Application of transcription factor fpt3 and its coding gene in regulating pathogenicity of false smut fungus

Abstract

This invention discloses the application of transcription factor FPT3 and its encoding gene in regulating the pathogenicity of *Fusarium graminearum*, belonging to the field of biotechnology. This invention establishes for the first time a direct link between the *Fusarium graminearum* transcription factor FPT3 and its pathogenicity during wheat infection, and utilizes PEG-mediated homologous recombination knockout technology to regulate the pathogenicity of *Fusarium graminearum*. FPT3 Genes are knocked out at specific sites to obtain FPT3 Gene knockout mutant. Compared with wild-type Fusarium pseudograss, the described FPT3 The gene knockout mutant exhibited significantly reduced pathogenicity after infecting wheat, indicating that FPT3 plays a key regulatory role in the infection and disease formation of *Fusarium graminearum* in wheat. Therefore, this invention not only reveals the biological function of FPT3 as a key virulence transcription factor in *Fusarium graminearum*, but also demonstrates that inhibiting FPT3 expression or activity can effectively weaken the infectivity of *Fusarium graminearum* in wheat.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a transcription factor FPT3 and its encoding gene in regulating the pathogenicity of Fusarium graminearum. Background Technology

[0002] Stem base rot, also known as wheat crown rot, is one of the most devastating soil-borne diseases in wheat production. With changes in planting systems and farming practices, this disease is showing a rapid increasing trend year by year. The main pathogen causing wheat stem base rot is *Fusarium pseudocarpa* (…). Fusarium pseudograminearum It mainly overwinters as mycelium and spores on diseased plant debris. Due to the current lack of resistant wheat varieties and highly effective chemical control agents, controlling wheat stem rot is quite difficult. Therefore, elucidating the pathogenic mechanism of key pathogenic factors of *Fusarium graminearum* is of great significance for the control of plant diseases caused by *Fusarium graminearum*.

[0003] Transcription factors are core elements regulating gene expression, widely involved in multiple processes in pathogenic fungi, including mycelial growth, sporulation, stress response, and pathogenicity. Among the identified pathogenicity-related genes of *Fusarium graminearum*, transcription factors act as upstream regulatory hubs, their encoded proteins directly binding to the promoter regions of target genes and synergistically regulating the expression of downstream genes. Their functional loss often has multifaceted effects on pathogens. Currently, research on transcription factors for *Fusarium graminearum* is relatively limited, with identified key genes mainly concentrated in the APSES, Zn2Cys6, and bZIP families. However, a large number of unidentified transcription factors remain, their functions and regulatory networks unclear. Therefore, there is an urgent need to develop new transcription factors that regulate the pathogenicity of *Fusarium graminearum*. Summary of the Invention

[0004] In view of the above-mentioned prior art, the purpose of this invention is to provide the application of transcription factor FPT3 and its encoding gene in regulating the pathogenicity of Fusarium graminearum.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides the application of transcription factor FPT3 in regulating the pathogenicity of Fusarium graminearum; the amino acid sequence of said transcription factor FPT3 is shown in SEQ ID NO.1.

[0006] This invention has found that the transcription factor FPT3 is significantly correlated with the pathogenicity of *Fusarium graminearum*, and that inactivation of FPT3 can reduce the pathogenicity of *Fusarium graminearum*. Therefore, drugs targeting FPT3 can be developed to control *Fusarium graminearum*.

[0007] A second aspect of the present invention provides the application of the gene encoding transcription factor FPT3 in regulating the pathogenicity of *Fusarium graminearum*; the gene encoding transcription factor FPT3 is... FPT3 A gene is a nucleic acid molecule as shown in (i) or (ii) 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.

[0008] In the above applications, by knocking out or silencing FPT3 Genes reduce the pathogenicity of Fusarium pseudograss.

[0009] Specifically, the gene encoding the transcription factor FPT3 can be knocked out or silenced using any of the following substances: e1) Targeted knockout FPT3 Nucleic acid molecules of genes; 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).

