 72℃ for 7 min, and stored at 10℃ to obtain the amplified product. The primer sequences are shown in Table 1. At the same time, the restriction endonuclease EcoRⅤ was selected to digest the test genome overnight at 37℃, and a small amount of the digestion product was taken the next morning for gel running verification. After the genome was completely cut, double volume of ethanol was added and placed at -20℃ for more than 1 h, and then centrifuged at 13000 rpm for 10 min to collect the precipitate. After the ethanol is completely evaporated, 20-50 μL of enzyme-free sterile water is added to dissolve the product. The product to be detected is detected by gel electrophoresis under a constant voltage of 25 V, and then the DNA probe is DIG-labeled according to the method and reagents in the instruction manual of the digoxin assay kit (Roche), and hybridization detection is performed.

[0091] 3) Obtaining knockout mutant complementation strains

[0092] The complementation vector pYF11-MoMTG1-GFP was constructed. First, the vector pYF11-GFP was linearized with restriction endonuclease XhoⅠ. For specific steps, see the NEB manual. Using Guy11 genomic DNA as a template, primers pYF11-MoMTG1C-F / pYF11-MoMTG1C-R were used to amplify the MoMTG1C fragment with a size of 4500 bp.

[0093] The PCR reaction system for MoMTG1C amplification is as follows:

[0094]

[0095] The PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, 72°C for 2 min30 s, for a total of 35 cycles; 72°C for 7 min, and stored at 10°C to obtain the amplified product MoMTG1C. Subsequently, the recombinant vector pYF11-MoMTG1-GFP was constructed using the homologous recombinase 2×ClonExpress Mix. For the specific in vitro homologous recombination method, please refer to the instruction manual (Vazyme, C115). The recombinant product was transferred into JM109 competent cells, and the obtained positive clones were sent to a biological company for sequencing after resistance screening. The sequencing results were compared and analyzed with the recombinant vector map sequence to obtain the recombinant vector pYF11-MoMTG1-GFP. The vector was transferred into the corresponding knockout mutant by PEG-mediated protoplast transformation, and the complementing transformants were preliminarily screened with bleomycin. Subsequently, the complementing transformants were further determined by combining resistance, GFP fluorescent label and related phenotypes. The primer sequences are shown in Table 1.

[0096] The results showed that after hybridization with Probe 1, wild-type Guy11 could show a single band, while mutant ΔMomtg1 could not observe a band at the corresponding size position. After hybridization with Probe 2, wild-type Guy11 could not observe a band, while mutant ΔMomtg1 could show a single band ( Figure 2 ). This indicates that a knockout mutant with a single copy of HPH has been successfully obtained.

[0097] Example 3, Growth rate determination

[0098] Prepare the activated test strains, use a hole puncher to punch bacterial discs at the edge of the colony, transfer them to 70 mm CM plates, and then invert them in a 28°C incubator. After 3-6 days, measure and record the colony diameters and take photos.

[0099] The results showed that compared with the wild-type Guy11, the growth rate of ΔMomtg1 on CM plates was significantly reduced, and the colony pigment on CM plates was also abnormal, while the complemented strain MoMTG1-C recovered to a level close to that of the wild-type ( Figure 3 Figure A in Figure 1).

[0100] Example 4: Determination of conidia production ability and observation of conidia morphology

[0101] Prepare the activated test strain, use a hole puncher to punch a bacterial plate at the edge of the colony and transfer it to a 70 mm SDC plate, place it in a 28°C incubator for 5-7 days, use a surgical blade to cut longitudinally along the edge of the colony to obtain a cross section, take it out and place it on a slide, induce it under a black light for 12-24 hours, and pay attention to moisture retention. Then place the slide containing the cross section of the spore-producing plate under an inverted microscope to take pictures, observe and record the spore production.

[0102] Prepare another SDC plate inoculated with the test strain, place it in a 28℃ incubator for 5-7 days, and then transfer it to a black light for induction for 3 days. After measuring and calculating the colony area, use sterile water to collect all the conidia as much as possible and count them with a hemocytometer to estimate the number of conidia in the spore suspension. Finally, calculate the number of spores per unit area of ​​the colony based on the obtained data. Take a small amount of the collected conidia, make a temporary slide, observe the spore morphology under an inverted microscope, and take pictures for record.

[0103] The results showed that the number of conidiophores of ΔMomtg1 was significantly reduced compared with the wild type, the spore production ability was reduced, and 75.2% of the conidia were deformed, which was significantly higher than that of the wild type and the complement strain ( Figure 3 Figure B in the figure).

