Use of a small g-protein regulator VdRasGAP of verticillium dahliae in resistance to cotton verticillium wilt

Abstract

The application discloses application of a small G protein regulator VdRasGAP of Verticillium dahliae in resisting cotton verticillium wilt, and provides application of inhibition of expression and / or activity of the VdRasGAP protein in any of the following aspects: (A1) reducing pathogenicity of the Verticillium dahliae; (A2) resisting plant verticillium wilt caused by the Verticillium dahliae infection. The application lays a theoretical foundation for in-depth research on pathogenic mechanism of the Verticillium dahliae, provides a new theoretical basis and an effective target for efficient prevention and treatment of the cotton verticillium wilt, and lays a foundation for further development of a new type of target fungicide.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of a small G protein regulator VdRasGAP encoded by Verticillium dahliae in resistance to Verticillium wilt in cotton. Background Technology

[0002] Cotton, the most widely planted cash crop in my country, is an important source of textile fibers. Its by-products are also high-quality oilseeds and feed, playing a vital role in the national economy.

[0003] Currently, the main disease affecting cotton production is Verticillium wilt. Verticillium wilt is a typical soil-borne vascular fungal disease and the most significant factor impacting cotton yield and quality. It has a broad host spectrum, infecting over 400 species of dicotyledonous plants, including cotton, eggplant, tomato, and potato, causing enormous economic losses to agricultural production.

[0004] In my country, *Verticillium dahliae* is the main pathogen causing Verticillium wilt in cotton. It belongs to the phylum Ascomycota, class Sordariomycetes, subclass Hypocreomycetidae, and genus *Verticillium*. It invades the plant through the roots, colonizes and proliferates in the vascular bundles, and its germinating conidia spread throughout the plant via transpiration, ultimately leading to wilting, yellowing, and even death. Its dormant form, a black microsclerotium, not only protects *Verticillium dahliae* from harsh external environments such as low temperatures but also allows it to remain dormant for decades in hostless soil.

[0005] Verticillium wilt, caused by Verticillium dahliae, is known as the "cancer" of cotton, and its sustainable control has always faced significant challenges. The main reasons for this are as follows:

[0006] (1) First, unlike rice blast fungus, which is an airborne pathogen, Verticillium dahliae lives in the soil and is a soil-borne pathogen, so it is impossible to use chemical pesticides on a large scale for control in the early stage of infection; (2) Second, Verticillium dahliae has a highly resistant dormant body—black microsclerotia. Microsclerotia can survive for more than ten years without a host, and once colonized, they are difficult to completely eradicate; (3) At the same time, the Verticillium wilt antigen of important crops related to national economy and people's livelihood—cotton, sunflower, potato, etc.—has not yet been found, and the progress of antigen-dependent traditional breeding or disease resistance gene-dependent molecular breeding is not satisfactory.

[0007] Studying the pathogenic mechanisms of pathogens can lay the foundation for finding new control strategies. However, compared with model fungi such as rice blast fungus, research on the pathogenic mechanisms of Verticillium dahliae is relatively lagging and requires further investigation. Summary of the Invention

[0008] The purpose of this invention is to provide an application of VdRasGAP, a small G protein regulator of Verticillium dahliae, in the resistance to Verticillium wilt of cotton.

[0009] In a first aspect, the present invention claims protection for the use of inhibiting the expression and / or activity of VdRasGAP protein in any of the following:

[0010] (A1) Reduces the pathogenicity of Verticillium dahliae;

[0011] (A2) Resistance to Verticillium wilt caused by Verticillium dahliae infection.

[0012] Secondly, the present invention claims protection for the use of substances that inhibit the expression and / or activity of VdRasGAP protein in any of the following:

[0013] (A1) Reduces the pathogenicity of Verticillium dahliae;

[0014] (A2) Resistance to Verticillium wilt caused by Verticillium dahliae infection.

[0015] Thirdly, the present invention claims protection for the use of inhibiting the expression and / or activity of VdRasGAP protein in any of the following:

[0016] (B1) Slows down the growth rate of Verticillium dahliae;

[0017] (B2) Reduce the disease index of plants infected with Verticillium dahliae;

[0018] (B3) Reduces the degree of vascular bundle browning in plants infected with Verticillium dahliae;

[0019] (B4) Reduces the colonization ability of Verticillium dahliae in plant vascular bundles.

[0020] Fourthly, the present invention claims protection for the use of substances that inhibit the expression and / or activity of VdRasGAP protein in any of the following:

[0021] (B1) Slows down the growth rate of Verticillium dahliae;

[0022] (B2) Reduce the disease index of plants infected with Verticillium dahliae;

[0023] (B3) Reduces the degree of vascular bundle browning in plants infected with Verticillium dahliae;

[0024] (B4) Reduces the colonization ability of Verticillium dahliae in plant vascular bundles.

[0025] In all the above aspects, inhibition of the expression and / or activity of the VdRasGAP protein can be achieved through at least one of the following six regulatory levels: 1) inhibiting the expression of the gene encoding the VdRasGAP protein at the gene transcription level; 2) inhibiting the expression of the gene encoding the VdRasGAP protein at the post-transcriptional level (i.e., inhibiting the expression of the VdRasGAP protein by splicing or processing the primary transcript of the relevant gene); 3) inhibiting the expression of the VdRasGAP protein at the RNA transport level (i.e., inhibiting the expression of the VdRasGAP protein by regulating the transport of the mRNA of the relevant gene from the nucleus to the cytoplasm); 4) inhibiting the expression of the VdRasGAP protein by regulating the translation level; 5) inhibiting the expression of the VdRasGAP protein by regulating the mRNA degradation level; 6) inhibiting the activity of the VdRasGAP protein by regulating the post-translational level. The same applies below.

[0026] Furthermore, the inhibition of VdRasGAP protein expression and / or activity can be achieved by knocking out or reducing the expression of the gene encoding the VdRasGAP protein in the Verticillium dahliae genome. Accordingly, the substance can be a substance capable of knocking out or reducing the expression of the gene encoding the VdRasGAP protein in the Verticillium dahliae genome.

[0027] Furthermore, the inhibition of the expression and / or activity of the VdRasGAP protein can be achieved by introducing a homologous recombination fragment or homologous recombination vector into *Verticillium dahliae* to knock out the gene encoding the VdRasGAP protein. Accordingly, the substance can be a homologous recombination fragment or homologous recombination vector to knock out the gene encoding the VdRasGAP protein.

