Fungal effector drug target Nep1 and high-throughput drug molecule screening and application based on this drug target

Abstract

The present invention belongs to the technical field of drug target materials, and specifically relates to a fungal effector drug target Nep1 and high-throughput drug molecule screening and application based on the drug target. The present invention provides a fungal drug target protein CiNep1, and an inhibitor binding functional domain contained therein, wherein the fungal inhibitor binding functional domain comprises one or more amino acid sites corresponding to the amino acid sequence shown in SEQ ID NO.1. At the same time, the present invention uses this binding functional domain as a basis to design a new type of chemical molecule, uses the inhibitor binding functional domain in the protein Nep1 to perform high-throughput drug molecule screening, and successfully screens out a fungal drug that can inhibit the fungus soybean red crown rot fungus ( Calonectria ilicicola The protein Nep1 provided by the present invention can effectively interfere with the interaction between pathogenic microorganisms and host plants, providing a new approach for the development of existing antifungal drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug target materials, and in particular relates to a fungal effector drug target Nep1 and high-throughput drug molecule screening and application based on the drug target. Background Art

[0002] Soybean red crown rot is one of the main diseases that causes premature aging of soybeans. It is more serious in some soybean producing areas in the Huanghuaihai area of ​​my country, and can reduce yields by 50% in epidemic years. It is spreading year by year. The sexual form of the pathogen is Holly Lichiko ( Calonectria ilicicola ), the asexual state is Parasitic Columnar Sporangium ( Cylindrocladium parasiticum ), a necrotrophic parasite that causes soil-borne diseases. Currently, there are no commercially available soybean resistant varieties or control agents for this disease, severely limiting the yield and quality of agricultural products. Therefore, by studying the pathogen's host interaction mechanism, we aim to identify new drug targets for soybean red crown rot and develop new drugs.

[0003] During the long-term interaction between pathogens and host plants, plants have evolved two immune systems: PTI and ETI. PTI is the process of recognizing microbial components through PRRs, thereby triggering plant immune responses. Therefore, this type of immune response is relatively broad-spectrum; ETI refers to the process of NLRs located within the cell sensing the effector factors of pathogens directly or indirectly through signaling molecules such as hormones, thereby triggering the plant's specific immunity. For example, in 2023, it was first reported that a new fungicide FY21001 was developed using the rice blast fungus effector MoErs1 as a template. FY21001 is a compound designed based on protein structure that can specifically bind to and inhibit the function of MoErs1 and has excellent control effects against rice blast in the field. This study revealed a method for creating new agents that are specific to pathogen effector proteins and are used for disease prevention and control. During the infection of soybeans by soybean red crown rot fungus, soybean red crown rot fungus recognizes host signals, forms penetrating hyphae, and releases a large number of effectors into plant cells or the apoplast during the infection process. This indicates that the effector of soybean red crown rot fungus may play an extremely important role in the process of soybean red crown rot fungus infecting soybean. Summary of the Invention

[0004] The present invention identified 17 apoplast effectors of soybean red crown rot fungus through mass spectrometry analysis and bioinformatics. Among them, the purified protein of one effector (Necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins Nep1) can successfully induce plant immunity and enhance plant disease resistance, indicating that this effector can be developed and utilized as a plant resistance elicitor; in addition, knocking out the gene encoding this effector can significantly reduce the pathogenicity of soybean red crown rot fungus.

[0005] In one aspect, the present invention provides a fungal inhibitor binding domain, which comprises one or more of the following amino acid sites corresponding to the amino acid sequence shown in SEQ ID NO. 1: 129ASP, 139GLY, 140HIS, 143ASP, 144TRP, 145GLU, 146SER, 166ALA, 167SER, 169HIS, 170GLY, 172PHE, 196ASN, 197HIS, 232LYS, 233ALA, 234ASN, 235CYS, 236PRO and 245ASN.

[0006] In certain embodiments, the amino acid sequence of the fungal inhibitor binding domain is any one of the following,

[0007] 1) consisting of amino acid 129 to amino acid 245 of the sequence shown in SEQ ID NO.1; or

[0008] 2) An amino acid sequence having more than 95% homology with the amino acid sequence shown in 1) obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification.

[0009] In certain embodiments, the fungus is Psoralea corylifolia ( Calonectria ilicicola ).

