Application of effector protein moupe7 in regulating the pathogenicity of magnaporthe oryzae

Abstract

The application discloses application of an effector protein MoUPE7 in regulating Magnaporthe oryzae pathogenicity. The application constructs a knock-out vector of the gene MoUPE7 and introduces the vector into Magnaporthe oryzae protoplast, analyzes conidial germination and pathogenicity of the obtained knock-out mutant, and finds that the knock-out of the gene MoUPE7 delays conidial germination and appressorium formation of Magnaporthe oryzae, increases the sensitivity to H2O2, and significantly reduces the pathogenicity to rice. That is, inhibiting the expression of the effector protein MoUPE7 can reduce the pathogenicity of Magnaporthe oryzae and reduce the influence of Magnaporthe oryzae on the yield and quality of rice, and the reagent capable of inhibiting the expression of the effector protein MoUPE7 can be used for the prevention and treatment of rice blast. The application not only enriches the pathogenic mechanism of Magnaporthe oryzae, but also is favorable to the development of green prevention and control products of rice blast.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology. More specifically, it relates to the application of the effector protein MoUPE7 in regulating the pathogenicity of rice blast fungus. Background Technology

[0002] Rice blast is caused by the rice blast fungus (Bacillus oryzae). Magnaporthe oryzae Rice blast is a widespread disease of rice, occurring throughout the entire growth cycle and in all parts of the rice plant. Based on the timing and location of infection, it can be classified into different types, such as seedling blast, leaf blast, node blast, neck blast, and grain blast. Rice blast affects rice yield and quality, seriously threatening food security.

[0003] The widespread spread of rice blast relies on the complete infection cycle of *Magnaporda oryzae*, which mainly includes four processes: conidia attaching to the host leaf surface; conidia germinating to produce germ tubes and forming appressoriums required for infection; appressorium maturation forming infection nails that penetrate the host epidermis; and the further expansion of infection hyphae within the host cells. This infection cycle contains many pathogenic factors. Thoroughly exploring the pathogenic genes related to *Magnaporda oryzae* will not only contribute to a comprehensive understanding of its pathogenic molecular mechanisms but also facilitate the formulation of green control strategies and the development of related control reagents.

[0004] Although there are reports of effector proteins involved in regulating the pathogenicity of rice blast fungus, the pathogenic molecular mechanism of rice blast fungus is still incomplete, and the function of the effector protein MoUPE7 is not yet clear. Summary of the Invention

[0005] This invention discovers that the effector protein MoUPE7 can regulate the pathogenicity of *Magnapordica oryzae*. Inhibition of MoUPE7 expression significantly reduces the pathogenicity of *Magnapordica oryzae*, and also affects the germination of conidia and appressorium formation, increasing its sensitivity to H2O2. Therefore, this invention provides the application of the effector protein MoUPE7 in regulating the pathogenicity of *Magnapordica oryzae*.

[0006] The first objective of this invention is to provide the application of the effector protein MoUPE7 in regulating the pathogenicity of rice blast fungus.

[0007] A second objective of this invention is to provide the application of a reagent that inhibits the expression of the effector protein MoUPE7 in reducing the pathogenicity of rice blast fungus.

[0008] A third objective of this invention is to provide the use of a reagent that inhibits the expression of the effector protein MoUPE7 in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0009] A fourth object of the present invention is to provide the use of a product for knocking out the gene encoding the effector protein MoUPE7 in reducing the pathogenicity of rice blast fungus.

[0010] A fifth object of the present invention is to provide the use of products for knocking out the gene encoding the effector protein MoUPE7 in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0011] The sixth object of the present invention is to provide the application of a reagent for inhibiting the expression of the effector protein MoUPE7 in the prevention and control of rice blast.

[0012] A seventh object of the present invention is to provide the use of a reagent for inhibiting the expression of the effector protein MoUPE7 in the preparation of products for the prevention and control of rice blast.

[0013] An eighth object of the present invention is to provide the use of a product for knocking out the gene encoding the effector protein MoUPE7 in the prevention and control of rice blast.

[0014] A ninth object of the present invention is to provide the use of the product for knocking out the gene encoding the effector protein MoUPE7 in the preparation of a product for the prevention and control of rice blast.

[0015] The above-mentioned objective of this invention is achieved through the following technical solution:

[0016] This invention provides an effector protein that can regulate the pathogenicity of rice blast fungus, named effector protein MoUPE7, whose amino acid sequence is shown in SEQ ID NO.1. This invention constructs a gene... MoUPE7 (or) MoUPE7 Gene ， A knockout vector encoding the effector protein MoUPE7 was introduced into rice blast fungus protoplasts, and the knockout gene was found to be effective. MoUPE7 This will delay the germination of rice blast fungus conidia and the formation of appressorium, increase its sensitivity to H2O2, and significantly reduce its pathogenicity to rice. Therefore, this invention applies for the application of the protective effector protein MoUPE7 in regulating the pathogenicity of rice blast fungus.

