Application of MoMtg1 protein and its encoding gene in regulating the growth, development and pathogenicity of rice blast fungus

Abstract

The present invention discloses the application of MoMtg1 protein and its encoding gene in regulating the growth and pathogenicity of rice blast fungus. MoMTG1 Gene deletion will lead to slow growth of rice blast fungus, reduced spore production, deformed conidia, and a significant decrease in pathogenicity to susceptible rice. MoMTG1 The small molecule compound mebendazole obtained by virtual screening has a good control effect on rice blast by targeting the encoded protein. MoMTG1 The encoded protein can be used as a drug target and has good application prospects in the development of new targeted fungicides and disease prevention and control.

Description

Technical Field

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

[0002] Rice blast fungus ( Magnaporthe oryzae ) is a devastating fungal disease in rice production, resulting in 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 the rice blast fungus mainly begins when the conidia contact the rice surface (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, which recognize host surface signals and form appressoria at their tips. These germ tubes then differentiate into infection spikes that penetrate the 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). Primary infection hyphae then expand within the host cells, forming typical spindle-shaped lesions on the surface of rice leaves after 4–5 days. Under favorable conditions, numerous aerial hyphae and conidiophores develop on the lesions, releasing numerous conidia, thus initiating the next round of infection (Dean RA., Talbot NJ., Ebbole DJ., et al. The genome sequence of the rice blast fungus). Magnaporthe grisea . Nature, 2005, 434: 980–986).

[0003] Currently, the main means of controlling related diseases is through planting disease-resistant varieties and applying chemical pesticides. However, the complex pathotypes of pathogens in the field and the rapid population variation often lead to the loss of disease resistance in disease-resistant varieties. Furthermore, the irrational application of chemical agents can lead to environmental pollution and the development of drug resistance in pathogens. Therefore, there is an urgent need to develop new targeted pesticides that are highly effective, low-toxic, highly specific, and less susceptible to drug resistance. Currently, the number of molecular targets for pathogens that can serve as new targeted pesticides is very limited, which is a bottleneck and challenge facing the development of targeted pesticides in my country. Therefore, revealing the development and pathogenicity mechanisms of pathogens at the molecular level, identifying key pathogenic proteins, and combining structural biology, pesticide science, and other disciplines to screen and design small molecule compounds are expected to provide important targets and potential lead compounds for the development of new targeted pesticides, thus breaking the bottleneck of new targeted pesticide development in my country.

[0004] In this study, we identified a hypothetical protein encoding gene, MoMTG1, from the rice blast fungus. Deletion of this gene slows the growth and development of the fungus and significantly reduces its pathogenicity to rice. This gene also has no homologous genes in humans, animals, or plants, suggesting its potential as a target for drug development. A small molecule compound, screened based on the structure of the MoMTG1-encoded protein, showed significant inhibitory effects on the development of rice blast. Therefore, the MoMtg1 protein has the potential to serve as a target for the development of novel targeted fungicides and to be applied in the control of plant pathogenic fungal diseases. Summary of the Invention

[0005] In view of the deficiencies of 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 the rice blast fungus.

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

[0009] (A1) Application in controlling rice blast;

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

[0011] (A3) Application in screening fungicides for 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 in the preparation of products for cultivating rice blast fungi with reduced pathogenicity;

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

[0016] (A8) Use in the preparation of 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 substituting 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 to 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 gene encoding the protein can be obtained by deleting one or several codons for amino acid residues from the DNA sequence shown in SEQ ID No. 2, and / or performing missense mutations of one or several base pairs, and / or linking the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0027] In specific embodiments, "homology" includes amino acid sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher homology to the amino acid sequence shown in SEQ ID No. 1 of the present invention.

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

[0029] (A1) Application in controlling rice blast;

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

[0031] (A3) Application in screening fungicides for 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 in the preparation of products for cultivating rice blast fungi with reduced pathogenicity;

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

[0036] (A8) Use in the preparation of 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;

[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 expressing (C1).

[0049] In a specific embodiment, the nucleic acid molecule (B1) is the gene 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 greater, or 85% or greater, or 90% or greater, or 95% or greater identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in SEQ ID No. 1 of the present invention. Identity can be evaluated by the naked eye or by 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 greater identity can be 80%, 85%, 90% or greater identity.

[0051] Loss of the MoMTG1 gene leads to slower vegetative growth, reduced asexual reproduction, and abnormal conidia morphology in the rice blast fungus. Further studies revealed that the ∆Momtg1 mutant significantly reduced its pathogenicity, with significantly fewer lesions on rice leaves and lesions that failed to expand normally. These results suggest that the MoMTG1 gene plays a crucial 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 functionally conserved and is a conserved pathogenic factor.