[0010] On the other hand, the present invention also provides a fungal drug target protein CiNep1, which comprises the fungal inhibitor binding functional domain.

[0011] In certain embodiments, the amino acid sequence of the fungal drug target protein CiNep1 is any one of the following:

[0012] 1) the amino acid sequence shown in SEQ ID NO. 1; or

[0013] 2) An amino acid sequence having more than 95% homology with the amino acid sequence shown in 1) obtained by substitution, deletion and / or addition of one or more amino acids and / or terminal modification.

[0014] In certain embodiments, the nucleotide sequence of the fungal drug target protein CiNep1 is any one of the following:

[0015] 1) the nucleotide sequence shown in SEQ ID NO. 2; or

[0016] 2) It can hybridize with the complementary sequence of the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions and encode a protein having the same function as the protein encoded by the nucleotide sequence shown in SEQ ID NO. 2.

[0017] On the other hand, the present invention also provides the use of the fungal inhibitor binding domain or the fungal drug target protein CiNep1 as a drug target in high-throughput screening of fungal inhibitor molecules.

[0018] In certain embodiments, the fungus is Psoralea corylifolia ( Calonectria ilicicola ).

[0019] On the other hand, the present invention also provides the use of the fungal drug target protein CiNep1 in inducing plant immunity or enhancing plant disease resistance.

[0020] In certain embodiments, the fungal drug target protein CiNep1 is introduced into plant tissues or cells.

[0021] In certain embodiments, the plant is tobacco or a dicot.

[0022] In certain embodiments, the dicot is soybean, cotton, or pepper.

[0023] On the other hand, the present invention also provides the use of small molecule inhibitors that can bind to the fungal inhibitor binding functional domain or inhibit the expression activity of the fungal drug target protein CiNep1 in the preparation of drugs for preventing and treating plant diseases or human and animal diseases caused by pathogenic fungi.

[0024] In certain embodiments, the inhibitor molecule is a compound having a core skeleton of a bisbenzothiazole-sulfide bicyclic structure or a derivative thereof.

[0025] In certain embodiments, the structural formula is .

[0026] In certain embodiments, the fungus is Psoralea corylifolia ( Calonectria ilicicola ).

[0027] In certain embodiments, the plant is tobacco or a dicot.

[0028] In certain embodiments, the dicot is soybean, cotton, or pepper.

[0029] In certain embodiments, the concentration of the inhibitor small molecule in the drug is 250-500 μg / mL.

[0030] In certain embodiments, the concentration of the inhibitor small molecule in the medicament is 500 μg / mL.

[0031] Compared with the existing technology, the present invention provides a necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins Nep1, a soy red crown rot pathogen. This protein can successfully induce plant immunity and enhance plant disease resistance. At the same time, the present invention also identified the fungal inhibitor binding domain of the protein as a fungal drug target. Based on this binding domain, a new chemical molecule design was carried out. The inhibitor binding domain in the protein Nep1 was used for high-throughput drug molecule screening, and the inhibitor binding domain in the protein Nep1 was successfully screened to inhibit the fungus soybean red crown rot. Calonectria ilicicola The protein Nep1 provided by the present invention can effectively interfere with the interaction between pathogenic microorganisms and host plants, providing a new approach for the development of existing antifungal drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Southern blot verification of ∆CiNep1 transformants; A is the upstream fragment verification of ∆CiNep1 transformants, and B is the downstream fragment verification of ∆CiNep1 transformants.

[0033] Figure 2 ΔCiNep1 pathogenicity assay.

[0034] Figure 3 The prokaryotic recombinant CiNep1 protein induces cell necrosis in dicotyledonous plants; A is the minimum concentration of the purified CiNep1 protein that can induce necrosis in Nicotiana benthamiana cells; B is the concentration at which the purified CiNep1 protein can induce necrosis in dicotyledonous plant cells.

[0035] Figure 4 The three-dimensional structure prediction of the target protein CiNep1; A is the rationality of the model tested using the Ramachandran plot; B is the 3D structural model of the drug target protein CiNep1.

[0036] Figure 5 The predicted binding pocket of the target protein CiNep1; A is the predicted docking pocket of the drug target protein CiNep1, and the red part is the position of the docking pocket; B is the distribution of amino acids around the docking pocket of the drug target protein CiNep1.

