Application of FPSE09041 protein and coding gene thereof in reducing pathogenicity of Fusarium pseudograminearum

Abstract

The invention relates to the technical field of biology, and discloses an FPSE09041 protein and an application of a coding gene of the FPSE09041 protein in reducing pathogenicity of fusarium pseudograminearum. The amino acid sequence of the FPSE09041 protein is as shown in SEQ ID NO. 2, and the coding gene of the FPSE09041 protein is as shown in SEQ ID NO. 1. By knocking out the FPSE09041 gene, the expression of the FPSE09041 protein is inhibited, and the pathogenicity of the fusarium pseudograminearum to host plants is reduced. The research finds that the FPSE09041 is an indispensable key factor for pathogenicity of the fusarium pseudograminearum, and the survival and infection capabilities of pathogenic bacteria can be obviously weakened due to function deficiency of the FPSE09041. Therefore, the FPSE09041 protein and the coding gene thereof can be used as ideal molecular targets for developing novel, efficient and high-specificity antifungal agents, and a new technical path is provided for green prevention and control of wheat stem rot.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the application of the FPSE_09041 protein and its encoding gene in reducing the pathogenicity of Fusarium graminearum. Background Technology

[0002] *Fusarium pseudograminearum* is a pathogenic fungus that causes important diseases such as wheat stem rot, seriously threatening the safe production of wheat and other crops. Currently, control methods for this disease are limited, and long-term use of chemical fungicides easily leads to the development of resistance. Therefore, in-depth analysis of the pathogenic mechanism of *Fusarium pseudograminearum* and the discovery of new molecular targets are of great significance for developing novel, efficient, and low-toxicity antifungal strategies. In recent years, studies have found that many key genes in fungi play important roles in regulating mycelial growth, spore formation, germination, and pathogenicity. However, the key proteins closely related to pathogenicity in *Fusarium pseudograminearum* and their functions remain unclear.

[0003] Acetolactate synthase (AHAS) is a key enzyme in the biosynthesis of branched-chain amino acids (valine, leucine, and isoleucine) in organisms and is also one of the core targets for metabolic regulation in plants and fungi. This enzyme depends on flavin adenine dinucleotide (FAD), thiamine pyrophosphate (THDP), and magnesium ions (Mg). 2+ As a cofactor, it catalyzes the conversion of pyruvate into acetolactate, and is an indispensable metabolic node in living organisms.

[0004] Acetolactate synthase (AHAs), as a pivotal link between carbon and nitrogen metabolism and the regulation of branched-chain amino acid synthesis, exhibits highly conserved structure and function in both plants and fungi, while also displaying species-specific regulatory characteristics. In recent years, its value as a potential target for antifungal drugs and antimicrobial agents has become increasingly prominent, especially against the backdrop of growing herbicide resistance and pathogen resistance. A deeper understanding of the structure and functional mechanisms of AHAs has significant theoretical and applied implications. It not only deepens our understanding of the regulation of biological metabolic networks but also provides a crucial structural biology foundation for the development of novel targeted antifungal drugs. Summary of the Invention

[0005] The purpose of this invention is to provide a key protein FPSE_09041 in Fusarium graminearum and its encoding gene, and to elucidate its function in regulating hyphal growth, spore formation, spore germination and pathogenicity, so as to provide a potential molecular target for the development of novel antifungal drugs.

[0006] To achieve the above objectives, the present invention provides the application of FPSE_09041 protein in reducing the pathogenicity of Fusarium graminearum. The amino acid sequence of FPSE_09041 protein is shown in SEQ ID NO.2, and the encoding gene of FPSE_09041 protein is shown in SEQ ID NO.1. Inhibiting the expression of FPSE_09041 protein reduces the pathogenicity of Fusarium graminearum to host plants.

[0007] The present invention also provides the application of the FPSE_09041 gene in reducing the pathogenicity of Fusarium graminearum. The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein encoded by the FPSE_09041 gene is shown in SEQ ID NO.2. Knocking out the FPSE_09041 gene reduces the expression of the FPSE_09041 protein, thereby decreasing the pathogenicity of Fusarium graminearum to the host plant.

