Application of α-L-fucosidase MoFco1 and compound 0989 in the control of rice blast

Abstract

This invention belongs to the field of plant protection and discloses the application of α-L-fucosidase MoFco1 in screening drugs for the control of rice blast, as well as the application of compound 0989 in the control of rice blast. This invention identifies α-L-fucosidase MoFco1, which is active against XXFG in rice blast fungus. Located in the apoplast space, it regulates the pathogenicity of the fungus by degrading hemicellulose oligosaccharides in rice cell walls, inhibiting host ROS accumulation, and regulating defense responses. Based on virtual screening of the MoFco1 structure predicted by AlphaFold3, compound 0989 was obtained. This compound specifically binds to MoFco1 and effectively inhibits the pathogenicity of rice blast fungus. This invention provides new targets and new agents for the green control of rice blast and has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of plant protection, specifically involving the application of α-L-fucosidase MoFco1 as a drug screening target, and the application of small molecule compound 0989 in the prevention and control of rice blast. Background Technology

[0002] Rice (Oryza sativa) is a vital global food crop, and its high and stable yields are directly related to the stability and development of the national economy. Rice blast, caused by Magnaphalthe oryzae, is the most devastating rice disease, seriously threatening rice yield and quality. Current control strategies mainly rely on resistant varieties and fungicides. However, the frequent field mutations of the rice blast fungus lead to weakened resistance in resistant varieties, and improper use of fungicides can cause environmental pollution and pathogen resistance. Therefore, there is an urgent need to develop novel fungicides with new targets.

[0003] Plant cell walls are a natural physical barrier against rice blast fungus infection. Fucosylation modification of the hemicellulose side chains, with the corresponding oligosaccharide structure XXFG, has been confirmed to exist in rice tissues. Fucosylation is jointly regulated by fucosyltransferase and fucosidase, playing a crucial role in plant immune responses. During rice blast fungus infection, cell wall degrading enzymes are released to degrade the rice cell wall, with α-L-fucosidase being an important member. Previous studies have shown that α-L-fucosidase FgFco1 in *Fusarium graminearum* is active against XXFG, but the relationship between this enzyme and the pathogenicity of rice blast fungus remains unclear. This invention identifies α-L-fucosidase MoFco1 in rice blast fungus, clarifies its pathogenic mechanism, and screens for inhibitors, providing a new strategy for rice blast control. Summary of the Invention

[0004] This invention identified a protein called MoFco1 (GeneID: MGG_03257) belonging to the GH29 α-L-fucosidase subfamily 2 in *Bacillus oryzae*, the causal agent of rice blast fungus. MoFco1 is highly conserved with *Fusarium graminearum* FgFco1 and exhibits specific enzymatic activity towards α-1,2-linked fucosylated substrates (2'-FL, XXFG). During the infection stage, MoFco1 is located in the rice apoplast space and participates in the pathogenicity of *Bacillus oryzae* as an apoplast effector; its enzymatic activity is crucial to the complete virulence of the pathogen. The absence of MoFco1 does not affect the growth and development of *Bacillus oryzae*, but it leads to decreased pathogen infectivity, increased host ROS accumulation, and upregulated expression of defense response genes.

[0005] Based on the structure of MoFco1 predicted by AlphaFold3, virtual screening was performed targeting its enzyme activity center region to obtain compound 0989. This compound can specifically bind to MoFco1, effectively inhibiting its enzyme activity and thus blocking the pathogenic process of rice blast fungus, without significant toxicity to rice. This invention provides the application of MoFco1 in screening drugs for rice blast control, and the application of compound 0989 in controlling rice blast, enhancing rice resistance, and preparing pesticides. The related pesticides can be applied by spraying and are suitable for rice blast control in rice, planting sites, and propagation materials. Therefore, this invention is proposed.

[0006] This invention first provides the application of α-L-fucosidase MoFco1 (GeneID: MGG_03257) in screening drugs for the prevention and control of rice blast caused by rice blast fungus.

[0007] This invention further provides a method for screening drugs to control rice blast caused by rice blast fungus, comprising the following steps: based on the protein structure model of α-L-fucosidase MoFco1 (GeneID: MGG_03257) and the structure model of substrate L-fucose / XXFG, the structure model of the test compound is docked with them, and batch docking analysis is performed using AutoDock Vina (preferably, PyMOL is used to visualize the docking model); if the test compound binds to the enzyme active center region of MoFco1 (key binding sites include LYS288, ASP242, HIS143, TRP327, GLU61 and TRP62), preventing the binding of the substrate to MoFco1, then it is identified as a candidate compound for controlling rice blast.