[0104] Example 5: Determination of pathogenicity of rice blast fungus

[0105] 1) Rice spray inoculation

[0106] Prepare conidia plates of the test strain, rinse with 2-3 mL of sterile water and collect conidia by filtering through a layer of magic filter cloth, count with a hemocytometer and adjust the concentration to 1×10 5 / mL, and add 1 / 10 volume of 2% gelatin solution and shake well. Take CO39 rice seedlings cultured in the greenhouse for 10-14 days and spray inoculate them according to the standard of 5 mL spore suspension of the test strain per pot of rice. After spray inoculation, the rice seedlings are first moisturized in a dark sealed environment at 28℃ for 24 hours, and then transferred to a 12 h / 12 ​​h light and dark alternating incubator, and pay attention to moisturizing during the period. After 5-7 days, the disease situation of rice plants was observed and counted, and the leaves were cut and laid flat on a moist surface and photographed.

[0107] 2) Barley drip inoculation

[0108] Prepare a conidia suspension of the test strain and adjust the concentration to 1 × 10 5 / mL and add gelatin solution. Prepare four-angled barley seedlings that have been cultured for 5-6 days, select leaves that are well grown and close to each other, cut them off, and fix both ends with wet filter paper in a round plastic dish with a diameter of 150 mm. Add the conidia suspension of the test strain to the barley leaves, 25 μL per drop, and then place them in a dark environment at 28℃ for 24 hours of moisturizing culture, and then transfer them to a 12 h / 12 ​​h light-dark alternating incubator, paying attention to moisturizing during this period. Observe and take pictures after 4-6 days.

[0109] The results showed that only sporadic lesions that could not expand normally were observed on the rice leaves inoculated with ΔMomtg1, while a large number of typical expanded lesions were produced on the rice leaves inoculated with Guy11 and MoMTG1-C. Similar results were observed in barley droplet inoculation, and the lesions caused by ΔMomtg1 infection were significantly smaller than those of the wild type and complemented strains ( Figure 4 ).

[0110] Example 6: Small molecule screening, validation and efficacy determination based on the predicted structure of MoMtg1

[0111] 1) Use AlphaFold3 to predict the three-dimensional structure of MoMtg1, and use the pTM value to evaluate the accuracy of the predicted results. A pTM score higher than 0.5 indicates that the overall predicted folding of the protein may be similar to the true structure. Then download the prediction results to obtain the target protein structure predicted by Alphafold3. The results are shown in the figure below. Figure 7 shown.

[0112] 2) Based on the predicted three-dimensional structure of MoMtg1, Autodock vina was used to perform virtual screening of small molecule compounds, from which a small molecule with strong affinity to mebendazole (MBDZ) was screened out. Subsequently, AutoDockTools was used to analyze the binding mode and binding site of MoMtg1 and MBDZ, and the results were visualized in Pymol software. The results showed that the predicted binding sites of MoMtg1 and mebendazole were phenylalanine Phe at position 304, serine Ser at position 305, aspartic acid Asp at position 307, arginine Arg at position 363, and valine Val at position 366, and the binding energy between the two was -8.6 kcal / mol ( Figure 5 Figure A in Figure 1).

[0113] 3) The binding relationship between MoMtg1 and MBDZ was verified by microcalorimetry experiments. The results showed that MoMtg1 and MBDZ bound in vitro, with a dissociation constant Kd of 0.79 μM, while the empty tag protein SUMO did not bind to MBDZ ( Figure 5In order to further explore the binding mechanism of the two and verify the authenticity of the predicted binding sites, all five predicted binding sites on MoMtg1 were mutated to alanine Ala, which is non-polar, hydrophobic and chemically inert. The structure of the protein MoMtg1 after point mutation was predicted using AlphaFold3 to obtain MoMtg1 bsm In Pymol, MoMtg1 and MoMtg1 bsm The structures of MoMtg1 and MoMtg1 were aligned. bsm The structures of MoMtg1 still have a very high similarity, with a root mean square deviation (RMSD) of 0.332 Å, indicating that the mutation of the binding site does not significantly affect the protein structure of MoMtg1 ( Figure 5 Based on this, the MoMtg1 bsm The binding was verified by MBDZ. The results showed that MoMtg1 bsm The binding ability to MBDZ was significantly reduced ( Figure 5 Figure C in the figure).

[0114] 4) The efficacy of mebendazole was determined by the rice spray experiment. First, a suitable concentration of the test small molecule aqueous solution was prepared, and 1 / 10 volume of 2% gelatin solution was added and evenly sprayed on the leaves of the rice seedlings to be tested, and then returned to the greenhouse for continued cultivation. The rice seedlings sprayed with sterile water with an equal amount of gelatin solution were used as controls. After 12 hours, the conidia suspension of the wild type Guy11 of the rice blast fungus was inoculated, or the small molecule of the appropriate concentration was mixed with the spore suspension and sprayed together to test whether the test small molecule agent had a preventive and therapeutic effect on the occurrence of rice blast.