[0017] The gene encoding the effector protein MoUPE7, i.e., the gene MoUPE7 Its application in regulating the pathogenicity of rice blast fungus should also be within the scope of protection of this invention.

[0018] Optionally, the gene MoUPE7 The gene symbol in the NCBI database is MGG_16869, and the sequence ID is XM_003712578.1.

[0019] This invention confirms gene knockout MoUPE7This can significantly reduce the pathogenicity of *Magnapordica oryzae* to rice. It is known that inhibiting the expression of the effector protein MoUPE7 can significantly reduce the pathogenicity of *Magnapordica oryzae* to rice. Therefore, this invention also claims protection for the use of reagents that inhibit the expression of the effector protein MoUPE7 in reducing the pathogenicity of *Magnapordica oryzae*.

[0020] The present invention also claims protection for the use of a reagent that inhibits the expression of the effector protein MoUPE7 in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0021] Optionally, the reagent is prepared using antisense gene technology or RNA interference technology to target genes. MoUPE7 Reagents required for expression inhibition.

[0022] Optionally, the reagent is siRNA, which can inhibit the rice blast fungus gene. MoUPE7 The expression aims to reduce the pathogenicity of rice blast fungus.

[0023] The present invention also claims protection for the use of products for knocking out the gene encoding the effector protein MoUPE7 in reducing the pathogenicity of rice blast fungus.

[0024] The present invention also claims protection for the use of products for knocking out the gene encoding the effector protein MoUPE7 in the preparation of products that reduce the pathogenicity of rice blast fungus.

[0025] Specifically, the product is a gene-related product. MoUPE7 Products required for functional knockout or knockout of the entire gene. Optionally, the product is a gene. MoUPE7 Knockout vector.

[0026] Optionally, the gene MoUPE7 The knockout vector was constructed using the filamentous fungal expression vector pCT74.

[0027] Specifically, the above application is for reducing the pathogenicity of rice blast fungus in rice.

[0028] Furthermore, the present invention also claims protection for the use of reagents for inhibiting the expression of the effector protein MoUPE7 or products for knocking out the gene encoding the effector protein MoUPE7 in delaying the germination of conidia and the formation of appressorium in *Blastomyces oryzae*.

[0029] The present invention also claims protection for the use of reagents for inhibiting the expression of the effector protein MoUPE7 or products for knocking out the gene encoding the effector protein MoUPE7 in improving the sensitivity of rice blast fungus to H2O2.

[0030] The present invention also claims protection for the use of a reagent that inhibits the expression of the effector protein MoUPE7 in the control of rice blast.

[0031] The present invention also claims protection for the use of a reagent that inhibits the expression of the effector protein MoUPE7 in the preparation of products for the prevention and control of rice blast.

[0032] The present invention also claims protection for the use of products for knocking out the gene encoding the effector protein MoUPE7 in the control of rice blast.

[0033] The present invention also claims protection for the use of products for knocking out the gene encoding the effector protein MoUPE7 in the preparation of products for the prevention and control of rice blast.

[0034] Specifically, the rice blast disease is caused by the rice blast fungus (Bacillus oryzae). Magnaporthe oryzae Caused by ).

[0035] The present invention has the following beneficial effects:

[0036] This invention constructs genes MoUPE7 The knockout vector was introduced into *Magnaporum oryzae* protoplasts. Analysis of the conidial germination, stress resistance, and pathogenicity of the resulting knockout mutants revealed that the knockout gene... MoUPE7 It delays the germination and attachment of blast fungus conidia and increases the fungus's sensitivity to H2O2, significantly reducing its pathogenicity to rice. In other words, inhibiting the expression of the effector protein MoUPE7 can reduce the pathogenicity of blast fungus and its impact on rice growth. Therefore, reagents that can inhibit the expression of the effector protein MoUPE7 can be used for the control of rice blast.

[0037] This invention not only enriches the understanding of the pathogenic mechanism of rice blast fungus, but also facilitates the development of green control products for rice blast. Attached Figure Description

[0038] Figure 1 Rice blast fungus MoUPE7 A schematic diagram of the construction of a gene knockout vector.

[0039] Figure 2 Partial hygromycin-resistant transformants HPH PCR analysis results of the gene; in the figure, M is the DL 2000 Marker; 1 is water; 2 is wild-type rice blast fungus; 3-6 are hygromycin-resistant transformants, respectively. MoUPE7 -25、 MoUPE7 -29、 MoUPE7 -37 and MoUPE7 -42.

