Application of FvSup1 protein and coding gene thereof in regulating pathogenicity of fusarium verticillium

Abstract

The invention discloses an application of FvSup1 protein and a coding gene thereof in regulation and control of pathogenicity of fusarium verticillium, and belongs to the technical field of biology. New functions of the fusarium verticillium unknown function protein FvSup1 and the coding gene FvSup1 thereof are researched and confirmed for the first time. The method comprises the following steps: constructing an FvSup1 gene knockout vector, and introducing the FvSup1 gene knockout vector into a fusarium verticillium protoplast to obtain a knockout mutant delta FvSup1; the method comprises the following steps: constructing a gene back-up vector, and introducing the gene back-up vector into a delta FvSup1 protoplast; and a complement mutant delta FvSup1-C is obtained. Pathogenicity determination shows that the pathogenicity of the knockout mutant delta FvSup1 is obviously reduced; and the pathogenicity of the complement mutant delta FvSup1-C can be recovered to a wild type level. The test proves that the FvSup1 is a pathogenic related gene of the fusarium verticillium, and a new target is provided for preventing and treating plant diseases caused by the fusarium verticillium.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the FvSup1 protein and its encoding gene in regulating the pathogenicity of Fusarium verticillatum. Background Technology

[0002] Fusarium ( Fusarium As a globally distributed plant pathogenic fungus, it can widely infect many important crops such as wheat, corn, and tomatoes, inducing devastating diseases such as Fusarium head blight, wilt, and stem rot, seriously threatening global food security, and urgently requiring the establishment of an efficient and feasible prevention and control system.

[0003] Currently, chemical control remains the primary means of controlling Fusarium diseases. However, the long-term and excessive use of chemical pesticides has not only led to increased pesticide resistance in pathogens and reduced control efficacy, forcing a continuous increase in pesticide usage, but has also caused serious environmental pollution and pesticide residue problems in agricultural products, posing a dual threat to ecological security and human health. Therefore, the development of novel green control agents and the cultivation of disease-resistant crop varieties have become the core development direction for Fusarium disease control, and the key to technological breakthroughs lies in the precise identification of the pathogen's core pathogenic targets. Although the whole-genome sequencing of many pathogenic Fusarium species has been successfully completed, pathogenic genes with clearly defined functions in the genomes are still relatively scarce.

[0004] Fusarium pseudoverticum ( Fusarium verticillioides *Fusarium* species *Verticillium* is a common pathogenic fungus that can cause various transmissible diseases in grain crops, especially maize. Fusarium verticillioides Although the whole genome sequencing of *Fusarium oxysporum* has been completed, the genome of this strain contains 14,179 protein-coding genes. According to annotation statistics from databases such as NCBI and JGI Mycocosm, approximately 40%-60% of these genes are labeled as "hypothetical protein" or "uncharacterized protein." Therefore, further exploration of *Fusarium oxysporum* is needed. Fusarium verticillioides Researching pathogenic genes and studying their functions will help to fully understand the pathogenic molecular mechanisms of Fusarium moniliforme and provide a theoretical basis for its prevention and control. Summary of the Invention

[0005] In view of the above-mentioned prior art, the purpose of this invention is to provide an application of the FvSup1 protein and its encoding gene in regulating the pathogenicity of Fusarium verticillatum.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A first aspect of the present invention provides the use of the FvSup1 protein in regulating the pathogenicity of Fusarium verticillatum, said FvSup1 protein being a protein as shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.3; (A2) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

[0007] In the above applications, the pathogenicity of Fusarium verticillata is reduced by decreasing the expression level and / or activity of the FvSup1 protein.

[0008] This invention is the first to discover that the FvSup1 protein is a novel key pathogenic factor of *Fusarium oxysporum*, capable of regulating the pathogenicity of the fungus. Using the FvSup1 protein as a target, new drugs for the prevention and control of plant diseases caused by *Fusarium oxysporum* can be developed.

[0009] In a second aspect, the present invention provides the use of the above-mentioned FvSup1 protein as a target in (1) or (2) below: (1) Develop drugs for the prevention and control of plant diseases; (2) Cultivate plant disease-resistant varieties.

[0010] In the above application, the plant disease is caused by Fusarium quinquefolium.

[0011] A third aspect of the invention provides FvSup1 The application of genes in regulating the pathogenicity of Fusarium verticillatum, the aforementioned FvSup1 Genes are nucleic acid molecules as shown in (i), (ii), or (iii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1; (iii) A nucleic acid molecule that has 90% or more identity with the nucleic acid molecule defined in (i) or (ii) and whose encoded protein is functionally equivalent to the protein shown in SEQ ID NO.1.

[0012] In the above applications, the nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA or mRNA.

[0013] The term "identity" used here refers to sequence similarity to native nucleic acid sequences. Identity can be evaluated using computer software, such as the BLAST algorithm (Altschul). et al. 1990. Journal of Molecular Biology 215:403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90:5873-5877).

[0014] In the aforementioned nucleic acid molecules, the 90% or more identity can be at least 90%, 92%, 93%, 95%, 96%, 98%, or 99% identity.

[0015] In the above applications, by knocking out or silencing FvSup1 Genes, or repression FvSup1 Gene transcription is used to reduce the pathogenicity of Fusarium verticillatum.