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

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

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

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

[0057] The present invention discovered that the small-molecule fungicide compound mebendazole, obtained through virtual screening based on the predicted structure of the MoMtg1 protein, specifically binds to MoMtg1 and significantly inhibits rice blast infection. This suggests that the MoMtg1 protein has the potential to be developed as a novel fungicide target and can be used in the screening and design of targeted fungicides. Furthermore, mebendazole may have promising applications in controlling 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 described above, wherein the reduction of the expression level and / or activity of the MoMtg1 protein described 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 a homologous recombination fragment 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 scope of protection of the present invention.

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

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

[0064] (E2) Application 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 to regulate the growth, development, and pathogenicity of the rice blast fungus. Knockout experiments on wild-type rice blast fungus (Magnaporthe oryzae) revealed that deletion of the MoMTG1 gene leads to slower vegetative growth, decreased asexual reproduction, and abnormal conidia morphology. The ∆Momtg1 mutant exhibits significantly reduced pathogenicity, with significantly fewer lesions on rice leaves, and lesions that fail to expand normally. This suggests that MoMTG1 is not only a key regulatory gene for rice blast pathogenicity, but also holds broad promise for the prevention and control of plant fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 : Phylogenetic 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 after 20 hours of induction on a glass slide.

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

[0071] Figure 5 :Molecular docking model 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 a mutation in the binding site bsm The three-dimensional structure of the two groups 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 mebendazole analyzed by microcalorimetry.

[0072] Figure 6 :The effect of small molecule mebendazole on controlling rice blast. Figure 6 Figure A shows that Guy11 conidia suspensions containing different concentrations of mebendazole were sprayed onto rice leaves. 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 an aqueous solution containing different concentrations of mebendazole was sprayed on rice leaves 12 hours later and then inoculated with the conidia suspension of Guy11. 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] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The reagents or instruments used without indicating the manufacturer are considered to be 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 are only present in the Ascomycota, mainly distributed in the Eurotiomycetes, Dothideomycetes, Leotiomycetes and the Sordariomycetes where MoMtg1 is located. They are also sporadically distributed in the 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] Based on the MoMTG1 gene (MGG_03546) database in the FungiDB database, approximately 1000 bp upstream and downstream of the MoMTG1 coding region were selected. Genomic DNA from Guy11 was used as a template and PCR amplified using primers MoMTG1ko-LF-1 / MoMTG1ko-LF-2 and MoMTG1ko-RF-1 / MoMTG1ko-RF-2 to obtain upper and lower homology arms. A 1346 bp fragment of the hygromycin phosphotransferase gene (HPH) was amplified using primers HPH-1 / HPH-2 using the pCX62 vector as a template. Then, the upper and lower homologous arm fragments of MoMTG1 and the hygromycin phosphotransferase gene (HPH) fragment obtained by amplification in the previous step were used as templates. The HPH fragment was sequentially connected to the upper and lower homologous arms by Overlap PCR. The primers used were MoMTG1ko-LF / MoMTG1ko-RF. 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] PCR reaction conditions were as follows: 95°C for 5 min, 35 cycles of 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min, followed by 72°C for 7 min and storage at 10°C to obtain amplified products. The primer sequences are shown in Table 1.

[0084] Overlap PCR reaction system is:

[0085]

[0086] PCR reaction conditions were as follows: 95°C for 5 min, 35 cycles of 95°C for 30 s, 54°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min and storage at 10°C to obtain amplified products. The primer sequences are shown in Table 1.

[0087]

[0088] For protoplast transformation of the rice blast fungus, first prepare mycelial pellets of the wild-type strain Guy11 in 75 mL of liquid CM medium. The pellets are collected by filtration and pressed dry with absorbent paper. The pellets are then transferred to a 0.7 M sodium chloride solution containing 0.2 g of chitinase and incubated at 30°C and 70 rpm for 2 h. The enzymatic hydrolyzate is filtered through a single layer of Miracloth (EMDMillipore Corp., 475855-1R) and collected by centrifugation at 3300 rpm to obtain Guy11 protoplasts. The protoplasts are resuspended in STC buffer and diluted to the appropriate concentration. The protoplasts are then observed under a 20x microscope to confirm their state and concentration. The pellets are then aliquoted into sterile 10 mL centrifuge tubes, 150 μl per tube. Subsequently, at least 4 μg of the knockout fragment or at least 2 μg of the plasmid are added to each tube, gently mixed, and allowed to 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 minutes. Then add 6 mL of liquid TB3 medium and incubate at 30°C, 70 rpm for 1-2 hours. After incubation, mix the incubated protoplasts with 40 mL of solid TB3 medium containing the appropriate antibiotic and invert the plate. For knockout cells, add hygromycin B to a final concentration of 150 μg / mL. After solidification, add 50 mL of solid TB3 medium containing double the concentration of the appropriate antibiotic. After the plate cools and solidifies, incubate inverted in a 28°C incubator for at least 4 days. Once a single colony appears on the plate, transfer a small amount of mycelial mass to a CM plate containing the same resistance for further verification.

