A drug target protein MoFrd1 and its application in agricultural disease prevention and control

Abstract

The present invention provides a drug target protein MoFrd1 and its application in agricultural disease control. The amino acid sequence of MoFrd1 is shown in SEQ ID No.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID No.4. Studies have shown that knocking out the MoFrd1 encoding gene significantly reduces the pathogenicity of rice blast fungus. Homologous proteins of MoFrd1 are widely present in plant pathogenic fungi and show high conservation in pathogenic mechanisms, but homologous proteins have not been found in plants and mammals, indicating its potential as a pathogen-specific drug target. Based on the key domains and functional sites of the MoFrd1 protein, it is expected to develop specific agents for controlling plant fungal pathogens through design or screening. The present invention provides new drug targets and control strategies for controlling fungal diseases of crops, and has important application value and market potential.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology and pesticide development, and specifically relates to a drug target protein MoFrd1 and an application thereof in agricultural disease prevention and control. Background Art

[0002] Plant diseases cause serious losses to crops such as rice, wheat, corn and soybeans. Magnaporthe oryzae ) is one of the most destructive diseases of cultivated rice and ranks first among the top ten fungal diseases of plants. The annual food loss caused by Fusarium graminearum ( Fusarium graminearum ) is a major fungal disease in wheat production, causing serious yield and quality losses. The fungal toxins produced also harm human and animal health and threaten food safety. Both rice blast and wheat scab are listed in the "List of Class I Crop Pests and Diseases" by my country's Ministry of Agriculture and Rural Affairs. These and other devastating crop diseases seriously threaten global food security.

[0003] Effective fungicide application is an important measure to ensure crop health and yield. However, the number of available fungicide targets is very limited, mainly focusing on functional pathways such as ergosterol biosynthesis, melanin biosynthesis, succinate dehydrogenase, β-tubulin and cytochrome bc1 complex. The singleness of this target leads to serious homogeneity of fungicide structures and a significant increase in the risk of drug resistance. Therefore, there is an urgent need to reveal the pathogenic mechanism of plant pathogenic fungi at the molecular level, discover new drug targets, and develop green fungicides with original structures and mechanisms of action. Summary of the invention

[0004] The purpose of the present invention is to provide a drug target protein MoFrd1 and application thereof in agricultural disease prevention and treatment.

[0005] The present invention provides a drug target protein, whose amino acid sequence is shown in SEQ ID No.1, SEQ ID NO.2 or SEQ ID NO.3.

[0006] Specifically, the amino acid sequence SEQ ID No.1 is from Magnaporthe grisea ( Magnaporthe oryzae ), SEQID No.2 from Fusarium graminearum ( Fusarium graminearum ), SEQ ID No.3 from Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ).

[0007] The present invention further provides a gene encoding the drug target protein.

[0008] Specifically, the nucleotide sequence of the encoding gene is shown as SEQ ID No.4, SEQ ID No.5 or SEQ ID No.6.

[0009] The present invention also provides a biological material comprising the coding gene, which is selected from the following:

[0010] (a) Expression cassette;

[0011] (b) recombinant vector;

[0012] (c) Recombinant microorganisms or transgenic cell lines.

[0013] The present invention also provides the use of the drug target protein or the encoding gene in designing or screening specific agents for controlling plant fungal pathogens, wherein the plant fungal pathogens are selected from rice blast fungus, Fusarium graminearum or Colletotrichum gloeosporioides.

[0014] The present invention also provides a method for designing or screening specific agents for controlling plant fungal pathogens, wherein the plant fungal pathogens are selected from rice blast fungus, Fusarium graminearum or Colletotrichum gloeosporioides, and the method uses the drug target protein as a target to screen or design inhibitors capable of inhibiting its function through high-throughput screening technology; or uses the encoding gene as a target to screen interfering substances targeting the expression of the encoding gene or the function of the expressed protein through gene editing tools or gene silencing technology.

[0015] Specifically, the inhibitor is a small molecule compound; the interfering substance is siRNA, shRNA or antisense oligonucleotide targeting the gene, or a gene editing tool, a protein degradation molecule or an inhibitor based on nanomaterials.

[0016] Among them, computer-aided drug design technology is used to screen small molecule compounds that may specifically bind to MoFrd1 in the compound database based on the protein structure and key functional site information of MoFrd1, and the binding of the compound to MoFrd1 is verified by microthermophoresis experiments. Preferably, the inhibitory effect of the compound on plant fungal pathogens and the safety of crops are further evaluated, and finally a drug with good prevention and control effect and environmental friendliness is screened out for the prevention and control of various crop diseases caused by plant fungal pathogens.