[0028] In an embodiment of the present invention, the nucleotide sequence of the homologous recombination fragment used to knock out the gene encoding the VdRasGAP protein is shown in SEQ ID No. 3; or, the homologous recombination vector used to knock out the gene encoding the VdRasGAP protein is a vector containing the DNA fragment shown in SEQ ID No. 3.

[0029] Fifthly, the present invention claims protection for any of the following methods:

[0030] Method I: A method for reducing the pathogenicity of Verticillium dahliae, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein.

[0031] Method II: A method for combating Verticillium wilt caused by Verticillium dahliae infection in plants, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein.

[0032] Method III: A method for slowing down the growth rate of Verticillium dahliae and / or reducing the disease index of Verticillium dahliae-infected plants and / or reducing the degree of vascular bundle browning in Verticillium dahliae-infected plants and / or reducing the colonization ability of Verticillium dahliae in plant vascular bundles, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein.

[0033] Method IV: A method for preparing Verticillium dahliae with reduced pathogenicity, comprising the steps of: inhibiting the expression and / or activity of VdRasGAP protein.

[0034] Inhibiting the expression and / or activity of VdRasGAP protein can be achieved by knocking out or reducing the expression of the gene encoding VdRasGAP protein in the Verticillium dahlia genome.

[0035] Furthermore, the inhibition of the expression and / or activity of the VdRasGAP protein is achieved by introducing a homologous recombination fragment or homologous recombination vector into Verticillium dahliae to knock out the gene encoding the VdRasGAP protein.

[0036] Furthermore, the nucleotide sequence of the homologous recombination fragment used to knock out the gene encoding the VdRasGAP protein is shown in SEQ ID No. 3; or, the homologous recombination vector used to knock out the gene encoding the VdRasGAP protein is a vector containing the DNA fragment shown in SEQ ID No. 3.

[0037] In the aforementioned relevant aspects, the VdRasGAP protein may be any of the following:

[0038] (C1) The protein with the amino acid sequence shown in SEQ ID No. 2;

[0039] (C2) is a protein derived from Verticillium dahliae that has 99%, 95%, 90%, 85%, or 80% identity with the protein defined in (C1) and is associated with the pathogenicity of Verticillium dahliae.

[0040] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid sequences can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing an identity search on a pair of amino acid sequences, the identity value (%) can then be obtained.

[0041] The 80% or more identity can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 85% or more identity can be at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 90% or more identity can be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity. The 95% or higher level of identity can be at least 95%, 96%, 97%, 98%, or 99% identity.

[0042] In the aforementioned relevant aspects, the gene encoding the VdRasGAP protein may be any of the following:

[0043] (D1) The DNA molecule shown in SEQ ID No. 1;

[0044] (D2) is a DNA molecule derived from Verticillium dahliae that has 99%, 95%, 90%, 85%, or 80% identity with the DNA sequence defined by (D1) and encodes the VdRasGAP protein.

[0045] For the genes mentioned above, nucleotide sequence identity can be determined using identity search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of nucleotide sequences to calculate the identity value (%), then the identity value can be obtained.

[0046] In the aforementioned genes, the 95% or higher identity can be at least 96%, 97%, or 98% identity. The 90% or higher identity can be at least 91%, 92%, 93%, or 94% identity. The 85% or higher identity can be at least 86%, 87%, 88%, or 89% identity. The 80% or higher identity can be at least 81%, 82%, 83%, or 84% identity.

[0047] In the aforementioned relevant aspects, the plant is a host plant of *Verticillium dahliae*, that is, a plant that can be infected by *Verticillium dahliae*. Further, the host plant is cotton.

[0048] Sixthly, the present invention also claims protection for the reduced pathogenicity of *Verticillium dahliae* prepared using method IV described in the fifth aspect above. This *Verticillium dahliae* can be used for in-depth research on the pathogenic mechanism of *Verticillium dahliae*.

[0049] This invention first constructs a *Verticillium dahliae* ΔVdRasGAP knockout mutant and a complemented mutant strain (a strain that restores the function of the VdRasGAP gene) targeting the aforementioned mutant. Using these two constructed strains and the wild-type V592 strain, colony morphology, colony growth rate, sporulation yield, and pathogenicity were tested. The results showed that knockout of the VdRasGAP gene slowed the growth rate of *Verticillium dahliae* strains, but had almost no effect on sporulation yield. Simultaneously, knockout of the VdRasGAP gene significantly reduced the disease index of cotton infection and the degree of browning of the vascular bundles in infected cotton, thus significantly reducing the pathogenicity of *Verticillium dahliae* to cotton, a finding confirmed by qPCR results. Since the main pathogen causing Verticillium dahliae is Verticillium dahliae, this invention can affect the growth and development of Verticillium dahliae and reduce its pathogenicity to cotton by knocking out the VdRasGAP gene. This will lay a theoretical foundation for the study of the pathogenic mechanism of Verticillium dahliae, provide a new biological target for the prevention and control of Verticillium dahliae, and contribute to the more effective control of Verticillium dahliae. Attached Figure Description

[0050] Figure 1 The results of colony PCR validation for the knockout strain are shown. A is a schematic diagram of the gene knockout principle and the primers for validating the mutant; HPT represents the gene encoding hygromycin B phosphotransferase. B shows the validation of the ΔVdRasGAP knockout mutant using PCR with the four primer pairs in A; M is a mixture of DNA molecular weight standards (positive control). C shows the transcription level of the VdRasGAP gene in V592, ΔVdRasGAP, and the complementary strain VdRasGAPcomp. Student's t-test was used, and double asterisks indicate that the mutant was statistically significantly different from V592 (P<0.01).

[0051] Figure 2 These are colony photographs of the wild-type strain, knockout strain, and replacement strain.

[0052] Figure 3 The growth characteristics of wild-type, knockout, and complement strains were identified. In this study, A represents the statistical results of colony diameters for the wild-type, knockout, and complement strains; B represents the results of investigating the effect of the VdRasGAP gene on sporulation of *Verticillium dahliae*. Student's t-test was used; a single asterisk indicates a statistically significant difference (P<0.05) between the mutant and V592, while NS indicates no significant difference compared to V592.

[0053] Figure 4 The results of the observation on the effects of the VdRasGAP gene on the morphology and growth rate of Verticillium dahliae conidia.