[0016] A fourth aspect of the invention provides regulation FvSup1 The use of a substance that expresses the gene or regulates the activity and / or content of FvSup1 protein in at least one of the following (1)-(3): (1) Regulates the pathogenicity of Fusarium verticillatum; (2) Preparation of drugs for the prevention and control of plant diseases; (3) Cultivate plant disease-resistant varieties; In the above application, the plant disease is caused by Fusarium quinquefolium.

[0017] In some preferred implementation schemes, regulation FvSup1 The substance that expresses the gene or regulates the activity and / or content of the FvSup1 protein is any one of the following: e1) Suppress, reduce, or silence FvSup1 Nucleic acid molecules involved in gene expression; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

[0018] A fifth aspect of the invention provides the use of the FvSup1 protein in at least one of the following (1)-(4): (1) Degradation of plant cell walls; (2) Degradation of xylan; (3) Prepare products that degrade plant cell walls; (4) Prepare products containing degraded xylan; The FvSup1 protein is any one of the proteins shown in (A1)-(A3) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.3; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); (A3) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

[0019] The beneficial effects of this invention are: (1) This invention is the first to study and confirm the unknown functional protein FvSup1 of Fusarium verticillatum and its encoding gene. FvSup1 New features. This invention builds upon existing technologies. FvSup1 A gene knockout vector was introduced into Fusarium verticillata protoplasts; the gene was then knocked out of Fusarium verticillata using homologous recombination to obtain the knockout mutant Δ. FvSup1 By constructing a gene complementation vector, it was introduced into Δ FvSup1 Protoplasts; Obtaining the complemented mutant Δ FvSup1 -C. Pathogenicity assays showed that the knockout mutant Δ FvSup1 Pathogenicity is significantly reduced; complement mutant Δ FvSup1 The pathogenicity of -C can be restored to the wild-type level. The above experiments demonstrate that... FvSup1 This study identified pathogenicity-related genes of Fusarium tumefaciens, providing new targets for the control of plant diseases caused by Fusarium tumefaciens.

[0020] (2) This invention also expressed the FvSup1 protein in vitro and investigated its degradation activity on plant cell walls and xylan. The results showed that the FvSup1 protein possesses highly efficient plant cell wall degrading enzyme activity. It is speculated that *Fusarium verticillata* utilizes the FvSup1 protein to disrupt the plant cell wall's anti-degradation barrier, thereby achieving infection. Targeting and inactivating the FvSup1 protein can reduce the pathogenicity of *Fusarium verticillata*. Attached Figure Description

[0021] Figure 1 Fusarium pseudoverticum FvSup1PCR verification results of gene knockout mutants; in the figure, lane 1 is the amplification product of the outer primer SEQ ID NO.17 / SEQ ID NO.18; lane 2 is the amplification product of the outer primer SEQ ID NO.19 / SEQ ID NO.20; lane 3 is the amplification product of the inner primer SEQ ID NO.15 / SEQ ID NO.16.

[0022] Figure 2 Fusarium quinquefolium Δ FvSup1 PCR validation results of the -C complement mutant; in the figure, lane 1 shows the amplification products of SEQ ID NO.15 / SEQ ID NO.16 using the genome of WT (Fv7600 strain) as a template, and lane 2 shows the amplification products of ΔC complement mutant. FvSup1 Using the genome as a template, the amplification products of SEQ ID NO.15 / SEQ ID NO.16, lane 2 is used for Δ FvSup1 The genome of -C is used as a template for the amplification products of SEQ ID NO.15 / SEQ ID NO.16.

[0023] Figure 3 Δ FvSup1 Knockout mutants and Δ FvSup1 Growth of the -C complemented mutant under different stress conditions; in the figure, A: Δ FvSup1 Knockout mutants and Δ FvSup1 -C Growth of the complemented mutant under different stress media; B, C, D, and E are statistical analyses of colony diameters on PDA medium and PDA medium supplemented with a final concentration of 0.2 g / L Congo Red, 20 μg / mL SDS, and 0.7 M NaCl, respectively.

[0024] Figure 4 Δ FvSup1 Knockout mutants and Δ FvSup1 Results of the investigation on conidial morphology and germination status of the C1 complemented mutant; in the figure, A, B, and C are WT, Δ, and Δ, respectively. FvSup1 Knockout mutants and Δ FvSup1 -C complement mutant conidial morphology; D, E, and F are WT, Δ, and Δ respectively. FvSup1 Knockout mutants and Δ FvSup1 Morphology of conidial germination in the -C complement mutant; scale bars for A, B, and C in the figure = 20 µm, and scale bars for D, E, and F = 50 µm.

[0025] Figure 5 Δ FvSup1 Knockout mutants and Δ FvSup1 Results of the investigation on the number of conidia in the -C complemented mutant.

[0026] Figure 6 Δ FvSup1 Knockout mutants and Δ FvSup1 -C complemented mutant pathogenicity test on maize: live inoculation diagram; In the diagram, A: root irrigation with only 50 mL of water as a control; B: 50 mL of Fusarium verticillatum Fv7600 spore suspension (spore concentration 1×10⁻⁶) was used. 6 Root irrigation (each cell / mL); C: 50 mL Δ FvSup1 Knockout mutant spore suspension (spore concentration of 1×10⁻⁶) 6 (each cell / mL) for root irrigation; D: 50 mL Δ FvSup1 -C-complemented mutant spore suspension (spore concentration 1×10⁻⁶) 6 (each cell / mL) for root irrigation; E: statistical results of disease index for treatments A, B, C, and D.