[0040] Figure 3 Partial hygromycin-resistant transformants MoUPE7PCR analysis results of the gene; in the figure, M is the DL 2000 Marker; 1 is water; 2 is wild-type rice blast fungus; 3-6 are hygromycin-resistant transformants, respectively. MoUPE7 -25、 MoUPE7 -29、 MoUPE7 -37 and MoUPE7 -42.

[0041] Figure 4 For HPH The fragments represent the results of Southern blot analysis of the probe against *Magnaporum oryzae* knockout transformants; 1 in the figure represents wild-type *Magnaporum oryzae*; 2–4 represent candidate positive transformants, in order. MoUPE7 -25、 MoUPE7 -29 and MoUPE7 -37.

[0042] Figure 5 For MoUPE7 The fragments represent the results of Southern blot analysis of the probe against *Magnaporum oryzae* knockout transformants; 1 in the figure represents wild-type *Magnaporum oryzae*; 2–4 represent candidate positive transformants, in order. MoUPE7 -25、 MoUPE7 -29 and MoUPE7 -37.

[0043] Figure 6 Some bleomycin-resistant transformants MoUPE7 PCR analysis results of the gene; in the figure, M is the DL 2000 Marker; 1 is water; 2 is wild-type rice blast fungus; 3-8 are bleomycin-resistant transformants Δ MoUPE7- 29 - com-2、Δ MoUPE7- 29 - com-5、Δ MoUPE7- 29 - com-8、Δ MoUPE7- 29 - com-17、Δ MoUPE7- 29 - com-19 and Δ MoUPE7- 29 - com-26.

[0044] Figure 7 Rice blast fungus knockout mutant MoUPE7and complement mutant MoUPE7 Observation results of conidial germination from -com.

[0045] Figure 8 Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 Results of glycogen staining observation of conidial germination from -com.

[0046] Figure 9 To knock out mutants MoUPE7 and complement mutant MoUPE7 Growth of -com under different stress conditions.

[0047] Figure 10 Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 -com's results on the pathogenicity of rice.

[0048] Figure 11 Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 -com provides statistical results on the disease index of rice. Detailed Implementation

[0049] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0050] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0051] The amino acid sequence of the effector protein MoUPE7 described in this invention is shown in SEQ ID NO.1. The gene encoding the effector protein MoUPE7, i.e. MoUPE7 The gene's gene symbol in the NCBI database is MGG_16869, and its sequence ID is XM_003712578.1.

[0052] The wild-type strain of *Magnapordica oryzae* used in this invention is the dominant race ZC13 ​​in Guangdong Province; the tested rice is the susceptible indica rice line CO39; the cloning vector is pMD18-T vector; the gene knockout vector is the filamentous fungal expression vector pCT74; and the gene complementation vector is pCTZN (derived from the pCT74 plasmid, i.e., the gene on pCT74...). SGFP and HPH Gene replaced with bleomycin resistance gene Zeocin ).

[0053] Example 1 Rice blast fungus MoUPE7 Gene knockout and replacement

[0054] Rice blast fungus MoUPE7 A schematic diagram of gene knockout vector construction is shown below. Figure 1 As shown, homologous recombination can... MoUPE7 Gene replacement HPH +GFP fragments are used to achieve gene knockout.

[0055] 1. Experimental Procedure

[0056] (1) Rice blast fungus MoUPE7 Amplification of upstream and downstream homologous arms of genes

[0057] According to rice blast fungus MoUPE7 The gene sequence was retrieved from the NCBI database, and its upstream and downstream sequences were identified. An 834 bp sequence was selected from the upstream sequence as the upstream homologous arm (homologous arm A), and a 907 bp sequence was selected from the downstream sequence as the downstream homologous arm (homologous arm B). Based on the selected upstream and downstream homologous arms, corresponding amplification primers were designed, appropriate restriction enzyme sites were introduced, and PCR amplification was performed to obtain *Magnaporthe oryzae*. MoUPE7 Upstream and downstream homologous arms of the gene are used to construct rice blast fungus. MoUPE7 Gene knockout vector.

[0058] This invention is designed for amplifying rice blast fungus. MoUPE7 The primer sequences for the upstream and downstream homologous arms of the gene are shown in Table 1.