[0008] The present invention also provides the application of nucleic acid that inhibits the expression of the FPSE_09041 gene in reducing the pathogenicity of Fusarium graminearum, wherein the nucleic acid is RNAi, antisense RNA, siRNA or shRNA, and the nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1.

[0009] The present invention also provides the application of expression cassettes, recombinant vectors or transgenic plant cell lines containing nucleic acids that inhibit the expression of the FPSE_09041 gene in reducing the pathogenicity of Fusarium graminearum, wherein the nucleic acid is RNAi, antisense RNA, siRNA or shRNA, and the nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1.

[0010] The above applications are specifically as follows: ① Regulate the mycelial growth of *Fusarium graminearum*; ② Regulate the spore germination rate of *Fusarium graminearum*; ③ As a target for screening agents that inhibit or kill Fusarium graminearum; ④ Application in the cultivation of transgenic plants resistant to wheat stem rot.

[0011] The present invention also provides the application of the FPSE_09041 gene or FPSE_09041 protein as a target in screening preparations that inhibit or kill Fusarium oxysporum. The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein is shown in SEQ ID NO.2.

[0012] The present invention also provides the application of an active substance that inhibits the expression of the FPSE_09041 gene or FPSE_09041 protein in the preparation of a formulation that inhibits or kills Fusarium oxysporum. The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein is shown in SEQ ID NO.2. The active substance that inhibits the expression of the FPSE_09041 gene or FPSE_09041 protein is a nucleic acid or an expression cassette, recombinant vector or transgenic plant cell line containing the nucleic acid, wherein the nucleic acid is RNAi, antisense RNA, siRNA or shRNA.

[0013] The present invention also provides a method for preparing a fungicide or bacteriostatic agent for Fusarium graminearum: co-culturing the test substance with Fusarium graminearum, screening out the test substance that inhibits the expression of the FPSE_09041 gene or inhibits the activity of the FPSE_09041 protein, and preparing a fungicide or bacteriostatic agent for Fusarium graminearum based on the test substance that inhibits the expression of the FPSE_09041 gene or inhibits the activity of the FPSE_09041 protein.

[0014] The present invention also provides a fungicide or bacteriostatic agent for Fusarium simulans, which inhibits the expression of the FPSE_09041 gene or inhibits the activity of the FPSE_09041 protein, thereby reducing the pathogenicity of Fusarium simulans to the host plant.

[0015] The advantages and positive effects of the FPSE_09041 protein and its encoding gene described in this invention in reducing the pathogenicity of Fusarium graminearum are as follows: This invention, through gene knockout and complementation experiments, confirms that the FPSE_09041 protein plays a key positive regulatory role in the growth, development, and pathogenicity of *Fusarium graminearum*. Specifically: ① Regarding mycelial growth: The FPSE_09041 deletion mutant (ΔFPSE_09041) showed a significant reduction in aerial mycelia and a decrease in mycelial growth rate; ② Regarding spore germination: the spore germination rate decreased by approximately 79.27%; ③ Pathogenicity: The mutant completely loses its ability to infect the base of wheat stems, thus losing its pathogenicity; ④ Reintroduction and restoration: After the FPSE_09041 gene was reintroduced into the mutant, mycelial growth, spore germination and pathogenicity were restored to the level of wild type.

[0016] The above results indicate that FPSE_09041 is an indispensable key factor in the pathogenicity of *Fusarium graminearum*, and its functional loss can significantly weaken the survival and infection ability of the pathogen. Therefore, the FPSE_09041 protein and its encoding gene can serve as ideal molecular targets for developing novel, efficient, and specific antifungal agents, providing a new technical pathway for the green control of wheat stem rot. This invention not only provides important gene resources for elucidating the pathogenic mechanism of *Fusarium graminearum*, but also provides new candidate targets for the targeted screening and design of fungicides, possessing significant theoretical value and application prospects.