[0017] The present invention also provides the use of the specific agent obtained by the above method in preventing and treating plant fungal diseases, wherein the plant fungal diseases are crop diseases caused by rice blast fungus, Fusarium graminearum or Colletotrichum gloeosporioides.

[0018] Specifically, the specific agent obtained is compound 2412, and its chemical structural formula is:

[0019] .

[0020] In a specific application, the crop is wheat or soybean.

[0021] The present invention found that MoFrd1 is an important pathogenic factor of rice blast fungus, and its gene knockout mutant has significantly reduced pathogenicity, and key functional sites such as H248, E268, R271, R292, H399 and R439 were identified. Importantly, homologous proteins of MoFrd1 are widely present in plant pathogenic fungi and show high conservation in pathogenic mechanisms, but homologous proteins have not been found in plants and mammals, indicating its great potential as a pathogen-specific drug target. In addition, the present invention provides the protein structure and key functional site information of MoFrd1, laying an important foundation for the rational design of pesticide molecules. Based on this target, by designing or screening pesticide molecules that specifically act on MoFrd1, it is expected to develop targeted new plant pathogenic fungus control agents. This can not only significantly improve the control effect of fungicides, but also effectively reduce the risk of pathogens developing drug resistance, providing an innovative solution for agricultural disease control. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 :Pyricularia grisea MoFRD1 Schematic diagram of gene knockout strategy.

[0023] Figure 2 :Pyricularia grisea MoFRD1 PCR verification results of gene knockout mutants.

[0024] Figure 3 : MoFRD1 The virulence of the gene knockout mutant was significantly reduced. Mofrd1 , gene complementation strain Δ Mofrd1-C The pathogenicity results of spraying conidia suspension on rice seedlings; on the right are the statistical results of the number of rice lesions.

[0025] Figure 4 :Evolutionary analysis of Frd1 proteins in different species, including fungi, bacteria, and protozoa.

[0026] Figure 5 :The three-dimensional structure of MoFrd1 protein predicted by AlphaFold3.

[0027] Figure 6 : Binding mode of compound 2412 and MoFrd1 protein.

[0028] Figure 7 : The control effect of compound 2142 at different concentrations on rice blast.

[0029] Figure 8 : Results of the test on the protective effect of compound 2412 against wheat fusarium scab.

[0030] Fig. 9 : Determination of the protective effect of compound 2412 against soybean anthracnose. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other implementation methods obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0032] The strains and reagents that can be used in some embodiments of the present invention are:

[0033] The wild-type strain Guy11 of Magnaporthe oryzae was maintained by our laboratory;

[0034] Competent Escherichia coli JM109 was purchased from Kangti Life Science;

[0035] DNA gel recovery kit (Vazyme);

[0036] Small-volume plasmid extraction kit (Vazyme);

[0037] Lyticase (Sigma);

[0038] Hygromycin (Yeasen);

[0039] Bleomycin (Invitrogen).

[0040]

[0041]

[0042] Example 1: Rice blast fungus MoFRD1 Acquisition of gene sequences and construction of knockout vectors

[0043] Fumarate reductase is considered to have the potential to be a drug target, but its specific function in plant pathogenic fungi is still unclear. The present invention obtained the gene encoding the fumarate reductase of Magnaporthe oryzae from the Fungal Genome Database (https: / / fungidb.org / fungidb / app) by comparing it with the fumarate reductase Frd1 of Saccharomyces cerevisiae. MoFRD1 The nucleotide sequence of (gene number MGG_03619) is 2147 bp in total (as shown in SEQ ID NO.4) and encodes 619 amino acids (as shown in SEQ ID NO.1).

[0044] The genomic DNA of wild-type strain Guy11 of rice blast fungus was used as template and the primer pairs listed in Table 1 were used to MoFRD1 PCR amplification of upstream and downstream fragments of the gene: the upstream fragment uses primers MoFRD1 -LF-F and MoFRD1 -LF-R, downstream fragment using primers MoFRD1 -RF-F and MoFRD1 -RF-R. Hygromycin resistance gene ( HPH ) fragments are composed of HPH The plasmid pCX62 containing the gene was used as a template and primers HPH -F and HPH -R amplification. Subsequently, the three fragments were connected by Overlap PCR technology to construct a knockout cassette with a length of about 3.4 kb ( Figure 1 ), used for protoplast transformation experiments.