[0010] As a preferred option, targeted knockout FPT3 The nucleic acid molecules of the gene are knockout fragment AH1 and knockout fragment H2B; the nucleotide sequence of knockout fragment AH1 is shown in SEQ ID NO.11, and the nucleotide sequence of knockout fragment H2B is shown in SEQ ID NO.12.

[0011] A third aspect of the invention provides the use of a substance that inhibits the activity of transcription factor FPT3 in either (1) or (2) below: (1) Control of plant diseases caused by Fusarium graminearum; (2) Prepare drugs to prevent and control Fusarium graminearum.

[0012] In the above applications, using transcription factor FPT3 as a target and selecting substances that inhibit the activity of transcription factor FPT3 can enable the prevention and control of plant diseases caused by Fusarium graminearum and the development of related therapeutic drugs.

[0013] In the above applications, substances that inhibit the activity of transcription factor FPT3 refer to substances that can reduce the content of transcription factor FPT3, block the binding of transcription factor FPT3 to its target gene promoters or regulatory sequences, weaken the transcriptional activation or transcriptional regulation function mediated by transcription factor FPT3, or reduce the expression level of downstream target genes of transcription factor FPT3. These substances can be selected from one or more of the following: small molecule compounds, natural product monomers, plant extracts, microbial metabolites, fermentation products, oligonucleotides, antisense nucleic acids, siRNA, shRNA, aptamers, peptides, antibodies, or functional fragments thereof.

[0014] A fourth aspect of the invention provides targeted knockout. FPT3 Application of genetic material in the prevention and control of plant diseases caused by Fusarium graminearum.

[0015] In the above applications, the targeted knockout FPT3 The preferred gene components are knockout fragment AH1 and knockout fragment H2B; the nucleotide sequence of knockout fragment AH1 is shown in SEQ ID NO.11, and the nucleotide sequence of knockout fragment H2B is shown in SEQ ID NO.12.

[0016] In the above applications, the plant disease is wheat stem base rot or Fusarium head blight. The beneficial effects of this invention are: This invention establishes for the first time a direct link between the Fusarium pseudobolum transcription factor FPT3 and its pathogenicity during wheat infection, and utilizes PEG-mediated homologous recombination knockout technology to inhibit the pathogenicity of Fusarium pseudobolum. FPT3 Genes are knocked out at specific sites to obtain FPT3 Gene knockout mutant. Compared with wild-type Fusarium pseudograss, the described FPT3 The gene knockout mutant exhibited significantly reduced pathogenicity after infecting wheat, indicating that FPT3 plays a key regulatory role in the infection and disease formation of *Fusarium graminearum* in wheat. Therefore, this invention not only reveals the biological function of FPT3 as a key virulence transcription factor in *Fusarium graminearum*, but also demonstrates that inhibiting FPT3 expression or activity can effectively weaken the infectivity of *Fusarium graminearum* in wheat.

[0017] Furthermore, targeting the transcription factor FPT3 and its encoding gene provides a clear molecular target and theoretical basis for screening and developing small molecule inhibitors against Fusarium graminearum. Therefore, this invention has significant application value in elucidating the pathogenic mechanism of Fusarium graminearum, discovering targets for wheat disease resistance, and developing novel small molecule inhibitors, providing a new technological foundation for the green, efficient, and precise control of Fusarium graminearum-related diseases in wheat. Attached Figure Description

[0018] Figure 1The Fusarium pseudocarpa transcription factor in Example 1 of this invention FPT3 A schematic diagram illustrating the construction of the knockout fragments required for knockout.

[0019] Figure 2 The Fusarium pseudocarpa transcription factor in Example 1 of this invention FPT3 PCR gel image of the fragment to be knocked out.

[0020] Figure 3 The Fusarium pseudocarpa transcription factor in Example 1 of this invention FPT3 Image of knockout PCR identification results; in the image, lane G represents FPT3 Gene amplification fragments; lane U represents the AH1 amplification fragment; lane D represents the H2B amplification fragment; lane H represents the hygromycin gene amplification fragment.

[0021] Figure 4 Example 2 of this invention: Fusarium pseudocarpa transcription factor FPT3 PCR gel image of the fragment to be added.

[0022] Figure 5 Example 2 of this invention: Fusarium pseudocarpa transcription factor FPT3 Image of the PCR identification results.

