Verticillium dahliae heat shock protein 90 ATP enzyme activating factor VdAHA1 as well as coding gene and application thereof

Abstract

The invention relates to the technical field of agricultural biology, in particular to a verticillium dahliae heat shock protein 90 ATP enzyme activating factor VdAHA1 as well as a coding gene and application of the verticillium dahliae heat shock protein 90 ATP enzyme activating factor VdAHA1. The invention provides a verticillium dahliae heat shock protein 90 ATP enzyme activating factor VdAHA1 related to high temperature resistance, drug resistance, environmental adaptability, ergosterol synthesis capability and pathogenicity of verticillium dahliae and a coding gene of the VdAHA1, and the VdAHA1 gene positively regulates the pathogenicity of verticillium dahliae and can be used as an important candidate gene for pathogenesis research of verticillium dahliae. The invention provides a theoretical basis for further understanding the pathogenic mechanism of pathogenic bacteria, also provides a new gene resource for verticillium dahliae pathogenic related genes, also provides a new idea for prevention and control of cotton verticillium wilt, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to the heat shock protein 90 ATPase activator VdAHA1 of Verticillium dahliae, its encoding gene, and its applications. Background Technology

[0002] Verticillium wilt, a disease of cotton, is caused by a soil-borne fungus, Verticillium dahliae. Verticillium dahliae The vascular bundle diseases caused by *Verticillium dahliae* (Kleb.) are known in the industry as the "cancer" of cotton. *Verticillium dahliae* is the main pathogen causing Verticillium wilt in cotton. It can infect more than 200 species of dicotyledonous plants, including cotton, leading to severe vascular bundle diseases. Notably, it can infect cotton at any stage of its growth cycle. Currently, there are no effective fungicides to control the spread of this pathogen within the plant's vascular bundles.

[0003] Verticillium dahliae exhibits frequent mutations and possesses dormant microsclerotia, allowing it to survive for extended periods in the soil, making its control extremely difficult. These microsclerotia also help the fungus resist various external adversities to ensure survival, and are also a significant primary source of infection for Verticillium wilt. In suitable cotton field soil conditions, the propagules or microsclerotia of Verticillium dahliae are induced to produce hyphae by root secretions. These hyphae rapidly grow and spread near or in contact with the cotton roots, invading the roots through root tips or wounds. During this process, the pathogen must overcome the host plant's cell walls, xylem, and other tissue barriers. To successfully infect the plant, Verticillium dahliae secretes effector proteins to evade or suppress the plant's immunity.

[0004] Heat shock proteins (HSPs) are a class of highly conserved stress proteins widely distributed in organisms, playing a vital role in growth, development, and adaptation to adversity. Heat shock protein 90 (Hsp90) is one of the first members of the heat shock protein (HSP) family to be identified, capable of responding to drastic temperature changes. Fungal Hsp90 participates in regulating fungal cell growth, development, sexual reproduction, and drug resistance through interactions with various client proteins, such as protein receptors, protein kinases, cofactors, and transcription factors.

[0005] Verticillium dahliae is very difficult to control due to its dormant microsclerotia and wide host range. Current research mainly focuses on exploring the molecular mechanisms of pathogenic genes to investigate the genes associated with the disease. Summary of the Invention

[0006] To address the aforementioned technical challenges, this invention first provides the application of Verticillium dahliae heat shock protein 90 ATPase activator VdAHA1 or a fusion protein of VdAHA1 protein, or its encoding gene, or biological materials containing its encoding gene in regulating the pathogenicity of Verticillium dahliae.

[0007] Prior to this invention, the heat shock protein 90 ATPase activator VdAHA1 and its encoding gene were unknown. VdAHA1 The function of *Verticillium dahliae* in development, adaptation, and pathogenicity is unknown to those skilled in the art, and its association with cotton Verticillium wilt is also unknown. This invention addresses this by knocking out and reintroducing the gene encoding heat shock protein 90 ATPase activator. VdAHA1 They discovered that this gene can regulate the pathogenicity of Verticillium dahliae.