[0027] Figure 7 Δ FvSup1 Knockout mutants and Δ FvSup1 -C complementation mutant pathogenicity test in maize: in vitro inoculation diagram; In the diagram, A: Results of WT inoculation 48 h; B: Results using Δ FvSup1 Figure showing the results of knockout mutant inoculation 48 h later; C: Δ FvSup1 -C: Results of inoculation with the complemented mutant 48 h later; D: Statistical results of lesion length in treatments A, B, and C; Scale bar = 20 mm in Figures A, B, and C.

[0028] Figure 8 SDS-PAGE analysis of FvSup1 protein.

[0029] Figure 9 Electron microscopy results of FvSup1 protein degrading the cell wall of corn stalks; in the figure, A is the electron microscopy result after 0 h of reaction; B is the electron microscopy result after 2 h of reaction; C is the electron microscopy result after 16 h of reaction; the scale bar in the figure is equal to 20 µm.

[0030] Figure 10 Results of the detection of xylan products degraded by FvSup1 protein. Detailed Implementation

[0031] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] As mentioned earlier, fully exploring the pathogenic genes of Fusarium verticillatum and conducting functional studies will help to fully understand the pathogenic molecular mechanism of Fusarium and provide new targets for the prevention and control of Fusarium.

[0033] Based on this, this invention conducted an in-depth study of unknown genes in the genome of *Fusarium verticillioides*. Among them, *Fusarium verticillioides* 7600 hypothetical protein (FVEG_01031) is a protein in *Fusarium verticillioides* with unknown function, named FvSup1, and its amino acid sequence is shown in SEQ ID NO.1; the coding gene of FvSup1... FvSup1 The nucleotide sequence is shown in SEQ ID NO.2.

[0034] This invention has conducted a series of verifications on FvSup1, and this invention has constructed a gene FvSup1 The knockout vector was introduced into Fusarium verticillata protoplasts, and the gene was knocked out from Fusarium verticillata using homologous recombination to obtain the knockout mutant Δ. FvSup1 Pathogenicity tests demonstrated that, compared to the wild-type *Fusarium verticillatum*, the knockout mutant Δ FvSup1 The pathogenicity is significantly reduced; FvSup1 After gene restoration, its pathogenicity returned to the wild-type level. This proves that: FvSup1 The gene is a pathogenic gene of Fusarium verticillatum, and using its encoded protein as a drug target may provide a new approach for the control of Fusarium verticillatum.

[0035] Furthermore, the present invention synthesized the FvSup1 protein through eukaryotic expression and investigated the ability of the FvSup1 protein to degrade plant cell walls and xylan. It was found that the FvSup1 protein has plant cell wall degrading enzyme activity and can degrade xylan into oligosaccharides with a degree of polymerization of 2-5.

[0036] To enable those skilled in the art to better understand the technical solution of this application, the technical solution of this application will be described in detail below with reference to specific embodiments.

[0037] The test materials used in the embodiments of this invention are all conventional test materials in the art and can be purchased through commercial channels. Experimental methods without specified detailed conditions are performed according to conventional test methods or the supplier's recommended operating instructions. Wherein: Fusarium pseudoverticum ( F. verticillioides The strain used was the standard strain of *Fusarium verticillatum*, Fv7600. The tested maize variety was B73. The gene knockout vector was PCB1003, and the gene complementation vector was pFL2.

[0038] CMC culture medium: 15g CMC (sodium carboxymethyl cellulose), 2g NaNO3, 1g KH2PO4, 0.5g MgSO4·7H2O, 1g yeast extract, and bring the volume to 1L with deionized water. CMC is insoluble in water at room temperature and needs to be aliquoted. Add 1.5g CMC to every 100mL of culture medium and autoclave at 121℃ for 25min.

[0039] YEPD medium (1L): 10g yeast extract, 10g tryptone, 20g glucose, bring to a final volume of 1L with deionized water, autoclave at 121℃ for 25min.

[0040] Enzymatic hydrolysate: 0.5g of lysin, 0.1g of lysozyme, and 0.5g of snail enzyme, diluted to 30mL with 1.2M KCl.

[0041] STC Buffer: 100g sucrose, 20mL 0.5M TRIS・HCl, 1.47g CaCl2・2H2O, bring to a final volume of 200mL with deionized water.

[0042] PTC solution: 400g PEG8000, diluted to 100mL with STC Buffer.

[0043] TB3 liquid culture medium: 3g yeast extract, 3g acid-hydrolyzed casein, 200g sucrose, and diluted to 1L with deionized water.

[0044] TB3 solid medium: Add 0.9g agar powder to every 100mL of TB3 liquid medium and autoclave at 121℃ for 25min.

[0045] MD medium: 13.4 g / L yeast basic nitrogen source; 0.4 mg / L biotin; 20 g / L glucose.

[0046] YPD medium: 1% (w / v) Yeast Extract, 2% (w / v) Peptone, 2% (w / v) Dextrose (glucose), 2% (w / v) Agar powder; all are mass / volume ratios, in g / 100ml.

[0047] BMGY medium: yeast extract: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 6.0 (or pH 7.0) phosphate buffer, glycerol: 1.0 mL, add distilled water to 100 mL.

[0048] BMMY medium: yeast extract: 1.0 g, peptone: 2.0 g, YNB: 1.34 g, 0.1 mol / L pH 6.0 (or pH 7.0) phosphate buffer, after high temperature sterilization, add 1 mL of methanol to every 100 mL of medium.