[0054] Figure 5 The results of the study on the effect of the VdRasGAP gene on the pathogenicity of *Verticillium dahliae* are presented. A shows photographs and cross-sectional views of cotton seedlings inoculated with wild-type, complement, and knockout strains; B shows the statistical results of the disease index of the wild-type, complement, and knockout strains; C shows the colonization of the knockout strain in the vascular bundles of cotton; and D shows the statistical results of the *Verticillium dahliae* biomass of the wild-type, complement, and knockout strains in cotton. Student's t-test was used. Double asterisks indicate a statistically significant difference (P<0.01) between the mutant and V592, while NS indicates no significant difference compared to V592. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0056] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0057] Strains: (1) Verticillium dahliae V592, a deciduous and highly pathogenic strain isolated from cotton. (2) Verticillium dahliae V592-GFP strain with green fluorescent label. Both strains were kindly provided by Researcher Guo Huishan of the State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences, and are described in the article "Han-Guang Wen, et al. Microbe-induced gene silencing boosts crop protection against soil-borne fungal pathogens. Nat Plants. 2023 Sep; 9(9):1409-1418. doi:10.1038 / s41477-023-01507-9. Epub 2023 Aug 31." They are available to the public from the applicant and can only be used to repeat the experiments of this invention and may not be used for other purposes. (3) Agrobacterium tumefacience competent cells AGL1, which were prepared by our laboratory. (4) Escherichia coli competent cells DH5α, which were prepared in our laboratory.

[0058] Plant material: The cultivar TM-1 of the highly susceptible cotton variety Gossypium hirsutum (reference: Wang G, Zhang D, Wang H, et al. Natural SNP Variation in GbOSM1 Promoter Enhances Verticillium Wilt Resistance in Cotton. Adv Sci (Weinh). 2024; 11(45):e2406522. doi:10.1002 / advs.202406522) was preserved in our laboratory.

[0059] Plasmid vectors: The *Verticillium dahliae* gene knockout vector PGKO2-Gateway and plasmid pUC-Hyg were provided by the laboratory of Professor Dai Xiaofeng at the Institute of Agricultural Product Processing, Chinese Academy of Agricultural Sciences. The pSULPH-mut-RG#PB vector was kindly donated by Researcher Guo Huishan at the State Key Laboratory of Plant Genomics, Institute of Microbiology, Chinese Academy of Sciences. The PGKO2-Gateway vector is described in the article "Chang Hyun Khang, et al. A dual selection based, targeted gene replacement tool for Magnaporthe grisea and Fusarium oxysporum. Fungal Genet Biol. 2005 Jun; 42(6):483-92. doi:10.1016 / j.fgb.2005.03.004.Epub 2005 Apr 25.", and the plasmid pUC-Hyg is described in the article "Zhidi Feng, et al. The Myosin5-mediated actomyosin motility system is required for Verticillium pathogenesis of cotton. Environmental The pSULPH-mut-RG#PB vector is described in the article “Chen Bin, Tian Juan, Feng Zhidi, et al. Construction and application of fluorescent labeling vector for Verticillium dahliae microfilaments [J]. Chinese Journal of Biotechnology, 2019, 35(08): 1520-1528. DOI: 10.13345 / j.cjb.190183.” The public can obtain the above plasmids from the applicant and they can only be used to repeat the experiments of this invention and may not be used for other purposes.

[0060] The culture medium formula used in the examples is as follows:

[0061] LB medium: Tryptone 5 g / L, Yeast Extract 10 g / L, Sodium Chloride (NaCl) 10 g / L. For solid medium, add 15 g / L agar powder. Autoclave at 121°C for 20 min.

[0062] YEP medium: Tryptone 10 g / L, Yeast Extract 10 g / L, Sodium Chloride (NaCl) 5 g / L. For solid medium, add 15 g / L agar. Autoclave at 121°C for 20 min.

[0063] Czapek's medium: Sodium nitrate (NaNO3) 2 g / L, dipotassium hydrogen phosphate trihydrate (K2HPO4·3H2O) 1.31 g / L, magnesium sulfate heptahydrate (MgSO4·7H2O) 1 g / L, potassium chloride (KCl) 1 g / L, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.00366 g / L, sucrose 30 g / L. pH 6.0, autoclaved at 113°C for 30 min.

[0064] PDA medium: Peel 200g potatoes, add 1L of water and boil for 30min. Filter through 4 layers of gauze, add water to 1L, then add 20g glucose. For solid medium, add 20g agar. Sterilize by high temperature and high pressure at 113℃ for 30min.

[0065] IM medium: Dipotassium hydrogen phosphate trihydrate (K₂HPO₄·3H₂O) 2.6 g / L, potassium dihydrogen phosphate (KH₂PO₄) 1.45 g / L, sodium chloride (NaCl) 0.15 g / L, magnesium sulfate heptahydrate (MgSO₄·7H₂O) 0.5 g / L, calcium chloride (CaCl₂) 0.05 g / L, ferrous sulfate heptahydrate (FeSO₄·7H₂O) 0.0025 g / L, ammonium nitrate (NH₄NO₃) 0.5 g / L, glucose 2.0 g / L, glycerol 5 ml / L, acetylsyleugenone (AS) (100 mg / ml) 2 ml / L, 2-(N-morpholino)ethanesulfonic acid (MES) (1 mol / L) 20 ml / L. For solid medium, add 15 g / L agar. Autoclave at 113°C for 30 min.

[0066] MM medium: Dipotassium hydrogen phosphate trihydrate (K₂HPO₄·3H₂O) 2.6 g / L, potassium dihydrogen phosphate (KH₂PO₄) 1.45 g / L, sodium chloride (NaCl) 0.15 g / L, magnesium sulfate heptahydrate (MgSO₄·7H₂O) 0.5 g / L, calcium chloride (CaCl₂) 0.05 g / L, ferrous sulfate heptahydrate (FeSO₄·7H₂O) 0.0025 g / L, ammonium nitrate (NH₄NO₃) 0.5 g / L, glucose 2.0 g / L. Sterilize at 113℃ for 30 min using a high-temperature autoclave.

[0067] 1 / 2 MS medium: sucrose 20 g / L, MS 2.2 g / L, agar 8 g / L. Adjust pH to 5.8, autoclave at 113°C for 30 min.

[0068] The amino acid sequence of the VdRasGAP protein of Verticillium dahliae involved in the following examples is shown in SEQ ID No. 2, and its gene sequence in the Verticillium dahliae genome is shown in SEQ ID No. 1.