[0037] Figure 6 is the chemical structural formula of the inhibitor small molecule.

[0038] Figure 7 This is a 3D conformational diagram of the inhibitor small molecule binding to the drug target protein CiNep1.

[0039] Figure 8 Schematic diagram of the amino acid 2D visualization of the binding of the inhibitory small molecule to the drug target protein CiNep1.

[0040] Figure 9 The protective effect of the inhibitor small molecule.

[0041] Figure 10 Small molecules that are inhibitors of therapeutic effects. DETAILED DESCRIPTION

[0042] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0043] Example 1 Construction of a knockout strain of CiNep1 and acquisition of mutants of soybean red crown rot pathogen

[0044] The present invention shows that the fungal drug target protein Nep1 is widely distributed in plant pathogenic microorganisms such as fungi, oomycetes and bacteria through sequence comparison, and has the characteristics of being developed as a new fungal drug sensitivity target protein. The fungal drug target protein Nep1 of the present invention is preferably derived from soybean red crown rot fungus ( Calonectria ilicicola ), the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of which is shown in SEQ ID NO.2.

[0045] Based on the principle of homologous double exchange, primers were designed to amplify 800-1200 bp homologous fragments upstream and downstream of CiNep1, about 200 bp away from the target gene at both ends. Corresponding primer adapters were added at the connection position, and nested primers were designed on this basis.

[0046] Using a three-segment fusion method, the three fragments were connected by touchdown PCR and then amplified using nested primers. The PCR amplification procedure was as follows: 94°C pre-denaturation for 3 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C for 2 minutes; then extension at 72°C for 10 minutes, followed by a hold at 4°C. The PCR amplification reaction system (50 μL) consisted of 30 μL of 2× Vazyme LAmp Master Mix (DyePlus), 1 μL of DNA (50 ng / μL), 2 μL each of primers F and R, and 20 μL of ddH2O. After agarose gel electrophoresis, the target band of the correct size was excised and recovered using a gel recovery kit. This fragment, the knockout vector fragment, was used for protoplast transformation.

[0047] From 7 days old CiConidia were collected from cultures grown in V8 medium and transferred to YEPD liquid medium containing 10 g of peptone, 3 g of yeast extract, and 20 g of glucose. After 24 hours of incubation at 25°C, young mycelia were filtered from the YEPD liquid medium, washed with 0.7 M sodium chloride, and then treated with lysozyme (10 mg / mL in 0.7 M sodium chloride, Sigma), cellulase (10 mg / mL in 0.7 M sodium chloride, Sigma), and snailase (10 mg / mL in 0.7 M sodium chloride, Sigma). After incubation at 30°C and 80 rpm for 3 hours, the enzyme solution was filtered through three layers of lens paper to remove mycelial residues, and the protoplasts in the filtrate were then washed twice with 0.7 M sodium chloride.

[0048] After protoplast transformation of knockout vector, the transformant genome was extracted and verified using verification primers. CiNep1 Southern blot verification results of transformants are as follows Figure 1 As shown, A is the upstream fragment verification of ∆CiNep1 transformant, B is the downstream fragment verification of ∆CiNep1 transformant, and ∆ CiNep1 strains.

[0049] The primers used in this example are shown in Table 1.

[0050] Table 1 Soybean red crown rot pathogens CiNep1 Primers used for knockout

[0051]

[0052] Example 2 Determination of Pathogenicity of ΔCiNep1

[0053] The activated strain Δ CiNep1 -12, Δ CiNep1 -25 and the parent strain were inoculated on V8 plates and cultured for 7 days. Conidia were collected in 50 mL centrifuge tubes and the concentration was adjusted to 10 6 / mL. 20 μL of the conidia suspension was inoculated onto the hypocotyls of 12-day-old soybean seedlings. The seedlings were then incubated in the dark in a climate chamber for 7 days. The lesion lengths of each test strain were recorded and photographed. Twenty soybean seedlings were inoculated with each strain. The experiment was repeated three times.

[0054] The experimental results are as follows Figure 2 As shown, the knockout mutant ∆ CiNep1 -12, ∆ CiNep1 -25 more sensitive strains Ci58 and Ci62 The pathogenicity to soybean is significantly reduced.