[0008] The structural models of the substrate L-fucose or XXFG were also used as controls to determine whether the candidate compound could specifically block the correct binding of the substrate to the active site of the MoFco1 enzyme; optionally, the study also included inhibition tests on rice blast fungus and / or control tests on rice blast fungus to determine its effectiveness.

[0009] Specifically, the protein structure model of MoFco1 was obtained from the AlphaFold protein structure database and was based on its amino acid sequence prediction.

[0010] The present invention also provides a method for controlling or preventing rice blast disease, comprising applying compound 0989, or an agricultural chemically active salt thereof (e.g., alkali addition salts of inorganic and organic bases, more specifically potassium, sodium, ammonium, dimethylamine, or isopropylamine salts) to rice, its planting site, or its propagation material.

[0011] Specifically, compound 0989 is prepared into a drug formulation selected from emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, poison bait, mother liquor, and mother powder; its content in the drug is 1-99% by weight, preferably 10-80% by weight, more preferably 15-50% by weight; the effective dose concentration of the drug is 0.1μM-1mM, and the application rate is 1 to 500g / ha, preferably 50-200g / ha.

[0012] The present invention also provides the use of compound 0989 or its agricultural chemically active salt for enhancing the resistance of rice to rice blast fungus; preferably, the agricultural chemically active salt is an alkali addition salt of inorganic base and organic base, more specifically its potassium salt, sodium salt, ammonium salt, dimethylamine salt, or isopropylamine salt.

[0013] The present invention also provides the use of compound 0989 or its agriculturally active salt in the preparation of pesticides for controlling or preventing rice from being infected by rice blast fungus or causing disease.

[0014] Specifically, the pesticide is a formulation of an emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, poison bait, mother liquor, and mother powder, and also includes pesticide-acceptable auxiliary ingredients.

[0015] More specifically, the pesticide is formulated for spray application; applied when rice blast is predicted to occur, or at the early stage of its occurrence.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) The function of MoFco1 as a key protein in the pathogenicity of rice blast fungus has been clarified, providing a new specific target for the development of fungicides;

[0018] (2) Compound 0989 was obtained by screening based on the MoFco1 structure. It has strong binding specificity, high safety for rice, and meets the requirements of green prevention and control.

[0019] (3) Compound 0989 has a novel mechanism of action. It works by inhibiting the activity of bacterial cell wall degradation enzymes and enhancing host immunity, and is less likely to cause pathogens to develop drug resistance.

[0020] (4) The identification of MoFco1 and the screening of inhibitors provide a theoretical basis for understanding the interaction mechanism between rice blast fungus and host, and have broad application prospects. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the family classification and domain analysis of MoFco1 in one embodiment of the present invention; wherein, A is an evolutionary tree showing MoFco1 and other organisms' α-L-fucosidases; B shows the differences in domains between the two subfamilies of MoFco1.

[0022] Figure 2 This is a schematic diagram illustrating the structural conservation verification of MoFco1 and FgFco1 in one embodiment of the present invention;

[0023] Figure 3 This is a schematic diagram illustrating the determination of α-1,2-fucosidase activity of MoFco1 in one embodiment of the present invention; wherein, A is the binding site of MoFco1 to L-fucose; B is the protein expressed by MoFco1 and MoFco16A in Pichia pastoris; C is the determination of the activity of MoFco1 against 2'-FL; and D is the determination of the activity of MoFco1 against XXFG.

[0024] Figure 4 This is a schematic diagram of the apoplast localization verification of MoFco1 during the infection stage in one embodiment of the present invention; wherein, A is the localization of MoFco1 during the infection stage; B is the co-localization of MoFco1 and apoplast protein Bas4 on the apoplast; C is the distribution of MoFco1 in the extracellular fluid after extracting rice intercellular fluid.

[0025] Figure 5 This is a schematic diagram illustrating the effect of MoFco1 deficiency on the growth and development of rice blast fungus in one embodiment of the present invention; wherein, A shows no significant change in ΔMofco1 growth on CM / MM medium; B shows that MoFco1 has no effect on conidia formation of rice blast fungus; C shows data analysis on conidia formation of mutant rice blast fungus; D shows the effect of MoFco1 on appressorium formation of rice blast fungus; and E shows the effect of MoFco1 on appressorium turgor pressure of rice blast fungus.