[0069] Example 1: Application of VdRasGAP, a small G protein regulator of Verticillium dahliae, in resistance to Verticillium wilt in cotton.

[0070] I. Extraction of total genomic DNA from Verticillium dahliae

[0071] Total genomic DNA was extracted from Verticillium dahliae V592 using the CTAB Plant Genomic DNA Rapid Extraction Kit from Biomed. The specific steps are as follows:

[0072] (1) Use a sterile toothpick to scrape an appropriate amount of Verticillium dahliae V592 bacterial block from the solid PDA culture medium and grind it thoroughly in a sterile mortar with liquid nitrogen;

[0073] (2) Transfer an appropriate amount of the ground powder into an EP tube, add 650 μL of 65°C preheated lysis buffer PL (with 2% β-mercaptoethanol added), then add 6 μL of 10 mg / ml RNase A, let stand at room temperature for 5 min, and vortex thoroughly to mix.

[0074] (3) Place the well-mixed sample in a 65℃ water bath and incubate for 30-60 minutes, inverting the sample every 10 minutes during the process.

[0075] (4) Add 700 μL of chloroform / isoamyl alcohol (volume ratio 24:1) to the sample, mix thoroughly, and centrifuge at 13000 rpm for 10 min.

[0076] (5) Carefully aspirate the supernatant and transfer it into a new EP tube;

[0077] (6) Add 1.5 times the volume of the supernatant of the binding solution PQ (with anhydrous ethanol added) into the EP tube and immediately mix by blowing and blowing.

[0078] (7) Add the mixture to the adsorption column AC, centrifuge at 13000 rpm for 1 min, repeat this step once and then discard the waste liquid;

[0079] (8) Add 500 μL of inhibitor removal solution to the adsorption column, centrifuge at 13000 rpm for 1 min, and discard the waste liquid;

[0080] (9) Add 500 μL of rinsing solution WB (with anhydrous ethanol added) to the adsorption column, centrifuge at 12000 rpm for 1 min, and discard the waste liquid;

[0081] (10) Place the adsorption column into an empty collection tube and centrifuge at 13000 rpm for 2 min. Then open the tube cap and let it stand at room temperature for 2 min to fully dry the washing solution.

[0082] (11) Transfer the adsorption column into a new EP tube, add 30-50 μL of preheated ddH2O to the adsorption membrane in the center of the adsorption column, let stand at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, and repeat the centrifugation once.

[0083] (12) After testing the concentration of the extracted DNA, store it at -20℃.

[0084] II. Construction of VdRasGAP gene knockout vector

[0085] The seamless cloning method is used, and the specific steps are as follows:

[0086] (1) Escherichia coli containing PGKO2-Gateway plasmid was inoculated into LB liquid medium containing 100 mg / L kanamycin, and Escherichia coli containing pUC-Hyg plasmid was inoculated into LB liquid medium containing 100 mg / L ampicillin. Both were cultured at 37°C and 220 rpm for 12 h with shaking. The bacterial cells were then collected and plasmids were extracted using a plasmid extraction kit.

[0087] (2) Using the DNA of wild-type Verticillium dahliae strain V592 extracted in step one as a template, the homologous arm fragment of approximately 1000 bp upstream of the VdRasGAP gene (sequence shown in SEQ ID No. 1) (including positions 1-1000 of SEQ ID No. 3) was amplified using homologous arm amplification primers VdRasGAP-1F and VdRasGAP-1R, and the fragment of approximately 1000 bp downstream of the VdRasGAP gene (including positions 2879-3878 of SEQ ID No. 3) was amplified using homologous arm amplification primers VdRasGAP-3F and VdRasGAP-3R.

[0088] VdRasGAP-1F:5'- cgacggtatc gataagctt c tatcttttgc gctctgtgc-3';

[0089] VdRasGAP-1R: 5'-ccaaaaatgc tccttcaaga tggcaaggag tcgcgtgtgg-3';

[0090] VdRasGAP-3F: 5'-ctgggttcgc aaagataata cccccatcga tgcgctcc-3';

[0091] VdRasGAP-3R: 5'- gcggtggcgg ccgctctaga acgtatattc gccgctactt-3'.

[0092] The underlined portion represents the sequence on the PGKO2-Gateway vector, which is used for homologous recombination with the vector.

[0093] Using pUC-Hyg plasmid as a template, the hygromycin phosphotransferase gene fragment was amplified using VdRasGAP-2F and VdRasGAP-2R primers.

[0094] VdRasGAP-2F: 5'-acgcgactcc ttgccatctt gaaggagcat ttttgggct-3';

[0095] VdRasGAP-2R: 5'-gagcgcatcg atgggggtat tatctttgcg aacccaggg-3'.

[0096] The amplification system is shown in Table 1. The amplification conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 1 min, 35 cycles; and a final extension at 72℃ for 5 min. PCR products were stored at -20℃ for later use.

[0097] Table 1. Gene amplification reaction system

[0098]

[0099]

[0100] (3) The PGKO2-Gateway plasmid was double-digested with HindIII and XbaI, and the linearized plasmid vector was recovered by gel extraction. The digestion system is shown in Table 2.

[0101] Table 2. Vector Enzyme Digestion System

[0102] Components 30μL system HindIII 1μL XbaI 1μL Cutsmart buffer 3μL PGKO2-Gateway plasmid 15μL <![CDATA[ddH2O]]> 10μL

[0103] The obtained linearized plasmid vector and the three PCR products obtained in step (2) (upstream homologous arm, downstream homologous arm, and hygromycin phosphotransferase gene fragment) were placed in a PCR tube and seamlessly ligated using a seamless cloning kit to obtain the knockout vector PGKO-up-Hyg-down. The seamless ligation system is shown in Table 3. The samples were gently mixed and reacted at 37°C for 30 min. After the reaction, the mixture was placed on ice for a few seconds and then stored at -20°C for later use.

[0104] Table 3. Homologous recombination fragment linkage system

[0105] Components 20μL series 5×CE II Buffer 4μL Linearized cloning vector 50-200ng Insertion of target fragment PCR product 20-200ng Exnase II 2μL <![CDATA[ddH2O]]> Add to 20μL

[0106] Structural description of the knockout vector PGKO-up-Hyg-down: The recombinant plasmid obtained by replacing the small fragment between HindIII and XbaI restriction sites in the PGKO2-Gateway plasmid with the DNA fragment shown in SEQ ID No. 3.

