A drug target protein FgCer1 and its application in agricultural disease control

Abstract

This invention relates to the application of the FgCer1 protein and its encoding gene in the preparation of drugs for controlling agricultural pathogenic fungi, belonging to the field of pesticide technology. This invention provides the application of the FgCer1 protein and its encoding gene as a target protein for fungicides in the preparation of drugs for controlling agricultural pathogenic fungi. Using the bacterial natural secondary metabolite HSAF as a probe, the target protein FgCer1 was identified, demonstrating its potential as a target for pathogenic fungal drugs. Based on key sites of the FgCer1 protein, through design or screening, it is hoped that specific drugs for controlling plant fungal diseases can be developed.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biopharmaceutical technology and pesticide development, specifically involving a drug target protein FgCer1 and its application in the prevention and control of agricultural diseases. Background Technology

[0002] Fusarium head blight, a global disease caused by Fusarium spp., seriously threatens wheat yield and quality. Infected wheat grains accumulate mycotoxins such as deoxynivalenol (DON), which in turn affects grain safety and human and animal health. Chemical control remains the most direct and effective means of controlling this disease. Fungicides such as carbendazim, tebuconazole, cyazofamid, and fluopyram have played a significant role in Fusarium head blight control. However, with the long-term and extensive use of fungicides, pathogens in the field have developed considerable resistance: pathogens have developed severe resistance to carbendazim, their sensitivity to tebuconazole has significantly decreased, and resistant strains have been detected; while cyazofamid and fluopyram, although highly effective, both have a high potential risk of resistance. Therefore, there is an urgent need to develop new, highly efficient fungicides and their novel targets of action.

[0003] In recent years, with the development of biotechnology, research on pesticide molecular targets and mechanisms of action has deepened, leading to the discovery of a number of new fungicide targets. For example, the oomycete fungicide fluthiazopyrone exerts its fungicidal effect by inhibiting oxidosterol-binding protein (OSBP); myosin-5, a novel target of cyazofamid, provides an important foundation for precise targeted fungicide development; and chitin synthase, indispensable in insects and fungi but absent in plants and mammals, has become an important target for creating highly efficient, safe, and eco-friendly pesticides. A new target can not only spawn dozens or even hundreds of pesticide varieties but also greatly alleviate the problem of pesticide resistance in existing pesticides.

[0004] Macrolides sulfadiazine (HSAF) are natural products isolated from the biocontrol bacterium *Lysobacter enzymogenes*. They exhibit strong antibacterial activity against various fungi and oomycetes (EC50 0.5-2.0 μg / mL), and possess thermostable properties and good environmental compatibility. HSAF consists of a unique macrolide system, a tetracarboxylic acid structure, and a 5,5,6-tricyclic skeleton, and its structure differs from currently commercially available fungicides. Current research on the mechanism of action of HSAF against pathogens is still in its early stages, which greatly opens up possibilities for the discovery of new targets.

[0005] In conclusion, continuously exploring new targets and creating lead compounds remains an urgent task in the research and development of green pesticides. In particular, for wheat scab, developing fungicides with novel mechanisms of action and their corresponding targets is of great significance for effectively controlling herbicide resistance and ensuring food security. Summary of the Invention

[0006] The purpose of this invention is to provide a drug target protein FgCer1 and its application in the prevention and control of agricultural diseases.

[0007] This invention provides a drug target protein FgCer1, the amino acid sequence of which is shown in SEQ ID No. 1.

[0008] Specifically, the target protein FgCer1 shown in SEQ ID No. 1 comes from Fusarium graminearum.

[0009] The present invention further provides the gene encoding the drug target protein.

[0010] Specifically, the nucleotide sequence of the encoding gene is shown in SEQ ID No. 2.

[0011] This invention also provides the application of substances that bind to the FgCer1 protein in the preparation of drugs for the prevention and control of agricultural fungal diseases.

[0012] In one specific instance, the substance that binds to the FgCer1 protein is a substance capable of binding to glutamine at position 208 and glutamate at position 272 of the FgCer1 protein.