[0115] The results showed that the number of lesions on rice leaves caused by Guy11 conidia suspension with 0.5 ppm MBDZ was significantly reduced compared with the control group, and the inhibitory effect was dose-dependent, that is, as the concentration of mebendazole increased, the number of lesions also decreased significantly ( Figure 6 At the same time, the test rice plants were sprayed with MBDZ aqueous solution at final concentrations of 0.5 ppm, 1 ppm and 5 ppm as pretreatment, and conidia spray inoculation was performed 12 hours later. The results showed that 6 days after inoculation, the number and area of ​​leaf lesions were significantly reduced compared with the DMSO control group, and the protective effect was concentration-dependent ( Figure 6 Figure B in the figure).

[0116] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. The protein is used in any of the following (A1)-(A10): (A1) Application in the control of rice blast; (A2) Use in the preparation of products for preventing and controlling rice blast; (A3) Application in screening fungicides against rice blast; (A4) Application in the preparation of products for screening rice blast fungicides; (A5) Application in breeding rice blast fungus with reduced pathogenicity; (A6) Use of the method in the preparation of a product for cultivating rice blast fungus with reduced pathogenicity; (A7) Application in the cultivation of rice blast fungus with reduced conidia yield; (A8) Use of the method in preparing a product for cultivating rice blast fungus with reduced conidia yield; (A9) Application in cultivating rice blast fungus with reduced growth rate; (A10) Use in the preparation of a product for cultivating rice blast fungus with a reduced growth rate; The amino acid sequence of the protein is shown in SEQ ID No.

1.

2. The use according to claim 1, characterized in that: The protein is derived from the rice blast fungus ( Magnaporthe oryzae ).

3. The use according to claim 1 or 2, characterized in that: The method of screening rice blast fungicides is to use the protein described in claim 1 as a drug target to screen rice blast fungicides.

4. The use according to claim 3, characterized in that: The bactericide is mebendazole.

5. The biological material related to the protein of claim 1 is used in any one of the following (A1)-(A10): (A1) Application in the control of rice blast; (A2) Use in the preparation of products for preventing and controlling rice blast; (A3) Application in screening fungicides against rice blast; (A4) Application in the preparation of products for screening rice blast fungicides; (A5) Application in breeding rice blast fungus with reduced pathogenicity; (A6) Use of the method in the preparation of a product for cultivating rice blast fungus with reduced pathogenicity; (A7) Application in the cultivation of rice blast fungus with reduced conidia yield; (A8) Use of the method in preparing a product for cultivating rice blast fungus with reduced conidia yield; (A9) Application in cultivating rice blast fungus with reduced growth rate; (A10) Use in preparing a product for cultivating rice blast fungus with a reduced growth rate.

6. The use according to claim 5, characterized in that: The biological material is any one of the following: (B1) a nucleic acid molecule encoding the protein of claim 1; (B2) an expression cassette containing the nucleic acid molecule described in (B1); (B3) a recombinant vector containing the nucleic acid molecule described in (B1), or a recombinant vector containing the expression cassette described in (B2); (B4) a recombinant microorganism containing the nucleic acid molecule described in (B1), or a recombinant microorganism containing the expression cassette described in (B2), or a recombinant microorganism containing the recombinant vector described in (B2); (B5) a transgenic plant cell line containing the nucleic acid molecule described in (B1), or a transgenic plant cell line containing the expression cassette described in (B2); (B6) transgenic plant tissue containing the nucleic acid molecule described in (B1), or transgenic plant tissue containing the expression cassette described in (B2); (B7) a transgenic plant organ containing the nucleic acid molecule described in (B1), or a transgenic plant organ containing the expression cassette described in (B2); (C1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein of claim 1 or 2; (C2) A gene encoding the nucleic acid molecule expressed in (C1).

7. The use according to claim 6, characterized in that The nucleic acid molecule sequence in (B1) is shown as SEQ ID No.

2.

8. A method for cultivating transgenic rice blast fungus with reduced pathogenicity and / or reduced conidia yield and / or reduced growth rate, characterized in that: The method comprises the step of obtaining a transgenic rice blast fungus by reducing the expression level and / or activity of the protein described in claim 1, wherein the reduction of the expression level and / or activity of the protein described in claim 1 is achieved by knocking out, inhibiting or silencing the gene encoding the protein described in claim 1 in the recipient rice blast fungus.

9. The method according to claim 8, characterized in that The homologous recombination method is used to knock out the gene encoding the protein described in claim 1 in the recipient rice blast fungus.

10. The method according to claim 9, characterized in that The method of knocking out the gene encoding the protein described in claim 1 in the recipient rice blast fungus by using homologous recombination is to introduce the homologous recombination fragment used for homologous recombination into the protoplasts of the recipient rice blast fungus.

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