[0023] Figure 6 In Example 3 of this invention, the wild-type Fp22 (WT) and knockout mutant of *Fusarium graminearum* were used. Fpt3-1 , Fpt3-2 The infection capacity of wheat by the inoculation method of the reinfected strains (Fpt3-C-1 and Fpt3-C-2) and the coleoptile inoculation method was compared; in the figure, A is the phenotype after inoculation; B is the statistical data of lesion length after inoculation.

[0024] Figure 7 In Example 3 of this invention, the wild-type Fp22 (WT) of *Fusarium graminearum* and its knockout mutant ( Fpt3-1 , Fpt3- 2 Comparison of wheat infection capacity using millet culture medium inoculation method; In the figure, A represents the phenotype after inoculation; B represents the disease index statistics after inoculation. Detailed Implementation

[0025] 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.

[0026] As mentioned earlier, elucidating the pathogenic mechanism of Fusarium graminearum and developing new transcription factors are of great significance for the prevention and control of plant diseases caused by Fusarium graminearum.

[0027] In view of this, this invention conducted an in-depth study of unknown genes in the genome of *Fusarium pseudograminearum*, among which the hypothetical protein FPSE_02717 [Fusarium pseudograminearum] is a transcription factor with unknown function in *Fusarium pseudograminearum*, and was named transcription factor FPT3. The amino acid sequence of transcription factor FPT3 is shown in SEQ ID NO.1, and is as follows: MSTLPPDHRKGVSRATENTVNMTVSQSTKAKPASKACNGKPKTKTQMHRRSRTGCYTCRLRRKKCDEGTPMCTACKHLGLQCEYKRPMWWSNNDMRRKHKEDIKMIIKRKKLSEKSSHNIQNSVTSSPPGLTHSLPTSATFTDPLDRTRSASIDSQFPGAFNFNSPPSGN EYGFGAPMHPEFMFGSYSPYEIDVKTERQMFVNDVPTVRESHISTFSTYHTPPPAGTVLPHGPLDGEWTEQVFQERKQSLSEETLNCNFFDFSHGPSTESRQIQLELDENDQSLLDHFIQFVLPTIFPILESNQHCSVGSDLVLPALQSNSAYLHCCLSVAAQHLKSHTNA PNEDLDNDIMRHRYATIWALCEALKKDENHQQILEATLGLIFFQSVVGRYDDGLLDIPWHQHFQAAISLVQKLDLHGMVTDPTRASMQTPFNMSLSSWIDILGATMKGRSPTFAHTYREKHLSQFNPSLGLRELMGCDDRVMYLISEIACLESLKKDGMDDFTLCQHVSAL GEQISLTEMGDAGPKMPFNANGSLSPKQLSKNMTMAFRIAARIYLCSLVPGFNPRQPSTMGLVEKLTNVLQHIPSGPNGFDRNLAWVYLIGGSTSVPGSTFRVFFDDRLVQLGDSARFGTMGRVSTLLHEVWAQNDSLAGVNTPGSTTEAAQLHIHWRDVMESKGWDFLLI The gene encoding transcription factor FPT3 FPT3 The nucleotide sequence of the gene is shown in SEQ ID NO.2, as follows:

[0028] To verify the function of transcription factor FPT3, this invention utilizes the principle of PEG-mediated homologous recombination to target the transcription factor of *Fusarium graminearum*. FPT3 In situ knockout was performed to obtain the knocked-out transcription factor. FPT3 Gene deletion mutant strains; and on FPT3 The pathogenicity of gene-deleted mutant strains was investigated, and it was found that compared with the wild type, FPT3 The gene deletion mutant strain showed a significantly reduced pathogenicity to wheat. This proves that: FPT3 The gene is a pathogenic gene of Fusarium graminearum, and its encoded protein, as a drug target, is expected to provide a new approach for the prevention and control of wheat stem rot.

[0029] 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.