[0049] Example 1: FvSup1 Knockout mutants and FvSup1 Construction of complement mutants 1. FvSup1 Construction of gene knockout vectors: According to the NCBI website FvSup1 The DNA sequences at both ends of the gene are selected. FvSup1 DNA sequences approximately 1.5 kb upstream and downstream of the coding region (shown in SEQ ID NO. 3 and SEQ ID NO. 4) were used as two homologous arms in the homologous recombination process to construct a knockout vector. This experiment used the PCB1003 vector, which carries a hygromycin phosphotransferase gene between approximately 1.5 kb upstream and downstream DNA sequences. HPH The upstream and downstream fragments were ligated using fusion PCR. HPH At both ends. Using the genome of strain Fv7600 as a template, the upstream fragment A was amplified using primers shown in SEQ ID NO. 5 and SEQ ID NO. 6; the downstream fragment B was amplified using primers shown in SEQ ID NO. 7 and SEQ ID NO. 8. Using plasmid PCB1003 as a template, the downstream fragment B was amplified using primers shown in SEQ ID NO. 9 and SEQ ID NO. 10. HPH The upstream fragment (h1); obtained by amplification using the primers shown in SEQ ID NO.11 and SEQ ID NO.12. HPH Downstream segment (h2).

[0050] The PCR reaction conditions were as follows: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 2 min, for a total of 35 cycles; 72℃ for 10 min. After purifying the PCR amplification products using the Tiangen Universal DNA Purification Kit (DP214-2), the knockout fragments A+h1 and h2+B were amplified using a two-step method with primer pairs (SEQ ID NO.5) / (SEQ ID NO.10) and (SEQ ID NO.11) / (SEQ ID NO.8) as templates.

[0051] Step 1: Reaction system (25 μL): 12.5 μL of 2×High Fidelity PCR Master Mix, 0.5 μL of fragment A / B, 0.5 μL of fragment h1 / h2, and ddH2O to 25 μL. Reaction program: Pre-denaturation 94℃, 2 min; denaturation 94℃, 15 s; annealing 58℃, 15 s; extension 72℃, 1 min / kb; 5 cycles; final extension 72℃, 5 min.

[0052] Step 2: Reaction system (50 μL): 25 μL of the product from step 1, 12.5 μL of 2×High Fidelity PCRMaster Mix, 1.0 μL of primer AF / YG-F (10 μM), 1.0 μL of primer HY-R / BR (10 μM), and ddH2O to a final volume of 50 μL. Reaction program: Pre-denaturation 94℃, 2 min; denaturation 94℃, 15 s; annealing 58℃, 15 s; extension 72℃, 1 min / kb; 30 cycles; final extension 72℃, 5 min. The PCR amplification products were recovered and purified using the Tiangen Universal DNA Purification Kit (DP214-2) for later use.

[0053] 2. FvSup1 Construction of gene complementation vector: Primer pairs SEQ ID NO.13 and SEQ ID NO.14 were designed to amplify a gene fragment containing the target gene and its first 1500 bp, with the pFL2 vector restriction site linked to the homologous arm, using the genomic DNA of Fv7600 as a template.

[0054] right FvSup1 After the gene complement fragment was recovered and purified, it was ligated into the pFL2 vector via homologous recombination. Following sequencing analysis and alignment, pFL2- was obtained. FvSup1 Gene complementation vector.

[0055] 3. Preparation of Fusarium verticillata protoplasts: (1) Obtaining conidia: Six fresh Fusarium oxysporum strains were punched with a sterile punch and transferred to 100 mL of CMC medium. The mixture was cultured at 25°C and 200 rpm for 5 days with shaking. The filtrate was filtered through three layers of lens paper, centrifuged at 5000 rpm for 5 min, and the supernatant was discarded to obtain a large number of conidia.

[0056] (2) Obtaining germ tubes: Add an appropriate amount of conidia to YEPD medium to induce germination and produce germ tubes. Culture at 25℃ and 200rpm for 16 h with shaking. After shaking culture, filter the germ tubes with three layers of lens paper, wash them twice with sterile water, and press the filter paper dry to obtain a large number of germ tubes.

[0057] (3) Obtaining protoplasts: Take a sterile 50 mL centrifuge tube, add 0.25 g germ tube, 2.5 mL of enzymatic hydrolysate and 2.5 mL of 1.2 M KCl solution, gently shake to mix, place on a water bath shaker, enzymatically hydrolyze at 30℃ and 90 rpm, gently invert and mix once every half hour, and the required amount of protoplasts can be obtained in about 1.5-2 h.

[0058] (4) Filter the enzyme digest using two layers of lens paper, centrifuge at 4000 rpm for 5 min, and discard the supernatant. Add an appropriate amount of STC Buffer to the protoplast precipitate to achieve a protoplast concentration of 1×10⁻⁶. 7 Approximately [number] cells / mL is sufficient.

[0059] 4. Transformation of Fusarium verticillata protoplasts: (1) Prepare four sterile 10 mL centrifuge tubes. Use a pipette to gently pipette 200 µL of protoplasts into the bottom of the centrifuge tubes, and then add 5 µL, 10 µL, 15 µL and 20 µL of A+h1 and h2+B fragments respectively (fragment concentration 200-400 ng / μL). Gently shake for 15 seconds and place in a clean bench for 20 min.