[0017] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0018] Figure 1 The PCR detection results of the *Fusarium graminearum* FPSE_09041 gene knockout mutant in this embodiment of the invention; Figure 2 This diagram shows the growth of *Fusarium graminearum* strain 2035 (WT), the FPSE_09041 gene knockout transformant series strains (09041-2, 09041-7), and the gene reversion mutant strain 09041-C in embodiments of the present invention. A represents colony growth (cultured on a PDA for 3 days); B is a bar graph of mycelial growth rate; and C represents conidial germination. Figure 3 The pathogenicity of *Fusarium graminearum* strain 2035 (WT), the FPSE_09041 gene knockout transformant series strains (09041-2, 09041-7), and the gene reversion mutant strain 09041-C in the base of wheat stems in this embodiment of the invention is shown in Figure A: wheat plant disease incidence; and Figure B: bar chart of wheat stem base rot disease severity index. Detailed Implementation

[0019] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0021] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0022] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to the methods of this invention.

[0023] Sources of materials and reagents in the embodiments of this invention: Fusarium wilt strain 2035: obtained from the Institute of Plant Protection, Hebei Academy of Agricultural and Forestry Sciences. The public can contact us to obtain it if needed. DNA Gel Recovery Kit (Bio-Teke); Miniature Plasmid Extraction Kit (OMEGA); PCR reagents: Taq polymerase (Thermo), FastPfu enzyme (Full Gold), restriction endonuclease Fermentas; Common reagents: Hygromycin B (Roche), Ampicillin (AMP), Genimycin G418, Alkaline phenol:chloroform:isoamyl alcohol (25:24:1), chloroform, isopropanol, anhydrous ethanol, 70% ethanol, cellulase, lysozyme, and driselase (Sigma).

[0024] Preparation of culture media and solutions: CMC medium (1L): 15.0g sodium carboxymethyl cellulose, 0.5g yeast extract, 1.0g peptone, sterilized at 121℃ for 20min; YEPD medium (1L): 3.0g yeast extract, 10g peptone, 20g glucose, adjust pH to 7.0 with 5M NaOH, sterilize at 121℃ for 20min; RM medium (1L): Casamino acid hydrolysate 1.0g, yeast extract 1.0g, sucrose 274g, pH adjusted to 7.0 with 5M NaOH, sterilized at 121℃ for 20min; STC buffer (500mL): 0.8M sorbitol, 50mM Tris-Cl, 50mM CaCl2, sterilized at 121℃ for 20min; PTC buffer (100mL): PEG8000 40g, add STC solution to 100mL, melt in microwave oven, sterilize at 121℃ for 20min; 0.7M NaCl solution (1L): Dissolve 40.95g NaCl in 1L of water and sterilize at 121℃ for 20min; Protoplast buffer (20mL): 20mg of lysozyme, 300mg of cellulase, 300mg of lysozyme, and 0.7M NaCl to make up to 20mL. Filter and sterilize using a bacterial filter.

[0025] Example 1: Construction of homologous recombination fragment of FPSE_09041 gene 1. Obtaining the protein and encoding gene of *Fusarium graminearum* FPSE_09041: The protein FPSE_09041 and its encoding gene (or cDNA) of *Fusarium graminearum* strain 2035 can be amplified using the DNA (or cDNA) of *Fusarium graminearum* strain 2035 as a template via the 09041-F / R primer pair. The material from which DNA or RNA is extracted can be *Fusarium graminearum* strain 2035 mycelium.

[0026] 09041F: 5'-ATGCTCCGAAGTCGCCCTA-3' (SEQ ID NO. 3); 09041R: 5'-TCAAGAACCGTGGAGACCA-3' (SEQ ID NO. 4).

[0027] The gene encoding *Fusarium graminearum* FPSE_09041, as shown in SEQ ID NO.1 of the sequence listing, consists of 2049 nucleotides; it encodes the protein FPSE_09041 shown in SEQ ID NO.2 of the sequence listing.