[0059] Table 1 Rice blast fungus MoUPE7 Amplification primers for upstream and downstream homologous arms of genes

[0060]

[0061] Genomic DNA was extracted from wild-type strains of *Magnapordica oryzae* using the OMEGA Fungal DNA Kit; using the obtained genomic DNA as a template, primers were then applied... MoUPE7 -AF and MoUPE7 -AR was used for PCR amplification to obtain MoUPE7Homologous arm A segment of the gene ( MoUPE7 -A); using primers MoUPE7 -BF and MoUPE7 -BR was used for PCR amplification to obtain MoUPE7 Homologous arm B segment of the gene ( MoUPE7 -B).

[0062] The reaction systems used for PCR amplification of upstream and downstream homologous arms are shown in Table 2.

[0063] Table 2 PCR reaction system

[0064]

[0065] The PCR reaction program was as follows: 94℃ for 5 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 1 min, for a total of 30 cycles; 72℃ for 10 min.

[0066] After the PCR reaction, a small sample was taken for agarose gel electrophoresis. Based on the electrophoresis results, the PCR amplification products were cleaned and recovered to obtain the desired product. MoUPE7 -A and MoUPE7 -B.

[0067] (2) MoUPE7 Construction of gene knockout vector

[0068] Refer to the TA cloning kit instructions, and... MoUPE7 -A and MoUPE7 -B was ligated to the T vector to obtain the recombinant plasmid pMD18T- MoUPE7 -A and pMD18T- MoUPE7 -B.

[0069] The general procedure is as follows: Take 1 µL of pMD18-T vector and add 4 µL of PCR recovered product ( MoUPE7 -A or MoUPE7 -B) and 5 µL solution I, ligated overnight at 16℃; 10 µL of the ligation product was added to 100 µL of *E. coli* DH5α competent cells and placed on ice for 30 min; heat-shocked in a 42℃ water bath for 90 s, then cooled on ice for 5 min; 800 µL of LB liquid medium was added, and cultured at 37℃ and 150 rpm with shaking for 45 min; centrifuged at 4000 rpm for 5 min, the supernatant was discarded, and 100 µL of bacterial culture was mixed with the precipitate and spread onto LB solid medium (containing 50 µg / mL Amp); cultured upside down at 37℃ for 8–12 h; positive transformants with Amp resistance were picked, recombinant plasmid DNA was extracted and sequenced to obtain the recombinant plasmid pMD18T- MoUPE7 -A and pMD18T-MoUPE7 -B.

[0070] Referencing recombinant plasmid pMD18T- MoUPE7 The construction process of -A, using Xba I and Eco RI on recombinant plasmid pMD18T- MoUPE7 The -B and pCT74 vectors were double-digested, and the recovered DNA was then extracted. MoUPE7 -B fragment and linearized pCT74 vector, ligated using T4 DNA ligase MoUPE7 The -B fragment was ligated into the pCT74 vector and transformed into E. coli DH5α competent cells to obtain the recombinant plasmid pCT74- MoUPE7 -B.

[0071] Similarly, using Kpn I and Apa I respectively targeted the recombinant plasmid pMD18T- MoUPE7 -A and recombinant plasmid pCT74- MoUPE7 -B is subjected to double enzyme digestion, and the enzyme is recovered. MoUPE7 -A fragment and linearized recombinant plasmid were ligated using T4 DNA ligase. MoUPE7 -A fragment and linearized pCT74- MoUPE7 -B ligation was performed, and the cells were transformed into *E. coli* DH5α competent cells; after enzyme digestion and identification, the gene knockout vector pCT74- was obtained. MoUPE7 -KO.

[0072] (3) MoUPE7 Amplification of gene complement fragments

[0073] exist MoUPE7 Approximately 1000 bp fragments were selected from the upstream and downstream sequences of the gene. Corresponding amplification primers were designed and suitable restriction enzyme sites were introduced for amplification. MoUPE7 Gene complementation fragments, construction MoUPE7 Gene complementation vector. Designed MoUPE7 The primers for amplifying the gene complement fragment are shown in Table 3.

[0074] Table 3 MoUPE7 Amplification primers for gene complement fragments

[0075]

[0076] Using the extracted genomic DNA of the wild-type strain of *Magnapordica oryzae* as a template, the primers shown in Table 3 were used for amplification. MoUPE7 The gene complement fragment is 3436 bp in size.

[0077] The reaction system used for PCR amplification was the same as in Table 2. The reaction program was as follows: 94℃ for 5 min; 94℃ for 5 min, 58℃ for 30 s, 72℃ for 4 min, for a total of 30 cycles; 72℃ for 10 min.

[0078] After the PCR reaction, a small sample was taken for agarose gel electrophoresis. Based on the electrophoresis results, the PCR amplification products were cleaned and recovered to obtain the desired product. MoUPE7 Gene complement fragment ( MoUPE7 -com).