[0030] 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 graminearum wild-type Fp22, described in the literature "Tai J, Zhang X, Gao X, Liu H, Xu M. High-Quality Genome Resource of Fusarium pseudograminearum Isolate Fp22-2 by Oxford Nanopore Long-Read Sequencing. Plant Dis. 2023 Jun;107(6):1925-1928.doi: 10.1094 / PDIS-09-22-2034-A. Epub 2023 Jun 7. PMID: 36510423."

[0031] The HTK plasmid is described in the literature “Twaruschek K, Spörhase P, Michlmayr H, Wiesenberger G and Adam G (2018) New Plasmids for Fusarium Transformation Allowing Positive-Negative Selection and Efficient Cre-loxP Mediated Marker Recycling. Front. Microbiol. 9:1954. doi: 10.3389 / fmicb.2018.01954”.

[0032] The public can obtain the above materials from the applicant to reproduce this invention.

[0033] The composition of CMC culture medium is as follows: each liter of CMC culture medium contains 15g Carboxymethycellulose, 1g NH4NO3, 1g KH2PO4, 0.5g MgSO4·7H2O, and 1g Yeast Extract.

[0034] The composition of the upper culture medium is as follows: each liter of upper culture medium contains 3g Yeast Extract, 3g acid-hydrolyzed casein, 200g sucrose, and 15g Agar.

[0035] The composition of the lower culture medium is as follows: each liter of lower culture medium contains 3g Yeast Extract, 3g acid-hydrolyzed casein, 200g sucrose, and 10g Agar.

[0036] Example 1: Preparation of FPT3 knockout mutant of Fusarium graminearum transcription factor 1. Construction FPT3 Gene knockout fragment FPT3 The construction approach for gene knockout fragments is as follows: Figure 1 As shown, using the HTK plasmid as a template, two pairs of primers were designed: H1-F (GGCTTGGCTGGAGCTAGTGGAGGTCAA; SEQ ID NO.3), H1-R (GGATGAGGGCCACGAACGCCAGCAC; SEQ ID NO.4) and H2-F (TCGGTCAATACACTACATGGCGTG; SEQ ID NO.5), H2-R (GTCGACTTAATAACACATTGCGGACGT; SEQ ID NO.6), to amplify the hygromycin resistance gene fragments H1 and H2 on the plasmid, respectively.

[0037] Then, using *Fusarium graminearum* Fp22 genomic DNA as a template, primers FPT3A-F (ATCAGAAAGTCAAAGCTCTCCG; SEQ ID NO.7) and FPT3A-R (TTGACCTCCACTAGCTCCAGCCAAGCCTGGGGTGTGTGATGTACAAGG; SEQ ID NO.8) were designed to amplify the genome. FPT3 The 984 bp upstream sequence of the gene was named fragment A; primers FPT3B-F (CGTCCGCAATGTGTTATTAAGTCGACCATTACTTCTGACGAACCTTCTTG; SEQ ID NO.9) and FPT3B-R (GCCATTGAATGTGGCCAGTG; SEQ ID NO.10) were designed to amplify the gene. FPT3 The 1033bp sequence downstream of the gene is named fragment B.

[0038] Reaction conditions: 95℃ for 3 min; 95℃ for 30 s, 58℃ for 40 s, 72℃ for 2 min, for a total of 35 cycles; the PCR product was identified by 1% agarose gel electrophoresis, then excised and purified, and dissolved in deionized water for later use.

[0039] Fragment A (984 bp upstream of transcription factor FPT3) and fragment H1 (1902 bp upstream of the hygromycin resistance gene fragment), fragment B (1033 bp downstream of transcription factor FPT3) and fragment H2 (1999 bp downstream of the hygromycin resistance gene fragment) were ligated using overlap PCR to obtain products AH1 (SEQ ID NO.11) and H2B (SEQ ID NO.12). To improve splicing efficiency, a segmented amplification method was used. The first step involved 15 cycles of amplification without primers under the following conditions: 95℃ for 3 min; 95℃ for 15 s, 58℃ for 2 min, and 72℃ for 2 min. The second step involved adding primers and continuing the reaction for 35 cycles under the following conditions: 95℃ for 3 min; 95℃ for 30 s, 58℃ for 1 min, and 72℃ for 2 min. The PCR products were identified by 1% agarose gel electrophoresis, purified by gel extraction, and dissolved in deionized water for later use.