[0060] (2) Take 700 µL of PTC and slowly add it along the wall of the centrifuge tube from the previous step. Then gently rotate and invert the tube to mix the PTC with the protoplast suspension in the tube until the mixture is clear and transparent without any water ripples. Add another 700 µL of PTC along the wall of the tube, gently rotate and invert the tube until there are no water ripples. After mixing, let it stand in a clean bench for 20 minutes.

[0061] (3) After standing, add 5 mL of TB3 liquid culture medium and 5 µL of 100 mg / mL Amp antibiotic solution to the centrifuge tube, mix by inverting, and seal. Incubate at 25°C and 200 rpm for 16 h.

[0062] (4) After the shaking culture is completed, add the liquid from the four centrifuge tubes to 200 mL of TB3 liquid culture medium at about 50 °C, and immediately add 100 µL of Amp at a concentration of 100 mg / mL and 200 µL of HPH antibiotic at a concentration of 100 mg / mL. After mixing, pour the mixture evenly into 12 culture dishes, seal them, and place them upside down in a constant temperature incubator at 25 °C for 24 h in the dark.

[0063] (5) Pour another layer of TB3 solid culture medium containing 100 µL of 100 mg / mL Amp and 200 µL of 100 mg / mL HPH antibiotic at about 50°C onto the above plate, seal it, and incubate it upside down in a 25°C incubator in the dark.

[0064] (6) After culturing for 2-5 days, transfer the transformants to a PDA culture medium plate with an HPH antibiotic concentration of 200 μg / mL using a sterile toothpick for antibiotic screening (9 transformants per plate).

[0065] 5. FvSup1 PCR validation analysis of knockout mutants: Following the method for crude extraction of *Fusarium verticillatum* genome, genomic DNA was extracted from positive transformants and analyzed by PCR. First, small pieces of mycelial material were excised and stored as backups. Then, half a colony of mycelium was scraped into a 2 mL EP tube, with a small amount of quartz sand added. 500 µL of 2% CTAB and 500 µL of phenol / chloroform were added to each tube. The tubes were incubated at 37°C and 250 rpm for 1–1.5 h on a shaker, centrifuged at 12000 rpm for 15 min, and the supernatant (approximately 400 µL) was collected. An equal volume of isopropanol was added to precipitate the precipitate, and the tubes were centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and the tubes were washed once with 1 mL of ice-cold 70% ethanol. Residual liquid was removed using a vacuum concentrator. The gDNA was dissolved in 20 µL of ddH₂O containing 10 μg / mL RNase and digested at 37°C for at least 30 min. The transformants were then validated by PCR. First, the probe primers (internal gene primers SEQ ID NO.15 and SEQ ID NO.16) and external gene primers (SEQ ID NO.17 / SEQ ID NO.18, SEQ ID NO.19 / SEQ ID NO.20) were used for validation. The PCR reaction system consisted of: 2 μL template DNA, 0.5 μL each primer, 10 μL 2×Taq plus Master Mix, and 7 μL ddH2O. The PCR conditions were: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 2 min, for a total of 35 cycles; and finally, 72℃ for 10 min to obtain the amplified product.

[0066] PCR amplification was performed using the probe and primers (SEQ ID NO.15 and SEQ ID NO.16). Knockout mutants showed no... FvSup1 Internal probe bands were observed; PCR amplification was performed using outer primers (SEQ ID NO.16 / SEQ ID NO.17, SEQ ID NO.18 / SEQ ID NO.19), and the knockout mutant showed an amplification band. The results indicate that this embodiment successfully constructed the gene. FvSup1 Knockout mutant Δ FvSup1 ( Figure 1 ).

[0067] 6. FvSup1 PCR validation analysis of complemented mutants Will FvSup1 Gene complementation vector introduction Δ FvSup1Protoplasts were used to obtain complemented transformants. Genomic DNA was extracted from the complemented transformants and analyzed by PCR. The gene fragment was amplified by PCR using primers SEQ ID NO.15 and SEQ ID NO.16. The PCR reaction system consisted of 2 μL template DNA, 0.5 μL each primer, 10 μL 2×Taq plus MasterMix, and 7 μL ddH2O. The PCR reaction conditions were: 94℃ for 5 min; 94℃ for 30 s, 56℃ for 30 s, 72℃ for 2 min, for a total of 35 cycles; and 72℃ for 10 min to obtain the amplification product. The complemented mutant yielded the desired band, as shown in Figure 2. This embodiment successfully constructed the gene. FvSup1 complement mutant Δ FvSup1 -C.

[0068] Example 2: FvSup1 Knockout mutants and FvSup1 An examination of the ability of complemented mutants to respond to different stresses. 1. Test method: The construction of Example 1 FvSup1 Knockout mutant Δ FvSup1 , FvSup1 complement mutant Δ FvSup1 -C and wild-type *Fusarium verticillatum* Fv7600 were inoculated onto PDA medium supplemented with 0.2 g / L Congo Red, 20 μg / mL SDS, 0.7 M NaCl, and 3% H2O2. The cultures were incubated at 28°C for 7 days, and colony growth and diameter were observed.

[0069] 2. Test Results: The results are as follows Figure 3 As shown, the results indicate that the *Fusarium verticillatum* knockout mutant Δ FvSup1 and complement mutant Δ FvSup1 -C showed no significant difference in response to different stresses compared to the wild type.