[0107] III. Transforming E. coli by knocking out the vector

[0108] (1) Thaw competent E. coli DH5α cells on ice and add the knockout vector PGKO-up-Hyg-down obtained in step 2. After gently tapping the centrifuge tube wall to mix, incubate on ice for 30 min, then heat shock in a metal bath at 42°C for 90 s, and then quickly transfer to ice to cool for 2 min. Add 700 μL of LB liquid medium and incubate at 37°C on a shaker at 200 rpm for 1 h. Then centrifuge the bacterial culture at high speed for 10 s, discard 500 μL of supernatant, resuspend the remaining bacterial culture and spread it evenly on LB agar plates containing 100 mg / L kanamycin, and incubate upside down in a 37°C incubator overnight.

[0109] (2) Select single clones into 500 μL of LB liquid medium containing 100 mg / L kanamycin, place them in a shaker at 37 °C and incubate at 200 rpm for 4 h. Take 2 μL of the mixture and use the system shown in Table 4 to identify positive clones using VdRasGAP-1F and VdRasGAP-3R primers (sequences are described above). (Amplification of a fragment of about 3878 bp is considered positive). The reaction conditions are: 95 °C pre-denaturation for 3 min; 95 °C denaturation for 15 s; 58 °C annealing for 15 s; 72 °C extension for 1 min, 35 cycles; and a final extension at 72 °C for 5 min.

[0110] (3) The clones that were initially identified as correct in the bacterial culture PCR were sent to the company for sequencing to obtain the finally confirmed positive clones.

[0111] Table 4. Gene Amplification Reaction System

[0112] Components 20μL system 2×Rapid Taq Master Mix 10μL Forward primer (10 μM) 1μL Reverse primer (10 μM) 1μL bacterial solution 2μL <![CDATA[ddH2O]]> 6μL

[0113] IV. Transforming the knockout vector into Agrobacterium

[0114] (1) Extract the plasmid from the positive clone obtained in step 3 using a plasmid extraction kit. Place 100 μL of Agrobacterium tumefaciens AGL1 competent cells on ice, thaw them, add 1 μL of the extracted plasmid, gently mix with a pipette, transfer to a pre-cooled electroporation cuvette, set the electroporator to 1.8 KV for electroporation, add 700 μL of liquid LB medium, and incubate at 28℃ on a shaker at 220 rpm for 1 hour.

[0115] (2) Take 200 μL of bacterial suspension and spread it evenly on a YEP solid plate containing 100 mg / L rifampicin and 100 mg / L kanamycin. Incubate upside down in an incubator at 28°C for 2 days. Then select single colonies and identify the transformants using the identification method described in step 3 (2). Store the correctly identified Agrobacterium at -20°C for later use.

[0116] V. Constructing a replenishment mechanism

[0117] (1) Using the DNA of wild-type Verticillium dahliae strain V592 extracted in step one as a template, PCR amplification was performed using primer pair ComVdRasGAP-1F / 1R and primer pair ComVdRasGAP-3F / 3R to obtain the upstream 2500bp and the ORF region 2502bp of the VdRasGAP gene; using position 2481-3232 of SEQ ID No.4 as a template, PCR amplification was performed using primer pair ComVdRasGAP-2F / 2R to obtain a 717bp green fluorescent tag fragment.

[0118] ComVdRasGAP-1F:5'- acgacggcca gtgccaagct t tccctgttg agccaggaca g-3';

[0119] ComVdRasGAP-1R: 5'-tcgcccttgc tcaccatgat ggcaaggagt cgcgtgtg-3';

[0120] ComVdRasGAP-3F: 5'-gcatggacga gctgtacaag atgtccgtga tgctgcaag-3';

[0121] ComVdRasGAP-3R: 5'- tccgaattca ctagtggatc c ctaccatcc ctttttccgc-3';

[0122] ComVdRasGAP-2F: 5'-cacgcgactc cttgccatca tggtgagcaa gggcgagga-3'.

[0123] ComVdRasGAP-2R: 5'-gcagcatcac ggacatcttg tacagctcgt ccatgcc-3'.

[0124] The underlined part is the sequence on the pSULPH-mut-RG#PB vector, which is used for homologous recombination with the vector.

[0125] The amplification system is shown in Table 1. The amplification conditions are as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 58℃ annealing for 15 s, 72℃ extension for 2 min, 35 cycles; and a final extension at 72℃ for 5 min. The PCR products were stored at -20℃ for later use.

[0126] (2) The pSULPH-mut-RG#PB vector plasmid was double-digested with restriction endonucleases HindIII and PstI. The digestion system is shown in Table 5.

[0127] Table 5. Vector Enzyme Digestion System

[0128] Components 30μL system HindIII 1μL PstI 1μL Cutsmart buffer 3μL pSULPH-mut-RG#PB vector plasmid 15μL <![CDATA[ddH2O]]> 10μL

[0129] Gel recovery involved seamlessly ligating the three PCR products from step (1) – the upstream 2500 bp and 2502 bp ORF region of the VdRasGAP gene, and the 717 bp green fluorescent tag fragment – ​​with the linearized plasmid vector using a seamless cloning kit to obtain the complement vector pComVdRasGAP. The ligation system is shown in Table 6.

[0130] Table 6. Homologous recombination fragment linkage system

[0131] Components 10μL series 5×CE II Buffer 2μL Linearized cloning vector 2μL Insert the target fragment (the PCR product obtained in step (1)). 1 μL each Exnase II 1μL <![CDATA[ddH2O]]> Add to 10μL

[0132] Structural description of the pComVdRasGAP vector: The recombinant plasmid obtained by replacing the small fragment between the HindIII and PstI restriction sites of the pSULPH-mut-RG#PB vector plasmid with the DNA fragment shown in SEQ ID No. 4.

[0133] VI. Transform the replenishment vector into Escherichia coli

[0134] The method is exactly the same as in step three. After plating and selecting single clones for colony PCR identification, positive clones are selected, and plasmids are extracted for sequencing. The plasmids that have been correctly identified by sequencing are transformed into Agrobacterium AGL1 using the method in step four, and the plasmids and bacterial culture are stored at -20℃ for later use.

[0135] VII. Transformation of the knockout strain into Verticillium dahliae

[0136] The ATMT-mediated genetic transformation method involves the following steps:

[0137] (1) The constructed plasmid PGKO-up-Hyg-down was transformed into Agrobacterium AGL1 strain by electroporation. After culturing for 1 hour, it was spread on YEP plates with corresponding resistance and placed in an incubator at 28℃ for 2 days.