[0040] The agarose gel electrophoresis results of products AH1 and H2B are as follows: Figure 2 As shown. Products AH1 and H2B were used as... FPT3 Gene knockout fragments.

[0041] 2. Knockout of Fusarium pseudobulb using PEG-mediated homologous recombination FPT3 Gene Wild-type strain Fp22 of *Fusarium graminearum* was cultured on PDA plates at 25°C for 3 days. Five mycelial cakes were then collected using a blue pipette tip and placed in 100ml of CMC culture medium (CMC, 1.5%). The culture was incubated at 175 rpm and 25°C for 3-5 days. The CMC culture was filtered through sterilized filter cloth into a 100ml sterile centrifuge tube and centrifuged at 10,000 rpm for 1 min. The supernatant was discarded, and pink conidia precipitate was visible at the bottom of the centrifuge tube. The precipitate was transferred to 100ml of YEPD culture medium and incubated at 175 rpm and 25°C for approximately 12 hours.

[0042] Collect young hyphae by filtering with sterile filter cloth. Rinse the hyphae with 1.2M KCl solution. Collect 0.5g of hyphae and place them in a 50mL centrifuge tube containing 10mL of enzymatic digestion solution. Gently shake to mix and incubate at 90rpm and 30℃ for 1-2 hours. Use a pipette to pipette 20μL of the mycelial digestion mixture to examine the digestion under a microscope. Once the mycelial digestion is complete (a large number of round protoplasts are visible), filter the digestion mixture through three layers of sterile lens paper and one layer of sterile filter cloth into a new sterile 50mL centrifuge tube. Centrifuge at 3500rpm for 5min at room temperature. Discard the supernatant and add 10mL of 1xSTC buffer to wash the precipitate. Resuspend the precipitate by pipette and centrifuge at 3500rpm for 5min at room temperature. Discard the supernatant and repeat the washing process once. Add an appropriate amount of 1xSTC buffer and adjust the protoplast concentration to 2×10⁻⁶ using a hemocytometer. 7 per ml.

[0043] 200 μL of the prepared protoplast suspension was transferred to a new 50 mL centrifuge tube, and 3 μg of the constructed AH1 and 3 μg of the constructed H2B were added to each tube for further processing. FPT3After gene knockout, gently mix by tilting the plate and let stand at room temperature for 20 minutes. Add 1 mL of sterile 40% PTC solution, mix by tilting the plate and let stand at room temperature for another 30-40 minutes. Add 5 mL of TB3 culture medium and incubate at 90 rpm and 25°C on a tilting shaker for 12 hours. Add hygromycin to a final concentration of 40 μg / mL and ampicillin (Amp+) to a final concentration of 50 μg / mL, along with 25 mL of melted and cooled lower culture medium to approximately 65°C. Mix well and spread evenly in sterile petri dishes. After the lower culture medium has cooled and solidified to room temperature, add 15 mL of upper culture medium containing hygromycin to a final concentration of 80 μg / mL and ampicillin to a final concentration of 50 μg / mL. Incubate at 25°C. After approximately 2-3 days of incubation, snowflake-like hyphae will grow to the surface of the upper culture medium. Single colonies are then picked and transferred to the upper culture medium or PDA plates containing a final concentration of 40 μg / ml hygromycin for further resistance selection. Colonies that grow normally after this second selection are transferred to fresh PDA medium and incubated for 2-3 days until a suitable amount of hyphae have grown. DNA is then extracted using the CTAB method to confirm the correct colonies. FPT3 Gene knockout mutants.

[0044] Using HTK plasmid as a template, primers H-detect-F (GCCGATCTTAGCCAGACGAG; SEQ ID NO. 13) and H-detect-R (GCGGTCGATGTGTCTGTCCTC; SEQ ID NO. 14) were designed for the identification of the hygromycin resistance gene in the knockout mutant. Using Fusarium graminearum Fp22 genomic DNA as a template, primers FPT3-detect-F (GAACACCGTCAACATGACTG; SEQ ID NO. 15) and FPT3-detect-R (CTCGGAATGTACTACCAGGGAC; SEQ ID NO. 16) were designed for the detection of the target gene. FPT3 Identification: Using Fusarium graminearum Fp22 genomic DNA and HTK plasmid as templates, primer pairs FPT3-detect-AF (CTAGTCAATGTCTTGGTCAAGTGG; SEQ ID NO.17), H1-R and H1-F, and FPT3-detect-BR (CAAGGATATCGCAAGTAAGCACAG; SEQ ID NO.18) were designed for the identification of AH1 and H2B.