[0026] Figure 6 This is a schematic diagram illustrating the verification of MoFco1's involvement in the pathogenicity of rice blast fungus in one embodiment of the present invention; wherein, A is the inoculation of rice leaves with MoFco1 mutant strain; B is the statistical analysis of lesion grading after inoculation of rice with MoFco1 mutant strain; C is the lesion area analysis after inoculation of rice with MoFco1 mutant strain; D is the injection of MoFco1 mutant strain into the leaf sheath of living rice; E is the state of infected hyphae observed after inoculation of rice leaf sheaths with MoFco1 mutant strain; F is the statistical analysis of the number of infected hyphae at different grades inoculated on rice leaf sheaths with MoFco1 mutant strain.

[0027] Figure 7 This is a schematic diagram illustrating the verification of MoFco1 response to host ROS accumulation in one embodiment of the present invention; wherein, A is the colony diameter of the mutant under different concentrations of diamide (Dia); B is the state of rice cells stained with DAB after MoFco1 mutant was inoculated into rice; C is the statistical data of DAB-stained cells after MoFco1 mutant was inoculated into rice; and D is the grading of infected hyphae after treatment with the reactive oxygen species inhibitor DPI.

[0028] Figure 8 This is a schematic diagram of virtual screening and MST verification of the binding of compound 0989 to MoFco1 in one embodiment of the present invention; wherein, A is the molecular docking result of MoFco1 and 0989; B is the molecular docking result of MoFco1 and 0104; C is the molecular docking result of MoFco1 and 1432; D is the evaluation of the binding of the three compounds to MoFco1 using microthermophoresis (MST).

[0029] Figure 9 This is a schematic diagram illustrating the activity determination of compound 0989 in inhibiting the pathogenicity of rice blast fungus in one embodiment of the present invention. In this diagram, A represents the pathogenicity test of the three compounds after treatment on rice leaves followed by inoculation with the pathogen; B represents the statistical analysis of lesion area after treatment on rice leaves followed by inoculation with the pathogen; and C represents the rice leaf death caused by treatment with 1432. Detailed Implementation

[0030] The present invention will be further described below through specific embodiments in order to better understand the present invention. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and do not constitute a limitation on the present invention.

[0031] Example 1: Identification of α-L-fucosidase MoFco1 from rice blast fungus

[0032] Based on the orthologous data of fucosidase from *Fusarium graminearum* FgFco1 (OG6_102401), a phylogenetic tree was constructed. The results showed that fungal α-L-fucosidases are divided into two subfamilies. Among *Bacillus oryzae* MGG_03257, MGG_00042, and MGG_00316, only MGG_03257 belongs to subfamily 2, the same as FgFco1, and is named MoFco1. Figure 1 (A). Analysis using the Pfam database shows that MoFco1 contains an α-L-fucosidase domain and a C-terminal fucosidase domain, consistent with subfamily 2 characteristics. Figure 1 (B)

[0033] Structure alignment was performed in PyMOL, and the root mean square deviation (RMSD) was 0.441. The quaternary structures of the proteins largely overlapped, confirming that MoFco1 and FgFco1 are highly conserved in sequence and structure (Table 1). Figure 2 ).

[0034] Table 1. BLAST results of FgFco1 in *Magnaporthe oryzae*.

[0035]

[0036] Example 2: Determination of α-1,2-fucosidase activity of MoFco1

[0037] To verify the enzymatic function of MoFco1, this embodiment constructed wild-type and mutant recombinant expression vectors, obtained purified protein through a Pichia pastoris heterologous expression system, and conducted enzyme activity assays using specific substrates.

[0038] Using L-fucose as a ligand, six interacting sites were identified through molecular docking: LYS288, ASP242, HIS143, TRP327, GLU61, and TRP62. Figure 3 (A). Subsequently, these 6 sites were mutated to alanine, denoted as MoFco16A. The coding sequences of MoFco1, MoFco16A, and FgFco1 were constructed into the pPIC9k vector, transformed into Pichia pastoris KM71, and induced to express. The target protein was obtained by His tag purification, and protein expression was confirmed by Coomassie brilliant blue staining. Figure 3 (B) Using 2'-FL and XXFG as substrates, the mixture was incubated in 50 mM sodium acetate buffer at pH 5.0 at 37°C for 12 hours. The amount of product generated was determined using an L-fucose assay kit.