[0079] (4) MoUPE7 Construction of gene complementation vector

[0080] use Xba I and Not I respectively to MoUPE7 The -com and pCTZN vectors were double-digested, and the recovered DNA was then recovered. MoUPE7 -com fragments and linearized pCTZN vector were ligated using T4 DNA ligase. MoUPE7 The -com fragment was ligated into a linearized pCTZN vector and transformed into *E. coli* DH5α competent cells; after enzyme digestion and identification, the desired product was obtained. MoUPE7 Gene complementation vector pCTZN- MoUPE7 -com.

[0081] (5) Preparation of rice blast fungus protoplasts

[0082] Wild-type *Magnapordica oryzae* strains were activated on Jianli medium (containing 5.0 g / L yeast extract, 22.0 g / L anhydrous glucose, and 17.0 g / L agar powder) plates and cultured upside down at 28°C for 10 days. Mycelial fragments from the Jianli medium were transferred to 50 mL of YPS medium (containing 6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein, and 10.0 g / L sucrose) and cultured at 28°C with shaking at 120 rpm for 2 days. Mycelia were filtered through a cell sieve, thoroughly ground in a mortar, and a suitable amount of mycelial fragments were transferred to 200 mL of YPS medium and cultured at 28°C with shaking at 120 rpm for 1 day. Mycelia were then filtered through a 200-mesh cell sieve, washed twice with ddH2O, and then rinsed with sterile 0.8 mol / L... Wash once with NaCl solution. Use sterile forceps to transfer the hyphae to a sterile petri dish (with four layers of filter paper underneath), then cover with multiple layers of sterile filter paper to blot the hyphae dry. Gently press the filter paper with forceps. Transfer the hyphae to a pre-weighed sterile EP tube and weigh again to obtain the hyphae weight. Add an appropriate amount of 15 mg / mL enzymatic hydrolysate to the EP tube, with a volume-to-mass ratio of 10:1. Digest at 30℃ and 80–110 rpm for 1 h with shaking. Collect the hydrolysate by filtering through four layers of sterile lint-free paper and transfer it to a pre-chilled 2 mL EP tube. Centrifuge at 4℃ and 3500 rpm for 10 min. Discard the supernatant and resuspend the precipitate in 1.5 mL of pre-chilled STC (containing 1.2 mol / L sorbitol, 10 mmol / L Tris-HCl, 50 mmol / L CaCl2, pH 7.5). Centrifuge at 4℃ and 5000 rpm for 10 min. Discard the supernatant and resuspend the precipitate in 1.5 mL of pre-chilled STC. Resuspend the precipitate in pre-chilled STC at mL and store on ice. After counting, dilute to an appropriate concentration. Calculate the protoplast concentration using a hemocytometer. Add an appropriate amount of pre-chilled STC to control the final protoplast concentration at 1 × 10⁻⁶. 7 ~1×10 8 Dispense per 200 μL tube at a rate of 1 / mL.

[0083] The protoplasts of the rice blast fungus knockout mutant were prepared according to the above-described preparation steps for rice blast fungus protoplasts.

[0084] (6) Transformation of rice blast fungus protoplasts

[0085] use Xba Ⅰ For the knockout vector pCT74- MoUPE7 -KO was used for single enzyme digestion to obtain the linear vector pCT74- MoUPE7 -KO. 5 μg of the linear vector pCT74- MoUPE7 -KJ is mixed with 200 µL of rice blast fungus protoplasts; or, the single-enzyme digested pCTZN- ​Mix the -com fragment with 200 µL of *Blastoma oryzae* knockout mutant protoplasts; incubate on ice for 20 min; add 1 mL of PTC conversion buffer (60% PEG4000, 50 mmol / L CaCl2, 10 mmol / L Tris-HCl, pH 7.5) dropwise while mixing, and incubate at room temperature for 20 min; centrifuge at 4℃ and 5000 rpm for 10 min; discard the supernatant, resuspend the pellet in 1 mL of regeneration liquid medium (containing 6.0 g / L yeast extract, 6.0 g / L hydrolyzed casein, and 200.0 g / L sucrose), transfer to a sterile 50 mL Corning tube, add 3 mL of regeneration liquid medium, and incubate at 28℃ and 100 rpm with gentle shaking for 16–18 h; add 4 mL of revived protoplasts to 30 mL of regeneration solid medium cooled to approximately 45℃ (the regeneration liquid medium contains 1.5% agar powder). In the experiment of transforming protoplasts by knocking out the vector, the regeneration medium contained 200 µg / mL hygromycin; in the experiment of transforming protoplasts by reintroducing the vector, the regeneration medium contained 150 µg / mL bleomycin. The mixture was poured into plates, and the solidified plates were incubated upside down in the dark at 28°C for 3 days. Single colonies of transformants containing hygromycin / bleomycin resistance were picked for identification.