[0045] If both of the following conditions are met: (1) the target gene cannot be identified FPT3 (1) bands of the hygromycin resistance gene were identified; (2) bands of the hygromycin resistance gene were identified; (3) bands of AH1 and H2B were identified; then it can be determined that FPT3The knockout mutant is correct.

[0046] The results are as follows Figure 3 As shown, this embodiment successfully constructed two FPT3 knockout mutants of Fusarium graminearum transcription factor (FPT3). Fpt3-1 , Fpt3-2 ).

[0047] Example 2: FPT3 Construction of gene complementation strains 1. Construction FPT3 Gene complementation fragment: Using Fusarium graminearum Fp22 genomic DNA as a template, primers FPT3SU-F (GTGAGGAGCTAGATCGCAATGG; SEQ ID NO.19) and FPT3SU-R (GTGTTGTCTAATCAGAGCGAGGC; SEQ ID NO.20) were designed to amplify... FPT3 Genes and their upstream and downstream sequences were constructed to obtain FPT3 The gene complement fragment, whose nucleotide sequence is shown in SEQ ID NO.21, is as follows:

[0048] Reaction conditions: 94℃ for 2 min; 94℃ for 30 s, 58℃ for 30 s, 68℃ for 60 s, for a total of 30 cycles; PCR products were identified by 1% agarose gel electrophoresis. Figure 4 The gel was then recovered and purified by cutting, and dissolved in deionized water for later use.

[0049] 2. Gene replacement: The FPT3 knockout mutant of Fusarium pseudocarpa transcription factor constructed in Example 1 Fpt3-2 For gene restoration targets, FPT3 Gene complementation fragment transferred into Fusarium pseudocarpa transcription factor FPT3 knockout mutant Fpt3-2 .

[0050] FPT3 The protoplasm and transformation method of the gene deletion mutant strain are described in Example 1. The resistance screening was changed to fluorouridine. The lower culture medium was antibiotic-free, and fluorouridine was added to the upper culture medium at a working concentration of 10 μg / mL.

[0051] Using Fusarium graminearum Fp22 genomic DNA as a template, primers FPT3-F (CAGATGCACCGTCGCTCAAG; SEQ ID NO.22) and FPT3-R (CCTTCTTGAGAGACTCAAGGCAG; SEQ ID NO.23) were designed for targeting the target gene. FPT3 The identification.

[0052] The results are as follows Figure 5 As shown, the system was successfully constructed. FPT3 Gene complement strains ( Fpt3 -C-1 and Fpt3 -C-2).

[0053] Example 3: FPT3 knockout mutant of Fusarium pseudocarpa transcription factor FPT3 Investigation on the pathogenicity of gene-reinforced strains to wheat 1. Test method: (1) Coleoptile inoculation method: Build Example 1 FPT3 Knockout mutants ( Fpt3-1 , Fpt3-2 Example 2 FPT3 Gene complement strains ( Fpt3 -C-1 and Fpt3 -C-2) Fpt3- Conidia were obtained by culturing strains C and wild-type Fusarium pseudocaryophyllum Fp22, and the conidia concentration was adjusted to 5 × 10⁻⁶. 6 per ml.

[0054] On the third day after wheat (Mingxian 169) was sown, the top third of the coleoptile was cut off with scissors, and 2 μL of spore suspension with adjusted concentration was dripped into the wound. Four days later, the length of the lesions was measured and the pathogenicity of Fusarium pseudograss was observed to be weakened after knocking out the lesions.