[0028] The FPSE_09041 protein or gene mentioned above can also be synthesized artificially.

[0029] The nucleotide sequence of the encoding gene of *Fusarium graminearum* FPSE_09041 is as follows (SEQ ID NO.1):

[0030] The amino acid sequence of the protein encoded by *Fusarium graminearum* FPSE_09041 is as follows (SEQ ID NO.2): .

[0031] 2. Obtain the homologous recombination knockout fragment and knockout cassette of the gene encoding Fusarium oxysporum FPSE_09041: Genomic DNA was extracted from Fusarium graminearum 2035 using a fungal genomic DNA extraction kit. Using the genomic DNA as a template, primers 09041-1F / 2R and 09041-3F / 4R were designed based on the coding gene FPSE_09041 (SEQ ID NO.1) to amplify homologous sequence fragments of approximately 1250 bp upstream (L) and 1178 bp downstream (R) of the target fragment, respectively.

[0032] 09041F1: 5'-AGCTGTGTCTGTGTTGTA-3' (SEQ ID NO.5); 09041R2: 5'-CAAAATAGGCATTGATGTGTTGACCTCCTGGAGTAGATGTATGGGTTT-3' (SEQ IDNO.6); 09041F3: 5'-CTCGTCCGAGGGCAAAGGAATAGAGTAGAGTGGGAGGAAGTAAAGA-3' (SEQ IDNO.7); 09041R4: 5'-TGGCAGTTATCTTAACAATTCT-3' (SEQ ID NO. 8).

[0033] Using the pHIG2RHPH2-GFP-GUS plasmid as a template, the hygromycin resistance (hph) gene fragment containing the promoter sequence was amplified using HYG-F and HYG-R primers.

[0034] HYG-F: 5'-GGAGGTCAACACATCAATGCCTATT-3' (SEQ ID NO.9); HYG-R: 5'-CTACTCTATTCCTTTGCCCT-3' (SEQ ID NO. 10).

[0035] The reaction system consisted of 25 μL of TransStart® FastPfu PCR SuperMix (2X) premix, 1 μL each of the two primers, 1 μL of DNA template, and 22 μL of ddH2O. The amplification program was 95℃ pre-denaturation for 2 min, one cycle, followed by 32 cycles, each consisting of 95℃ for 20 s, 55℃ for 20 s, 72℃ for 1 min, and 72℃ extension for 50 min, one cycle.

[0036] PCR products were separated by agarose gel electrophoresis, and the target band was recovered (Bio-Teke gel recovery kit). The gel-recovered products of the L, R, and hph fragments were mixed and ligated into a single knockout fragment using the double-joint method for later use.

[0037] The first step of double-joint PCR: Amplification was performed by mixing the L and R fragments and the hph fragment using PrimeStar from Takara Bio. The PCR system consisted of: 2×Buffer: 12.5 μL, dNTPs: 2 μL, fragment L: 1 μL, fragment R: 1 μL, hph: 1 μL, PrimeStar enzyme: 0.25 μL, and ddH2O to a final volume of 25 μL. The PCR program was: 95℃ for 1 min; 95℃ for 30 s, 58℃ for 1 min 40 s, 72℃ for 5 min, 13 cycles; 72℃ for 10 min.

[0038] Double-joint PCR Step 2: Using the PCR product from the previous step as a template, add primer pair 1F / 4R. The PCR system is as follows: 5×Buffer: 10 μL, 09041-1F / 4R: 1 μL each, dNTPs: 4 μL, Fastpfu enzyme: 1 μL, PCR product from the previous step: 0.3 μL, ddH2O to a final volume of 50 μL. The PCR program is: 95℃ for 2 min; 95℃ for 20 s, 55℃ for 20 s, 72℃ for 2 min, 32 cycles; 72℃ for 5 min.

[0039] After the PCR program is completed, 1.2% agarose gel electrophoresis is performed. After the target band is detected correctly, the PCR product is concentrated (>150μg). The concentrated product is the homologous recombination knockout fragment of the FPSE_09041 encoding gene.