[0008] Furthermore, the present invention provides the application of Verticillium dahliae heat shock protein 90 ATPase activator VdAHA1 or a fusion protein of VdAHA1 protein, or its encoding gene, or biological materials containing its encoding gene in at least one of the following aspects: (1) Regulating the high-temperature adaptability of Verticillium dahliae; (2) Regulating the drug resistance of Verticillium dahliae; (3) Regulate the ergosterol content of Verticillium dahliae; (4) Regulating the environmental adaptability of Verticillium dahliae; (5) Regulate the pathogenicity of Verticillium dahliae to plants; (6) Plant disease control.

[0009] In some implementations, the pathogenicity, high-temperature adaptability, drug resistance, ergosterol content, environmental adaptability, or pathogenicity to plants of Verticillium dahliae can be regulated by adjusting the content or activity of the heat shock protein 90 ATPase activator VdAHA1 or its encoding gene.

[0010] In some implementations, by reducing the content or activity of the heat shock protein 90 ATPase activator VdAHA1 or its encoding gene in Verticillium dahliae, the pathogenicity, heat adaptability, drug resistance, ergosterol content, environmental adaptability, pathogenicity to plants, or plant diseases can be reduced.

[0011] In some implementations, knocking out the gene encoding the VdAHA1 ATPase activator of Verticillium dahliae can reduce the pathogenicity, heat tolerance, drug resistance, ergosterol content, environmental adaptability, or pathogenicity to plants of Verticillium dahliae.

[0012] In some implementations, the content or activity of the heat shock protein 90 ATPase activator VdAHA1 or its encoding gene in Verticillium dahliae is regulated by transgenic technology or gene editing technology.

[0013] In some embodiments, the high-temperature adaptability is heat resistance; and / or, the drug resistance is resistance to ketoconazole, itraconazole, or fluconazole; and / or, the pathogenicity to plants is pathogenicity to cotton; and / or, the plant disease is cotton verticillium wilt.

[0014] In some implementations, the cotton is a variety susceptible to Verticillium wilt.

[0015] In some embodiments, the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues.

[0016] In some embodiments, the fusion protein of the VdAHA1 protein is obtained by attaching a tag, an enzyme cleavage site, and / or a linker peptide sequence to the N-terminus and / or C-terminus of the amino acid sequence of the VdAHA1 protein.

[0017] In some embodiments, the amino acid sequence of the Verticillium dahliae heat shock protein 90 ATPase activator VdAHA1 is shown in SEQ ID NO.2.

[0018] In some embodiments, the nucleic acid sequence of the encoding gene is shown in SEQ ID NO.1.

[0019] Furthermore, the present invention provides a method for reducing the pathogenicity of *Verticillium dahliae*, reducing its high-temperature adaptability, reducing its drug resistance, reducing its ergosterol content, reducing its environmental adaptability, or reducing its pathogenicity to plants, comprising: knocking out the heat shock protein 90 ATPase activator in *Verticillium dahliae*. VdAHA1 Gene.

[0020] Furthermore, the present invention provides a method for controlling cotton Verticillium wilt, comprising: knocking out heat shock protein 90 ATPase activator in Verticillium dahliae. VdAHA1 Gene.

[0021] In some implementations, the heat shock protein 90 ATPase activator in Verticillium dahliae Vd080 is knocked out. VdAHA1 Gene.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a heat shock protein 90 ATPase activator VdAHA1 and its encoding gene, which are related to the heat resistance, drug resistance, environmental adaptability, ergosterol synthesis ability, and pathogenicity of Verticillium dahliae. VdAHA1 The gene positively regulates the pathogenicity of *Verticillium dahliae*, and it can serve as an important candidate gene for studying the pathogenic mechanism of *Verticillium dahliae*. This invention provides a theoretical basis for further understanding the pathogenic mechanism of the pathogen, provides new gene resources for pathogenic genes related to *Verticillium dahliae*, and also provides new ideas for the control of cotton Verticillium wilt, with broad application prospects. Attached Figure Description

[0023] Figure 1 It is Verticillium dahliae VdAHA1 Schematic diagram of gene knockout and replacement principles.