[0089] 2) Southern blot analysis of knockout mutants

[0090] Genomic DNA was extracted from wild-type Guy11 and candidate knockout mutants. Primers were designed based on sequence information and used to amplify the corresponding DNA hybridization probes. Primers HPH-1 / HPH-2 were used to amplify a 1346-bp hygromycin probe, Probe 2, and primers MoMTG1ko-F / MoMTG1ko-R were used to amplify a 400-bp gene probe, Probe 1. PCR reaction conditions were: 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 56°C for 30 s, and 68°C for 30 s, followed by 72°C for 7 min and storage at 10°C. Amplification products were obtained. Primer sequences are shown in Table 1. The test genomes were digested overnight at 37°C with the restriction endonuclease EcoRV. A small amount of the digested product was run on a gel the following morning for verification. After complete genome fragmentation, two volumes of ethanol were added and the fragments were incubated at -20°C for at least 1 h. The precipitate was then collected by centrifugation at 13,000 rpm for 10 min. After the ethanol has completely evaporated, add 20-50 μL of enzyme-free sterile water to dissolve the product. The product to be tested is examined by gel electrophoresis at a constant voltage of 25 V. Subsequently, the DNA probe is DIG-labeled according to the methods and reagents specified in the Digoxigenin Assay Kit (Roche) and hybridization detection is performed.

[0091] 3) Obtaining knockout mutant complementation strains

[0092] To construct the complementing vector pYF11-MoMTG1-GFP, first linearize the pYF11-GFP vector with the restriction endonuclease Xho I. For detailed steps, refer to the NEB product manual. Using Guy11 genomic DNA as a template, primers pYF11-MoMTG1C-F and pYF11-MoMTG1C-R were used to amplify the 4500 bp MoMTG1C fragment.

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

[0094]

[0095] PCR reaction conditions were as follows: 95°C for 5 min; 95°C for 30 s, 57°C for 30 s, and 72°C for 2 min 30 s, for a total of 35 cycles; 72°C for 7 min and storage at 10°C. The amplified product, MoMTG1C, was obtained. Subsequently, the recombinant vector pYF11-MoMTG1-GFP was constructed using the homologous recombinase 2× ClonExpress Mix. For detailed in vitro homologous recombination methods, refer to the manufacturer's instructions (Vazyme, C115). The recombinant product was transformed into JM109 competent cells. After resistance screening, positive clones were sent to a biotechnology company for sequencing. Sequencing results were compared with the recombinant vector sequence map to obtain the recombinant vector pYF11-MoMTG1-GFP. The vector was then transformed into the corresponding knockout mutant using PEG-mediated protoplast transformation. Recombinant transformants were initially screened with bleomycin. Recombinant transformants were then identified based on resistance, GFP fluorescent tagging, and relevant phenotypes. 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 be observed at the corresponding size position. After hybridization with Probe 2, wild-type Guy11 could not be observed, 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 out 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 also showed abnormalities, while the complemented strain MoMTG1-C recovered to a level close to that of the wild-type ( Figure 3 Figure A in the figure).

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

[0101] Prepare the activated test strain. Use a microporator to punch a plate at the edge of the colony and transfer it to a 70 mm SDC plate. Incubate in a 28°C incubator for 5-7 days. Use a scalpel blade to cut the colony longitudinally along the edge to obtain a cross-section. Remove the cross-section and place it on a glass slide. Induce under black light for 12-24 hours, ensuring moisture retention. Then, place the slide containing the cross-section of the sporulating plate under an inverted microscope to photograph and observe and record sporulation.

[0102] Prepare separate SDC plates inoculated with the test strain and incubate them in a 28°C incubator for 5-7 days, then induce them under black light for 3 days. After measuring and calculating the colony area, collect as many conidia as possible with sterile water and count them using a hemocytometer to estimate the number of conidia in the spore suspension. Finally, calculate the number of conidia per unit area based on this data. A small amount of the collected conidia is aspirated and prepared as a temporary slide. Observe the spore morphology under an inverted microscope and photograph it.