[0138] (2) Pick a single clone from the plate and place it in 5 mL of YEP liquid medium with the corresponding resistance, and incubate overnight in a shaker at 28°C.

[0139] (3) Take 50 μL of Agrobacterium and put it into 10 mL of MM medium (containing 100 μg / mL Kan and 100 μg / mL Rif), and incubate it in a shaker at 28℃ for 1 day.

[0140] (4) Centrifuge the cultured bacterial solution at 4000 rpm for 15 min. Discard the supernatant and gently resuspend the culture in 20 mL of IM liquid medium (containing 100 μg / mL Kan, 40 mmol / L MES and 200 μmol / L AS).

[0141] (5) Repeat step (4) once.

[0142] (6) Resuspend the bacterial cells in IM liquid medium (containing 100 μg / mL Kan, 40 mmol / L MES and 200 μmol / L AS) and adjust the OD600 to 0.2-0.3.

[0143] (7) Place the bacterial culture in a shaker at 28°C and incubate until the OD600 is about 0.6, which takes about 4 hours.

[0144] (8) Use a sterile toothpick to scrape the bacterial blocks of Verticillium dahliae strain V592 and strain V592-GFP from the PDA plate into 50 mL of Czapek liquid medium, place it in a shaker at 26℃ and 150 rpm for 4-5 days.

[0145] (9) Filter the bacterial solution with sterile eight-layer gauze, centrifuge at 3800 rpm for 15 min, and discard the supernatant.

[0146] (10) Add sterile water to dilute the conidia concentration to 10. 7 The concentration of conidia is approximately 100 / mL.

[0147] (11) Place the sterilized microporous filter membrane on an IM plate (containing 100 μg / mL Kan, 40 mmol / L MES and 200 μmol / LAS) and dry it thoroughly in a laminar flow hood.

[0148] (12) Take 700 μL of Agrobacterium and Verticillium dahliae into centrifuge tubes and mix them by pipetting. Then take 200 μL of the mixture and spread it evenly on IM solid medium (four replicates in total). Incubate in an inverted incubator at 26°C for 2 days.

[0149] (13) Transfer the microporous filter membrane to a PDA plate with corresponding resistance for transformant screening.

[0150] (14) After about a week, positive transformants will grow. Use a sterile toothpick to pick the transformants onto a PDA plate with the corresponding resistance for resistance screening.

[0151] (15) Verify the transformants by PCR, RT-qPCR or microscopic observation.

[0152] (16) Inoculate the correct transformants onto new PDA plates, and perform colony PCR identification, RT-qPCR and strain preservation after a period of time.

[0153] Colony PCR identification:

[0154] PCR amplification was performed using the detection primers testVdRasGAP-1F and testVdRasGAP-1R, testVdRasGAP-2F and testVdRasGAP-2R, testVdRasGAP-3F and testVdRasGAP-3R, and testVdRasGAP-4F and testVdRasGAP-4R, respectively, using the amplification system shown in Table 1 and the amplification program described in step two (2).

[0155] testVdRasGAP-1F: 5'-tgcaaaacct cgcgaacaag-3';

[0156] testVdRasGAP-1R:5'-gggaatcgac cgcatgatct-3'.

[0157] testVdRasGAP-2F: 5'-ttgaaggagc atttttgggc-3';

[0158] testVdRasGAP-2R: 5'-ttatctttgc gaacccaggg-3'.

[0159] testVdRasGAP-3F: 5'-gcatgtccga cgcttcattc-3';

[0160] testVdRasGAP-3R: 5'-gctgatctga ccagttgcct-3'.

[0161] testVdRasGAP-4F: 5'-actgtcgggc gtacacaaat-3';

[0162] testVdRasGAP-4R:5'-ggtcatgtgt ggtccttcgt-3'.

[0163] The positions of each primer in the knockout mutant strain are as follows: Figure 1 As shown in Figure A, the amplification results are shown below. Figure 1 The results showed that all four primer pairs amplified the correct bands.

[0164] RT-qPCR identification:

[0165] qRT-PCR amplification was performed using RT-qPCR primers Qpcr-VdRasGAP-F and Qpcr-VdRasGAP-R, VdTub1-RT-F and VdTub1-RT-R. Details are as follows:

[0166] 1) Take 1 pg-500 ng of total RNA, add 2 μL of 4×gDNA wiper mix from the HiScript IIQ RT SuperMix for qPCR kit, and then add RNase-free ddH2O to make up to 8 μL. Gently mix and place in a metal bath at 42°C for 2 min.

[0167] 2) Add 2 μL of 5×HiScript II qRT SuperMix II enzyme from the HiScript IIQ RT SuperMix for qPCR kit to the liquid in step 1, mix well and place in the PCR instrument. Set the program: 50℃, 30 min, 85℃, 5 s.

[0168] 3) Reverse transcribed cDNA was obtained using the Toyobo SYBR Green Master Mix kit, following the instructions for qRT-PCR: 15 μL total volume, 7.5 μL SYBR Permix Ex Taq II, 0.4 μL primers, 2 μL template cDNA, and 5.1 μL ddH2O. Tubulin-F / R of *Verticillium dahliae* was used as an internal control for qRT-PCR analysis. The reaction program was: 95℃ for 3 min, 95℃ for 20 s, 60℃ for 30 s, and 72℃ for 20 s, for 45 cycles. Each reaction was repeated at least three times, and the average value was calculated. -△△CT calculate.

[0169] Qpcr-VdRasGAP-F:5'-atcatgcggt cgattccctc-3';

[0170] Qpcr-VdRasGAP-R:5'-tgcctttacg caccatgact-3'.

[0171] VdTub1-RT-F: 5'-gcaagctcgc cgtcaaca-3';

[0172] VdTub1-RT-R:5'-tggcggagca ggtcaggta-3'.

[0173] Amplification results are shown below Figure 1 The results showed that the expression level of VdRasGAP in the knockout mutant was almost zero, indicating that the knockout strain was successfully created.

[0174] The empirically verified VdRasGAP knockout strains obtained in this step are designated as ΔVdRasGAP if they do not contain GFP, and ΔVdRasGAP-GFP if they contain GFP.

[0175] 8. Transform the complementation vector into the VdRasGAP knockout strain.

[0176] The method is exactly the same as step seven, except that the pComVdRasGAP complement vector obtained in step five is transferred into the validated knockout strain ΔVdRasGAP in step seven, thus obtaining the complement strain of the knockout strain.