[0086] (7) ​ PCR validation analysis of knockout mutants

[0087] Genomic DNA was extracted from the hygromycin-resistant transformants according to the instructions of the OMEGA Fungal DNA Kit, and then subjected to PCR verification analysis. For verification, primers were first used... ​ -F / ​ -R to proceed ​ PCR amplification of gene fragments; then using primers ​ -F / ​ -R can amplify ​ Transformants of gene fragments undergo ​ PCR amplification analysis of gene fragments.

[0088] The sequences of the primers used for PCR amplification are shown below:

[0089] ​ -F: 5′-TTCTGCGGGCGATTTGTGTA-3′,

[0090] ​ -R: 5′-AAAAAGCCTGAACTCACCGC-3′;

[0091] ​-F: 5′-CATCCGTATAAAGAGGCTGGC -3′,

[0092] ​ -R: 5′-AACGTACATCTCGGTCCCC -3′;

[0093] The reaction conditions used for PCR validation analysis are shown in Table 4.

[0094] Table 4 Reaction conditions used for PCR validation analysis

[0095]

[0096] The reaction procedure used for PCR validation analysis was as follows: 3 min at 98℃; 10 s at 98℃, 10 s at 60℃, 10 s at 72℃, for a total of 30 cycles; 2 min at 72℃.

[0097] (8) ​ PCR validation analysis of complemented mutants

[0098] Genomic DNA was extracted from bleomycin-resistant transformants according to the instructions of the OMEGA Fungal DNA Kit and then analyzed by PCR. Primers were used to... ​ -F / ​ -R performs gene fragmentation ​ PCR amplification.

[0099] The reaction system used for PCR validation analysis was the same as in Table 4, except that the primers used in the system were different. ​ -F / ​ -R.

[0100] The reaction procedure used for PCR validation analysis was as follows: 3 min at 98℃; 10 s at 98℃, 10 s at 57℃, 10 s at 72℃, for a total of 30 cycles; 2 min at 72℃.

[0101] (9) ​ Southern blot analysis of knockout mutants

[0102] Use primers ​ -F / ​ -R amplification of the target gene probe, using primers ​ -F / ​ -R amplification ​ Gene probes, with the obtained target gene probes and ​ Gene probes are used ​Southern blot analysis of the knockout mutant. The Southern blot hybridization procedure was performed according to the instructions of DIG High Prime DNA Labeling and Detection Starter Kit I (Roche LOT28309220).

[0103] PCR amplification of target genes and ​ The reaction systems used for the gene probes are shown in Table 5.

[0104] Table 5 PCR amplification of target genes and ​ Reaction system used for gene probes

[0105]

[0106] 2. Experimental Results

[0107] Using PEG-mediated protoplast transformation, the gene knockout vector pCT74- ​ -KO transformation was performed on wild-type protoplasts of *Magnapordium oryzae*, and hygromycin was used for screening, resulting in 42 transformants with hygromycin resistance. ​ Gene-specific primers were used to perform PCR validation analysis on four of the transformants, and the results are as follows: ​ As shown. By ​ It can be seen that amplification was achieved in all four transformants. ​ Genes. Based on this, further utilization... ​ Gene-specific primers were used to amplify the above PCR results. ​ The four transformants of the gene were subjected to ​ The PCR validation analysis showed the following results: ​ As shown. By ​ It can be seen that none of the four transformants amplified to [the desired result]. ​ Genes. The above results indicate that all four selected transformants were positive transformants, meaning that this invention has successfully obtained... ​ Gene knockout mutant ​ .

[0108] This invention is based on ​ Genes and ​ Using genes as probes, Southern blot was employed to analyze candidate positive transformants verified by PCR. ​ The gene knockout mutant was further validated. ​ The fragment was used as a probe, and the results of Southern blot analysis of the candidate positive transformants are as follows: ​ As shown; ​The fragment was used as a probe, and the results of Southern blot analysis of the candidate positive transformants are as follows: ​ As shown. By ​ and ​ It can be seen that, with ​ Using the gene as a probe for hybridization, all three positive transformants detected hybridized to produce a target band of approximately 6500 bp, while the wild-type rice blast fungus did not produce a band. ​ When the gene was used as a probe for hybridization, it was found that none of the three positive transformants could hybridize to produce the target band of approximately 8091 bp, indicating that the three transformants... ​ The gene was indeed successfully knocked out.