[0040] Example 2: Construction and validation of the FPSE_09041 gene knockout mutant The FPSE_09041 gene was knocked out using homologous recombination, and the homozygous mutant Δ was obtained by PCR verification. FPSE_ 09041 .

[0041] 1. Obtaining the FPSE_09041 gene knockout mutant: Wild-type *Fusarium graminearum* strain 2035 was inoculated into a 50 mL Erlenmeyer flask containing 30 mL of YEPD liquid medium and cultured at 25°C and 150 rpm for 24 hours to induce mycelial pellet formation. The mycelial pellets were then broken up using a nucleic acid extraction shaker and transferred to a 250 mL Erlenmeyer flask containing 100 mL of YEPD liquid medium. The mixture was incubated at 25°C and 150 rpm for 8 hours. Mycelia were collected and lysed using 20 mL of protoplast buffer per gram of mycelia. The mixture was then incubated at 30°C and 90 rpm for 6 hours. Protoplasts were collected, washed once with 0.7 M NaCl by centrifugation, and then washed once with STC Buffer by centrifugation. The protoplasts were then resuspended in STC Buffer to a concentration of 2-5 × 10⁻⁵. 7 per mL.

[0042] Add 5 μg of the gene homologous recombination knockout fragment constructed in Example 1, invert and mix well, then incubate on ice for 30 min. Add 400 μL of 40% PTC to the tube, invert and mix well, and let stand in the dark for 18 min. Add 20 mL of RM medium, shake overnight at room temperature, and pour 20 mL of RM medium into 100 mL of PDA medium (containing hygromycin 250 μg / mL). Quickly transfer to a plate and incubate in the dark at 25°C. Once transformants with single colonies have grown, extract transformant DNA using a fungal genomic DNA extraction kit. Using the transformant DNA as a template, perform PCR detection with four pairs of primers. 09041-5F: 5'-AGATGATGCTCAGACACGA-3' (SEQ ID NO. 11); 09041-6R: 5'-TAGGCTGCTTGGCTATCT-3' (SEQ ID NO. 12); 09041-7F: 5'-AGAATGTCATCGACTCATCTA-3' (SEQ ID NO. 13); 090418R: 5'-TACACAGGATTCATCACCAGAT-3' (SEQ ID NO. 14); H852: 5'-AACTCACCGCGACGTCTGTC-3' (SEQ ID NO. 15); H850: 5'-TTGTCCGTCAGGACATTGTT-3' (SEQ ID NO. 16); H855R: 5'-GCTGATCTGACCAGTTGC-3' (SEQ ID NO. 17); H856F: 5'-GTCGATGCGACGCAATCGT-3' (SEQ ID NO. 18).

[0043] Detection fragments such as Figure 1 As shown, 09041-5F / 6R detects whether each transformant contains the target gene; H850 / H852 detects whether the hph gene has been introduced into each transformant; 09041-7F / H855R detects whether homologous recombination has occurred upstream of the target gene; and H856F / 09041-8R detects whether homologous recombination has occurred downstream of the target gene. Therefore, only when 09041-5F / 6R fails to detect the target band, while the other three primer pairs detect the correct bands, can the transformant be determined as a positive transformant, and a positive transformant strain is obtained.

[0044] 2. Functional verification of the gene encoding FPSE_09041: Using wild-type *Fusarium graminearum* strain 2035 as a template, primer 09041-CF / CR amplified the complement gene fragment containing a full-length gene fragment 1-1.5 kb upstream of the start codon of the target gene and without a stop codon. The obtained complement fragment and the pFL2 vector digested with XhoI were co-transformed into yeast strain XK1-25. The cells were plated on SD-Trp medium and detected as single colonies using primer 09041-5F / 6R. After successful detection, the cells were shaken to extract yeast plasmids using a yeast plasmid extraction kit. The recombinant yeast plasmids were transformed into *E. coli* for propagation. The recombinant plasmids were then transformed into the FPSE_09041 gene knockout mutant according to step 1.2, and transformants were screened using G418. The final concentration of G418 in the bottom agar was 300 μg / mL, and the final concentration of G418 in the top agar was 500 μg / mL. Transformants were detected by PCR using primers 09041-5F / 6R. Transformants that were positive by PCR and showed a phenotypic reversion to wild type on PDA plates were used for phenotypic verification.