[0039] The results showed that MoFco1 exhibited significant α-L-fucosidase activity for both substrates, comparable to the enzyme activity level of the positive control FgFco1. Figure 3 (C); while MoFco1 6A The enzyme activity was completely lost due to mutations at key sites, confirming that these six sites are essential for MoFco1 to exert its enzyme function. This also confirmed that MoFco1 possesses α-1,2-fucosidase specificity and can hydrolyze XXFG oligosaccharides in rice cell wall hemicellulose. Figure 3 (D).

[0040] Example 3: Apoplast localization verification of MoFco1

[0041] To clarify the role of MoFco1 in the infection process of rice blast fungus, a homologous recombination strategy was used to construct the ΔMofco1 knockout mutant. Simultaneously, MoFCO1-GFP and MoFCO1-GFP mutants were also constructed. ΔSP -GFP (missing signal peptide) and MoFCO1-GFP / BAS4-RFP (Bas4 is an apoplast marker protein) complementation vector were used to obtain recombinant rice blast fungus strains via protoplast transformation. Figure 4 (A)

[0042] The conidial suspensions of each recombinant strain (concentration 3.5 × 10⁻⁶) were prepared. 5MoFco1 (samples / mL) was added dropwise to the detached leaf sheath inner epidermis of 14-day-old CO39 rice plants and cultured in the dark at 28°C for 24 hours. Fluorescence signals were observed using a laser scanning confocal microscope (ZEISS LSM 980 with Airyscan2). Co-localization of MoFco1 and Bas4 was observed in the GFP and RFP channels of the confocal microscope. The results showed that the green fluorescence of MoFco1-GFP was localized in the apoplast space (EIHM). The fusion of the GFP fluorescence of MoFco1 and the RFP fluorescence of Bas4 (in the Merge channel) resulted in yellow light, indicating co-localization. Meanwhile, MoFco1... ΔSP -GFP's green fluorescence was confined to the cytoplasm of the infected hyphae, and no extracellular fluorescence signal was detected. Figure 4 (B)

[0043] To verify the secretory characteristics of MoFco1, conidia (5 × 10⁻⁶) of the ΔMofco1 / MoFCO1-GFP strain were collected. 5 The strain was sprayed onto CO39 rice leaves with a concentration of [number] cells / mL. After 48 hours, the leaves were cut, rinsed with sterile water, dried, and placed in centrifuge tubes. A small amount of sterile glass beads was added, and the tubes were centrifuged at 1000 rpm for 10 minutes to collect the intercellular fluid. Western blot analysis showed a specific band in the intercellular fluid using a chemiluminescence imaging system. Simultaneously, the strain was inoculated into CM liquid medium and cultured at 28°C and 180 rpm for 2 days. Mycelia were removed by filtration, and the culture medium was collected. After enrichment using a His affinity chromatography column, Western blot analysis also detected the MoFco1-GFP band, confirming that MoFco1 has secretory capacity and is an effector located on the apoplast. Figure 4 (C)

[0044] Example 4: Effects of MoFco1 deletion on the growth and development of rice blast fungus

[0045] To clarify whether MoFco1 is involved in the growth and development of rice blast fungus, this example uses Guy11, ΔMofco1, and ΔMofco1 / MoFCO1 as research objects, and systematically measures key phenotypes such as colony growth, sporulation capacity, appressorium formation, and turgor pressure.

[0046] First, mycelial blocks of each strain were inoculated into the center of CM and MM agar plates, with three biological replicates for each treatment. The plates were incubated in the dark at 28°C for 7 days. Colony diameter was measured, and colony area was calculated using ImageJ software. The results showed no significant differences among the strains on CM and MM agar plates (P>0.05), indicating that the absence of MoFco1 does not affect the vegetative growth of *Bacillus oryzae*. Figure 5 (A)

[0047] Mycelial blocks of each strain were inoculated onto SDC sporulation medium plates and incubated in the dark at 28°C for 4 days. The mycelium on the surface of the medium was gently scraped off with a sterile spatula, and sporulation was induced under black light for another 3 days. 5 mL of sterile water was added to each plate, and the surface of the medium was gently brushed with a sterile brush to collect the spore suspension. 10 μL of the filtered spore suspension was diluted 10-fold with sterile water and added to a hemocytometer. The spores were counted under an optical microscope (10×40x) to calculate the sporulation yield per plate. The results showed no significant difference among the three strains (P>0.05), and microscopic observation showed that the conidia produced by each strain were morphologically normal, with no deformities or abnormal germination. Figure 5 (B)