[0024] Figure 2 It is Verticillium dahliae VdAHA1 PCR verification results of genomic DNA from gene knockout and complement mutants.

[0025] Figure 3 It is Verticillium dahliae VdAHA1 Results of high-temperature resistance testing for gene knockout and complement mutants.

[0026] Figure 4 It is Verticillium dahliae VdAHA1 Results of drug resistance testing for gene knockout and complement mutants.

[0027] Figure 5 It is Verticillium dahliae VdAHA1 Results of stress resistance testing for gene knockout and complement mutants.

[0028] Figure 6 It is Verticillium dahliae VdAHA1 Results of microsclerotium changes and melanin-related gene expression levels in gene knockout and complement mutants.

[0029] Figure 7 It is Verticillium dahliae VdAHA1 Results of ergosterol content detection in gene knockout and complement mutants.

[0030] Figure 8 The susceptible cotton varieties were inoculated with wild-type Verticillium dahliae and Verticillium dahliae. VdAHA1 The disease incidence of gene knockout and complement mutants, the browning of cotton plant xylem, and the detection results of fungal biomass in cotton plant xylem. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. In the embodiments provided in this specification, where specific techniques or conditions are not specified, they are performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0032] This invention relates to molecular biology experiments. Unless otherwise specified, reference can be made to the book *Molecular Cloning* (J. Sambrook, E.F. Fritsch, and T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used and guiding reference books for those skilled in the art when performing experiments related to molecular biology. Furthermore, depending on the experimental purpose, those skilled in the art may conduct corresponding experiments under the guidance of the operating manuals accompanying various commercially available kits or entrust them to specialized companies, such as gene sequencing, plasmid sequencing, and molecular weight determination.

[0033] Example 1: Verticillium dahliae VdAHA1 Obtaining and identifying knockout and complement mutants Using the highly pathogenic Verticillium dahliae strain Vd080 as material, select VdAHA1 The gene contains upstream (Up-1.5Kbp) and downstream (Down-1.5Kbp) sequences. These sequences were amplified using B303-VdAHA1-UP-F / R and B303-VdAHA1-DOWN-F / R amplification. VdAHA1 Upstream and downstream homologous arms of the gene. The Hyg fragment was obtained by amplification using B303-VdAHA1-Hyg-F / R. These three fragments were then constructed into the B303 vector via homologous recombination to obtain... VdAHA1 Knock out the vector Up-Hyg-Down-B303-Hyg. Transform the vector via protoplast conversion. VdAHA1 The knockout vector Up-Hyg-Down-B303-Hyg was transformed into Verticillium dahliae strain Vd080, and the resulting strains were selected for resistance. VdAHA1 Gene knockout mutant.

[0034] exist VdAHA1 Based on gene knockout mutants, further construction... VdAHA1 Gene complementation mutant, the steps are as follows: Amplification was performed using BIA1302-VdAHA1-CF / R to obtain... VdAHA1The gene and its 1.5Kbp promoter were amplified using BIA1302-VdAHA1-RFP-F / R to obtain the RFP fragment. These two fragments were then constructed into the BIA1302 vector via homologous recombination, and further transformed into protoplasts to obtain... VdAHA1 Gene complementation mutant.

[0035] The primers used in the above construction process are shown in Table 1. Further extraction... VdAHA1 Gene knockout mutants and VdAHA1 The genomic DNA of the gene complementation mutant was verified using PCR. For example... Figure 1 , Figure 2 As shown, the results indicate that two knockout mutants and two complement mutants were obtained in this experiment, denoted as ΔVdAHA1-1, ΔVdAHA1-2, C-ΔVdAHA1-1, and C-ΔVdAHA1-2, respectively.