[0013] The present invention also provides the use of the drug target protein FgCer1 or the encoding gene described herein in the design or screening of inhibitors for the prevention and control of plant pathogenic fungi.

[0014] This invention also provides a method for designing or screening inhibitors for the prevention and control of plant pathogenic fungi, using the drug target protein FgCer1 as a target and screening for substances that can bind to it using high-throughput screening technology.

[0015] Specifically, the plant pathogenic fungus described in this invention is selected from Fusarium graminearum.

[0016] Specifically, the substance that binds to the FgCer1 protein is a small molecule compound, including but not limited to any of the following:

[0017] .

[0018] The inhibitors for the control of plant pathogenic fungi described in this invention can be designed using conventional methods in the field, such as computer-aided drug design technology, based on the protein structure and key functional site information of FgCer1, by screening small molecule compounds that may specifically bind to FgCer1 in a compound database, and further evaluating the inhibitory effect of the compounds on plant pathogenic fungi.

[0019] The benefits of this invention are as follows: This invention provides information on the protein structure and key functional sites of FgCer1, offering an important basis for the rational design of pesticide molecules. Based on this target, by designing or screening pesticide molecules that specifically act on FgCer1, novel targeted fungicides can be developed, providing new options for plant disease control. Attached Figure Description

[0020] Figure 1 PCR verification results for Fusarium graminearum FgCer1 gene knockout mutant and complement.

[0021] Figure 2 Sensitivity of FgCer1 gene knockout mutant to HSAF.

[0022] Figure 3 The interaction sites between FgCer1 and HSAF are shown; where A is the molecular docking model of HSAF and FgCer1, and B is the SPR detection of the binding of FgCer1 and mutant proteins to HSAF.

[0023] Figure 4 To screen the binding models of compounds with FgCer1; Figure A represents Fs-1, Figure B represents Fs-2, and Figure C represents Fs-3.

[0024] Figure 5 To screen compounds for their inhibitory activity against Fusarium graminearum. Detailed Implementation

[0025] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0026] Unless otherwise specified, the Fusarium graminearum used in the following examples is preserved in our laboratory and is open to public access.

[0027] Example 1: Identification of FgCer1 as a target of HSAF

[0028] (1) Construction of FgCer1 knockout box

[0029] Using genomic DNA from *Fusarium graminearum* PH-1 as a template, the upstream fragment was amplified using primers FgCer1-UF and FgCer1-UR (Table 1), and the downstream fragment was amplified using primers FgCer1-DF and FgCer1-DR. Hygromycin fragments were amplified using KzHPH-F and KzHPH-R, and the upstream, downstream, and HPH fragments were fused using Supermix and the corresponding fusion program. The fusion products were used as templates to amplify fragments using FgCer1-CF and FgCer1-CR, and the purified PCR product was the FgCer1 knockout cassette. The PCR product was then used for protoplast transformation.

[0030] (2) Protoplast transformation

[0031] First, protoplasts were prepared. Fusarium graminearum PH-1 hyphae were placed in a 50 mL sterile Erlenmeyer flask, and 30 mL of YEPD medium was added. The flask was incubated overnight at 25°C and 175 rpm with shaking. The culture was filtered through a sterile filter cloth, and the hyphae were collected. The filter cloth was rinsed twice with 0.7 M NaCl. The hyphae were then transferred to a 50 mL sterile Erlenmeyer flask, and 10 mL of cell wall lysis buffer was added per 1 g of hyphae. The flask was incubated at 30°C and 90 rpm for 1.5–2 h. After hyphae lysis, the mixture was filtered through a sterile filter cloth into a sterile 50 mL centrifuge tube. The tube was centrifuged at 7000 rpm for 5 min, the supernatant was discarded, and 10 mL of STC buffer was added to the centrifuge tube to gently wash the precipitate. The tube was then centrifuged at 5000 rpm for 5 min. 0.5–1 mL of STC buffer was added according to the amount of precipitate, and the mixture was thoroughly mixed by pipetting. The concentration of protoplasts was determined under a microscope using a hemocytometer, and the concentration was adjusted to 2-5 × 10⁻⁶ per milliliter. 5 One protoplast is recommended. Aliquot 200 μL of protoplast solution into 50 mL centrifuge tubes and incubate on ice for at least 1 h. Add 40 μL of the above PCR fusion fragment to each protoplast solution and incubate for 30 min. Slowly drip 1 mL of SPTC into the centrifuge tube (this can be done in two separate drips, 500 μL each time, with a 1-minute interval), gently mix, and incubate for at least 30 min. Then add the mixture to 200 mL of RM medium and pour into a culture dish. Incubate at 25°C overnight. The next day, pour an equal volume of 100 μg / mL SRM medium to cover the RM medium and incubate for 3 days.