[0048] The conidial suspensions of each strain were adjusted to a concentration of 2×10⁻⁶. 4 20 μL of appressorium was added to a hydrophobic glass slide and placed in a culture dish lined with moist filter paper. The slide was then incubated at 28°C in the dark under humidified conditions. After 3 and 9 hours, the number of appressoriums formed was observed and counted under an optical microscope, and the appressorium formation rate (appressorium number / total spore number × 100%) was calculated. The results showed that no appressoriums formed in any strain after 3 hours; after 9 hours, there was no significant difference in the appressorium formation rate among Guy11, ΔMofco1, and the replacement strain (P>0.05). Figure 5 (C). To determine the turgor pressure of appressorium, appressorium slides cultured for 9 hours were immersed in sterile water systems containing 1M, 2M, 3M, 4M, and 5M glycerol, respectively, and left at room temperature for 30 minutes. The collapse of appressorium was observed, and the collapse rate (number of collapsed appressoriums / total number of appressoriums × 100%) was calculated. The results showed that at each glycerol concentration, the appressorium collapse rate of ΔMofco1 was not significantly different from that of Guy11 and the supplemented strain (P>0.05), indicating that the absence of MoFco1 did not affect the formation of turgor pressure in appressorium (C). Figure 5 (D). Further analysis of the infection status of ΔMofco1 ( Figure 5 (E), when spores were injected into rice leaf sheaths, the infection level was found to be significantly reduced.

[0049] The above results indicate that MoFco1 does not participate in the growth and development processes of rice blast fungus, such as vegetative growth, conidia production, appressorium formation, and turgor pressure maintenance. Its function is mainly concentrated in the interaction stage between the fungus and the host.

[0050] Culture medium preparation method:

[0051] 1. CM Culture Medium: Measure 50 mL of 20× nitrate stock solution (120 g sodium nitrate, 10.4 g potassium chloride, 10.4 g magnesium sulfate heptahydrate, 30.4 g potassium dihydrogen phosphate, dissolved in distilled water to 1 L), and 50 mL of 1000× trace element stock solution (2.2 g zinc sulfate heptahydrate, 1.1 g boric acid, 0.5 g manganese chloride tetrahydrate, 0.5 g ferric sulfate heptahydrate, 0.17 g cobalt chloride hexahydrate, 0.16 g copper sulfate pentahydrate, 0.15 g sodium manganate dihydrate, 5 g... 1 mL of tetrasodium EDTA (dissolved in distilled water to 100 mL), 1 mL of vitamin stock solution (0.01 g biotin, 0.01 g vitamin B6, 0.01 g vitamin B1, 0.01 g riboflavin, 0.01 g para-aminobenzoic acid, 0.01 g niacin, dissolved in distilled water to 100 mL), 10 g glucose, 2 g peptone, 1 g yeast extract, 1 g casein amino acids, and 15 g agar powder were added. The solution was then diluted to 1 L with distilled water, dispensed, sterilized at 121 °C for 20 minutes, and cooled for later use.

[0052] 2. SDC medium: Weigh 120g of rice straw, cut it into 2-3cm pieces, add 1L of water and boil for 60 minutes. Filter with double-layer gauze, add 40g of corn flour and 15g of agar powder to the filtrate, continue to boil for 10 minutes until completely dissolved, add water to make up to 1L, stir and mix well, dispense, sterilize at 121℃ for 20 minutes, cool and pour into plates for later use.

[0053] 3. MM medium: Weigh 1.5g sodium nitrate, 0.5g potassium chloride, 0.5g magnesium sulfate heptahydrate, 1.5g potassium dihydrogen phosphate, 0.01g ferrous sulfate, 10g glucose, and 15g agar powder, add distilled water to a final volume of 1L, stir to dissolve, dispense, sterilize at 121℃ for 20 minutes, and cool for later use (does not contain complex organic nitrogen sources).

[0054] Example 5: Verification of MoFco1's role in the pathogenicity of rice blast fungus

[0055] This embodiment systematically evaluates the pathogenicity of each strain through three methods: spray inoculation, leaf sheath injection, and detached leaf sheath infection.