[0036] Table 1. Construction of Verticillium dahliae VdAHA1 Primer information used for knockout, complementation vectors, and mutant PCR validation

[0037] Example 2: Determining the high-temperature resistance of knockout and complement mutants using temperature gradients Spore suspensions of knockout and addition mutant strains and wild-type strains were collected from Czapek's liquid medium (2 g / L NaNO3, 0.5 g / L KCl, 0.02 g / L FeSO4·7H2O, 0.5 g / L MgSO4·7H2O, 30 g / L sucrose, and separately sterilized 1.31 g / L K2HPO4) after 5 days of culture. The spore concentration was adjusted to 1 × 10⁻⁶. 7 CFU / mL. Knockout, complement, and wild-type strains were cultured on PDA medium and incubated in the dark at 15°C, 25°C, and 30°C for 14 days. [See...] Figure 3 Each experiment was repeated three times. Heat resistance was compared by measuring colony diameter.

[0038] The results showed that the heat resistance of the knockout mutant was significantly reduced.

[0039] Example 3: Determination of resistance to different drugs in knockout, complement mutants and wild-type strains Ketoconazole (25 mg / L), itraconazole (0.1 mg / L), and fluconazole (2 mg / L) were added to Czapek's solid medium, respectively. *Verticillium dahliae* mycelial discs were inoculated, and colony diameter was measured to analyze WT (whole wheat area). ΔVdAHA1-1 , ΔVdAHA1-2 , C-ΔVdAHA1-1 and C-ΔVdAHA1-2 Resistance to different drugs, seeFigure 4 Each experiment was repeated 3 times.

[0040] The results showed that the drug resistance of the knockout mutant was significantly reduced.

[0041] Example 4: Detection of stress resistance in knockout and complement mutants and wild-type strains Knockout, complement, and wild-type strains were cultured on PDA medium containing 0.9 mol / L KCl, 0.9 mol / L NaCl, 0.9 mol / L sorbitol (SBT), 0.004% SDS, 0.02% CR, and 0.02% CFW, respectively. (See attached table.) Figure 5 Each experiment was repeated three times. Stress resistance was compared by measuring colony diameter.

[0042] The results showed that the stress resistance of the knockout mutant was significantly reduced in PDA medium containing all the above additives.

[0043] Example 5: Detection of microsclerotia and melanin in knockout, complementation mutants and wild-type strains. Knockout and complement mutants, as well as wild-type strains, were cultured on BMM medium (0.2 g / L NaNO3, 0.52 g / L KCl, 0.52 g / L MgSO4·7H2O, 1.52 g / L KH2PO4, 3 μmol / L thiamine, 0.1 μmol / L biotin, 5 g / L glucose, 15 g / L agar, pH = 11.5) with a microporous filter membrane for 30 days. (See attached image.) Figure 6 Each experiment was repeated 3 times.

[0044] To compare the changes in melanin gene expression levels among different strains, this experiment used qRT-PCR to determine the expression levels of melanin-related genes in different strains of *Verticillium dahliae*. RNA was extracted from different strains after 7 days of shake culture, and cDNA was obtained by reverse transcription. The expression levels were then analyzed using *Verticillium dahliae*-specific... Vdβt The gene is the target fragment for trihydroxynaphthalene reductase ( VaflM ), Cyclospora dehydratase ( VdSCD ), biopigment synthesis protein Ayg1 ( Vayg1 ) to perform relative quantitative determination (e.g. Figure 6 The information on the quantitative fluorescence primers is shown in Table 2.

[0045] The results showed that the knockout mutant contained trihydroxynaphthalene reductase ( VaflM ), Cyclospora dehydratase ( VdSCD ), biopigment synthesis protein Ayg1 ( Vayg1 The expression level of ) was significantly reduced.