[0070] Example 3: FvSup1 Knockout mutants and FvSup1 Investigation of the number of conidia in the complemented mutant 1. Test method: The construction of Example 1 FvSup1 Knockout mutant Δ FvSup , FvSup1 complement mutant Δ FvSup1-C and wild-type *Fusarium verticillatum* Fv7600 were inoculated onto PDA medium and grown at 28°C for 5 days. Six fresh mycelial discs were inoculated at the edge of each colony into 100 mL of CMC medium and incubated at 28°C and 200 rpm for 5 days. Sporulation was counted using a hemocytometer, and morphological observation and photography were performed. Each experiment was repeated three times, with three replicates per experiment. The final results were averaged and significance analysis was performed.

[0071] Wild-type Fusarium oxysporum f. v7600 and knockout mutant Δ FvSup1 and complement mutant Δ FvSup1 -C conidial concentration was adjusted to 20-25 spores per small square in a microscopic hemocytometer. The conidia were inoculated into 100 mL of YEPD medium and incubated at 28℃ and 200 rpm for 16 h. The spore germination status was observed. Each experiment was repeated 3 times, with 3 replicates for each experiment.

[0072] 2. Test Results: WT, Δ FvSup1 Knockout mutants and Δ FvSup1 -C complemented mutant conidial morphology and conidial germination morphology, such as Figure 4 As shown; the results of the investigation of conidial quantity are as follows Figure 5 As shown. The results indicate that the *Fusarium verticillatum* knockout mutant Δ FvSup1 and complement mutant Δ FvSup1 -C showed no significant difference in conidial morphology, germination status, or quantity compared to the wild type.

[0073] Example 4: FvSup1 Knockout mutants and FvSup1 Pathogenicity testing of complemented mutants in maize 1. Test method: (1) Live inoculation: The construction of Example 1 FvSup1 Knockout mutant Δ FvSup1 , FvSup1 complement mutant Δ FvSup1 -C and wild-type *Fusarium verticillatum* Fv7600 were inoculated into CMC medium and incubated at 28°C and 200 rpm for 5 days. The spore concentration was adjusted to 1×10⁻⁶. 6 Quantity / mL, for later use.

[0074] Two-week-old maize plants with similar growth were used as the experimental subjects, and the following treatments were set up: Treatment A: Use only 50 mL of clean water to irrigate the roots as a control; Treatment B: Use 50 mL of wild-type Fusarium oxysporum f. v7600 spore suspension (spore concentration 1×10⁻⁶). 6 Root irrigation (each cell / mL); Treatment C: 50 mL Δ FvSup1 Knockout mutant spore suspension (spore concentration of 1×10⁻⁶) 6 Root irrigation (each cell / mL); Treatment D: 50 mL Δ FvSup1 -C Replenishment of mutant spore suspension (spore concentration 1×10⁻⁶) 6 (each cell / mL) for root irrigation.

[0075] Thirty corn plants were placed in each treatment at 28℃ and 90% humidity for 48 hours to observe the disease incidence. Disease severity was graded according to existing technology (“Research on Identification and Integrated Control Technology of Corn Stem Rot Pathogen” [J]. Seed Science and Technology, 2025, 43 (16):134-136.): Grade 0: no disease; Grade 1: slight discoloration at the base of the stem; Grade 2: lesions covering less than 1 / 4 of the stem base; Grade 3: lesions covering 1 / 4 to 1 / 2 of the stem base; Grade 4: lesions covering 1 / 2 to 3 / 4 of the stem base; Grade 5: lesions covering more than 3 / 4 of the stem base or plant lodging.

[0076] The disease index is calculated based on the number of diseased plants at each level, using the following formula: Disease index = Σ(Number of diseased plants at each level × Corresponding level) ÷ (Total number of plants surveyed × Highest level) × 100 (2) In vitro inoculation: The construction of Example 1 FvSup1 Knockout mutant Δ FvSup1 , FvSup1 complement mutant Δ FvSup1 -C and wild-type Fusarium oxysporum f. v7600 were inoculated into PDA medium and cultured at 28°C and 200 rpm for 5 days for later use.

[0077] Two-week-old etiolated maize with similar growth was chosen as the experimental subject. The reasons for selecting etiolated maize for in vitro inoculation are as follows: 1) It can eliminate the interference of structures such as chlorophyll and cuticle and secondary metabolites, making the infection conditions more uniform and reducing experimental errors; 2) Erythrogenic seedlings have tender tissues, allowing pathogens to infect them more quickly, and symptoms are easier to observe and quantify, which can effectively improve the sensitivity and repeatability of in vitro inoculation experiments.

[0078] Cut corn plants 1 cm above the ground, and use a punch to take 30 fresh mycelium cakes from each of the above-mentioned plates. Inoculate the cakes 2 cm away from the cut, with 30 corn plants per treatment. Place the plates at 28℃ and 90% humidity, and observe the length of corn lesions after 48 hours.

[0079] 2. Test Results: (1) Results of live inoculation: Results of live inoculation experiments as follows Figure 6 As shown, the results indicate that the deletion of FvSup1 significantly reduces the pathogenicity of Fusarium verticillatum; after the gene is reintroduced, its pathogenicity is restored.

[0080] (2) Results of in vitro inoculation: Results of in vitro inoculation experiments as follows Figure 7 As shown, the results indicate that: FvSup1 The deletion of this gene significantly reduced the pathogenicity of Fusarium verticillatum; its pathogenicity was restored after the gene was reintroduced.