[0045] Table 1 Primers used in the examples

[0046]

[0047] Embodiment 2: MoFRD1 Obtaining gene knockout mutants

[0048] (1) Inoculate the wild-type strain Guy11 on a CM plate and culture it in a dark incubator at 28°C for 3-5 days.

[0049] (2) Use a scalpel to cut a small bacterial mass from the edge of a fresh colony, inoculate it into CM liquid culture medium, and culture it in a shaker at 28°C and 160 rpm for 2 days.

[0050] (3) Collect the mycelium by filtering through sterile Micracloth and press dry with absorbent paper to remove excess water.

[0051] (4) Place the collected mycelium in a 50 mL centrifuge tube containing 20 mL of enzymatic hydrolysis solution (0.2 g of lytic enzyme dissolved in 20 mL of 0.7 M NaCl solution, filtered through a bacterial filter for later use) and incubate the tube in a shaker at 28°C and 70 rpm for 2-3 h.

[0052] (5) Filter the mycelial hydrolysate through sterile Micracloth into a 50 mL centrifuge tube, rinse with 0.7 M NaCl, centrifuge at 4°C, 3600 rpm for 8 min, discard the supernatant, and collect the protoplast precipitate.

[0053] (6) Add 10 mL of STC solution to resuspend the protoplasts. Centrifuge at 3600 rpm at 4°C for 8 min and discard the supernatant.

[0054] (7) According to the number of protoplasts precipitated, add appropriate amount of STC solution to resuspend the protoplasts to adjust the protoplast concentration. Aliquot the protoplasts (150 μL / tube) into 10 mL centrifuge tubes for later use.

[0055] (8) Add 3 μg of target DNA fragment, flick to mix, and place on ice for 25 min.

[0056] (9) Add 1 mL of PTC solution dropwise along the tube wall, gently rotate the centrifuge tube to mix, and let it stand on ice for 20 min.

[0057] (10) Add 7 mL of TB3 liquid medium and revive the cells in a shaker at 28°C and 70 rpm for 2-3 h.

[0058] (11) Add the above culture medium to a 50 mL centrifuge tube and add TB3 solid culture medium containing 150 μg / mL hygromycin to 50 mL. Shake gently to mix and invert the plate to dry.

[0059] (12) Cover the culture dish with a layer of TB3 solid culture medium containing 300 μg / mL hygromycin, seal the dish after drying, and incubate it in a dark incubator at 28°C for 5-7 days.

[0060] (13) Pick a single colony transformant grown on the transformation plate and transfer it to a CM plate containing hygromycin resistance and culture it in a 28°C incubator in the dark for 3 days.

[0061] (14) Pick an appropriate amount of mycelium to extract genomic DNA.

[0062] (15) Using the bold genomic DNA as template, the primers and Figure 1 The method shown was PCR validated ( Figure 2 ).

[0063] Example 3: Determination of pathogenicity of rice blast fungus

[0064] (1) Inoculate the test strain on a CM plate and culture it in a dark incubator at 28°C for 3-5 days.

[0065] (2) Use a scalpel to cut small bacterial pieces from the edge of a fresh colony, inoculate them on an SDC plate, and culture them in a dark incubator at 28°C for 5 days.

[0066] (3) Use a scalpel to scrape off the aerial hyphae on the surface of the culture medium and culture under a black light for 3 days to induce spore production.

[0067] (4) Add sterile ddH2O and gently scrape the surface of the culture medium with the bottom of a 1.5 mL centrifuge tube to suspend the spores.

[0068] (5) Use a pipette to draw up the spore suspension, collect it through two layers of sterile Miracloth into a 15 mL conical centrifuge tube, and centrifuge at 5000 rpm for 5 min.

[0069] (6) Resuspend the spores in sterile ddH2O and repeat the centrifugation twice.

[0070] (7) Suspend the spores in sterile ddH2O, count the spore concentration under a microscope using a hemocytometer, and adjust the spore concentration to 5 × 10 4 Pieces / mL.

[0071] (8) Use a small spray bottle to evenly spray 5 mL of the above spore suspension containing 0.2% gelatin onto 2-week-old susceptible rice CO39 leaves.

[0072] (9) Place the inoculated rice in an incubator at 25°C and 90% humidity. First, incubate in the dark for 24 h, and then incubate in alternating light and dark conditions for 12 h each for 5–7 d.