[0046] Table 2. Primer information for quantitative fluorescence detection

[0047] Example 6: Detection of changes in ergosterol content in knockout, complement mutants, and wild-type strains. Spore suspensions of WT, ΔVdAHA1-1, ΔVdAHA1-2, C-ΔVdAHA1-1, and C-ΔVdAHA1-2 were cultured in PDB liquid medium in the dark for 7 days at 25°C and 150 rpm. After filtration through gauze, the filtered spores and hyphae were dried and ground into powder. Approximately 0.5 g of the sample was weighed and added to 0.5 mL of 10% KOH methanol solution. The mixture was ultrasonically extracted for 15 min and then incubated in a water bath at 70°C for 1 h. 0.2 mL of deionized water and 0.5 mL of cyclohexane were added, shaken for 1 min, and centrifuged at 1000 g for 5 min. Another 0.5 mL of cyclohexane was added, shaken for 1 min, and centrifuged at 1000 g for 5 min. 0.5 mL of the cyclohexane layer was collected and concentrated under vacuum at 40°C until dry. The solution was reconstituted with 100 μL of methanol, filtered through a 0.45 μm microporous membrane, and analyzed by HPLC. (See...) Figure 7 Each experiment was repeated three times.

[0048] The results showed that the ergosterol content in the knockout mutant was significantly reduced.

[0049] Example 7 Pathogenicity Identification of Knockout, Completion Mutants and Wild-Type Strains All strains (WT, ΔVdAHA1-1, ΔVdAHA1-2, C-ΔVdAHA1-1, and C-ΔVdAHA1-2) were incubated in Czapek's liquid medium at 25°C and 150 rpm for 5 days, and then diluted to 1 × 10⁻⁶. 7 CFU / mL, used for subsequent experiments.

[0050] Jimian 11 was planted in vermiculite-sand-paper pots. After the first true leaf fully unfolded, the roots were damaged and the seedlings were inoculated with spore suspensions of WT, ΔVdAHA1-1, ΔVdAHA1-2, C-ΔVdAHA1-1, and C-ΔVdAHA1-2, and soaked for 15 minutes. Disease surveys were conducted multiple times 10–25 days after inoculation. Each strain was tested three times individually, with three replicates. Based on the symptoms of cotyledons and true leaves, the number of diseased plants was counted, and seedlings were classified into grades 0, 1, 2, 3, and 4 for disease assessment. The assessment data were calculated using the disease index formula, as follows: The susceptible cultivar Jimian 11 was inoculated with different mutant strains of Verticillium dahliae. After disease onset, the disease severity was evaluated. Results showed that inoculation with knockout mutants was more effective. ΔVdAHA1-1 ,ΔVdAHA1-2 The disease indices were 22.16 ± 0.90 and 23.31 ± 0.70, respectively; the disease index for inoculation with wild-type Verticillium dahliae was 64.22 ± 1.56; the disease index for inoculation with complement mutant was... C-ΔVdAHA1-1 and C-ΔVdAHA1-2 The disease severity indices were 57.18 ± 1.51 and 59.06 ± 1.12, respectively.

[0051] These results indicate that vaccination ΔVdAHA1-1 , ΔVdAHA1-2 The cotton seedlings of this strain showed milder Verticillium wilt symptoms. Figure 8 ).

[0052] 7.1 Browning of the xylem in cotton plants after inoculation To investigate the browning of the xylem in cotton plants inoculated with different bacterial strains, cotton stems approximately 1 cm from the cotyledon level were collected 21 days after disease onset. Longitudinal sections of the same part of the stems from different plants were performed and observed under a stereomicroscope. Figure 8 The experiment was repeated 3 times.

[0053] The results showed that the knockout mutant reduced the damage to the vascular bundles of cotton.