[0055] Example 3 Purification of soybean red crown rot pathogen CiNep1

[0056] (1) CiNep1 After cloning the nucleotide sequence of SEQ ID NO.2, the prokaryotic protein was expressed using Escherichia coli BL21 and recombined with the pGEX-4T-2 linear vector using the homologous recombination method;

[0057] (2) After obtaining the circularized vector, transform it into the competent cell of TreliefTM5α in E. coli, and sequence the positive transformants;

[0058] (3) After shaking the correct transformants, extract the plasmid, and then transform the plasmid into the competent E. coli BL21 cells;

[0059] (4) Select a single clone and shake culture it in 7 mL of liquid LB containing the corresponding antibiotics; (5) Inoculate it into liquid LB containing the corresponding antibiotics at a ratio of 1:50 for propagation;

[0060] (6) Add IPTG to a final concentration of 0.1 mM for expression;

[0061] (7) Centrifuge at 5000 rpm for 10 min, remove the precipitate, wash it three times with 1× PBS, concentrate it 10 times, and store it on ice for later use;

[0062] (8) Add PMSF to a final concentration of 100 mM and sonicate on ice until the solution becomes transparent;

[0063] (9) The speed was set to 13400 g. After centrifugation for 20 min, SDS-PAGE was used to detect the protein expression and solubility in the supernatant and precipitate.

[0064] (10) The supernatant was filtered and adjusted to pH 7.5. Protein purification was performed using AKTA, and then the protein CiNep1 was obtained by passing it through a Glutathione 4FF Chromatography Column and a desalting column.

[0065] Example 4: Purified CiNep1 protein induces cell death in dicotyledonous plants

[0066] The purified protein CiNep1 was injected into tobacco at a concentration series (7 μM, 700 nM, 350 nM, 70 nM, 14 nM, 1.4 nM, and 0.7 nM). After 48 hours of greenhouse culture, the necrosis of tobacco cells was observed. Buffer and 7 μM GST were added as blank controls during this process. The purified CiNep1 protein at a concentration of 70 nM was injected into soybean, cotton, pepper, wheat, and corn leaves. After 48 hours of greenhouse culture, the necrosis of each crop cell was observed.

[0067] Cell necrosis Figure 3 As shown, the minimum concentration of purified CiNep1 protein that could induce tobacco cell death was 1.4 nM ( Figure 3 A). 70 nM of purified CiNep1 protein was injected into soybean, cotton, pepper, wheat, and corn leaves. The experimental results showed that the purified CiNep1 protein could successfully induce cell death in dicotyledonous plants such as soybean, cotton, and pepper, but could not induce cell death in monocotyledonous plants such as corn and wheat ( Figure 3 B).

[0068] Example 5 Prediction of the soybean red crown rot pathogen CiNep1 model

[0069] The target protein sequence was modeled into a three-dimensional structure, and 5 models were obtained. The model with the highest score was selected for subsequent research. To further clarify the accuracy of the results, the Ramachandran plot was used to further evaluate the quality of the prediction results. Figure 4 As can be seen from A, the number of amino acid residues falling in the permissible region accounts for 97.1% of the total number of amino acid residues. Combined with PLDDT scoring and PROCHECK test, the Ranked0 model meets the parameter requirements ( Figure 4 B) can proceed to the next step of research.

[0070] Example 6 High-throughput screening of small molecules for inhibitors of CiNep1 against soybean red crown rot pathogen

[0071] Based on the 3D molecular structure of CiNep1, the active pocket of the target protein was first predicted. Figure 5 As can be seen in A, the red part is the location of the target protein binding pocket; further analysis shows that there are 20 amino acid residues around the docking pocket of the target protein CiNep1, namely 129Asp, 139Gly, 140His, 143Asp, 144Trp, 145Glu, 146Ser, 166Ala, 167Ser, 169His, 170Gly, 172Phe, 196Asn, 197His, 232Lys, 233Ala, 234Asn, 235Cys, 236Pro, 245Asn ( Figure 5 B) This protein pocket will be used for the next virtual screening.