[0081] The core difference between live inoculation and in vitro inoculation lies in the different physiological states of the host and the infection environment. Live inoculation involves irrigating intact maize plants, and the infection process is completed within the plant, simulating a natural infection scenario and being influenced by factors such as the plant's own immune regulation and physiological metabolism. In vitro inoculation uses ectopic B73 tissue, and the infection process is carried out in a controlled environment outside the plant, eliminating interference from the plant's overall physiological regulatory system.

[0082] The reasons for examining the two inoculation methods are as follows: in vivo inoculation can reflect the true pathogenicity of pathogens under natural conditions, while in vitro inoculation can specifically assess the direct effects of pathogenic virulence factors on host tissues. Combining the two methods achieves complementary verification, providing key experimental evidence for elucidating the functional mechanism of target proteins in the pathogenic process.

[0083] Example 5: Eukaryotic expression of FvSup1 protein 1. pPIC9K / FvSup1 Construction of yeast eukaryotic expression plasmids: Take 10 μL of Escherichia coli culture containing pPIC9K plasmid and culture it in LB liquid medium containing kanamycin at 37°C with shaking for 8 h. Extract the plasmid according to the instructions of the plasmid mini-extraction kit.

[0084] Select restriction endonucleases Eco RI and Not I. Perform double digestion of the plasmid to expose the ligation ends of the fragment. If the reaction is complete, recover the digested products by electrophoresis and gel electrophoresis of the remaining reaction solution.

[0085] Construct a complete expression vector according to the Ligation-Free Cloning System kit instructions, and attach expressions to both ends. Eco RI and Not I restriction site FvSup1 The gene amplification product was ligated into the double-digested yeast expression vector pPIC9K.

[0086] FvSup1 The gene amplification product was obtained as follows: RNA was extracted from the hyphae of the standard strain Fv7600 of *Fusarium verticillatum*, and a gene amplification product was designed. FvSup1 Gene-specific primers (SEQ ID NO.21 and SEQ ID NO.22) were used for RT-PCR amplification to obtain primers with ends bearing [missing information]. Eco RI and Not I restriction site FvSup1 The amplification product of the gene will FvSup1 The gene amplification product was ligated into the double-digested yeast expression vector pPIC9K to obtain the yeast expression plasmid pPIC9K / carrying the target gene. FvSup1 .

[0087] Yeast expression plasmid pPIC9K / FvSup1 Transplanted with E. coli E. coli After T1 competent cells were cultured at 28°C for 12 h, single colonies were picked and cultured in 1 mL of LB liquid medium containing 50 μg / mL kanamycin at 37°C with shaking for 6 h. 1.5 μL of the bacterial culture was used as a template for PCR verification and the product was sent for sequencing. If the sequence result was correct, the yeast expression vector pPIC9K / FvSup1 carrying the target gene was obtained.

[0088] 2. Obtain yeast engineered strains containing the pPIC9K / FvSup1 expression plasmid: (1) Linearization and dephosphorylation of expression vectors Take 10 μL of yeast expression vector pPIC9K / FvSup1 Escherichia coli culture was incubated in 15 mL of LB broth containing 50 μg / mL kanamycin at 37°C with shaking for 8 h. Plasmids were extracted according to the instructions of the plasmid mini-prep kit and their concentration was determined. Linearized restriction endonucleases were selected. Sac I and dephosphorylase AP were reacted according to the instructions of the restriction endonuclease kit.

[0089] (2) Electrocution Before electroporation transformation, a usable GS115 strain and fresh competent yeast cells must be obtained. Aliquot 80 μL of the prepared competent yeast cells into each tube, add the linearized expression vector plasmid, mix gently, and incubate on ice for 5 min. Transfer the mixture to a pre-chilled electroporation cuvette, electroporate at 300 V for 15 ms, immediately add 1 mol / L sorbitol solution (on ice), mix well, and incubate at 28°C for 60 min. Spread 300 μL onto MD medium and incubate at 28°C for 2–3 days.

[0090] (3) Screening and validation of engineered yeast strains Single colonies were selected and screened on YPD medium containing 150 μg / mL and 180 μg / mL kanamycin. Colonies that grew rapidly on both concentration plates were selected and verified by PCR using 5'AOX1 (SEQ ID NO.23) and 3'AOX1 (SEQ ID NO.24) as primers. The products were then sequenced. If the fragment size and sequencing results were correct, the bacterium could be used as a fermentation engineer.

[0091] 3. Eukaryotic expression, isolation, and purification of FvSup1 protein: (1) Fermentation of engineered bacteria The correctly screened and identified fermentation engineered bacteria were streaked onto YPD medium and cultured at 28°C for 2 days. Afterward, the activated engineered bacteria were picked and inoculated into BMGY medium. The culture was incubated at 28°C and 200 rpm with shaking for 22-24 hours. After centrifugation at 4200 rpm, the bacterial culture was transferred to BMMY medium to begin induction of expression. 1 mL of methanol was added every 12 hours for 7 consecutive days. The supernatant crude enzyme solution was then collected at 4°C / 8000 rpm / 15 min.

[0092] (2) Ammonium sulfate precipitation and dialysis of crude enzyme solution Add dried ammonium sulfate powder to the crude enzyme solution until 80% saturation, then incubate overnight at 4°C. Centrifuge at 4°C / 8000 rpm / 15 min, discard the supernatant, add 5 mL of PBS (pH=7.4) buffer, and after all the precipitated protein has dissolved, transfer to a dialysis bag and dialyze in PBS buffer for 12 h. Centrifuge at 4°C / 8000 rpm / 15 min to collect the supernatant.