[0109] Using the method of random insertion, ​ Gene complementation vector ​ -Com-pCTZN transformed into rice blast fungus knockout mutant ​ ( MoUPE7 Protoplasts (-29) were screened using bleomycin, yielding 7 bleomycin-resistant transformants. The genetic DNA of these transformants was extracted and processed using... MoUPE7 Gene-specific primers were used to perform PCR validation analysis on these transformants, and the results are as follows: Figure 6 As shown. By Figure 6 It was found that among the obtained bleomycin-resistant transformants, 6 transformants were able to amplify to the target gene fragment, indicating that these 6 transformants contained... MoUPE7 Gene analysis confirmed that these six transformants were positive transformants, meaning that this invention has successfully obtained... Moupe7 complement mutant of the gene Moupe7- com.

[0110] Example 2: Phenotypic observation and growth rate determination of rice blast fungus knockout mutant and complement mutant

[0111] Wild-type and knockout mutant of rice blast fungus MoUPE7 and complement mutant MoUPE7 -com were inoculated onto Jianli medium and cultured at 28°C in the dark. The colony morphology was observed daily, and the colony diameter was measured on day 10.

[0112] Observations showed that, compared with the wild-type rice blast fungus, the knockout mutant... MoUPE7 The colony morphology and growth rate were not significantly different from those of the wild-type strain of rice blast fungus.

[0113] Example 3: Observation on conidia production and germination of rice blast fungus knockout mutant and complement mutant.

[0114] 1. Collection and germination observation of conidia

[0115] Moisten the activated rice blast fungus (wild type, knockout mutant) with ddH2O (3 mL~4 mL / plate). MoUPE7 Or complement mutant MoUPE7 On the surface of the *Bacillus oryzae* colonies, crush the mycelia with a sterilized spoon. Transfer the mycelial solution to tomato-oat medium (40 g of raw oats boiled in water for 1 h, filtered, then mixed with 150 mL of tomato juice, 0.06 g of calcium carbonate, and 2.5%–3% agar powder, and then diluted to 1 L with ddH2O) using a pipette, approximately 500 µL of mycelial solution per plate. Spread the mycelial solution evenly onto the plate using a glass rod. Incubate at 28°C under light for 24 h, then invert for 4–7 days. Add 5 mL of ddH2O to the tomato-oat medium using a pipette, and scrape off the colonies with a spoon. Collect the spore solution through a sterile four-layer lint-free filter. Adjust the spore concentration to 1 × 10⁻⁶. 6 Add 0.05% Tween 20 to the culture medium; transfer 20 µL of spore solution to a hydrophobic glass slide using a pipette; incubate in a humidified environment at 28℃ in the dark, and take samples at 2 h, 4 h, 6 h, 8 h, 10 h and 12 h to observe the germination of conidia.

[0116] The comparison shows that the knockout mutant MoUPE7 The sporulation rate was not significantly different from that of the wild-type strain of rice blast fungus.

[0117] Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 The germination observation results of conidia from -com are as follows: Figure 7 As shown. By Figure 7 It can be seen that the wild-type and Δ type of rice blast fungus MoUPE7 The -29-com strain germinated and produced germ tubes 2 hours after contact with a hydrophobic glass slide. After 6 hours, the tips of the germ tubes began to swell, forming small appressoria; by 8 hours, the appressoria had enlarged, darkened in color, and gradually matured. Meanwhile, Δ... MoUPE7 Two hours after contact with the hydrophobic slide, the strain formed short germ tubes; after 8 hours, the tips of the germ tubes began to swell, forming small appressoria; after 10 hours, the appressoria became larger but lighter in color; after 12 hours, the appressoria darkened in color, indicating... MoUPE7 Gene knockout affects the germination of conidia and the normal growth of appressorium.

[0118] 2. Observation of glycogen staining in conidia

[0119] The spores of the test strain were eluted with sterile ddH2O and the concentration was adjusted to 1×10⁻⁶. 6 Cells / mL were inoculated onto glass slides, kept moist in petri dishes, and incubated in the dark. Samples were taken at 2 h, 4 h, 6 h, 8 h, and 10 h for staining and observation. Glycogen staining was performed using KI / I2 solution (60 mg / mL KI, 10 mg / mL I2). After staining for 1 min, the samples were observed directly under a regular optical microscope.

[0120] Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 The results of glycogen staining observation of conidial germination from -com are as follows: Figure 8 As shown. By Figure 8 It can be seen that wild type and Δ MoUPE7 After 6 hours of contact with a hydrophobic glass slide, the glycogen in strain -29-com began to transfer from conidia to appressoria; after 10 hours, most of the glycogen in the conidia had been transferred to the appressoria. Meanwhile, Δ... MoUPE7 Eight hours after the strain was exposed to a hydrophobic glass slide, glycogen from conidia began to transfer into appressoria; after 10 hours, glycogen was still observed in both conidia and appressoria, indicating that... MoUPE7 Gene knockout slows down the process of glycogen transfer from rice blast fungus conidia to appressorium.