[0055] (2) Millet culture medium inoculation method: Boil an appropriate amount of millet in boiling water for two minutes, drain, and place 40g of millet into 100ml Erlenmeyer flasks. Sterilize in an autoclave at high temperature to prepare millet culture medium. The wild-type strain of *Fusarium graminearum* Fp22 was activated beforehand on a PDA plate. FPT3 Knockout mutants ( Fpt3-1 , Fpt3-2 After the millet culture medium has been sterilized and brought to room temperature, use a blue pipette tip to pick up three pieces of mycelium and place them into the sterilized millet culture medium. Incubate at a constant temperature of 25°C for 7 days. During the incubation process, shake the millet culture medium every day to disperse the mycelium and prevent it from clumping, so that the mycelium can grow more evenly. Once the mycelium has fully grown, pour out the millet culture medium and let it air dry in a ventilated place.

[0056] Wheat (Mingxian 169) seeds were sown in petri dishes and germinated for 2 days. Once the wheat seeds showed signs of germination, they were transplanted into soil. Approximately three days after planting, 0.4g of the prepared wild-type strain of *Fusarium graminearum* Fp22 was weighed out. FPT3 Knockout mutants ( Fpt3-1 , Fpt3-2 Millet culture medium containing mycelium was evenly sprinkled on the base of wheat stems. After 14 days of inoculation, the differences in wheat stem base rot caused by knockout mutants and wild-type strains were observed and statistically analyzed, and the disease index was calculated.

[0057] Disease index = 100 × ∑ (number of diseased wheat at each level × representative value at each level) / (total number of wheat surveyed × highest representative value) The disease grading standards are as follows: Grade 0: The plant is not diseased; Grade 1: The stem in the ground is obviously brown or the first leaf sheath shows mild symptoms; Grade 3: The first leaf sheath is noticeably brown; Level 5: The first leaf sheath turns black or the second leaf sheath turns brown; Level 7: The third leaf sheath shows signs of browning, or the plant is slow to grow or is near death because it is not diseased; Level 9: The plant died due to disease.

[0058] 2. Experimental Results: wild-type Fp22 of *Fusarium graminearum*, knockout mutant and FPT3 The results of the comparison of the infection ability of gene-reinforced strains on wheat via coleoptile inoculation are as follows: Figure 6As shown, the results indicate that compared with the wild-type Fusarium graminearum Fp22, FPT3 The length of lesions in wheat infected with gene knockout mutants was significantly reduced, and the complementation... FPT3 The length of lesions in wheat after genetic modification is basically the same as that in the wild type.

[0059] The results of comparing the infection ability of wild-type Fusarium graminearum Fp22 and its knockout mutant on wheat using millet culture medium inoculation method are as follows: Figure 7 As shown, the results indicate that compared with the wild-type Fusarium graminearum Fp22, FPT3 The disease index of wheat infected with gene knockout mutants was significantly reduced.

[0060] The above results demonstrate that FPT3 is a key transcription factor in *Fusarium graminearum* infection of wheat, causing wheat stem rot, and plays an important role in the infection process. The FPT3 transcription factor in *Fusarium graminearum* is... FPT3 Gene knockout or deletion can reduce the pathogenicity of Fusarium pseudograss.

[0061] 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 transcription factor FPT3 inactivation in reducing the pathogenicity of Fusarium graminearum, characterized in that, The amino acid sequence of the transcription factor FPT3 is shown in SEQ ID NO.

1.

2. The application of the gene encoding the transcription factor FPT3 by knockout or silencing in regulating the pathogenicity of Fusarium graminearum, characterized in that, The gene encoding the transcription factor FPT3 is FPT3 A gene is a nucleic acid molecule as shown in (i) or (ii) 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.

3. The application according to claim 2, characterized in that, Knockout or silence of the gene encoding transcription factor FPT3 using any of the following substances: e1) Targeted knockout FPT3 Nucleic acid molecules of genes; 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).

4. The application according to claim 3, characterized in that, Targeted knockout FPT3 The nucleic acid molecules of the gene are knockout fragment AH1 and knockout fragment H2B; the nucleotide sequence of knockout fragment AH1 is shown in SEQ ID NO.11, and the nucleotide sequence of knockout fragment H2B is shown in SEQ ID NO.12.

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