[0056] Combine Guy11, ΔMofco1, ΔMofco1 / MoFCO1, ΔMofco1 / MoFCO1 6A -GFP strains were inoculated into SDC medium, and after inducing sporulation under black light, the spore suspension was collected and the concentration was adjusted to 5 × 10⁻⁶ with sterile water. 5 The concentration of spores per mL was increased by adding 0.2% (w / v) gelatin to enhance spore adhesion. A spray inoculation method was used, where the spore suspension was evenly sprayed onto the surface of rice leaves. Three pots of rice were used for each treatment. Immediately after inoculation, the plants were placed in a dark, humidified incubator for 24 hours, followed by 7 days of incubation in a light-dark alternating incubator. Lesion grades were recorded according to the rice blast lesion grading standard, and lesions were statistically analyzed at a depth of 4 cm.2 The number of lesions of different grades within the leaves was measured, and the lesion area was measured using ImageJ. The results showed that after Guy11 inoculation, rice leaves exhibited numerous type 4 and 5 lesions, ΔMofco1 and ΔMofco1 / MoFCO1. 6A -GFP inoculation significantly limited lesion expansion, with type 2 and 3 lesions predominating, while the number of type 4 and 5 lesions decreased significantly; the number and area of ​​lesions in ΔMofco1 / MoFCO1 were not significantly different from those in Guy11 (P>0.05). Figure 6 (A and B in the middle).

[0057] The in vivo leaf sheath injection method was used. Fresh leaf sheaths of CO39 rice were taken, and a concentration of 3.5 × 10⁻⁶ was injected using a sterile injection needle. 5 A spore suspension of 1 spore / mL was injected into the inner side of the leaf sheath. After 5 days of incubation at 28°C in the dark under humid conditions, the inner epidermis of the leaf sheath was peeled off, and the lesion expansion was observed under an optical microscope. The results showed that the lesions of Guy11 and the reinfected strains spread irregularly and had a larger infection area; while those of ΔMofco1 and ΔMofco1 / MoFCO1... 6A -GFP lesions are confined to the vicinity of the injection site, and the extent of their spread is significantly reduced. Figure 6 (C and D in the middle).

[0058] The in vitro leaf sheath infection method was used. In vitro leaf sheaths of CO39 rice were taken, the inner epidermis was peeled off, and the sheaths were placed in a culture dish containing sterile water. A spore suspension (3.5 × 10⁻⁶) was added. 5 The fungal hyphae (IH) at 100 infection sites were observed under an optical microscope after incubation at 28°C in the dark for 36 hours. They were classified according to a grading standard: Type I (no penetration, only spore attachment), Type II (primary IH formation, not penetrating cells), Type III (secondary IH formation, not extending to adjacent cells), and Type IV (IH extending to adjacent cells, forming an infection network). Statistical results confirmed that the enzyme activity of MoFco1 is crucial for effective infection and lesion expansion of rice blast fungus; the absence of MoFco1 or loss of its enzyme activity leads to a significant decrease in pathogenicity. Figure 6 (E and F in the middle).

[0059] Example 6: Mechanism analysis of MoFco1 response to host ROS accumulation and inhibition of defense response

[0060] This embodiment of the study investigated the molecular mechanism by which MoFco1 regulates the pathogenicity of rice blast fungus by examining both the host ROS accumulation response and the inhibition of the defense response. First, the fungus's tolerance to ROS was analyzed using a diamide (Dia) stress experiment. Dia is a thiol oxidant that can induce intracellular ROS accumulation. Mycelial blocks of each strain were inoculated into CM medium at concentrations of 0.25 mM, 0.5 mM, 1 mM, and 2 mM, respectively, and cultured at 28°C for 7 days. Colony diameters were measured, and the growth inhibition rate was calculated as ((control colony diameter - treated colony diameter) / control colony diameter × 100%). The results showed that the growth inhibition rate of all strains increased with increasing Dia concentration, but the inhibition rate of ΔMofco1 was significantly higher than that of Guy11 and the reintroduced strain, indicating that ΔMofco1 is more sensitive to ROS stress, and that MoFco1 participates in the fungus's response to ROS. Figure 7 (A)