[0093] (3) Isolation and purification of proteases The positively charged nickel ions in the HisTrap FF cru de (nickel column) packing material have an affinity for histidine, thus enabling the adsorption of target proteins containing a 6×His tag. The crude protein enzyme solution, after dialysis to remove salt ions, was separated and purified using a UNICORN 5.11 instrument. The collected protease solution was dialyzed with deionized water, aliquoted, and stored at -80°C or used immediately at 4°C.

[0094] The molecular weight of the protease was determined by SDS-PAGE denaturing electrophoresis. The procedure and staining / destaining process were performed according to the gel preparation kit instructions. After electrophoresis, the stacking gel was removed, and the separating gel was stained and destained. After destaining, the gel was placed on a film viewing lamp for observation and analysis to determine if the desired enzyme was expressed and whether the separation and purification were successful. The results showed that FvSup1 protein (…) was successfully obtained. Figure 8 ).

[0095] Example 6: Investigation of FvSup1 protein degradation of plant cell walls 1. Test method: (1) Degradation of corn stalks: 300 µL of 0.2 mg / mL FvSup1 protein (prepared in Example 5) was mixed with 0.003 g of corn stalks and reacted at 50 °C for 0 h, 2 h and 16 h before being observed by SEM4000X scanning electron microscopy.

[0096] (2) Investigation on xylan degradation: 100 µL of 0.2 mg / mL FvSup1 protein (prepared in Example 5) was mixed with 100 µL of 0.2% xylan (the main hemicellulose component of maize cell wall) solution and reacted at 50 °C for 6 h. The degradation products were then detected by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS).

[0097] MALDI-TOF MS was performed using an AB Sciex 5800 matrix-assisted laser excitation ionization mass spectrometer with CHCA as the matrix solution. 1 μL of desalted sample was mixed with 1 μL of matrix solution and spotted onto a 384-well MALDI target plate, then air-dried at room temperature. The mass-to-charge ratio (m / z) acquired by the mass spectrometer ranged from 100 to 750.

[0098] 2. Test Results: Electron micrographs of FvSup1 protein reacted with corn straw at 0h, 2h, and 16h, as shown below. Figure 9 As shown, the results indicate that the FvSup1 protein can effectively degrade the cell wall of corn stalks.

[0099] The detection results of the products of FvSup1 protein degradation of xylan are as follows: Figure 10 As shown, the results indicate that the FvSup1 protein can effectively degrade xylan into oligosaccharides with a degree of polymerization of 2-5.

[0100] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of FvSup1 protein in regulating the pathogenicity of Fusarium verticillatum, wherein the FvSup1 protein is the protein shown in (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.3; (A2) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

2. The application according to claim 1, characterized in that, The pathogenicity of Fusarium verticillata can be reduced by decreasing the expression level and / or activity of the FvSup1 protein.

3. Applications of FvSup1 protein as a target in the following (1) or (2): (1) Develop drugs for the prevention and control of plant diseases; (2) Cultivate plant disease-resistant varieties.

4. The application according to claim 3, characterized in that, The plant disease is caused by Fusarium quinquefolium.

5. FvSup1 The application of genes in regulating the pathogenicity of Fusarium verticillatum, the aforementioned FvSup1 Genes are nucleic acid molecules as shown in (i), (ii), or (iii) below: (i) Nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO.2; (ii) Nucleic acid molecules other than (i) that encode the amino acid sequence shown in SEQ ID NO.1; (iii) A nucleic acid molecule that has 90% or more identity with the nucleic acid molecule defined in (i) or (ii) and whose encoded protein is functionally equivalent to the protein shown in SEQ ID NO.

1.

6. The application according to claim 5, characterized in that, By knocking out or silencing FvSup1 Genes, or repression FvSup1 Gene transcription is used to reduce the pathogenicity of Fusarium verticillatum.

7. Regulation FvSup1 The use of a substance that expresses the gene or regulates the activity and / or content of FvSup1 protein in at least one of the following (1)-(3): (1) Regulates the pathogenicity of Fusarium verticillatum; (2) Preparation of drugs for the prevention and control of plant diseases; (3) Cultivate plant disease-resistant varieties.

8. The application according to claim 7, characterized in that, The plant disease is caused by Fusarium quinquefolium.

9. The application according to claim 7, characterized in that, Regulation FvSup1 The substance that expresses the gene or regulates the activity and / or content of the FvSup1 protein is any one of the following: e1) Suppress, reduce, or silence FvSup1 Nucleic acid molecules involved in gene expression; e2) An expression cassette containing the nucleic acid molecule described in e1); e3) A recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) Recombinant microorganisms containing the nucleic acid molecules described in e1), or recombinant microorganisms containing the expression cassette described in e2), or recombinant microorganisms containing the recombinant vector described in e3).

10. The use of FvSup1 protein in at least one of the following (1)-(4): (1) Degradation of plant cell walls; (2) Degradation of xylan; (3) Prepare products that degrade plant cell walls; (4) Prepare products containing degraded xylan; The FvSup1 protein is any one of the proteins shown in (A1)-(A3) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.3; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1); (A3) A protein having the same function as the amino acid sequence shown in SEQ ID NO.1, by substitution and / or deletion and / or addition of one or more amino acid residues.

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