[0177] The reinstated strain was identified using RT-PCR (see step seven). Amplification results are shown below. Figure 1 The results showed that, compared with the knockout mutant ΔVdRasGAP, the expression level of VdRasGAP in the complement strain was significantly increased, approaching that of the wild-type V592.

[0178] The empirically verified correct supplementary strain obtained in this step is denoted as VdRasGAPCom.

[0179] IX. Determining the effect of the VdRasGAP gene on the colony morphology and growth rate of Verticillium dahliae.

[0180] Tested strains: Verticillium dahliae V592, knockout strain ΔVdRasGAP, and replenishment strain VdRasGAPCom.

[0181] Use a sterile punch with a diameter of 0.5 cm to make holes near the edge of the colony. Place the bacterial block on a new PDA plate and incubate for 14 days. Measure the colony diameter every day using the cross-sectional method.

[0182] In this invention, the knockout strain ΔVdRasGAP and the replenishment strain VdRasGAPCom were cultured with the wild-type strain V592 on PDA medium for 14 days.

[0183] Colony morphology is shown Figure 2 The colony diameter statistics are shown in [the original text]. Figure 3 As can be seen from Figure A, the colony morphology of the wild-type and the complement strains is basically the same, but the growth rate of the knockout strain is significantly slower than that of the wild-type and the complement strains.

[0184] 10. Determining the effect of the VdRasGAP gene on sporulation of Verticillium dahliae

[0185] Using a punch, holes were made at the edges of colonies of wild-type strain V592, knockout strain ΔVdRasGAP, and replenishment strain VdRasGAPCom after 7 days of growth. Four bacterial blocks were placed in 15mL centrifuge tubes containing 2mL of sterile water. After thorough shaking, 10μL of the spore suspension was added to a hemocytometer to count conidia. Each treatment was performed in triplicate.

[0186] See results Figure 3 As shown in Figure B, the sporulation of the knockout strain ΔVdRasGAP was not significantly different from that of the wild-type V592 and the complement strain, indicating that the absence of VdRasGAP does not affect the sporulation capacity of Verticillium dahliae.

[0187] XI. Observe the effects of the VdRasGAP gene on the morphology and growth rate of Verticillium dahliae conidia.

[0188] Tested strains: wild-type strain V592, knockout strain ΔVdRasGAP, and replenishment strain VdRasGAPCom.

[0189] The colonies grown on the PDA medium were washed with liquid Czapek's medium to obtain a conidial solution. The washed conidial solution was dropped onto a glass slide in a chamber and then incubated at 26°C. 2 μL of 10 mg / mL CFW dye was added to the conidial solution at 0 h, 8 h, and 16 h, and the samples were observed under a microscope.

[0190] See results Figure 4 It can be seen that, compared with wild-type V592 and the replacement strain, the conidia morphology of the knockout strain ΔVdRasGAP is more spherical, and its growth rate is significantly slower than that of wild-type V592 and the replacement strain. This indicates that the absence of VdRasGAP will change the morphology of Verticillium dahliae conidia and slow down the growth rate.

[0191] 12. Investigate the effect of the VdRasGAP gene on the pathogenicity of Verticillium dahliae.

[0192] Hydroponic cultivation of cotton: TM-1 cotton seeds are delinted with concentrated sulfuric acid, then washed with sterile water to remove excess sulfuric acid. The seeds are then soaked in sterile water at 28°C for 2 days until they begin to germinate. The germinated seeds are then spread on moist vermiculite, and germination occurs in approximately 2 days. The germinated cotton seedlings are then placed in hydroponic containers and cultured until they have two leaves and a central bud, at which point they are inoculated.

[0193] Verticillium dahliae infection process: A suitable amount of mycelium was cut from a PDA plate using a sterile toothpick and placed in Czapek's liquid medium. The plate was incubated at 26°C and 150 rpm for 5 days. Afterwards, the bacterial suspension was filtered through eight layers of gauze, centrifuged at 4000 rpm for 15 minutes, and the conidia were diluted with sterile water to a concentration of 10. 7 The concentration of conidia was approximately 100 μL. The root-dipping method was used for infection; cotton roots were placed in the bacterial solution for 10 minutes before being transferred to a hydroponic container. 1 / 10 MS medium was added to the water every week. Pathogenicity data, root cross-section microscopy observation, and fungal biomass determination were performed after 30 days of infection. Five replicates were set up for each sample, and the experiment was repeated three times. The tested strains included wild-type strain V592 or V592-GFP, knockout strains ΔVdRasGAP and ΔVdRasGAP-GFP, and the complement strain VdRasGAPCom.

[0194] Cotton pathogenicity statistics: The severity of cotton Verticillium wilt was classified into five levels from most severe to least severe: Level 4, Level 3, Level 2, Level 1, and Level 0. Grading standards: Level 4 (75-100% of leaves showing symptoms, excluding the lower limit but including the upper limit), Level 3 (50-75% or more of leaves showing symptoms, excluding the lower limit but including the upper limit), Level 2 (25-50% or more of leaves showing symptoms, excluding the lower limit but including the upper limit), Level 1 (0-25% of leaves showing symptoms, excluding the lower limit but including the upper limit), and Level 0 (no symptoms). Pathogenicity was statistically analyzed on cotton plants infected for 30 days to determine if there was a significant difference in pathogenicity compared to the wild type.

[0195] Biomass analysis: Cotton taproots infected with *Verticillium dahliae* for 30 days were used as material. DNA was extracted using a plant genomic DNA rapid extraction kit. The cotton 18S gene was used as an internal reference gene. Using the *Verticillium dahliae*-specific primer pair VdEF1-α-F / R, real-time PCR was employed to determine fungal biomass and quantitatively analyze the colonization of the pathogen in inoculated cotton plants. Additionally, GFP fluorescence PI staining was used to observe the colonization of the knockout strain ΔVdRasGAP-GFP and the wild-type V592-GFP in the cotton vascular bundles.

[0196] 18S-F: 5'-cggctaccac atccaaggaa-3';

[0197] 18S-R: 5'-tgtcactacc tccccgtgtc a-3';

[0198] VdEF1-α-F: 5'-cggctaccac atccaaggaa-3';

[0199] VdEF1-α-R: 5'-tgtcactacc tccccgtgtc a-3'.

[0200] The above experiment also included a negative control (Mock) of healthy cotton TM-1 that was not infected with Verticillium dahliae.