[0072] After software analysis and optimization, CiNep1 The protein is set as the receptor and the inhibitor small molecule is set as the ligand. The software evaluates the binding between the drug molecule and the target protein through the binding energy after docking. It is generally believed that the lower the binding energy between the ligand and the receptor, the more stable the binding. The obtained model can show the binding site and the corresponding interaction force. After multiple rounds of virtual screening, potential compounds are finally obtained, such as Figure 6 , showing the structure of the small molecule inhibitor, whose English name is 6-[(1,3-benzothiazol-2-ylsulfanyl) methyl]-2-[(4,6-dimethylquinazolin-2-yl)amino]pyrimidin-4(1H)-one, Smiles is Cc1ccc2nc(Nc3nc (CSc4nc5ccccc5s4)cc(=O)[nH]3)nc(C)c2c1, Pubchem accession number is 135425015, and chemical formula is C 22 H 18 N6OS2, with a molecular weight of 466.55, was synthesized in the laboratory and used for antibacterial activity assay.

[0073] In order to further clarify the binding conformation of small molecule inhibitors, the software analysis showed that the small molecule inhibitors completely fell into CiNep1 In the binding pocket of proteins, such as Figure 7 Further analysis revealed that small molecule inhibitors CiNep1 The amino acid residues W144, E145, S146, G170, and N234 of the protein form hydrogen bonds ( Figure 8 ).

[0074] Example 7 Determination of antibacterial activity of small molecule inhibitors

[0075] In order to determine the antibacterial activity of small molecule compounds, yellow soybean seedlings were treated with protective treatment before inoculation with soybean red crown rot fungus and therapeutic treatment after inoculation. In this example, the concentration of the inhibitor small molecule was adjusted to 250 μg / mL and 500 μg / mL with water.

[0076] For the protection treatment, the hypocotyls of etiolated soybean seedlings were soaked with the above two concentrations of inhibitor small molecules, dried, and cultured in a 25°C climate chamber in the dark for 24 h. Then, 10 μL of soybean red crown rot conidia suspension (10 6 / mL) and continue culturing for 5 days.

[0077] For the treatment, 10 μL of conidia suspension was first inoculated into the hypocotyls of etiolated soybean seedlings and cultured in the dark in a climate chamber at 25°C for 24 h. Then, the hypocotyls of etiolated soybean seedlings were soaked with the above-mentioned two concentrations of small molecule compounds, dried, and then cultured in the dark in a climate chamber for another 4 days.

[0078] The control in the above experiments was the hypocotyls of etiolated soybean seedlings treated with water. Each treatment in this experiment was replicated 21 times, and the experiment was repeated 3 times.

[0079] The results are as follows Figure 9-10 As shown, from Figure 9 It can be seen from the results that the protection efficiency of small molecule inhibitors increases with the increase of the concentration of small molecule compounds. The protection efficiencies at 250 μg / mL and 500 μg / mL are 60.02% and 81.10%, respectively. Figure 10 It can be seen that the therapeutic efficiency of small molecule inhibitors increases with the increase of the concentration of small molecule compounds, and the protection efficiencies at 250 μg / mL and 500 μg / mL are 22.13% and 50.42%, respectively.

[0080] While the specific embodiments of the present invention have been described in detail above, these are intended to be exemplary only, and the present invention is not limited thereto. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, any equivalent changes and modifications made without departing from the spirit and scope of the present invention are intended to be encompassed within the scope of the present invention.

Claims

1. An isolated polypeptide CiNep1, characterized in that The amino acid sequence of the polypeptide is shown in SEQ ID NO.

1.

2. A nucleic acid encoding the polypeptide CiNep1 according to claim 1, characterized in that The sequence of the nucleic acid is shown in SEQ ID NO.

2.

3. Use of the polypeptide CiNep1 according to claim 1 in high-throughput screening of fungal inhibitor molecules, characterized in that: The fungus is soybean red crown rot ( Calonectria ilicicola ), the application is to perform high-throughput screening of fungal inhibitor molecules using 144TRP, 145GLU, 146SER, 170GLY and 234ASN corresponding to the amino acid sequence shown in SEQ ID NO.1 as drug targets.

4. The use according to claim 3, characterized in that High-throughput screening of fungal inhibitor molecules was performed using the amino acid sequence shown in SEQ ID NO.1 as a drug target.

5. Use of a small molecule inhibitor capable of binding to or inhibiting the expression activity of the polypeptide CiNep1 according to claim 1 in the preparation of an inhibitor for preventing and treating soybean diseases caused by soybean red crown rot fungus, characterized in that: The structural formula of the small molecule inhibitor is .

Images