[0061] The host ROS accumulation was detected using DAB (3,3′-diaminobenzidine tetrahydrochloride) staining. The results showed that MoFco1 deficiency led to enhanced host ROS accumulation. Figure 7 (B and C). To further confirm the effect of ROS accumulation on pathogen infection, detached leaf sheaths were treated with DPI (diphenyl iodide chloride). DPI is an irreversible NADPH oxidase inhibitor that can inhibit plant ROS production. Detached leaf sheaths were immersed in sterile water containing 10 μM DPI and treated at room temperature for 5 hours. Spore suspensions of each strain were then inoculated, and the infection hyphae were graded after 36 hours. The results showed that without DPI treatment, the number of type III and IV IHs in Guy was 36 and 30, respectively, accounting for only 66%. In ΔMofco1, the number of type III and IV IHs was 22 and 4, respectively, accounting for 26%. The ΔMofco1 / MoFCO1 ratio... 6A -GFP had 26 and 5 type III and IV IHs, respectively, accounting for 31%. Compared with Guy11, ΔMofco1 and ΔMofco1 / MoFCO1... 6A -GFP showed a significant reduction in the number of type III and IV IHs. However, after DPI treatment, the proportion of type III and IV IHs in Guy11 was 78%, while in ΔMofco1 it was 58%, and the ΔMofco1 / MoFCO1 ratio was [not specified]. 6A -GFP type III and type IV IHs accounted for 63%. Compared with Guy11, ΔMofco1 and ΔMofco1 / MoFCO1 6A The difference in the proportion of type III and type IV IH in GFP was significantly reduced. This indicates that DPI treatment restored ΔMofco1 and ΔMofco1 / MoFCO1. 6A -GFP infection deficiency, MoFco1 participates in inhibiting ROS accumulation in rice ( Figure 7 (D).

[0062] Example 7: Virtual screening and binding verification of compound 0989

[0063] To obtain inhibitors targeting MoFco1, this embodiment conducted virtual screening based on the MoFco1 protein structure and verified the binding specificity of the compounds using microthermophoresis (MST). First, the MoFco1 protein structure file was obtained from the AlphaFold3 database. The protein structure was preprocessed using AutoDock Tools. Lead compounds meeting the criteria were screened from the ZINC database, and the compound structures were converted to PDBQT format using Open Babel.

[0064] A Python script was written to drive AutoDock Vina for batch molecular docking. The three compounds with the strongest binding affinity (1432, 0989, and 0104, purchased from the ZINC database ZINC000005330953(1432), ZINC000000267143(0989), and ZINC000000045612(0104)) were selected as candidate inhibitors. The interaction between the candidate compounds and MoFco1 was analyzed using AutoDock Tools. It was found that compound 0989 can form stable hydrogen bonds and hydrophobic interactions with the active site region of MoFco1. Figure 8 Compound A); Compound 1432 interacts with only two key sites ( Figure 8 Compound B); Compound 0104 did not effectively bind to the enzyme's active site ( Figure 8 (C). Using the PyMOL visualization docking model, it was further confirmed that compound 0989 binds tightly to the active enzyme center region of MoFco1, with a high degree of spatial conformational matching.

[0065] The structural formulas of the three compounds are as follows:

[0066]

[0067] 0989 structural formula

[0068]

[0069] 0104 structural formula

[0070]

[0071] 1432 structural formula

[0072] To verify the binding affinity of the compounds to MoFco1, MST (Mechanical, Stimulating, and Transformation) technology was used. The results showed that compound 0989 exhibited strong binding affinity to MoFco1; compound 1432 showed weak binding affinity; and no obvious binding signal was detected in compound 0104, indicating that it could not bind to MoFco1. These results confirm that compound 0989 can specifically bind to the active site region of MoFco1 and is a potential MoFco1 inhibitor. Figure 8 (D).

[0073] Example 8: Activity assay of compound 0989 in inhibiting the pathogenicity of rice blast fungus

[0074] Fourteen-day-old CO39 rice seedlings were selected as the test plants. Compounds 1432, 0989, and 0104 were purchased from the corresponding suppliers in the ZINC database. Stock solutions were prepared by dissolving them in DMSO. Three biological replicates were set up for each treatment.

[0075] Treatment Method 1 (Pesticide Application Before Inoculation): Use a small sprayer to evenly spray the diluted compound solution onto the surface of the rice leaves, ensuring that both the upper and lower surfaces of the leaves are in contact with the pesticide. 24 hours after application, spray with a suspension of conidial spores of rice blast fungus Guy11 (5×10⁻⁶). 5 (each cell / mL contains 0.2% gelatin).

[0076] Treatment Method 2 (Simultaneous Treatment of Agent and Spores): Add the compound stock solution to the conidial suspension, adjusting the final compound concentration to 100 μM and the spore concentration to 5 × 10⁻⁶. 5 The concentration of the inoculated rice was 100% per mL, and the mixture was sprayed onto the rice leaves immediately after thorough mixing. After inoculation, the rice was placed in a dark, humidified incubator (100% relative humidity) for 24 hours, and then transferred to a light-dark alternating incubator for 5 days to observe the disease incidence on the rice leaves.