[0201] See results Figure 5 It can be seen that:

[0202] Thirty days after indoor artificial inoculation, cotton seedlings inoculated with wild-type and reinjected strains showed obvious symptoms of yellowing, wilting, and even death, while cotton seedlings inoculated with knockout strains showed no obvious symptoms and their phenotype was similar to the negative control (Mock). Figure 5 (A)

[0203] Cross-sectional observation of cotton stems 30 days after inoculation revealed severe browning in both wild-type V592 strain and the replacement strain, while the stems infected with the knockout strain showed almost no browning, similar to the negative control (Mock). Figure 5 (A)

[0204] Statistical results of the disease index showed that the pathogenicity of the knockout strain was significantly lower than that of the wild-type strain. Figure 5 (B) When inoculated with the knockout strain, 15.6% of cotton seedlings showed grade 2-4 wilting symptoms, while 84.4% of cotton seedlings inoculated with the reinjection strain showed grade 2-4 wilting symptoms, which was significantly higher than with the knockout strain. When inoculated with wild-type V592, 100% of cotton seedlings showed grade 2-4 wilting symptoms, with the reinjection strain showing slightly lower levels than wild-type V592.

[0205] The colonization numbers of wild-type strain, knockout strain ΔVdRasGAP, and complement strain VdRasGAPCom on the cotton taproot were verified by real-time PCR. The results are shown in [Figure number missing]. Figure 5 The results showed that the colonization ability of the knockout strain was significantly reduced compared to the wild-type strain after the deletion of VdRasGAP. Furthermore, the colonization of the knockout strain ΔVdRasGAP-GFP and the wild-type V592-GFP in cotton vascular bundles was observed, further confirming this finding. Figure 5 (C)

[0206] The results above indicate that VdRasGAP is an important gene for Verticillium dahliae invasion of cotton, and the deletion of this gene can significantly reduce the pathogenicity of Verticillium dahliae to the host cotton.

[0207] Therefore, this invention primarily reveals the role of the VdRasGAP gene in the pathogenicity of *Verticillium dahliae* in cotton, demonstrating that the VdRasGAP gene has a significant positive regulatory effect on both mycelial growth and pathogenicity of *Verticillium dahliae*. This lays a theoretical foundation for in-depth research into the pathogenic mechanism of *Verticillium dahliae*, provides new theoretical basis and effective targets for the efficient control of cotton Verticillium wilt, and also lays the foundation for further development of novel targeted fungicides.

[0208] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. Inhibition of VdRasGAP protein expression and / or activity in any of the following: (A1) Reduces the pathogenicity of Verticillium dahliae; (A2) Resistance to Verticillium wilt caused by Verticillium dahliae infection.

2. The use of substances that inhibit the expression and / or activity of VdRasGAP protein in any of the following: (A1) Reduces the pathogenicity of Verticillium dahliae; (A2) Resistance to Verticillium wilt caused by Verticillium dahliae infection.

3. Inhibition of VdRasGAP protein expression and / or activity in any of the following: (B1) Slows down the growth rate of Verticillium dahliae; (B2) Reduce the disease index of plants infected with Verticillium dahliae; (B3) Reduces the degree of vascular bundle browning in plants infected with Verticillium dahliae; (B4) Reduces the colonization ability of Verticillium dahliae in plant vascular bundles.

4. The use of substances that inhibit the expression and / or activity of VdRasGAP protein in any of the following: (B1) Slows down the growth rate of Verticillium dahliae; (B2) Reduce the disease index of plants infected with Verticillium dahliae; (B3) Reduces the degree of vascular bundle browning in plants infected with Verticillium dahliae; (B4) Reduces the colonization ability of Verticillium dahliae in plant vascular bundles.

5. Any of the following methods: Method I: A method for reducing the pathogenicity of Verticillium dahliae, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein; Method II: A method for combating Verticillium wilt in plants caused by Verticillium dahliae infection, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein; Method III: A method for slowing down the growth rate of Verticillium dahliae and / or reducing the disease index of Verticillium dahliae-infected plants and / or reducing the degree of vascular bundle browning in Verticillium dahliae-infected plants and / or reducing the colonization ability of Verticillium dahliae in plant vascular bundles, comprising the following steps: inhibiting the expression and / or activity of VdRasGAP protein; Method IV: A method for preparing Verticillium dahliae with reduced pathogenicity, comprising the steps of: inhibiting the expression and / or activity of VdRasGAP protein.

6. The application or method according to any one of claims 1-5, characterized in that: Inhibition of VdRasGAP protein expression and / or activity is achieved by knocking out or reducing the expression of the gene encoding the VdRasGAP protein in the Verticillium dahliae genome; or, the substance is a substance capable of knocking out or reducing the expression of the gene encoding the VdRasGAP protein in the Verticillium dahliae genome. Furthermore, the inhibition of the expression and / or activity of the VdRasGAP protein is achieved by introducing a homologous recombination fragment or homologous recombination vector into Verticillium dahliae for knocking out the gene encoding the VdRasGAP protein; or, the substance is a homologous recombination fragment or homologous recombination vector for knocking out the gene encoding the VdRasGAP protein. Furthermore, the nucleotide sequence of the homologous recombination fragment used to knock out the gene encoding the VdRasGAP protein is as shown in SEQ ID No. 3; or, the homologous recombination vector used to knock out the gene encoding the VdRasGAP protein is a vector containing the DNA fragment shown in SEQ ID No.

3.

7. The application or method according to any one of claims 1-6, characterized in that: The VdRasGAP protein is any one of the following: (C1) The protein with the amino acid sequence shown in SEQ ID No. 2; (C2) is a protein derived from Verticillium dahliae that has 99%, 95%, 90%, 85%, or 80% identity with the protein defined in (C1) and is associated with the pathogenicity of Verticillium dahliae.

8. The application or method according to any one of claims 1-7, characterized in that: The gene encoding the VdRasGAP protein is any one of the following: (D1) The DNA molecule shown in SEQ ID No. 1; (D2) is a DNA molecule derived from Verticillium dahliae that has 99%, 95%, 90%, 85%, or 80% identity with the DNA sequence defined by (D1) and encodes the VdRasGAP protein.

9. The application or method according to any one of claims 1-8, characterized in that: The plant in question is a host plant of Verticillium dahliae; Furthermore, the host plant is cotton.

10. Verticillium dahliae with reduced pathogenicity prepared by method IV of any one of claims 5-9.

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