[0121] Example 4: Analysis of stress resistance of rice blast fungus knockout mutants and complement mutants

[0122] Wild-type, knockout mutant MoUPE7 and complement mutant MoUPE7 -com were inoculated into different types of culture medium containing different stress factors (including 0.8 mol / L NaCl, 0.8 mol / L Sorbitol, 0.2 g / L CR, 0.05 g / L CFW, 0.01% SDS and 20 mmol / L H2O2), and cultured in a 28℃ incubator in the dark for 10 days. The morphology of the colonies was observed and the diameter and inhibition rate of the colonies were counted.

[0123] Knockout mutant MoUPE7 and complement mutant MoUPE7 -com growth under different stress conditions, such as Figure 9 As shown. By Figure 9It can be seen that, compared with the wild type of rice blast fungus, Δ MoUPE7 Sensitivity to 20 mmol / L H₂O₂ increased (colon diameter decreased significantly), but there was no significant difference in sensitivity to 0.8 mol / L NaCl, 0.8 mol / L Sorbitol, 0.01% SDS, 0.2 g / LCR, and 0.05 g / L CFW. In the early stages of pathogen infection of host plants, plants produce large amounts of ROS (including hydrogen peroxide (H₂O₂) and superoxide radicals (O₂). 2- ) and hydroxyl radicals (OH) - (etc.) inhibit the growth of pathogens, MoUPE7 Increased sensitivity to H2O2 is detrimental to its infection of rice.

[0124] Example 5: Pathogenicity analysis of rice blast fungus knockout mutants and complement mutants

[0125] Collect wild-type and knockout mutants of rice blast fungus MoUPE7 and complement mutant MoUPE7 The conidial solution from -com was adjusted to a concentration of 5 × 10⁻⁶ using ddH₂O. 4 The concentration of Tween 20 was increased to 0.05% by adding spores per mL. The spores were then sprayed onto rice seedlings at the four-leaf stage. The seedlings were cultured in the dark for 24 hours at 28°C, followed by 12 hours of light and 12 hours of darkness. The seedlings were kept moist throughout the process. The disease incidence on the rice leaves was investigated and photographed after 5 days.

[0126] Rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 -com's results on the pathogenicity testing of rice are as follows Figure 10 As shown. With Figure 10 The corresponding rice blast fungus knockout mutant MoUPE7 and complement mutant MoUPE7 -com's statistical results on the disease index of rice are as follows Figure 11 As shown. By Figure 10 and Figure 11 It can be seen that, compared with rice leaves inoculated with wild-type rice blast fungus, the leaves inoculated with Δ MoUPE7 The disease spots on the rice leaves were smaller, while those inoculated with MoUPE7 The lesions on the leaves of rice from -com are not significantly different from those of the wild type of rice blast fungus, indicating that... MoUPE7 -com's pathogenicity has returned to wild-type levels, which also indicates MoUPE7 Knockout of genes leads to a decrease in the pathogenicity of rice blast fungus.

[0127] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of effector protein MoUPE7 in reducing the pathogenicity of rice blast fungus, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.1; the application is to inhibit the expression of the protein in *Magnaporum oryzae* or to knock out the gene encoding the protein in *Magnaporum oryzae*.

2. The application of the reagent that inhibits the expression of the effector protein MoUPE7 as described in claim 1 in reducing the pathogenicity of rice blast fungus.

3. The use of the reagent that inhibits the expression of the effector protein MoUPE7 as described in claim 1 in the preparation of products that reduce the pathogenicity of rice blast fungus.

4. The use of a product for knocking out the gene encoding the effector protein MoUPE7 of claim 1 in reducing the pathogenicity of rice blast fungus.

5. Use of the product for knocking out the gene encoding the effector protein MoUPE7 of claim 1 in the preparation of a product that reduces the pathogenicity of rice blast fungus.

6. The application of the reagent for inhibiting the expression of the effector protein MoUPE7 as described in claim 1 in the prevention and control of rice blast.

7. The use of a reagent that inhibits the expression of the effector protein MoUPE7 as described in claim 1 in the preparation of a product for the prevention and control of rice blast.

8. The use of a product for knocking out the gene encoding the effector protein MoUPE7 of claim 1 in the prevention and control of rice blast.

9. Use of the product for knocking out the gene encoding the effector protein MoUPE7 of claim 1 in the preparation of a product for the prevention and control of rice blast.

10. The application according to any one of claims 6 to 9, characterized in that, The rice blast disease is caused by the rice blast fungus.

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