[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 the conidia using a hemocytometer and adjust the concentration to 1×10 5 spores / mL, and 1 / 10 volume of 2% gelatin solution was added and shaken thoroughly. CO39 rice seedlings cultured in the greenhouse for 10-14 days were spray-inoculated with 5 mL of the test strain spore suspension per pot. After spray inoculation, the rice seedlings were incubated in a dark, sealed environment at 28°C for 24 hours, then transferred to a 12-hour light / dark cycle incubator, maintaining moisture during this period. After 5-7 days, the rice plants were observed and counted for disease status. Leaves were cut, 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 5Prepare 5-6 day-old four-row barley seedlings. Select well-grown, close-fitting leaves, cut them, and secure the ends with moistened filter paper in a 150 mm diameter round plastic dish. Add 25 μL of the conidia suspension of the test strain to the barley leaves, then incubate in a dark environment at 28°C for 24 hours. Then, transfer to a 12 h / 12 ​​h light / dark cycle incubator, ensuring moisture retention. Observe and photograph after 4-6 days.

[0109] The results showed that only sporadic lesions that could not expand normally were observed on rice leaves inoculated with ΔMomtg1, while a large number of typical expanding lesions were produced on rice leaves inoculated with Guy11 and MoMTG1-C. Similar results were observed in barley droplet inoculation, with lesions caused by ΔMomtg1 infection being 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. The accuracy of the prediction is evaluated by the pTM value. A pTM score higher than 0.5 indicates that the overall predicted fold of the protein is likely to be similar to the true structure. Then download the prediction results to obtain the target protein structure predicted by Alphafold3. The results are as follows 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 for MBDZ was screened. Subsequently, the binding mode and binding site of MoMtg1 and MBDZ were analyzed using AutoDock Tools and visualized in Pymol software. The results showed that the predicted binding sites of MoMtg1 and MBDZ are 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, with a binding energy of -8.6 kcal / mol ( Figure 5 Figure A in the figure).

[0113] 3) Microcalorimetry experiments were used to verify the binding relationship between MoMtg1 and MBDZ. The results showed that MoMtg1 and MBDZ bind 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 between 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, and the MoMtg1 bsm The structure files of MoMtg1 and MoMtg1 were analyzed in Pymol. bsm The structures of MoMtg1 and MoMtg1 were aligned. bsm The structures of the two proteins 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 Figure B in the figure). Based on this, the MoMtg1 bsm 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 through a rice spray test. First, an appropriate concentration of the test small molecule in water was prepared. A 1 / 10 volume of 2% gelatin solution was added and evenly sprayed onto the leaves of the test rice seedlings. The seedlings were returned to the greenhouse for further incubation. Rice seedlings sprayed with sterile water containing an equal volume of gelatin solution served as controls. Twelve hours later, a conidia suspension of the wild-type Guy11 rice blast fungus was inoculated. Alternatively, an appropriate concentration of the small molecule was mixed with the conidia suspension and sprayed together to test the effectiveness of the test small molecule against rice blast.

[0115] The results showed that the number of lesions on rice leaves caused by the Guy11 conidia suspension supplemented 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 (Figure A in the figure). Meanwhile, the rice plants were pre-treated with MBDZ aqueous solutions at final concentrations of 0.5 ppm, 1 ppm, and 5 ppm, and inoculated with conidia 12 hours later. The results showed that six 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 applications (A1)-(A4): (A1) Application in controlling rice blast; (A2) Use in the preparation of products for preventing and controlling rice blast; (A3) Application in screening fungicides for rice blast; (A4) Application in the preparation of products for screening rice blast fungicides; The amino acid sequence of the protein is shown in SEQ ID No. 1; The application is achieved by inhibiting, reducing or silencing the expression of the gene encoding the protein.

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

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

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

5. Use of a biomaterial related to the protein of claim 1 in any one of the following (A1) to (A4): (A1) Application in controlling rice blast; (A2) Use in the preparation of products for preventing and controlling rice blast; (A3) Application in screening fungicides for rice blast; (A4) Application in the preparation of products for screening rice blast fungicides; The application is achieved by inhibiting, reducing or silencing the expression of the gene encoding the protein.

6. The use according to claim 5, characterized in that The biological material is any one of the following: (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 expressing (C1).

7. The use according to claim 1, characterized in that The nucleic acid sequence encoding the protein of claim 1 is shown in SEQ ID No.

2.

8. A method for reducing the pathogenicity and / or conidia yield and / or growth rate of rice blast fungus, 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 of claim 1 in the recipient rice blast fungus by using homologous recombination is to introduce a homologous recombination fragment for homologous recombination into the protoplast of the recipient rice blast fungus.

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