[0045] 09041-CF: 5'-TCTCATCACCATCACCATCACAGAATGTCATCGACTCATCTA-3' (SEQ IDNO.19); 09041-CR: 5'-TCGCCCTTGCTCACCCTCGAAGAACCGTGGAGACCACT-3' (SEQ ID NO. 20).

[0046] Example 3: Biological trait analysis of the FPSE_09041 encoding gene mutant 1. Measurement of mycelial growth rate: Wild-type *Fusarium graminearum* strain 2035, the knockout mutant, and the replacement strain were activated on PDA plates and incubated at room temperature for 3-4 days. Mycelial discs were created using a 5mm diameter punch at equal distances from the colony center; three discs were created for both wild-type and mutant strains. Each mycelial disc was inoculated into the center of a 9cm agar plate containing 20mL of PDA medium, with the disc upside down and ensuring even contact between the disc and the plate. Three replicates were performed for each type. The plates were inverted and incubated at room temperature for 72 hours, and the diameter was measured.

[0047] The results showed that, compared with the wild-type *Fusarium graminearum* strain 2035 (WT), the mycelial growth rate of the FPSE_09041 knockout mutants (09041-2, 09041-7) was significantly reduced. Figure 2 The growth rate of the reverse mutant (09041-C) was restored (in both A and B). The experimental results indicate that the FPSE_09041 protein is involved in regulating the hyphal growth of *Fusarium graminearum*.

[0048] 2. Conidia production assay: Using a 5mm diameter punch, collect mycelial cakes from wild-type *Fusarium oxysporum* strains and mutant plates activated for 3-4 days. Transfer 5 mycelial cakes to Erlenmeyer flasks containing 100mL CMC medium, with 3 replicates per strain. Incubate at 25℃ and 150rpm for 5 days, then measure the spore concentration in the suspension using a hemocytometer.

[0049] like Figure 2 As shown in Figure C, compared with wild-type Fusarium pseudograss strain 2035 (WT) and the complemented strain FPSE_09041, the germination rate of conidia of FPSE_09041 knockout mutants (09041-2, 09041-7) was significantly reduced under induction conditions, indicating that the FPSE_09041 protein affects the germination of Fusarium pseudograss conidia.

[0050] 3. Pathogenicity determination: The mutant and wild-type strains were cultured on PDA plates for 7 days, respectively. The resulting bacterial trays were then perforated using a 5mm diameter punch and placed in 100mL of CMC liquid medium. The trays were incubated at 25℃ and 150rpm for 5-7 days. The spore suspension concentration for each strain was 1×10⁻⁶. 5 For wheat inoculation. Wheat seeds were soaked in spore suspension for 15 minutes, then the suspension was discarded, and the seeds were sown in petri dishes containing sterile filter paper and sterile water. Each treatment was repeated 3 times, with 10 seeds per repeat. The seeds were incubated in a light-dark alternating incubation room at 22°C for 12 hours. When the wild-type strain showed severe disease, the disease index was investigated and calculated.

[0051] The results show that ( Figure 3 The disease index of wild-type strain 2035 reached 96%, while the disease index of the mutant was zero. The pathogenicity of FPSE_09041 gene knockout was significantly reduced. The disease index of the FPSE_09041 complement strain recovered to the wild-type level, which indicates that the FPSE_09041 protein is involved in the pathogenicity of the bacteria.