[0032] (3) Mutant screening and validation

[0033] Single colonies grown on the selection medium were transferred to PDA plates containing 100 μg / mL hygromycin and cultured for 3 days. Colonies were then picked for further validation. Genomic DNA was extracted, and the target gene and the size of its upper and lower arm binding regions were verified using appropriate primer pairs. Figure 1 The absence of the target gene, but the presence of both upper and lower binding arms, indicates a mutant. FgCer1-MF and FgCer1-MR primers were used to verify the absence of the target gene. The FgCer1 target gene is 4475 bp in size, and the mutant target gene is absent compared to wild-type PH-1 and its complement. FgCer1-UF and YzHPH-R primers verified the binding region between the upstream fragment of the target gene and hygromycin, measuring 2038 bp. YzHPH-F and FgCer1-DR primers verified the binding region between the upstream fragment of the mutant and hygromycin, measuring 2030 bp. Compared to wild-type PH-1 and its complement, the mutant exhibits both upper and lower binding domains.

[0034] (4) Sensitivity detection of mutants to HSAF

[0035] Wild-type and mutant strains were inoculated onto PDA medium for 3 days. After inoculating the culture medium with 5 mm holes along the hyphal margin, the culture was transferred to YBA medium supplemented with different concentrations of HSAF. Each treatment was repeated in triplicate. The control group (without HSAF) was used. Colony diameter was recorded every 12 hours. In the control group, photos were taken after the hyphae had fully colonized the medium. Figure 2 As shown, the ΔFgCer1 mutant showed significantly reduced HSAF sensitivity compared to the wild-type and complement strains.

[0036] Table 1 Primers used in the examples

[0037] .

[0038] Example 2: Interaction model between FgCer1 protein and HSAF

[0039] FgCer1 is a ceramide synthase. To further verify that FgCer1 is a target of HSAF, this invention conducted an in vitro interaction experiment between FgCer1 and HSAF. FgCer1 protein was expressed, isolated, and purified. The affinity coefficient between FgCer1 and HSAF was determined using surface plasmon resonance (SPR) technology. HSAF was diluted to the printing concentration using DMSO as the stationary phase printing solution. The printing solution was printed onto a 3D photocrosslinked chip using a Biodot™ AD1520 chip array printer. Four replicates were printed for each sample, and four positive control spots (rapamycin) were printed at the four corners. PBST (pH=7.4, 0.1% Tween 20) was added to the FgCer1 protein sample stock solution to dilute it to five concentration gradients: 10 nM, 40 nM, 160 nM, 640 nM, and 2560 nM. Mobile phase samples of different concentration gradients were loaded sequentially from low to high concentration. Based on the real-time detection results from the SPR device, the kinetic curve of the interaction was fitted and the affinity parameters were output. The results show that FgCer1 and HSAF are strongly bound, with a dissociation constant of 1.52 × 10⁻⁶. -7 M ( Figure 3 ).