[0054] 7.2 Fungal biomass detection To compare the changes in pathogen biomass in cotton seedlings inoculated with different strains, this experiment used qRT-PCR to determine the biomass of *Verticillium dahliae* from the stems of different cotton seedlings. Total DNA was extracted from the stems of cotton seedlings 20 days after inoculation, using *Verticillium dahliae*-specific DNA as a criterion. Vdβt The gene is the target fragment, and the cotton housekeeping gene is also used. GhAct For household management segments, relatively quantitative measurements were performed (e.g.) Figure 8 ).

[0055] The results showed that: inoculation ΔVdAHA1-1 , ΔVdAHA1-2 In cotton seedlings of the strain Vdβt A significant decrease in gene expression indicates VdAHA1 Gene deletion mutants proliferate and spread more slowly in the roots of the host cotton, resulting in a decrease in fungal colonization.

[0056] 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 the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of Verticillium dahliae heat shock protein 90 ATPase activator VdAHA1 or a fusion protein of VdAHA1 protein, or its encoding gene, or biological materials containing its encoding gene in regulating the pathogenicity of Verticillium dahliae.

2. The application of Verticillium dahliae heat shock protein 90 ATPase activator VdAHA1 or a fusion protein of VdAHA1 protein, or its encoding gene, or biological material containing its encoding gene, in at least one of the following aspects: (1) Regulating the high-temperature adaptability of Verticillium dahliae; (2) Regulating the drug resistance of Verticillium dahliae; (3) Regulate the ergosterol content of Verticillium dahliae; (4) Regulating the environmental adaptability of Verticillium dahliae; (5) Regulate the pathogenicity of Verticillium dahliae to plants; (6) Plant disease control.

3. The application according to claim 1, characterized in that, By regulating the content or activity of the heat shock protein 90 ATPase activator VdAHA1 or its encoding gene in Verticillium dahliae, the pathogenicity, high-temperature adaptability, drug resistance, ergosterol content, environmental adaptability, or pathogenicity to plants of Verticillium dahliae can be controlled.

4. The application according to claim 3, characterized in that, By reducing the content or activity of the heat shock protein 90 ATPase activator VdAHA1 or its encoding gene in Verticillium dahliae, the pathogenicity, high-temperature adaptability, drug resistance, ergosterol content, environmental adaptability, and pathogenicity to plants can be reduced or plant diseases can be controlled.

5. The application according to claim 3, characterized in that, The content or activity of VdAHA1, the heat shock protein 90 ATPase activator of Verticillium dahliae, or its encoding gene can be regulated by transgenic technology or gene editing technology.

6. The application according to claim 3, characterized in that, The high-temperature adaptability refers to high-temperature resistance; and / or, the drug resistance refers to resistance to ketoconazole, itraconazole, or fluconazole; and / or, the pathogenicity to plants refers to pathogenicity to cotton; and / or, the plant disease refers to cotton Verticillium wilt.

7. The application according to any one of claims 1 to 6, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, engineered bacteria, or non-renewable plant cells or tissues. And / or, the fusion protein of the VdAHA1 protein is obtained by attaching a tag, an enzyme cleavage site and / or a linking peptide sequence to the N-terminus and / or C-terminus of the amino acid sequence of the VdAHA1 protein.

8. The application according to claim 1, characterized in that, The amino acid sequence of the *Verticillium dahliae* heat shock protein 90 ATPase activator VdAHA1 is shown in SEQ ID NO.2; And / or, the nucleic acid sequence of the encoding gene is shown in SEQ ID NO.

1.

9. A method for reducing the pathogenicity of *Verticillium dahliae*, reducing its high-temperature adaptability, reducing its drug resistance, reducing its ergosterol content, reducing its environmental adaptability, or reducing its pathogenicity to plants, characterized in that... include: Knockout of heat shock protein 90 ATPase activator in Verticillium dahliae VdAHA1 Gene.

10. A method for controlling cotton Verticillium wilt, characterized in that, include: Knockout of heat shock protein 90 ATPase activator in Verticillium dahliae VdAHA1 Gene.

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