[0073] (10) Conduct statistical analysis on the incidence and take photos to record it. Figure 3 ).

[0074] Example 4: Phylogenetic analysis of MoFrd1

[0075] (1) Fumarate reductase is considered to have the potential to be a drug target, but its specific function in plant pathogenic fungi is still unclear. In order to explore its evolutionary relationship in different species, this paper conducted a phylogenetic analysis of the fumarate reductase of Magnaporthe oryzae MoFrd1 and other species.

[0076] (2) The amino acid sequence of MoFrd1 (shown in SEQ ID NO.1) was obtained from the fungal genome database. The amino acid sequences of homologous proteins of other related species were also downloaded, covering different species such as fungi, bacteria, and protozoa, including but not limited to the amino acid sequences shown in SEQ ID NO.2 from Fusarium graminearum and SEQ ID NO.3 from Colletotrichum gloeosporioides. The corresponding nucleotide sequences of the encoding genes are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0077] (3) Use the CLUSTALW tool to perform multiple sequence alignment of MoFrd1 and other homologous sequences.

[0078] (4) The phylogenetic tree was constructed using the neighbor-joining method using MEGA 7.0 software. Figure 4 ).

[0079] Example 5: Method for screening small molecule compounds using MoFrd1 protein as a target

[0080] (1) The three-dimensional structure of MoFrd1 protein predicted by AlphaFold3 ( Figure 5 ).

[0081] (2) Based on the experimental results, determine the active site of MoFrd1 protein.

[0082] (3) Obtain small molecule compounds from compound libraries such as ZINC, PubChem, and ChemDiv.

[0083] (4) Use AutoDock Vina for small molecule compound screening.

[0084] (5) Candidate compounds were screened according to the docking affinity, with priority given to small molecules with low binding energy (usually ≤ -7 kcal / mol). One of the compounds obtained was compound 2412.

[0085] (6) PyMOL was used to visualize the docking results and analyze the specific amino acid residues and interaction types of compound 2412 binding to MoFrd1 ( Figure 6 ).

[0086] (7) The binding of compound 2412 to MoFrd1 was verified by microscale thermophoresis (MST).

[0087] (8) Evaluation of the efficacy of compound 2412 against rice blast ( Figure 7 ) and safety to crops.

[0088] Example 6: Broad-spectrum efficacy determination of compound 2412

[0089] (1) Determination of efficacy against wheat fusarium head blight. Compound 2412 was used to pre-spray the flowering spikelets of the wheat variety "Fielder", and then 10 μL of conidia suspension of Fusarium graminearum (1×10 5 spores / mL) were injected into the florets in the middle of the wheat ear. The inoculated ears were covered with plastic bags to maintain 100% relative humidity for 2 days. After removing the plastic bags, the humidity in the growth chamber was adjusted to 70%. The number of spikelets showing typical fusarium symptoms was counted 14 days after inoculation ( Figure 8 ).

[0090] (2) Determination of the efficacy against soybean anthracnose. Compound 2412 was used to pre-spray the leaves of soybean variety “Hefeng 47”, and then 20 μL of conidia suspension of Colletotrichum gloeosporioides (2×10 5 spores / mL) were inoculated on soybean leaves. The inoculated soybean leaves were placed in a 25°C moisturizing incubator. The lesion size was measured 5 days after inoculation ( Fig. 9 ).

Claims

1. Use of a compound in the preparation of a specific agent for preventing and treating plant fungal diseases caused by plant fungal pathogens, wherein the plant fungal pathogens are selected from the group consisting of Pyricularia grisea, Fusarium graminearum, and Colletotrichum gloeosporioides; The compound is obtained by the following method: using the drug target protein with an amino acid sequence such as SEQ ID No. 1, SEQ ID NO. 2 or SEQ ID NO. 3 as a target, screening or designing an inhibitor capable of inhibiting its function by high-throughput screening technology; or using the coding gene of the drug target protein as a target, screening for interfering substances targeting the expression of the coding gene of the drug target protein or the function of the expressed protein by gene editing tools or gene silencing technology; And the compound has the chemical structural formula: 。 2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the drug target protein is shown as SEQ ID No.4, SEQ ID NO.5 or SEQ ID NO.

6.

3. The use according to claim 2, characterized in that: The plant fungal disease is a rice, wheat or soybean disease caused by rice blast fungus, Fusarium graminearum or Colletotrichum gloeosporioides.

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