[0077] ImageJ software was used to measure the lesion area, and the control effect was calculated (control effect (%) = (control lesion area of ​​rice - treatment lesion area of ​​rice) / control lesion area of ​​rice × 100). The results showed that, in the first method, compared with the DMSO control group, the lesion expansion in the 0989 treatment group was restricted, and the lesion area was significantly reduced. In the 0104 and 1432 treatment groups, the restriction on lesion expansion was not obvious, and the lesion area did not change significantly. Figure 9 (A and B). In the second treatment, compared to the control, the 0989 and 0104 treatment groups showed limited lesion expansion and significantly reduced lesion area, while the 1432 treatment group showed no significant limitation in lesion expansion and no significant change in lesion area. Furthermore, for the second treatment, the 1432 treatment led to leaf death in rice plants (…). Figure 9(C). The above results confirm that compound 0989 can effectively reduce the pathogenicity of rice blast fungus by specifically binding to MoFco1 and inhibiting its enzyme activity. It also has high safety for rice and is a potential agent for the control of rice blast.

[0078] As demonstrated by the above implementation, the α-L-fucosidase MoFco1 identified in this invention is a key pathogenic protein of rice blast fungus, exhibiting high specificity and well-defined function as a target for fungicide screening. The screened compound 0989, designed based on the MoFco1 structure, possesses advantages such as high binding specificity, good safety for rice, no significant phytotoxicity, and a novel mechanism of action. This invention provides a new target and novel candidate agent for the green control of rice blast, possessing significant practical application value in agricultural production and broad prospects for further research and development into a commercial fungicide.

Claims

1. Application of α-L-fucosidase MoFco1 (GeneID: MGG_03257) in screening drugs for the prevention and control of rice blast caused by rice blast fungus.

2. A method for screening a drug for preventing and treating rice blast caused by Magnaporthe grisea, characterized by, The process includes the following steps: Based on the protein structure model of α-L-fucosidase MoFco1 (GeneID: MGG_03257) and the structure model of the substrate L-fucose / XXFG, the structure model of the test compound is docked with them, and batch docking analysis is performed using AutoDock Vina (preferably, PyMOL is used to visualize the docking model); if the test compound binds to the enzyme active center region of MoFco1 (key binding sites include LYS288, ASP242, HIS143, TRP327, GLU61 and TRP62), preventing the binding of the substrate to MoFco1, then it is identified as a candidate compound for the control of rice blast.

3. The method of claim 2, wherein, The structural models of the substrate L-fucose or XXFG were also used as controls to determine whether the candidate compound could specifically block the correct binding of the substrate to the active site of the MoFco1 enzyme; optionally, the study also included inhibition tests on rice blast fungus and / or control tests on rice blast fungus to determine its effectiveness.

4. The method of claim 2, wherein, The protein structure model of MoFco1 was obtained from the AlphaFold protein structure database and was based on its amino acid sequence prediction.

5. A method of controlling or preventing Pyricularia oryzae causing rice blast in rice, characterized by, This includes applying compound 0989, or its agriculturally active salts (such as base addition salts of inorganic and organic bases, more specifically their potassium, sodium, ammonium, dimethylamine, and isopropylamine salts) to rice, its growing sites, or its propagation material.

6. The method of claim 5, wherein, The compound 0989 is prepared into a drug formulation selected from emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, poison bait, mother liquor, and mother powder; its content in the drug is 1-99% by weight, preferably 10-80% by weight, more preferably 15-50% by weight; the effective dose concentration of the drug is 0.1μM-1mM, and the application rate is 1 to 500g / ha, preferably 50-200g / ha.

7. The use of compound 0989 or its agrochemically active salt for enhancing the resistance of rice to rice blast fungus; preferably, the agrochemically active salt is an alkali addition salt of inorganic base and organic base, more specifically its potassium salt, sodium salt, ammonium salt, dimethylamine salt, or isopropylamine salt.

8. The use of compound 0989 or its agriculturally active salt in the preparation of pesticides for controlling or preventing rice from being infected by rice blast fungus or causing disease.

9. The application as described in claim 8, characterized in that, The pesticide is a formulation of an emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, aqueous solution, poison bait, mother liquor, and mother powder, and also includes pesticide-acceptable auxiliary ingredients.

10. The application as described in claim 9, characterized in that, The pesticide is formulated for spray application; it is applied when rice blast is predicted to occur, or at the early stage of its occurrence.

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