[0040] The target protein FgCer1 was modeled using AlphaFold software, and molecular docking was performed using AutoDockVina software. Key interaction sites between FgCer1 and HSAF were predicted. The amino acids forming hydrogen bonds included glutamine (Gln) at position 208, glutamate (Glu) at position 272, and histidine (His) at position 285. Figure 3 A). The FgCer1 point mutant protein was obtained, and the dissociation constants of the mutant proteins ΔGln208, ΔGlu272, and ΔHis285 with HSAF, as determined by SPR, were 4.95 × 10⁻⁶. -5 M, 2.41×10 -5 M, 9.56×10 -7 Mutations in M, Gln208, and Glu272 weakened the binding of FgCer1 to HSAF, clearly identifying Gln208 and Glu272 as key binding sites. Figure 3 B).

[0041] Example 3: Screening of small molecule compounds using FgCer1 protein as a target

[0042] The three-dimensional structure of the bait protein was constructed using AlphaFold3. Autodock Vina was used for the first high-throughput screening of a selected compound library, resulting in Vina scores for 116,806 compounds. 1% of the Max Affinity values ​​from the high-throughput screening were then used for the second round of virtual screening. Autodock Vina was used for the second round of virtual screening, and 10% of the Max Affinity values ​​from the virtual screening were used for the third round of refined screening. Finally, a third round of refined screening was performed to obtain the final Max Affinity values. Autodock Vina's scoring criteria have four gradients: very good binding (score of -10 kcal / mol or lower); good binding (score between -7 and -10 kcal / mol); moderate binding (score typically between -5 and -7 kcal / mol); and weak binding (score typically above -5 kcal / mol). Of the 127 compounds selected for refined screening using Autodock Vina, 21 had protein binding scores less than -10 kcal / mol, representing very good scores. Three compounds, Fs-1, Fs-2, and Fs-3, were analyzed to obtain their docking results with FgCer1, with binding constants of -10.263 kcal / mol, -10.181 kcal / mol, and -10.020 kcal / mol, respectively. Figure 4 ).

[0043] Example 4 Activity determination of small molecule compounds

[0044] 100 μg / mL of the compound screened in Example 3 was added to a PDA plate. Fusarium graminearum hyphae were taken using a 5 mm punch and inoculated into the center of the drug-containing PDA plate. The plates were then incubated at 26°C. When the hyphae of the control group reached the edge of the culture dish, the culture dish was removed, and the diameter of each colony was measured and recorded.

[0045] The results are as follows Figure 5 As shown, the inhibition rates of Fs-1, Fs-2, and Fs-3 against Fusarium graminearum were 38.18%, 44.24%, and 40%, respectively. Compounds that can bind to protein FgCer1 have good inhibitory effects on Fusarium graminearum, indicating that protein FgCer1 can be used for screening drugs that inhibit Fusarium graminearum.

Claims

1. The application of substances containing FgCer1 protein in the preparation of drugs for the prevention and control of agricultural fungal diseases, wherein the amino acid sequence of FgCer1 protein is shown in SEQ ID No.

1.

2. The application as described in claim 1, characterized in that, The substance that binds to the FgCer1 protein is a substance that can bind to glutamine at position 208 and glutamate at position 272 of the FgCer1 protein.

3. The application as described in claim 1, characterized in that, The substance that binds to the FgCer1 protein is selected from any of the following: 。 4. The application as described in claim 1, characterized in that, Agricultural fungal diseases are caused by Fusarium graminearum.

5. The application of FgCer1 protein in screening or designing drugs that can be used to control agricultural fungal diseases, wherein the amino acid sequence of FgCer1 protein is shown in SEQ ID No.

1.

6. The application as described in claim 1, characterized in that, Agricultural fungal diseases are caused by Fusarium graminearum.

7. The application as described in claim 1, characterized in that, The specific screening method involves using the FgCer1 protein shown in SEQ ID No. 1 as a target, and screening or designing substances that can bind to the FgCer1 protein through high-throughput screening technology.

8. The gene encoding the FgCer1 protein shown in SEQ ID No.

1.

9. The gene according to claim 8, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

2.

10. The use of the gene of claim 8 or 9 in screening or designing drugs that can be used to control agricultural fungal diseases, preferably, the agricultural fungal disease is a disease caused by Fusarium graminearum.

Images