[0052] Therefore, this invention, through gene knockout and complementation experiments, confirms that the FPSE_09041 protein plays a key positive regulatory role in the growth, development, and pathogenicity of *Fusarium graminearum*. Specifically, this is manifested in: ① Regarding mycelial growth: The FPSE_09041 deletion mutant (ΔFPSE_09041) showed a significant reduction in aerial mycelia and a decrease in mycelial growth rate; ② Regarding spore germination: the spore germination rate decreased by approximately 79.27%; ③ Pathogenicity: The mutant completely loses its ability to infect the base of wheat stems, thus losing its pathogenicity; ④ Reintroduction and restoration: After the FPSE_09041 gene was reintroduced into the mutant, mycelial growth, spore germination and pathogenicity were restored to the level of wild type.

[0053] The above results indicate that FPSE_09041 is an indispensable key factor in the pathogenicity of *Fusarium graminearum*, and its functional loss can significantly weaken the survival and infection ability of the pathogen. Therefore, the FPSE_09041 protein and its encoding gene can serve as ideal molecular targets for developing novel, efficient, and specific antifungal agents, providing a new technical pathway for the green control of wheat stem rot. This invention not only provides important gene resources for elucidating the pathogenic mechanism of *Fusarium graminearum*, but also provides new candidate targets for the targeted screening and design of fungicides, possessing significant theoretical value and application prospects.

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of FPSE_09041 protein in reducing the pathogenicity of Fusarium graminearum, characterized by: The amino acid sequence of the FPSE_09041 protein is shown in SEQ ID NO.2, and the gene encoding the FPSE_09041 protein is shown in SEQ ID NO.

1. Inhibiting the expression of FPSE_09041 protein reduces the pathogenicity of Fusarium graminearum to host plants.

2. The application of the FPSE_09041 gene in reducing the pathogenicity of Fusarium graminearum, characterized by: The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein encoded by the FPSE_09041 gene is shown in SEQ ID NO.

2. Knocking out the FPSE_09041 gene reduces the expression of the FPSE_09041 protein, thereby decreasing the pathogenicity of Fusarium graminearum to the host plant.

3. The application of nucleic acids that inhibit FPSE_09041 gene expression in reducing the pathogenicity of Fusarium graminearum, characterized by: The nucleic acid is RNAi, antisense RNA, siRNA or shRNA, and the nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.

1.

4. The application of expression cassettes, recombinant vectors, or transgenic plant cell lines containing nucleic acids that inhibit the expression of the FPSE_09041 gene in reducing the pathogenicity of Fusarium graminearum, characterized in that: The nucleic acid is RNAi, antisense RNA, siRNA or shRNA, and the nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.

1.

5. The application of the FPSE_09041 gene or FPSE_09041 protein as a target in screening agents that inhibit or kill Fusarium oxysporum, characterized in that: The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein is shown in SEQ ID NO.

2.

6. The application of an active substance that inhibits the expression of the FPSE_09041 gene or FPSE_09041 protein in the preparation of formulations that inhibit or kill Fusarium oxysporum, characterized in that: The nucleotide sequence of the FPSE_09041 gene is shown in SEQ ID NO.1, and the amino acid sequence of the FPSE_09041 protein is shown in SEQ ID NO.

2. The active substance that inhibits the expression of the FPSE_09041 gene or FPSE_09041 protein is a nucleic acid or an expression cassette, recombinant vector or transgenic plant cell line containing the nucleic acid, wherein the nucleic acid is RNAi, antisense RNA, siRNA or shRNA.

7. A method for preparing a fungicide or bacteriostatic agent for Fusarium oxysporum, characterized in that: The test samples were co-cultured with Fusarium graminearum to screen for test samples that inhibited the expression of the FPSE_09041 gene or the activity of the FPSE_09041 protein. Based on the test samples that inhibited the expression of the FPSE_09041 gene or the activity of the FPSE_09041 protein, fungicides or bacteriostatic agents for Fusarium graminearum were prepared.

8. A fungicide or inhibitor of Fusarium oxysporum, characterized in that: Fungicides or bacteriostatic agents of Fusarium oxysporum can inhibit the expression of the FPSE_09041 gene or suppress the activity of the FPSE_09041 protein, thereby reducing the pathogenicity of Fusarium oxysporum to host plants.

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